U.S. patent application number 17/665441 was filed with the patent office on 2022-06-23 for biosynthesis of cannabinoids and cannabinoid precursors.
This patent application is currently assigned to Ginkgo Bioworks, Inc.. The applicant listed for this patent is Ginkgo Bioworks, Inc.. Invention is credited to Kim Cecelia Anderson, Elena Brevnova, Dylan Alexander Carlin, Brian Carvalho, Nicholas Flores, Katrina Forrest, Massimo Merighi, Gabriel Rodriguez, Emily E. Wrenbeck.
Application Number | 20220195472 17/665441 |
Document ID | / |
Family ID | |
Filed Date | 2022-06-23 |
United States Patent
Application |
20220195472 |
Kind Code |
A1 |
Anderson; Kim Cecelia ; et
al. |
June 23, 2022 |
BIOSYNTHESIS OF CANNABINOIDS AND CANNABINOID PRECURSORS
Abstract
Aspects of the disclosure relate to biosynthesis of cannabinoids
and cannabinoid precursors in recombinant cells and in vitro.
Inventors: |
Anderson; Kim Cecelia;
(Boston, MA) ; Brevnova; Elena; (Boston, MA)
; Carlin; Dylan Alexander; (Boston, MA) ;
Carvalho; Brian; (Boston, MA) ; Flores; Nicholas;
(Boston, MA) ; Forrest; Katrina; (Boston, MA)
; Merighi; Massimo; (Boston, MA) ; Rodriguez;
Gabriel; (Boston, MA) ; Wrenbeck; Emily E.;
(Boston, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Ginkgo Bioworks, Inc. |
Boston |
MA |
US |
|
|
Assignee: |
Ginkgo Bioworks, Inc.
Boston
MA
|
Appl. No.: |
17/665441 |
Filed: |
February 4, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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17078872 |
Oct 23, 2020 |
11274320 |
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17665441 |
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PCT/US2020/019760 |
Feb 25, 2020 |
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17078872 |
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62810938 |
Feb 26, 2019 |
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62810367 |
Feb 25, 2019 |
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International
Class: |
C12P 17/06 20060101
C12P017/06; C12N 1/16 20060101 C12N001/16; C12N 9/04 20060101
C12N009/04; C12N 9/10 20060101 C12N009/10; C12N 9/88 20060101
C12N009/88; C12N 15/52 20060101 C12N015/52 |
Claims
1.-71. (canceled)
72. A yeast cell for producing a cannabinoid compound or a
cannabinoid precursor that comprises a heterologous polynucleotide
encoding a polyketide synthase (PKS), wherein the PKS comprises an
amino acid sequence that has at least 95% sequence identity to SEQ
ID NO: 7 or wherein the PKS comprises a conservatively substituted
version of SEQ ID NO: 7.
73. The yeast cell of claim 72, wherein relative to the sequence of
SEQ ID NO: 7, the PKS comprises an amino acid substitution at a
residue corresponding to position 28, 34, 50, 70, 71, 76, 88, 100,
151, 203, 219, 285, 359, and/or 385 in SEQ ID NO: 7.
74. The yeast cell of claim 72, wherein the PKS comprises: a) the
amino acid P at a residue corresponding to position 28 in SEQ ID
NO: 7; b) the amino acid Q at a residue corresponding to position
34 in SEQ ID NO: 7; c) the amino acid N at a residue corresponding
to position 50 in SEQ ID NO: 7; d) the amino acid M at a residue
corresponding to position 70 in SEQ ID NO: 7; e) the amino acid Y
at a residue corresponding to position 71 in SEQ ID NO: 7; f) the
amino acid I at a residue corresponding to position 76 in SEQ ID
NO: 7; g) the amino acid A at a residue corresponding to position
88 in SEQ ID NO: 7; h) the amino acid P or T at a residue
corresponding to position 100 in SEQ ID NO: 7; i) the amino acid P
at a residue corresponding to position 151 in SEQ ID NO: 7; j) the
amino acid K at a residue corresponding to position 203 in SEQ ID
NO: 7; k) the amino acid C at a residue corresponding to position
219 in SEQ ID NO: 7; l) the amino acid A at a residue corresponding
to position 285 in SEQ ID NO: 7; m) the amino acid M at a residue
corresponding to position 359 in SEQ ID NO: 7; and/or n) the amino
acid M at a residue corresponding to position 385 in SEQ ID NO:
7.
75. The yeast cell of claim 72, wherein the PKS comprises one or
more of the following amino acid substitutions relative to SEQ ID
NO: 7: V71Y and/or F70M.
76. The yeast cell of claim 72, wherein the PKS comprises: a) C at
a residue corresponding to position 164 in SEQ ID NO: 7; b) H at a
residue corresponding to position 304 in SEQ ID NO: 7; and/or c) N
at a residue corresponding to position 337 in SEQ ID NO: 7.
77. The yeast cell of 72, wherein the heterologous polynucleotide
comprises a nucleotide sequence that has at least 95% sequence
identity to SEQ ID NO: 38 or 176.
78. The yeast cell of claim 72, wherein the yeast cell is a
Saccharomyces cell, a Yarrowia cell, a Pichia cell or a
Komagataella cell.
79. The yeast cell of claim 78, wherein the yeast cell is a
Saccharomyces cell.
80. The yeast cell of claim 79, wherein the Saccharomyces cell is a
Saccharomyces cerevisiae cell.
81. The yeast cell of claim 78, wherein the yeast cell is a
Yarrowia cell.
82. The yeast cell of claim 72, wherein the yeast cell further
comprises one or more heterologous polynucleotides encoding one or
more of: an acyl activating enzyme (AAE), a polyketide cyclase
(PKC), a prenyltransferase (PT), and/or a terminal synthase
(TS).
83. The yeast cell of claim 72, wherein the yeast cell is capable
of producing a cannabinoid compound or a cannabinoid precursor,
wherein the cannabinoid compound is a compound of Formulas 8, 9,
10, or 11: ##STR00222## or a pharmaceutically acceptable salt,
solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer,
isotopically labeled derivative, or prodrug thereof; wherein the
cannabinoid precursor is a compound of Formulas 4, 5, or 6:
##STR00223## or a pharmaceutically acceptable salt, solvate,
hydrate, polymorph, co-crystal, tautomer, stereoisomer,
isotopically labeled derivative, or prodrug thereof; wherein R is
straight-chain unsubstituted C.sub.1-20 alkyl,
84. The yeast cell of claim 72, wherein the yeast cell is capable
of producing 3,5,7-trioxododecanoyl-CoA, olivetol, olivetolic acid,
cannabigerolic acid (8.alpha.), cannabidiolic acid (9a),
tetrahydrocannabinolic acid (10.alpha.), and/or cannabichromenic
acid (11a).
85. The yeast cell of claim 84, wherein the yeast cell is capable
of producing more 3,5,7-trioxododecanoyl-CoA, olivetol, and/or
olivetolic acid than a yeast cell that expresses a PKS that
comprises SEQ ID NO: 5.
86. The yeast cell of claim 82, wherein the PKC is an olivetolic
acid cyclase (OAC).
87. The yeast cell of claim 82, wherein the PT is a cannabigerolic
acid synthase (CBGAS).
88. The yeast cell of claim 82, wherein the TS is a
tetrahydrocannabinolic acid synthase (THCAS), a cannabidiolic acid
synthase (CBDAS), and/or a cannabichromenic acid synthase
(CBCAS).
89. The yeast cell of claim 72, wherein the PKS comprises SEQ ID
NO: 7.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of International
Application Serial Number PCT/US2020/019760, filed Feb. 25, 2020,
entitled "BIOSYNTHESIS OF CANNABINOIDS AND CANNABINOID PRECURSORS,"
which claims the benefit under 35 U.S.C. .sctn. 119(e) of U.S.
Provisional Application Ser. No. 62/810,367, filed Feb. 25, 2019,
entitled "BIOSYNTHESIS OF CANNABINOIDS AND CANNABINOID PRECURSORS,"
and U.S. Provisional Application Ser. No. 62/810,938, filed Feb.
26, 2019, entitled "BIOSYNTHESIS OF CANNABINOIDS AND CANNABINOID
PRECURSORS," the disclosure of each of which is incorporated by
reference herein in its entirety.
REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA
EFS-WEB
[0002] The instant application contains a Sequence Listing which
has been submitted in text format via EFS-Web and is hereby
incorporated by reference in its entirety. Said text copy, created
on Oct. 23, 2020, is named G091970030US07-SEQ-FL.txt and is
1,806,581 bytes in size.
FIELD OF INVENTION
[0003] The present disclosure relates to the biosynthesis of
cannabinoids and cannabinoid precursors in recombinant cells.
BACKGROUND
[0004] Cannabinoids are chemical compounds that may act as ligands
for endocannabinoid receptors and have multiple medical
applications. Traditionally, cannabinoids have been isolated from
plants of the genus Cannabis. The use of plants for producing
cannabinoids is inefficient, however, with isolated products
restricted to the primary endogenous Cannabis compounds, and the
cultivation of Cannabis plants is restricted in many jurisdictions.
Cannabinoids can also be produced through chemical synthesis (see,
e.g., U.S. Pat. No. 7,323,576 to Souza et al). However, such
methods suffer from low yields and high cost. Production of
cannabinoids, cannabinoid analogs, and cannabinoid precursors using
engineered organisms may provide an advantageous approach to meet
the increasing demand for these compounds.
SUMMARY
[0005] Aspects of the present disclosure provide methods for
production of cannabinoids and cannabinoid precursors from fatty
acid substrates using genetically modified host cells.
[0006] Aspects of the present disclosure provide host cells that
comprises a heterologous polynucleotide encoding a polyketide
synthase (PKS), wherein the PKS comprises a sequence that is at
least 90% identical to SEQ ID NO: 7. In some embodiments, relative
to the sequence of SEQ ID NO: 7, the PKS comprises an amino acid
substitution at a residue corresponding to position 34, 50, 70, 71,
76, 100, 151, 203, 219, 285, 359, and/or 385 in SEQ ID NO: 7. In
some embodiments, the PKS comprises: the amino acid Q at a residue
corresponding to position 34 in SEQ ID NO: 7; the amino acid N at a
residue corresponding to position 50 in SEQ ID NO: 7; the amino
acid M at a residue corresponding to position 70 in SEQ ID NO: 7;
the amino acid Y at a residue corresponding to position 71 in SEQ
ID NO: 7; the amino acid I at a residue corresponding to position
76 in SEQ ID NO: 7; the amino acid P or T at a residue
corresponding to position 100 in SEQ ID NO: 7; the amino acid P at
a residue corresponding to position 151 in SEQ ID NO: 7; the amino
acid K at a residue corresponding to position 203 in SEQ ID NO: 7;
the amino acid C at a residue corresponding to position 219 in SEQ
ID NO: 7; the amino acid A at a residue corresponding to position
285 in SEQ ID NO: 7; the amino acid M at a residue corresponding to
position 359 in SEQ ID NO: 7; and/or the amino acid M at a residue
corresponding to position 385 in SEQ ID NO: 7.
[0007] In some embodiments, the PKS is capable of producing:
a) a compound of Formula (4):
##STR00001##
b) a compound of Formula (5):
##STR00002##
c) a compound of Formula (6):
##STR00003##
[0008] In some embodiments,
a) the compound of Formula (4) is the compound for Formula
(4a):
##STR00004##
b) the compound of Formula (5) is the compound for Formula
(5a):
##STR00005##
[0009] and/or
c) the compound of Formula (6) is the compound of Formula (6a):
##STR00006##
[0010] In some embodiments, the host cell produces more of a
compound of Formula (5) than a host cell that comprises a
heterologous polynucleotide encoding a PKS that comprises the
sequence of SEQ ID NO: 7. In some embodiments, the PKS comprises
one or more of the following amino acid substitutions relative to
SEQ ID NO: 7: V71Y and F70M. In some embodiments, the PKS
comprises: C at a residue corresponding to position 164 in SEQ ID
NO: 7; H at a residue corresponding to position 304 in SEQ ID NO:
7; and/or N at a residue corresponding to position 337 in SEQ ID
NO: 7. In some embodiments, the PKS comprises SEQ ID NO: 7. In some
embodiments, the PKS comprises SEQ ID NO: 15 or 145. In some
embodiments, the heterologous polynucleotide comprises a sequence
that is at least 90% identical to SEQ ID NOs: 38 or 176.
[0011] Aspects of the present disclosure relate to host cell that
comprises a heterologous polynucleotide encoding a polyketide
synthase (PKS), wherein the PKS comprises a sequence that is at
least 90% identical to SEQ ID NO: 714. In some embodiments, the PKS
is capable of producing:
[0012] a. a compound of Formula (4):
##STR00007##
[0013] b. a compound of Formula (5):
##STR00008##
[0014] c. a compound of Formula (6):
##STR00009##
[0015] In some embodiments,
[0016] a) the compound of Formula (4) is the compound for Formula
(4a):
##STR00010##
[0017] b) the compound of Formula (5) is the compound for Formula
(5a):
##STR00011##
[0018] and/or
[0019] c) the compound of Formula (6) is the compound of Formula
(6a):
##STR00012##
[0020] Further aspects of the present disclosure provide host cells
that comprises a heterologous polynucleotide encoding a polyketide
synthase (PKS), wherein relative to the sequence of SEQ ID NO: 5
the PKS comprises one or more amino acid substitutions within the
active site of the PKS, and wherein the host cell is capable of
producing a compound of Formula (4), (5), or (6).
[0021] In some embodiments, relative to SEQ ID NO: 5, the PKS
comprises an amino acid substitution at one or more of the
following positions in SEQ ID NO: 5: 17, 23, 25, 51, 54, 64, 95,
123, 125, 153, 196, 201, 207, 241, 247, 267, 273, 277, 296, 307,
320, 324, 326, 328, 334, 335C, and 375. In some embodiments,
relative to SEQ ID NO:5, the PKS comprises: T17K, I23C, L25R, K51R,
D54R, F64Y, V95A, T123C, A125S, Y153G, E196K, L201C, I207L, L241I,
T247A, M267K, M267G, 1273V, L277M, T296A, V307I, D320A, V324I,
S326R, H328Y, S334P, S334A, T335C, R375T, or any combination
thereof. In some embodiments, relative to SEQ ID NO: 5, the PKS
further comprises an amino acid substitution at one or more of the
following positions in SEQ ID NO: 5: 284, 100, 116, 278, 108, 348,
71, 92, 128, 100, 135, 229, 128, and 128. In some embodiments,
relative to SEQ ID NO:5, the PKS comprises: I284Y, K100L, K116R,
I278E, K108D, L348S, K71R, V92G, T128V, K100M, Y135V, P229A, T128A,
T128I, or any combination thereof. In some embodiments,
[0022] a) the compound of Formula (4) is the compound of Formula
(4a):
##STR00013##
[0023] b) the compound of Formula (5) is the compound of Formula
(5a):
##STR00014##
[0024] and/or
[0025] c) the compound of Formula (6) is the compound of Formula
(6a):
##STR00015##
[0026] In some embodiments, the host cell produces more of a
compound of Formula (5) than a host cell that comprises a
heterologous polynucleotide encoding a PKS that comprises the
sequence of SEQ ID NO: 5. In some embodiments, the PKS comprises at
least 90% to any one SEQ ID NOs: 207-249. In some embodiments, the
heterologous polynucleotide comprises a sequence that is at least
90% identical to SEQ ID NOs: 250-292.
[0027] Further aspects of the present disclosure relate to host
cells that comprises a heterologous polynucleotide encoding a
polyketide synthase (PKS), wherein relative to the sequence of SEQ
ID NO: 5 the PKS comprises the amino acid substitution T335C. In
some embodiments, the PKS is at least 90% identical to SEQ ID NO:
207. In some embodiments, the PKS comprises SEQ ID NO: 207.
[0028] Further aspects of the present disclosure relate to host
cells that comprises a heterologous polynucleotide encoding a
polyketide synthase (PKS), wherein the PKS comprises the amino acid
C at a residue corresponding to position 335 of SEQ ID NO: 5, and
wherein the host cell is capable of producing more of a compound of
Formula (5) than a host cell that comprises a heterologous
polynucleotide encoding a PKS comprising SEQ ID NO: 5.
[0029] In some embodiments, the PKS comprises a sequence that is at
least 90% identical to any one of SEQ ID NOs: 7, 13, 145, 8, and
15. In some embodiments, the PKS comprises a sequence that is at
least 90% identical to SEQ ID NO: 5. In some embodiments, the
compound of Formula (5) is the compound of Formula (5a):
##STR00016##
[0030] In some embodiments, the heterologous polynucleotide
comprises a sequence that is at least 90% identical to SEQ ID NO:
250 or 706.
[0031] Further aspects of the present disclosure provide host cells
that comprises a heterologous polynucleotide encoding a polyketide
synthase (PKS), wherein the PKS is capable of reacting a compound
of Formula (2) with a compound of Formula (3):
##STR00017##
to produce a compound of Formula (6):
##STR00018##
[0032] In some embodiments, the PKS comprises a sequence that is at
least 90% identical to SEQ ID NO: 6. In some embodiments, the PKS
comprises the amino acid W at a residue corresponding to position
339 of SEQ ID NO: 6. In some embodiments, the PKS comprises: C at a
residue corresponding to position 164 in SEQ ID NO: 6; H at a
residue corresponding to position 304 in SEQ ID NO: 6; and/or N at
a residue corresponding to position 337 in SEQ ID NO: 6. In some
embodiments, the PKS is capable of producing:
a compound of Formula (4):
##STR00019##
[0033] b) or a compound of Formula (5):
##STR00020##
[0034] In some embodiments,
[0035] the compound of Formula (6) is a compound for Formula
(6a):
##STR00021##
[0036] In some embodiments,
[0037] a) the compound of Formula (4) is a compound for Formula
(4a):
##STR00022##
[0038] b) the compound of Formula (5) is a compound for Formula
(5a):
##STR00023##
[0039] In some embodiments,
[0040] a. the compound of Formula (2) is a compound of Formula
(2a):
##STR00024##
[0041] b. the compound of Formula (3) is a compound of Formula
(3a):
##STR00025##
[0042] In some embodiments, the host cell produces a ratio of
compound (6) to compound (5) that is higher than the ratio produced
by a host cell that comprises a heterologous polynucleotide
encoding a PKS that comprises the sequence of SEQ ID NO: 6. In some
embodiments, the PKS comprises SEQ ID NO: 6. In some embodiments,
the heterologous polynucleotide comprises a sequence that is at
least 90% identical to SEQ ID NO: 37 or 186.
[0043] Further aspects of the disclosure provide host cells that
comprises a heterologous polynucleotide encoding an acyl activating
enzyme (AAE), wherein the AAE comprising a sequence that is at
least 90% identical to a sequence selected from SEQ ID NOs: 63-69,
141-142, and 707-708.
[0044] Further aspects of the disclosure provide host cell that
comprises a heterologous polynucleotide comprising a sequence that
is at least 90% identical to a sequence selected from SEQ ID NOs:
70-76 and 712-713.
[0045] Further aspects of the disclosure provide host cells that
comprises a heterologous polynucleotide encoding an acyl activating
enzyme (AAE), wherein the AAE comprises: the amino acid sequence
SGAAPLG (SEQ ID NO: 114); the amino acid sequence AYLGMSSGTSGG (SEQ
ID NO: 115); the amino acid sequence DQPA (SEQ ID NO: 116); the
amino acid sequence QVAPAELE (SEQ ID NO: 117); the amino acid
sequence VVID (SEQ ID NO: 118); and/or the amino acid sequence
SGKILRRLLR (SEQ ID NO: 119). In some embodiments, the host cell
produces at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or
100% more hexanoyl-coenzyme A in the presence of hexanoic acid and
Coenzyme A relative to a recombinant host cell that does not
comprise a heterologous gene encoding an AAE and/or the host cell
produces at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or
100% more butanoyl-coenzyme A in the presence of butyric acid and
Coenzyme A relative to a recombinant host cell that does not
comprise a heterologous gene encoding an AAE.
[0046] In some embodiments, the AAE comprises: the amino acid
sequence SGAAPLG (SEQ ID NO: 114) at residues corresponding to
positions 319-325 in SEQ ID NO:64; the amino acid sequence
AYLGMSSGTSGG (SEQ ID NO: 115) at residues corresponding to
positions 194-205 in SEQ ID NO:64; the amino acid sequence DQPA
(SEQ ID NO: 116) at residues corresponding to positions 398-401 in
SEQ ID NO:64; the amino acid sequence QVAPAELE (SEQ ID NO: 117) at
residues corresponding to positions 495-502 in SEQ ID NO:64; the
amino acid sequence VVID (SEQ ID NO: 118) at residues corresponding
to positions 564-567 in SEQ ID NO:64; and/or the amino acid
sequence SGKILRRLLR (SEQ ID NO: 119) at residues corresponding to
positions 574-583 in SEQ ID NO:64.
[0047] Further aspects of the disclosure provide host cells that
comprises a heterologous polynucleotide encoding an acyl activating
enzyme (AAE), wherein the AAE comprises: an amino acid sequence
with no more than three amino acid substitutions at residues
corresponding to positions 428-440 in SEQ ID NO:64; or an amino
acid sequence with no more than one amino acid substitution at
residues corresponding to positions 482-491 in SEQ ID NO:64,
wherein the host cell produces at least 10%, 20%, 30%, 40%, 50%,
60%, 70%, 80%, 90%, or 100% more hexanoyl-coenzyme A in the
presence of hexanoic acid and Coenzyme A relative to a recombinant
host cell that does not comprise a heterologous gene encoding an
AAE; and/or wherein the host cell produces at least 10%, 20%, 30%,
40%, 50%, 60%, 70%, 80%, 90%, or 100% more butanoyl-coenzyme A in
the presence of butyric acid and Coenzyme A relative to a
recombinant host cell that does not comprise a heterologous gene
encoding an AAE.
[0048] In some embodiments, the AAE comprises: I or V at a residue
corresponding to position 432 in SEQ ID NO:64; S or D at a residue
corresponding to position 434 in SEQ ID NO:64; K or N at a residue
corresponding to position 438 in SEQ ID NO:64; and/or L or M at a
residue corresponding to position 488 in SEQ ID NO:64. In some
embodiments, the AAE comprises: the amino acid sequence
RGPQIMSGYHKNP (SEQ ID NO: 120); the amino acid sequence
RGPQVMDGYHNNP (SEQ ID NO: 121); the amino acid sequence
RGPQIMDGYHKNP (SEQ ID NO: 122); the amino acid sequence VDRTKELIKS
(SEQ ID NO: 123); and/or the amino acid sequence VDRTKEMIKS (SEQ ID
NO: 124). In some embodiments, the AAE comprises a sequence that is
at least 90% identical to a sequence selected from SEQ ID NOs:
63-69, 141-142, and 707-708. In some embodiments, the AAE comprises
at least one conservative substitution relative to the sequence of
SEQ ID NO:64. In some embodiments, the host cell further comprises
one or more heterologous polynucleotides encoding one or more of: a
polyketide synthase (PKS), a polyketide cyclase (PKC); a
prenyltransferase (PT); and/or a terminal synthase (TS).
[0049] Further aspects of the present disclosure provide methods
comprising culturing any of the host cells of the disclosure. In
some embodiments, the host cell is cultured in media comprising
sodium hexanoate. In some embodiments, the host cell is a plant
cell, an algal cell, a yeast cell, a bacterial cell, or an animal
cell. In certain embodiments, the host cell is a yeast cell. In
some embodiments, the yeast cell is a Saccharomyces cell, a
Yarrowia cell, a Pichia cell or a Komagataella cell. In certain
embodiments, wherein the Saccharomyces cell is a Saccharomyces
cerevisiae cell. In some embodiments, the host cell is a bacterial
cell. In certain embodiments, the bacterial cell is an E. coli
cell. In some embodiments, the host cell further comprises one or
more heterologous polynucleotides encoding one or more of: an acyl
activating enzyme (AAE), a polyketide cyclase (PKC), a
prenyltransferase (PT), and/or a terminal synthase (TS).
[0050] Further aspects of the present disclosure provide
non-naturally occurring nucleic acid encoding a polyketide synthase
(PKS), wherein the non-naturally occurring nucleic acid comprises
at least 90% identity to SEQ ID NOs: 32-62, 93-108, 172-206,
250-292, 421-548, 628-705 or 706. Further aspects of the present
disclosure provide vectors comprising non-naturally occurring
nucleic acids of the disclosure. Further aspects of the present
disclosure provide expression cassettes comprising non-naturally
occurring nucleic acids of the disclosure. Further aspects of the
present disclosure provide host cells transformed with
non-naturally occurring nucleic acids, vector, or expression
cassettes of the present disclosure. Further aspects of the present
disclosure provide host cells that comprise non-naturally occurring
nucleic acids, vector, or expression cassettes of the present
disclosure.
[0051] Each of the limitations of the invention can encompass
various embodiments of the invention. It is, therefore, anticipated
that each of the limitations of the invention involving any one
element or combinations of elements can be included in each aspect
of the invention. This disclosure is not limited in its application
to the details of construction and the arrangement of components
set forth in the following description or illustrated in the
drawings. The invention is capable of other embodiments and of
being practiced or of being carried out in various ways. Also, the
phraseology and terminology used in this application is for the
purpose of description and should not be regarded as limiting. The
use of "including," "comprising," or "having," "containing,"
"involving," and variations thereof, is meant to encompass the
items listed thereafter and equivalents thereof as well as
additional items.
BRIEF DESCRIPTION OF DRAWINGS
[0052] The accompanying drawings are not intended to be drawn to
scale. In the drawings, each identical or nearly identical
component that is illustrated in various figures is represented by
a like numeral. For purposes of clarity, not every component may be
labeled in every drawing. In the drawings:
[0053] FIG. 1 is a schematic depicting the native Cannabis
biosynthetic pathway for production of cannabinoid compounds,
including five enzymatic steps mediated by: (R1a) acyl activating
enzymes (AAE); (R2a) olivetol synthase enzymes (OLS); (R3a)
olivetolic acid cyclase enzymes (OAC); (R4a) cannabigerolic acid
synthase enzymes (CBGAS); and (R5a) terminal synthase enzymes (TS).
Formulae 1a-11a correspond to hexanoic acid (1a), hexanoyl-CoA
(2a), malonyl-CoA (3a), 3,5,7-trioxododecanoyl-CoA (4a), olivetol
(5a), olivetolic acid (6a), geranyl pyrophosphate (7a),
cannabigerolic acid (8a), cannabidiolic acid (9a),
tetrahydrocannabinolic acid (10a), and cannabichromenic acid (11a).
Hexanoic acid is an exemplary carboxylic acid substrate; other
carboxylic acids may also be used (e.g., butyric acid, isovaleric
acid, octanoic acid, decanoic acid, etc.; see e.g., FIG. 3 below).
The enzymes that catalyze the synthesis of
3,5,7-trioxododecanoyl-CoA and olivetolic acid are shown in R2a and
R3a, respectively, and can include multi-functional enzymes that
catalyze the synthesis of 3,5,7-trioxododecanoyl-CoA and olivetolic
acid. The enzymes cannabidiolic acid synthase (CBDAS),
tetrahydrocannabinolic acid synthase (THCAS), and cannabichromenic
acid synthase (CBCAS) that catalyze the synthesis of cannabidiolic
acid, tetrahydrocannabinolic acid, and cannabichromenic acid,
respectively, are shown in step R5a. FIG. 1 is adapted from
Carvalho et al. "Designing Microorganisms for Heterologous
Biosynthesis of Cannabinoids" (2017) FEMS Yeast Research June 1;
17(4), which is incorporated by reference in its entirety.
[0054] FIG. 2 is a schematic depicting a heterologous biosynthetic
pathway for production of cannabinoid compounds, including five
enzymatic steps mediated by: (R1) acyl activating enzymes (AAE);
(R2) polyketide synthase enzymes (PKS) or bifunctional polyketide
synthase-polyketide cyclase enzymes (PKS-PKC); (R3) polyketide
cyclase enzymes (PKC) or bifunctional PKS-PKC enzymes; (R4)
prenyltransferase enzymes (PT); and (R5) terminal synthase enzymes
(TS). Any carboxylic acid of varying chain lengths, structures
(e.g., aliphatic, alicyclic, or aromatic) and functionalization
(e.g., hydroxylic-, keto-, amino-, thiol-, aryl-, or alogeno-) may
also be used as precursor substrates (e.g., thiopropionic acid,
hydroxy phenyl acetic acid, norleucine, bromodecanoic acid, butyric
acid, isovaleric acid, octanoic acid, decanoic acid, etc).
[0055] FIG. 3 is a non-exclusive representation of select putative
precursors for the cannabinoid pathway in FIG. 2.
[0056] FIG. 4 is a graph showing activity of E. coli strains
expressing candidate AAEs as measured by a
5,5'-dithiobis-(2-nitrobenzoic acid) ("DTNB") assay. Lysates of E.
coli expressing candidate AAEs were assayed for ligation activity
of free CoA to either butyrate or hexanoate. Activity was
quantified by measuring the decrease in absorbance at 412 nm,
corresponding to a decrease of free CoA in solution. Error bars
represent the standard deviation of 2 independent measurements.
Negative control strain t49568 expresses an aldehyde dehydrogenase
protein from Y. lipolytica (corresponding to Uniprot ID
Q6C5T1).
[0057] FIGS. 5A-5B show a plasmid used to express AAE and OLS
proteins in S. cerevisiae. The coding sequence for the enzyme being
expressed (labeled "Library gene") is driven by the GAL1 promoter.
The plasmid contains markers for both yeast (URA3) and bacteria
(ampR), as well as origins of replication for yeast (2 micron),
bacteria (pBR322), and phage (f1).
[0058] FIG. 6 is a graph showing activity of S. cerevisiae strains
expressing candidate AAEs as measured by a DTNB assay. Lysates of
S. cerevisiae expressing candidate AAEs were assayed for ligation
activity of free CoA to hexanoate. Activity was quantified by
measuring the decrease in absorbance at 412 nm, corresponding to a
decrease of free CoA in solution. Error bars represent the standard
deviation of 3 independent measurements. Negative control strain
t390338 expresses GFP.
[0059] FIGS. 7A-7C show a sequence alignment of acyl activating
enzymes (AAEs). An alignment of t51477 (SEQ ID NO: 65), t49578 (SEQ
ID NO: 63), t49594 (SEQ ID NO: 64), t392878 (SEQ ID NO: 68),
t392879 (SEQ ID NO: 69), t55127 (SEQ ID NO: 66), and t55128 (SEQ ID
NO: 67) is shown. The sequence alignment was conducted using
Clustal Omega. See, e.g., Chojnacki et al., Nucleic Acids Res. 2017
Jul. 3; 45(W1):W550-W553.
[0060] FIG. 8 is a graph showing olivetol production by S.
cerevisiae strains expressing OLS candidate enzymes. Peak areas
obtained via LC/MS quantification were normalized to an internal
standard for olivetol. Normalized peak areas were further
normalized to a positive control strain (t339582) contained on each
plate. As explained in Example 2 and in Table 6, OLS candidate
enzymes within the library depicted in this Figure, and the
positive control OLSs depicted in this Figure, were later found to
contain a deletion in the nucleotide sequence encoding the OLS
proteins, which led to the production of truncated proteins.
Accordingly, all candidate OLS enzymes in this library, and the
positive controls, were also tested independently in a new library
containing only full-length OLS sequences, described in Example
3.
[0061] FIG. 9 is a graph showing olivetolic acid (OA) production by
S. cerevisiae strains expressing OLS candidate enzymes. Peak areas
obtained via LC/MS quantification were normalized to an internal
standard for OA. Normalized peak areas were further normalized to a
positive control strain (t339582) contained on each plate. As
explained in Example 2 and in Table 6, OLS candidate enzymes within
the library depicted in this Figure, and the positive control OLSs
depicted in this Figure, were later found to contain a deletion in
the nucleotide sequence encoding the OLS proteins, which led to the
production of truncated proteins. Accordingly, all candidate OLS
enzymes in this library, and the positive controls, were also
tested independently in a new library containing only full-length
OLS sequences, described in Example 3.
[0062] FIG. 10 is a graph showing normalized OA versus olivetol
production by S. cerevisiae strains expressing OLS candidate
enzymes. Peak areas obtained via LC/MS quantification for olivetol
and OA were normalized to an internal standard for olivetol or OA,
respectively. The regression line shown in FIG. 10 represents a 1:1
ratio of olivetol and olivetolic acid. Normalized peak areas were
further normalized to positive control strain (t339582) contained
on each plate. The strain t395094 demonstrated significantly
increased olivetol production compared to the positive controls,
while the strain t393974 showed enhanced production of OA over the
positive control strains. The enhanced production of OA over
olivetol by t393974 suggests that this enzyme possesses
bifunctional PKS-PKC activity. As explained in Example 2 and in
Table 6, OLS candidate enzymes within the library depicted in this
Figure, and the positive control OLSs depicted in this Figure, were
later found to contain a deletion in the nucleotide sequence
encoding the OLS proteins, which led to the production of truncated
proteins. Accordingly, all candidate OLS enzymes in this library,
and the positive controls, were also tested independently in a new
library containing only full-length OLS sequences, described in
Example 3.
[0063] FIGS. 11A-11H show a sequence alignment of olivetol
synthases (OLSs). An alignment of t394911 (SEQ ID NO: 28), t393974
(SEQ ID NO: 6), t393720 (SEQ ID NO: 27), t394336 (SEQ ID NO: 8),
t393991 (SEQ ID NO: 7), t395011 (SEQ ID NO: 15), t339568 (SEQ ID
NO: 5), t339579 (SEQ ID NO: 30), t339582 (SEQ ID NO: 31), t394457
(SEQ ID NO: 10), t394521 (SEQ ID NO: 11), t394436 (SEQ ID NO: 26),
t395094 (SEQ ID NO: 17), t394087 (SEQ ID NO: 1), t395023 (SEQ ID
NO: 29), t395103 (SEQ ID NO: 18), t394687 (SEQ ID NO: 2), t393835
(SEQ ID NO: 19), t394037 (SEQ ID NO: 22), t394905 (SEQ ID NO: 13),
t393563 (SEQ ID NO: 4), t394981 (SEQ ID NO: 14), t394790 (SEQ ID
NO: 12), t394797 (SEQ ID NO: 16), t394091 (SEQ ID NO: 21), t394043
(SEQ ID NO: 24), t394404 (SEQ ID NO: 25), t393495 (SEQ ID NO: 3),
t394547 (SEQ ID NO: 9), t394115 (SEQ ID NO: 20), and t394279 (SEQ
ID NO: 23) is shown. The sequence alignment was conducted using
Clustal Omega. See, e.g., Chojnacki et al., Nucleic Acids Res. 2017
Jul. 3; 45(W1):W550-W553.
[0064] FIG. 12A-12B are graphs showing olivetol production (FIG.
12A) and olivetolic acid production (FIG. 12B) from S. cerevisiae
strains expressing OLS candidate enzymes. Strains shown were
determined to be hits from the primary screen of the library of OLS
candidates screened in Example 3.
[0065] FIG. 13 is a graph showing olivetol production by S.
cerevisiae strains expressing C. sativa OLS (CsOLS) point-mutant
variants. Concentrations of olivetol in .mu.g/L were determined via
LC/MS quantification. The strain t405417 (having a T335C point
mutation relative to the CsOLS set forth in SEQ ID NO: 5)
demonstrated the highest olivetol production. Error bars represent
the standard deviation of 4 independent measurements.
[0066] FIGS. 14A-14B are graphs showing olivetol production from S.
cerevisiae strains expressing single point mutation and multiple
point mutation variants based on a Cymbidium hybrid cultivar OLS
template (ChOLS) (FIG. 14A) and a Corchorus olitorius OLS (CoOLS)
template (FIG. 14B). Strains shown were screened in a secondary
screen as described in Example 5. Olivetol titers were normalized
to the mean olivetol titer produced by the positive control strain
t527346 (FIG. 14A), and t606797 (FIG. 14B).
[0067] FIG. 15 is a graph showing olivetol production by a
prototrophic S. cerevisiae strain expressing candidate OLS enzymes.
Concentrations of olivetol in .mu.g/L were determined via LC/MS
quantification. Performance of OLS candidate enzymes exhibiting
higher olivetol production than C. sativa OLS positive controls is
shown. The strains t485662, t485672, and t496073 demonstrated
comparable olivetol production to the CsOLS T335C point-mutant
positive control.
[0068] FIG. 16 is a three-dimensional homology model showing
residues within about 8 angstroms of any of the residues within the
catalytic triad of the C. sativa OLS comprising SEQ ID NO: 5 and/or
within about 8 angstroms of a docked substrate within the C. sativa
OLS comprising SEQ ID NO: 5. Only residues at which an amino acid
substitution resulted in production of at least 10 mg/L olivetol
are shown with their electron clouds in light gray. The active site
was defined to include a docked molecule of hexanoyl-CoA (OLS
substrate) plus the catalytic triad. The top model was rotated 900
to produce the bottom model.
[0069] FIG. 17 is a three-dimensional homology model showing
residues within about 12 angstroms of any of the residues within
the catalytic triad of the C. sativa OLS comprising SEQ ID NO: 5
and/or within about 12 angstroms of a docked substrate within the
C. sativa OLS comprising SEQ ID NO: 5. Only residues at which an
amino acid substitution resulted in production of at least 10 mg/L
olivetol are shown with their electron clouds. The active site was
defined to include a docked molecule of hexanoyl-CoA (OLS
substrate) plus the catalytic triad. The top model was rotated
90.degree. to produce the bottom model.
DETAILED DESCRIPTION
[0070] This disclosure provides methods for production of
cannabinoids and cannabinoid precursors from fatty acid substrates
using genetically modified host cells. Methods include heterologous
expression of enzymes including acyl activating enzymes (AAE) and
polyketide synthase enzymes (PKS) such as olivetol synthase enzymes
(OLS). The disclosure describes identification of AAE and OLS
enzymes that can be functionally expressed in eukaryotic (e.g., S.
cerevisiae) and prokaryotic (E. coli) host cells such as S.
cerevisiae and E. coli. As demonstrated in Example 1, novel AAE
enzymes were identified that are capable of using hexanoate and
butyrate as substrates to produce cannabinoid precursors. As
demonstrated in Examples 2-3, novel OLS enzymes were identified
that are capable of producing olivetol and olivetolic acid.
Examples 4-6 further demonstrate enhanced production of olivetol
and/or olivetolic acid by protein engineering of OLS enzymes. The
novel enzymes described in this disclosure may be useful in
increasing the efficiency and purity of cannabinoid production.
Definitions
[0071] While the following terms are believed to be well understood
by one of ordinary skill in the art, the following definitions are
set forth to facilitate explanation of the disclosed subject
matter.
[0072] The term "a" or "an" refers to one or more of an entity,
i.e., can identify a referent as plural. Thus, the terms "a" or
"an," "one or more" and "at least one" are used interchangeably in
this application. In addition, reference to "an element" by the
indefinite article "a" or "an" does not exclude the possibility
that more than one of the elements is present, unless the context
clearly requires that there is one and only one of the
elements.
[0073] The terms "microorganism" or "microbe" should be taken
broadly. These terms are used interchangeably and include, but are
not limited to, the two prokaryotic domains, Bacteria and Archaea,
as well as certain eukaryotic fungi and protists. In some
embodiments, the disclosure may refer to the "microorganisms" or
"microbes" of lists/tables and figures present in the disclosure.
This characterization can refer to not only the identified
taxonomic genera of the tables and figures, but also the identified
taxonomic species, as well as the various novel and newly
identified or designed strains of any organism in the tables or
figures. The same characterization holds true for the recitation of
these terms in other parts of the specification, such as in the
Examples.
[0074] The term "prokaryotes" is recognized in the art and refers
to cells that contain no nucleus or other cell organelles. The
prokaryotes are generally classified in one of two domains, the
Bacteria and the Archaea.
[0075] "Bacteria" or "eubacteria" refers to a domain of prokaryotic
organisms. Bacteria include at least 11 distinct groups as follows:
(1) Gram-positive (gram+) bacteria, of which there are two major
subdivisions: (a) high G+C group (Actinomycetes, Mycobacteria,
Micrococcus, others) and (b) low G+C group (Bacillus, Clostridia,
Lactobacillus, Staphylococci, Streptococci, Mycoplasmas); (2)
Proteobacteria, e.g., Purple photosynthetic+non-photosynthetic
Gram-negative bacteria (includes most "common" Gram-negative
bacteria); (3) Cyanobacteria, e.g., oxygenic phototrophs; (4)
Spirochetes and related species; (5) Planctomyces; (6) Bacteroides,
Flavobacteria; (7) Chlamydia; (8) Green sulfur bacteria; (9) Green
non-sulfur bacteria (also anaerobic phototrophs); (10)
Radioresistant micrococci and relatives; and (11) Thermotoga and
Thermosipho thermophiles.
[0076] The term "Archaea" refers to a taxonomic classification of
prokaryotic organisms with certain properties that make them
distinct from Bacteria in physiology and phylogeny.
[0077] The term "Cannabis" refers to a genus in the family
Cannabaceae. Cannabis is a dioecious plant. Glandular structures
located on female flowers of Cannabis, called trichomes, accumulate
relatively high amounts of a class of terpeno-phenolic compounds
known as phytocannabinoids (described in further detail below).
Cannabis has conventionally been cultivated for production of fibre
and seed (commonly referred to as "hemp-type"), or for production
of intoxicants (commonly referred to as "drug-type"). In drug-type
Cannabis, the trichomes contain relatively high amounts of
tetrahydrocannabinolic acid (THCA), which can convert to
tetrahydrocannabinol (THC) via a decarboxylation reaction, for
example upon combustion of dried Cannabis flowers, to provide an
intoxicating effect. Drug-type Cannabis often contains other
cannabinoids in lesser amounts. In contrast, hemp-type Cannabis
contains relatively low concentrations of THCA, often less than
0.3% THC by dry weight, accounting for the ability of THCA to
convert to THC. Hemp-type Cannabis may contain non-THC and non-THCA
cannabinoids, such as cannabidiolic acid (CBDA), cannabidiol (CBD),
and other cannabinoids. Presently, there is a lack of consensus
regarding the taxonomic organization of the species within the
genus. Unless context dictates otherwise, the term "Cannabis" is
intended to include all putative species within the genus, such as,
without limitation, Cannabis sativa, Cannabis indica, and Cannabis
ruderalis and without regard to whether the Cannabis is hemp-type
or drug-type.
[0078] The term "cyclase activity" in reference to a polyketide
synthase (PKS) enzyme (e.g., an olivetol synthase (OLS) enzyme) or
a polyketide cyclase (PKC) enzyme (e.g., an olivetolic acid cyclase
(OAC) enzyme), refers to the activity of catalyzing the cyclization
of an oxo fatty acyl-CoA (e.g., 3,5,7-trioxododecanoyl-COA,
3,5,7-trioxodecanoyl-COA) to the corresponding intramolecular
cyclization product (e.g., olivetolic acid, divarinic acid). In
some embodiments, the PKS catalyzes the C2-C7 aldol condensation of
an acyl-COA with three additional ketide moieties added
thereto.
[0079] A "cytosolic" or "soluble" enzyme refers to an enzyme that
is predominantly localized (or predicted to be localized) in the
cytosol of a host cell.
[0080] A "eukaryote" is any organism whose cells contain a nucleus
and other organelles enclosed within membranes. Eukaryotes belong
to the taxon Eukarya or Eukaryota. The defining feature that sets
eukaryotic cells apart from prokaryotic cells (i.e., bacteria and
archaea) is that they have membrane-bound organelles, especially
the nucleus, which contains the genetic material, and is enclosed
by the nuclear envelope.
[0081] The term "host cell" refers to a cell that can be used to
express a polynucleotide, such as a polynucleotide that encodes an
enzyme used in biosynthesis of cannabinoids or cannabinoid
precursors. The terms "genetically modified host cell,"
"recombinant host cell," and "recombinant strain" are used
interchangeably and refer to host cells that have been genetically
modified by, e.g., cloning and transformation methods, or by other
methods known in the art (e.g., selective editing methods, such as
CRISPR). Thus, the terms include a host cell (e.g., bacterial cell,
yeast cell, fungal cell, insect cell, plant cell, mammalian cell,
human cell, etc.) that has been genetically altered, modified, or
engineered, so that it exhibits an altered, modified, or different
genotype and/or phenotype, as compared to the naturally-occurring
cell from which it was derived. It is understood that in some
embodiments, the terms refer not only to the particular recombinant
host cell in question, but also to the progeny or potential progeny
of such a host cell.
[0082] The term "control host cell," or the term "control" when
used in relation to a host cell, refers to an appropriate
comparator host cell for determining the effect of a genetic
modification or experimental treatment. In some embodiments, the
control host cell is a wild type cell. In other embodiments, a
control host cell is genetically identical to the genetically
modified host cell, except for the genetic modification(s)
differentiating the genetically modified or experimental treatment
host cell. In some embodiments, the control host cell has been
genetically modified to express a wild type or otherwise known
variant of an enzyme being tested for activity in other test host
cells.
[0083] The term "heterologous" with respect to a polynucleotide,
such as a polynucleotide comprising a gene, is used interchangeably
with the term "exogenous" and the term "recombinant" and refers to
a polynucleotide that has been artificially supplied to a
biological system, a polynucleotide that has been modified within a
biological system, or a polynucleotide whose expression or
regulation has been manipulated within a biological system. A
heterologous polynucleotide that is introduced into or expressed in
a host cell may be a polynucleotide that comes from a different
organism or species than the host cell, or may be a synthetic
polynucleotide, or may be a polynucleotide that is also
endogenously expressed in the same organism or species as the host
cell. For example, a polynucleotide that is endogenously expressed
in a host cell may be considered heterologous when it is situated
non-naturally in the host cell; expressed recombinantly in the host
cell, either stably or transiently; modified within the host cell;
selectively edited within the host cell; expressed in a copy number
that differs from the naturally occurring copy number within the
host cell; or expressed in a non-natural way within the host cell,
such as by manipulating regulatory regions that control expression
of the polynucleotide. In some embodiments, a heterologous
polynucleotide is a polynucleotide that is endogenously expressed
in a host cell but whose expression is driven by a promoter that
does not naturally regulate expression of the polynucleotide. In
other embodiments, a heterologous polynucleotide is a
polynucleotide that is endogenously expressed in a host cell and
whose expression is driven by a promoter that does naturally
regulate expression of the polynucleotide, but the promoter or
another regulatory region is modified. In some embodiments, the
promoter is recombinantly activated or repressed. For example,
gene-editing based techniques may be used to regulate expression of
a polynucleotide, including an endogenous polynucleotide, from a
promoter, including an endogenous promoter. See, e.g., Chavez et
al., Nat Methods. 2016 July; 13(7): 563-567. A heterologous
polynucleotide may comprise a wild-type sequence or a mutant
sequence as compared with a reference polynucleotide sequence.
[0084] The term "at least a portion" or "at least a fragment" of a
nucleic acid or polypeptide means a portion having the minimal size
characteristics of such sequences, or any larger fragment of the
full length molecule, up to and including the full length molecule.
A fragment of a polynucleotide of the disclosure may encode a
biologically active portion of an enzyme, such as a catalytic
domain. A biologically active portion of a genetic regulatory
element may comprise a portion or fragment of a full length genetic
regulatory element and have the same type of activity as the full
length genetic regulatory element, although the level of activity
of the biologically active portion of the genetic regulatory
element may vary compared to the level of activity of the full
length genetic regulatory element.
[0085] A coding sequence and a regulatory sequence are said to be
"operably joined" or "operably linked" when the coding sequence and
the regulatory sequence are covalently linked and the expression or
transcription of the coding sequence is under the influence or
control of the regulatory sequence. If the coding sequence is to be
translated into a functional protein, the coding sequence and the
regulatory sequence are said to be operably joined if induction of
a promoter in the 5' regulatory sequence results in transcription
of the coding sequence and if the nature of the linkage between the
coding sequence and the regulatory sequence does not (1) result in
the introduction of a frame-shift mutation, (2) interfere with the
ability of the promoter region to direct the transcription of the
coding sequence, or (3) interfere with the ability of the
corresponding RNA transcript to be translated into a protein.
[0086] The term "volumetric productivity" or "production rate"
refers to the amount of product formed per volume of medium per
unit of time. Volumetric productivity can be reported in gram per
liter per hour (g/L/h).
[0087] The term "specific productivity" of a product refers to the
rate of formation of the product normalized by unit volume or mass
or biomass and has the physical dimension of a quantity of
substance per unit time per unit mass or volume [MT.sup.-1M.sup.-1
or MT.sup.-1L.sup.-3, where M is mass or moles, T is time, L is
length].
[0088] The term "biomass specific productivity" refers to the
specific productivity in gram product per gram of cell dry weight
(CDW) per hour (g/g CDW/h) or in mmol of product per gram of cell
dry weight (CDW) per hour (mmol/g CDW/h). Using the relation of CDW
to OD600 for the given microorganism, specific productivity can
also be expressed as gram product per liter culture medium per
optical density of the culture broth at 600 nm (OD) per hour
(g/L/h/OD). Also, if the elemental composition of the biomass is
known, biomass specific productivity can be expressed in mmol of
product per C-mole (carbon mole) of biomass per hour
(mmol/C-mol/h).
[0089] The term "yield" refers to the amount of product obtained
per unit weight of a certain substrate and may be expressed as g
product per g substrate (g/g) or moles of product per mole of
substrate (mol/mol). Yield may also be expressed as a percentage of
the theoretical yield. "Theoretical yield" is defined as the
maximum amount of product that can be generated per a given amount
of substrate as dictated by the stoichiometry of the metabolic
pathway used to make the product and may be expressed as g product
per g substrate (g/g) or moles of product per mole of substrate
(mol/mol).
[0090] The term "titer" refers to the strength of a solution or the
concentration of a substance in solution. For example, the titer of
a product of interest (e.g., small molecule, peptide, synthetic
compound, fuel, alcohol, etc.) in a fermentation broth is described
as g of product of interest in solution per liter of fermentation
broth or cell-free broth (g/L) or as g of product of interest in
solution per kg of fermentation broth or cell-free broth
(g/Kg).
[0091] The term "total titer" refers to the sum of all products of
interest produced in a process, including but not limited to the
products of interest in solution, the products of interest in gas
phase if applicable, and any products of interest removed from the
process and recovered relative to the initial volume in the process
or the operating volume in the process. For example, the total
titer of products of interest (e.g., small molecule, peptide,
synthetic compound, fuel, alcohol, etc.) in a fermentation broth is
described as g of products of interest in solution per liter of
fermentation broth or cell-free broth (g/L) or as g of products of
interest in solution per kg of fermentation broth or cell-free
broth (g/Kg).
[0092] The term "amino acid" refers to organic compounds that
comprise an amino group, --NH2, and a carboxyl group, --COOH. The
term "amino acid" includes both naturally occurring and unnatural
amino acids. Nomenclature for the twenty common amino acids is as
follows: alanine (ala or A); arginine (arg or R); asparagine (asn
or N); aspartic acid (asp or D); cysteine (cys or C); glutamine
(gln or Q); glutamic acid (glu or E); glycine (gly or G); histidine
(his or H); isoleucine (ile or I); leucine (leu or L); lysine (lys
or K); methionine (met or M); phenylalanine (phe or F); proline
(pro or P); serine (ser or S); threonine (thr or T); tryptophan
(trp or W); tyrosine (tyr or Y); and valine (val or V).
Non-limiting examples of unnatural amino acids include homo-amino
acids, proline and pyruvic acid derivatives, 3-substituted alanine
derivatives, glycine derivatives, ring-substituted phenylalanine
derivatives, ring-substituted tyrosine derivatives, linear core
amino acids, amino acids with protecting groups including Fmoc,
Boc, and Cbz, .beta.-amino acids (.beta.3 and .beta.2), and
N-methyl amino acids.
[0093] The term "aliphatic" refers to alkyl, alkenyl, alkynyl, and
carbocyclic groups. Likewise, the term "heteroaliphatic" refers to
heteroalkyl, heteroalkenyl, heteroalkynyl, and heterocyclic
groups.
[0094] The term "alkyl" refers to a radical of, or a substituent
that is, a straight-chain or branched saturated hydrocarbon group
having from 1 to 20 carbon atoms ("C1-20 alkyl"). In certain
embodiments, the term "alkyl" refers to a radical of, or a
substituent that is, a straight-chain or branched saturated
hydrocarbon group having from 1 to 10 carbon atoms ("C.sub.1-10
alkyl"). In some embodiments, an alkyl group has 1 to 9 carbon
atoms ("C.sub.1-9 alkyl"). In some embodiments, an alkyl group has
1 to 8 carbon atoms ("C.sub.1-8 alkyl"). In some embodiments, an
alkyl group has 1 to 7 carbon atoms ("C.sub.1-7 alkyl"). In some
embodiments, an alkyl group has 1 to 6 carbon atoms ("C.sub.1-6
alkyl"). In some embodiments, an alkyl group has 2 to 6 carbon
atoms ("C.sub.2-6 alkyl"). In some embodiments, an alkyl group has
3 to 5 carbon atoms ("C.sub.3-5 alkyl"). In some embodiments, an
alkyl group has 5 carbon atoms ("C.sub.5 alkyl"). In some
embodiments, the alkyl group has 3 carbon atoms ("C3 alkyl"). In
some embodiments, an alkyl group has 1 to 5 carbon atoms
("C.sub.1-5 alkyl"). In some embodiments, an alkyl group has 1 to 4
carbon atoms ("C.sub.1-4 alkyl"). In some embodiments, an alkyl
group has 1 to 3 carbon atoms ("C.sub.1-3 alkyl"). In some
embodiments, an alkyl group has 1 to 2 carbon atoms ("C.sub.1-2
alkyl"). In some embodiments, an alkyl group has 1 carbon atom
("C.sub.1 alkyl").
[0095] Examples of C.sub.1-6 alkyl groups include methyl (C.sub.1),
ethyl (C.sub.2), propyl (C.sub.3) (e.g., n-propyl, isopropyl),
butyl (C.sub.4) (e.g., n-butyl, tert-butyl, sec-butyl, iso-butyl),
pentyl (C.sub.5) (e.g., n-pentyl, 3-pentanyl, amyl, neopentyl,
3-methyl-2-butanyl, tertiary amyl), and hexyl (C.sub.6) (e.g.,
n-hexyl). Additional examples of alkyl groups include n-heptyl
(C.sub.7), n-octyl (C.sub.8), and the like. Unless otherwise
specified, each instance of an alkyl group is independently
unsubstituted (an "unsubstituted alkyl") or substituted (a
"substituted alkyl") with one or more substituents (e.g., halogen,
such as F). In certain embodiments, the alkyl group is an
unsubstituted C.sub.1-10 alkyl (such as unsubstituted C.sub.1-6
alkyl, e.g., --CH.sub.3 (Me), unsubstituted ethyl (Et),
unsubstituted propyl (Pr, e.g., unsubstituted n-propyl (n-Pr),
unsubstituted isopropyl (i-Pr)), unsubstituted butyl (Bu, e.g.,
unsubstituted n-butyl (n-Bu), unsubstituted tert-butyl (tert-Bu or
t-Bu), unsubstituted sec-butyl (sec-Bu), unsubstituted isobutyl
(i-Bu)). In certain embodiments, the alkyl group is a substituted
C.sub.1-10 alkyl (such as substituted C.sub.1-6 alkyl, e.g.,
--CF.sub.3, benzyl).
[0096] The term "acyl" refers to a group having the general formula
--C(.dbd.O)R.sup.X1, --C(.dbd.O)OR.sup.X1,
--C(.dbd.O)--O--C(.dbd.O)R.sup.X1, --C(.dbd.O)SR.sup.X1,
--C(.dbd.O)N(R.sup.X1).sub.2, --C(.dbd.S)R.sup.X1,
--C(.dbd.S)N(R.sup.X1).sub.2, and --C(.dbd.S)S(R.sup.X1),
--C(.dbd.NR.sup.X1)R.sup.X1, --C(.dbd.NR.sup.X1)OR.sup.X1,
--C(.dbd.NR.sup.X1)SR.sup.X1, and
--C(.dbd.NR.sup.X1)N(R.sup.X1).sub.2, wherein R.sup.X1 is hydrogen;
halogen; substituted or unsubstituted hydroxyl; substituted or
unsubstituted thiol; substituted or unsubstituted amino;
substituted or unsubstituted acyl, cyclic or acyclic, substituted
or unsubstituted, branched or unbranched aliphatic; cyclic or
acyclic, substituted or unsubstituted, branched or unbranched
heteroaliphatic; cyclic or acyclic, substituted or unsubstituted,
branched or unbranched alkyl; cyclic or acyclic, substituted or
unsubstituted, branched or unbranched alkenyl; substituted or
unsubstituted alkynyl; substituted or unsubstituted aryl,
substituted or unsubstituted heteroaryl, aliphaticoxy,
heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy,
heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy,
heteroalkylthioxy, arylthioxy, heteroarylthioxy, mono- or
di-aliphaticamino, mono- or di-heteroaliphaticamino, mono- or
di-alkylamino, mono- or di-heteroalkylamino, mono- or di-arylamino,
or mono- or di-heteroarylamino; or two R.sup.X1 groups taken
together form a 5- to 6-membered heterocyclic ring. Exemplary acyl
groups include aldehydes (--CHO), carboxylic acids (--CO.sub.2H),
ketones, acyl halides, esters, amides, imines, carbonates,
carbamates, and ureas. Acyl substituents include, but are not
limited to, any of the substituents described in this application
that result in the formation of a stable moiety (e.g., aliphatic,
alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl,
heteroaryl, acyl, oxo, imino, thiooxo, cyano, isocyano, amino,
azido, nitro, hydroxyl, thiol, halo, aliphaticamino,
heteroaliphaticamino, alkylamino, heteroalkylamino, arylamino,
heteroarylamino, alkylaryl, arylalkyl, aliphaticoxy,
heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy,
heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy,
heteroalkylthioxy, arylthioxy, heteroarylthioxy, acyloxy, and the
like, each of which may or may not be further substituted).
[0097] "Alkenyl" refers to a radical of, or a substituent that is,
a straight-chain or branched hydrocarbon group having from 2 to 20
carbon atoms, one or more carbon-carbon double bonds, and no triple
bonds ("C.sub.2-20 alkenyl"). In some embodiments, an alkenyl group
has 2 to 10 carbon atoms ("C.sub.2-10 alkenyl"). In some
embodiments, an alkenyl group has 2 to 9 carbon atoms ("C.sub.2-9
alkenyl"). In some embodiments, an alkenyl group has 2 to 8 carbon
atoms ("C.sub.2-8 alkenyl"). In some embodiments, an alkenyl group
has 2 to 7 carbon atoms ("C.sub.2-7 alkenyl"). In some embodiments,
an alkenyl group has 2 to 6 carbon atoms ("C.sub.2-6 alkenyl"). In
some embodiments, an alkenyl group has 2 to 5 carbon atoms
("C.sub.2-5 alkenyl"). In some embodiments, an alkenyl group has 2
to 4 carbon atoms ("C.sub.2-4 alkenyl"). In some embodiments, an
alkenyl group has 2 to 3 carbon atoms ("C.sub.2-3 alkenyl"). In
some embodiments, an alkenyl group has 2 carbon atoms ("C.sub.2
alkenyl"). The one or more carbon-carbon double bonds can be
internal (such as in 2-butenyl) or terminal (such as in 1-butenyl).
Examples of C.sub.2-4 alkenyl groups include ethenyl (C.sub.2),
1-propenyl (C.sub.3), 2-propenyl (C.sub.3), 1-butenyl (C.sub.4),
2-butenyl (C.sub.4), butadienyl (C.sub.4), and the like. Examples
of C.sub.2-6 alkenyl groups include the aforementioned C.sub.2-4
alkenyl groups as well as pentenyl (C.sub.5), pentadienyl
(C.sub.5), hexenyl (C.sub.6), and the like. Additional examples of
alkenyl include heptenyl (C.sub.7), octenyl (C.sub.8), octatrienyl
(C.sub.8), and the like. Unless otherwise specified, each instance
of an alkenyl group is independently optionally substituted, i.e.,
unsubstituted (an "unsubstituted alkenyl") or substituted (a
"substituted alkenyl") with one or more substituents. In certain
embodiments, the alkenyl group is unsubstituted C.sub.2-10 alkenyl.
In certain embodiments, the alkenyl group is substituted C.sub.2-10
alkenyl.
[0098] "Alkynyl" refers to a radical of, or a substituent that is,
a straight-chain or branched hydrocarbon group having from 2 to 20
carbon atoms, one or more carbon-carbon triple bonds, and
optionally one or more double bonds ("C.sub.2-20 alkynyl"). In some
embodiments, an alkynyl group has 2 to 10 carbon atoms ("C.sub.2-10
alkynyl"). In some embodiments, an alkynyl group has 2 to 9 carbon
atoms ("C.sub.2-9 alkynyl"). In some embodiments, an alkynyl group
has 2 to 8 carbon atoms ("C.sub.2-8 alkynyl"). In some embodiments,
an alkynyl group has 2 to 7 carbon atoms ("C.sub.2-7 alkynyl"). In
some embodiments, an alkynyl group has 2 to 6 carbon atoms
("C.sub.2-6 alkynyl"). In some embodiments, an alkynyl group has 2
to 5 carbon atoms ("C.sub.2-5 alkynyl"). In some embodiments, an
alkynyl group has 2 to 4 carbon atoms ("C.sub.2-4 alkynyl"). In
some embodiments, an alkynyl group has 2 to 3 carbon atoms
("C.sub.2-3 alkynyl"). In some embodiments, an alkynyl group has 2
carbon atoms ("C.sub.2 alkynyl"). The one or more carbon-carbon
triple bonds can be internal (such as in 2-butynyl) or terminal
(such as in 1-butynyl). Examples of C.sub.2-4 alkynyl groups
include, without limitation, ethynyl (C.sub.2), 1-propynyl
(C.sub.3), 2-propynyl (C.sub.3), 1-butynyl (C.sub.4), 2-butynyl
(C.sub.4), and the like. Examples of C.sub.2-6 alkenyl groups
include the aforementioned C.sub.2-4 alkynyl groups as well as
pentynyl (C.sub.5), hexynyl (C.sub.6), and the like. Additional
examples of alkynyl include heptynyl (C.sub.7), octynyl (C.sub.8),
and the like. Unless otherwise specified, each instance of an
alkynyl group is independently optionally substituted, i.e.,
unsubstituted (an "unsubstituted alkynyl") or substituted (a
"substituted alkynyl") with one or more substituents. In certain
embodiments, the alkynyl group is unsubstituted C.sub.2-10 alkynyl.
In certain embodiments, the alkynyl group is substituted C.sub.2-10
alkynyl.
[0099] "Carbocyclyl" or "carbocyclic" refers to a radical of a
non-aromatic cyclic hydrocarbon group having from 3 to 10 ring
carbon atoms ("C.sub.3-10 carbocyclyl") and zero heteroatoms in the
non-aromatic ring system. In some embodiments, a carbocyclyl group
has 3 to 8 ring carbon atoms ("C.sub.3-8 carbocyclyl"). In some
embodiments, a carbocyclyl group has 3 to 6 ring carbon atoms
("C.sub.3-6 carbocyclyl"). In some embodiments, a carbocyclyl group
has 3 to 6 ring carbon atoms ("C.sub.3-6 carbocyclyl"). In some
embodiments, a carbocyclyl group has 5 to 10 ring carbon atoms
("C.sub.5-10 carbocyclyl"). Exemplary C.sub.3-6 carbocyclyl groups
include, without limitation, cyclopropyl (C.sub.3), cyclopropenyl
(C.sub.3), cyclobutyl (C.sub.4), cyclobutenyl (C.sub.4),
cyclopentyl (C.sub.5), cyclopentenyl (C.sub.5), cyclohexyl
(C.sub.6), cyclohexenyl (C.sub.6), cyclohexadienyl (C.sub.6), and
the like. Exemplary C.sub.3-8 carbocyclyl groups include, without
limitation, the aforementioned C.sub.3-6 carbocyclyl groups as well
as cycloheptyl (C.sub.7), cycloheptenyl (C.sub.7), cycloheptadienyl
(C.sub.7), cycloheptatrienyl (C.sub.7), cyclooctyl (C.sub.8),
cyclooctenyl (C.sub.8), bicyclo[2.2.1]heptanyl (C.sub.7),
bicyclo[2.2.2]octanyl (C.sub.8), and the like. Exemplary C.sub.3-10
carbocyclyl groups include, without limitation, the aforementioned
C.sub.3-8 carbocyclyl groups as well as cyclononyl (C.sub.9),
cyclononenyl (C.sub.9), cyclodecyl (C.sub.10), cyclodecenyl
(C.sub.10), octahydro-1H-indenyl (C.sub.9), decahydronaphthalenyl
(C.sub.10), spiro[4.5]decanyl (C.sub.10), and the like. As the
foregoing examples illustrate, in certain embodiments, the
carbocyclyl group is either monocyclic ("monocyclic carbocyclyl")
or contain a fused, bridged or spiro ring system such as a bicyclic
system ("bicyclic carbocyclyl") and can be saturated or can be
partially unsaturated. "Carbocyclyl" also includes ring systems
wherein the carbocyclic ring, as defined above, is fused with one
or more aryl or heteroaryl groups wherein the point of attachment
is on the carbocyclic ring, and in such instances, the number of
carbons continue to designate the number of carbons in the
carbocyclic ring system. Unless otherwise specified, each instance
of a carbocyclyl group is independently optionally substituted,
i.e., unsubstituted (an "unsubstituted carbocyclyl") or substituted
(a "substituted carbocyclyl") with one or more substituents. In
certain embodiments, the carbocyclyl group is unsubstituted
C.sub.3-10 carbocyclyl. In certain embodiments, the carbocyclyl
group is a substituted C.sub.3-10 carbocyclyl.
[0100] In some embodiments, "carbocyclyl" is a monocyclic,
saturated carbocyclyl group having from 3 to 10 ring carbon atoms
("C.sub.3-10 cycloalkyl"). In some embodiments, a cycloalkyl group
has 3 to 8 ring carbon atoms ("C.sub.3-8 cycloalkyl"). In some
embodiments, a cycloalkyl group has 3 to 6 ring carbon atoms
("C.sub.3-6 cycloalkyl"). In some embodiments, a cycloalkyl group
has 5 to 6 ring carbon atoms ("C.sub.5-6 cycloalkyl"). In some
embodiments, a cycloalkyl group has 5 to 10 ring carbon atoms
("C.sub.5-10 cycloalkyl"). Examples of C.sub.5-6 cycloalkyl groups
include cyclopentyl (C.sub.5) and cyclohexyl (C.sub.5). Examples of
C.sub.3-6 cycloalkyl groups include the aforementioned C.sub.5-6
cycloalkyl groups as well as cyclopropyl (C.sub.3) and cyclobutyl
(C.sub.4). Examples of C.sub.3-8 cycloalkyl groups include the
aforementioned C.sub.3-6 cycloalkyl groups as well as cycloheptyl
(C.sub.7) and cyclooctyl (C.sub.8). Unless otherwise specified,
each instance of a cycloalkyl group is independently unsubstituted
(an "unsubstituted cycloalkyl") or substituted (a "substituted
cycloalkyl") with one or more substituents. In certain embodiments,
the cycloalkyl group is unsubstituted C.sub.3-10 cycloalkyl. In
certain embodiments, the cycloalkyl group is substituted C.sub.3-10
cycloalkyl.
[0101] "Aryl" refers to a radical of a monocyclic or polycyclic
(e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g.,
having 6, 10, or 14 pi electrons shared in a cyclic array) having
6-14 ring carbon atoms and zero heteroatoms provided in the
aromatic ring system ("C.sub.6-14 aryl"). In some embodiments, an
aryl group has six ring carbon atoms ("C.sub.6 aryl"; e.g.,
phenyl). In some embodiments, an aryl group has ten ring carbon
atoms ("C.sub.10 aryl"; e.g., naphthyl such as 1-naphthyl and
2-naphthyl). In some embodiments, an aryl group has fourteen ring
carbon atoms ("C.sub.14 aryl"; e.g., anthracyl). "Aryl" also
includes ring systems wherein the aryl ring, as defined above, is
fused with one or more carbocyclyl or heterocyclyl groups wherein
the radical or point of attachment is on the aryl ring, and in such
instances, the number of carbon atoms continue to designate the
number of carbon atoms in the aryl ring system. Unless otherwise
specified, each instance of an aryl group is independently
optionally substituted, i.e., unsubstituted (an "unsubstituted
aryl") or substituted (a "substituted aryl") with one or more
substituents. In certain embodiments, the aryl group is
unsubstituted C.sub.6-14 aryl. In certain embodiments, the aryl
group is substituted C.sub.6-14 aryl.
[0102] "Aralkyl" is a subset of alkyl and aryl and refers to an
optionally substituted alkyl group substituted by an optionally
substituted aryl group. In certain embodiments, the aralkyl is
optionally substituted benzyl. In certain embodiments, the aralkyl
is benzyl. In certain embodiments, the aralkyl is optionally
substituted phenethyl. In certain embodiments, the aralkyl is
phenethyl. In certain embodiments, the aralkyl is 7-phenylheptanyl.
In certain embodiments, the aralkyl is C7 alkyl substituted by an
optionally substituted aryl group (e.g., phenyl). In certain
embodiments, the aralkyl is a C7-C10 alkyl group substituted by an
optionally substituted aryl group (e.g., phenyl).
[0103] "Partially unsaturated" refers to a group that includes at
least one double or triple bond. A "partially unsaturated" ring
system is further intended to encompass rings having multiple sites
of unsaturation but is not intended to include aromatic groups
(e.g., aryl or heteroaryl groups) as defined in this application.
Likewise, "saturated" refers to a group that does not contain a
double or triple bond, i.e., contains all single bonds.
[0104] The term "optionally substituted" means substituted or
unsubstituted.
[0105] Alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl,
and heteroaryl groups are optionally substituted (e.g.,
"substituted" or "unsubstituted" alkyl, "substituted" or
"unsubstituted" alkenyl, "substituted" or "unsubstituted" alkynyl,
"substituted" or "unsubstituted" carbocyclyl, "substituted" or
"unsubstituted" heterocyclyl, "substituted" or "unsubstituted" aryl
or "substituted" or "unsubstituted" heteroaryl group). In general,
the term "substituted," whether preceded by the term "optionally"
or not, means that at least one hydrogen present on a group (e.g.,
a carbon or nitrogen atom) is replaced with a permissible
substituent, e.g., a substituent which upon substitution results in
a stable compound, e.g., a compound which does not spontaneously
undergo transformation such as by rearrangement, cyclization,
elimination, or other reaction. Unless otherwise indicated, a
"substituted" group has a substituent at one or more substitutable
positions of the group, and when more than one position in any
given structure is substituted, the substituent is either the same
or different at each position. The term "substituted" is
contemplated to include substitution with all permissible
substituents of organic compounds, any of the substituents
described in this application that results in the formation of a
stable compound. The present invention contemplates any and all
such combinations in order to arrive at a stable compound. For
purposes of this invention, heteroatoms such as nitrogen may have
hydrogen substituents and/or any suitable substituent as described
in this application which satisfy the valencies of the heteroatoms
and results in the formation of a stable moiety.
[0106] Exemplary carbon atom substituents include, but are not
limited to, halogen, --CN, --NO.sub.2, --N.sub.3, --SO.sub.2H,
--SO.sub.3H, --OH, --OR.sup.aa, --ON(R.sup.bb).sub.2,
--N(R.sup.bb).sub.2, --N(R.sup.bb).sub.3.sup.+X.sup.-,
--N(OR.sup.cc)R.sup.bb, --SH, --SR.sup.aa, --SSR.sup.cc,
--C(.dbd.O)R.sup.aa, --CO.sub.2H, --CHO, --C(OR.sup.cc).sub.2,
--CO.sub.2R.sup.aa, --OC(.dbd.O)R.sup.aa, --OCO.sub.2R.sup.aa,
--C(.dbd.O)N(R.sup.bb).sub.2, --OC(.dbd.O)N(R.sup.bb).sub.2,
--NR.sup.bbC(.dbd.O)R.sup.aa, --NR.sup.bbCO.sub.2R.sup.aa,
--NR.sup.bbC(.dbd.O)N(R.sup.bb).sub.2, --C(.dbd.NR.sup.bb)R.sup.aa,
--C(.dbd.NR.sup.bb)OR.sup.aa, --OC(.dbd.NR.sup.bb)R.sup.aa,
--OC(.dbd.NR.sup.bb)OR.sup.aa,
--C(.dbd.NR.sup.bb)N(R.sup.bb).sub.2,
--OC(.dbd.NR.sup.bb)N(R.sup.bb).sub.2,
--NR.sup.bbC(.dbd.NR.sup.bb)N(R.sup.bb).sub.2,
--C(.dbd.O)NR.sup.bbSO.sub.2R.sup.aa, --NR.sup.bbSO.sub.2R.sup.aa,
SO.sub.2N(R.sup.bb).sub.2, --SO.sub.2R.sup.aa, --SO.sub.2OR.sup.aa,
--OSO.sub.2R.sup.aa, --S(.dbd.O)R.sup.aa, --OS(.dbd.O)R.sup.aa,
--Si(R.sup.aa).sub.3,
--OSi(R.sup.aa).sub.3--C(.dbd.S)N(R.sup.bb).sub.2,
--C(.dbd.O)SR.sup.aa, --C(.dbd.S)SR.sup.aa, --SC(.dbd.S)SR.sup.aa,
--SC(.dbd.O)SR.sup.aa, --OC(.dbd.O)SR.sup.aa,
--SC(.dbd.O)OR.sup.aa, --SC(.dbd.O)R.sup.aa,
--P(.dbd.O)(R.sup.aa).sub.2, --P(.dbd.O)(OR.sup.cc).sub.2,
--OP(.dbd.O)(R.sup.aa).sub.2, OP(.dbd.O)(OR.sup.cc).sub.2,
--P(.dbd.O)(N(R.sup.bb).sub.2).sub.2,
--OP(.dbd.O)(N(R.sup.bb).sub.2).sub.2,
--NR.sup.bbP(.dbd.O)(R.sup.aa).sub.2,
--NR.sup.bbP(.dbd.O)(OR.sup.cc).sub.2,
--NR.sup.bbP(.dbd.O)(N(R.sup.bb).sub.2).sub.2,
--P(R.sup.cc).sub.2--P(OR.sup.cc).sub.2,
--P(R.sup.cc).sub.3.sup.+X.sup.-,
--P(OR.sup.cc).sub.3.sup.+X.sup.-, --P(R.sup.cc).sub.4,
--P(OR.sup.cc).sub.4, --OP(R.sup.cc).sub.2,
--OP(R.sup.cc).sub.3.sup.+X.sup.-, --OP(OR.sup.cc).sub.2,
--OP(OR.sup.cc).sub.3.sup.+X.sup.-, --OP(R.sup.cc).sub.4,
--OP(OR.sup.cc).sub.4, --B(R.sup.aa).sub.2, --B(OR.sup.cc).sub.2,
--BR.sup.aa(OR.sup.cc), C.sub.1-10 alkyl, C.sub.1-10 perhaloalkyl,
C.sub.2-10 alkenyl, C.sub.2-10 alkynyl, heteroC.sub.1-10 alkyl,
heteroC.sub.2-10 alkenyl, heteroC.sub.2-10 alkynyl, C.sub.3-10
carbocyclyl, 3-14 membered heterocyclyl, C.sub.6-14 aryl, and 5-14
membered heteroaryl;
wherein:
[0107] each instance of R.sup.aa is, independently, selected from
C.sub.1-10 alkyl, C.sub.1-10 perhaloalkyl, C.sub.2-10 alkenyl,
C.sub.2-10 alkynyl, heteroC.sub.1-10 alkyl, heteroC.sub.2-10
alkenyl, heteroC.sub.2-10 alkynyl, C.sub.3-10 carbocyclyl, 3-14
membered heterocyclyl, C.sub.6-14 aryl, and 5-14 membered
heteroaryl, or two R.sup.aa groups are joined to form a 3-14
membered heterocyclyl or 5-14 membered heteroaryl ring, wherein
each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,
heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is
independently substituted with 0, 1, 2, 3, 4, or 5 R.sup.dd
groups;
[0108] each instance of R.sup.bb is, independently, selected from
hydrogen, --OH, --OR.sup.aa, --N(R.sup.cc).sub.2, --CN,
--C(.dbd.O)R.sup.aa, --C(.dbd.O)N(R.sup.cc).sub.2,
--CO.sub.2R.sup.aa, --SO.sub.2R.sup.aa,
--C(.dbd.NR.sup.cc)OR.sup.aa, --C(.dbd.NR.sup.cc)N(R.sup.cc).sub.2,
--SO.sub.2N(R.sup.cc).sub.2, --SO.sub.2R.sup.cc,
--SO.sub.2OR.sup.cc, --SOR.sup.aa, --C(.dbd.S)N(R.sup.cc).sub.2,
--C(.dbd.O)SR.sup.cc, --C(.dbd.S)SR.sup.cc,
--P(.dbd.O)(R.sup.aa).sub.2, --P(.dbd.O)(OR.sup.cc).sub.2,
--P(.dbd.O)(N(R.sup.cc).sub.2).sub.2, C.sub.1-10 alkyl, C.sub.1-10
perhaloalkyl, C.sub.2-10 alkenyl, C.sub.2-10 alkynyl,
heteroC.sub.1-10 alkyl, heteroC.sub.2-10 alkenyl, heteroC.sub.2-10
alkynyl, C.sub.3-10 carbocyclyl, 3-14 membered heterocyclyl,
C.sub.6-14 aryl, and 5-14 membered heteroaryl, or two R.sup.bb
groups are joined to form a 3-14 membered heterocyclyl or 5-14
membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl,
heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl,
heterocyclyl, aryl, and heteroaryl is independently substituted
with 0, 1, 2, 3, 4, or 5 R.sup.dd groups; wherein X.sup.- is a
counterion;
[0109] each instance of R.sup.cc is, independently, selected from
hydrogen, C.sub.1-10 alkyl, C.sub.1-10 perhaloalkyl, C.sub.2-10
alkenyl, C.sub.2-10 alkynyl, heteroC.sub.1-10 alkyl,
heteroC.sub.2-10 alkenyl, heteroC.sub.2-10 alkynyl, C.sub.3-10
carbocyclyl, 3-14 membered heterocyclyl, C.sub.6-14 aryl, and 5-14
membered heteroaryl, or two R.sup.cc groups are joined to form a
3-14 membered heterocyclyl or 5-14 membered heteroaryl ring,
wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,
heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is
independently substituted with 0, 1, 2, 3, 4, or 5 R.sup.dd
groups;
[0110] each instance of R.sup.dd is, independently, selected from
halogen, --CN, --NO.sub.2, --N.sub.3, --SO.sub.2H, --SO.sub.3H,
--OH, --OR.sup.ee, --ON(R.sup.ff).sub.2, --N(R.sup.ff).sub.2,
--N(R.sup.ff).sub.3.sup.+X.sup.-, --N(OR.sup.ee)R.sup.ff, --SH,
--SR.sup.ee, --SSR.sup.ee, --C(.dbd.O)R.sup.ee, --CO.sub.2H,
--CO.sub.2R.sup.ee, --OC(.dbd.O)R.sup.ee, --OCO.sub.2R.sup.ee,
--C(.dbd.O)N(R.sup.ff).sub.2, --OC(.dbd.O)N(R.sup.ff).sub.2,
--NR.sup.ffC(.dbd.O)R.sup.ee, --NR.sup.ffCO.sub.2R.sup.ee,
--NR.sup.ffC(.dbd.O)N(R.sup.ff).sub.2,
--C(.dbd.NR.sup.ff)OR.sup.ee, --OC(.dbd.NR.sup.ff)R.sup.ee,
--OC(.dbd.NR.sup.ff)OR.sup.ee,
--C(.dbd.NR.sup.ff)N(R.sup.ff).sub.2,
--OC(.dbd.NR)N(R.sup.ff).sub.2,
--NR.sup.ffC(.dbd.NR.sup.ff)N(Re).sub.2,
--NR.sup.ffSO.sub.2R.sup.ee, --SO.sub.2N(R.sup.ff).sub.2,
--SO.sub.2R.sup.ee, --SO.sub.2OR.sup.ee, --OSO.sub.2R.sup.ee,
--S(.dbd.O)R.sup.ee, --Si(R.sup.ee).sub.3, --OSi(R.sup.ee).sub.3,
--C(.dbd.S)N(R.sup.ff).sub.2, --C(.dbd.O)SR.sup.ee,
--C(.dbd.S)SR.sup.ee, --SC(.dbd.S)SR.sup.ee,
--P(.dbd.O)(OR.sup.ee).sub.2, --P(.dbd.O)(R.sup.ee).sub.2,
--OP(.dbd.O)(R.sup.ee).sub.2, --OP(.dbd.O)(OR.sup.ee).sub.2,
C.sub.1-6 alkyl, C.sub.1-6 perhaloalkyl, C.sub.2-6 alkenyl,
C.sub.2-6 alkynyl, heteroC.sub.1-6alkyl, heteroC.sub.2-6alkenyl,
heteroC.sub.2-6alkynyl, C.sub.3-10 carbocyclyl, 3-10 membered
heterocyclyl, C.sub.6-10 aryl, 5-10 membered heteroaryl, wherein
each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,
heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is
independently substituted with 0, 1, 2, 3, 4, or 5 R.sup.gg groups,
or two geminal R.sup.dd substituents can be joined to form .dbd.O
or .dbd.S; wherein X.sup.- is a counterion; [0111] each instance of
R.sup.ee is, independently, selected from C.sub.1-6 alkyl,
C.sub.1-6 perhaloalkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl,
heteroC.sub.1-6 alkyl, heteroC.sub.2-6alkenyl, heteroC.sub.2-6
alkynyl, C.sub.3-10 carbocyclyl, C.sub.6-10 aryl, 3-10 membered
heterocyclyl, and 3-10 membered heteroaryl, wherein each alkyl,
alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl,
carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently
substituted with 0, 1, 2, 3, 4, or 5 R.sup.gg groups;
[0112] each instance of R.sup.ff is, independently, selected from
hydrogen, C.sub.1-6 alkyl, C.sub.1-6 perhaloalkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, heteroC.sub.1-6alkyl,
heteroC.sub.2-6alkenyl, heteroC.sub.2-6alkynyl, C.sub.3-10
carbocyclyl, 3-10 membered heterocyclyl, C.sub.6-10 aryl and 5-10
membered heteroaryl, or two R.sup.ff groups are joined to form a
3-10 membered heterocyclyl or 5-10 membered heteroaryl ring,
wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,
heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is
independently substituted with 0, 1, 2, 3, 4, or 5 R.sup.gg groups;
and
[0113] each instance of R.sup.gg is, independently, halogen, --CN,
--NO.sub.2, --N.sub.3, --SO.sub.2H, --SO.sub.3H, --OH, --OC.sub.1-6
alkyl, --ON(C.sub.1-6 alkyl).sub.2, --N(C.sub.1-6 alkyl).sub.2,
--N(C.sub.1-6 alkyl).sub.3.sup.+X.sup.-, --NH(C.sub.1-6
alkyl).sub.2.sup.+X.sup.-, --NH.sub.2(C.sub.1-6 alkyl)
.sup.+X.sup.-, --NH.sub.3.sup.+X.sup.-, --N(OC.sub.1-6
alkyl)(C.sub.1-6 alkyl), --N(OH)(C.sub.1-6 alkyl), --NH(OH), --SH,
--SC.sub.1-6 alkyl, --SS(C.sub.1-6 alkyl), --C(.dbd.O)(C.sub.1-6
alkyl), --CO.sub.2H, --CO.sub.2(C.sub.1-6 alkyl),
--OC(.dbd.O)(C.sub.1-6 alkyl), --OCO.sub.2(C.sub.1-6 alkyl),
--C(.dbd.O)NH.sub.2, --C(.dbd.O)(C.sub.1-6 alkyl).sub.2,
--OC(.dbd.O)NH(C.sub.1-6 alkyl), --NHC(.dbd.O)(C.sub.1-6 alkyl),
--N(C.sub.1-6 alkyl)C(.dbd.O)(C.sub.1-6 alkyl),
--NHCO.sub.2(C.sub.1-6 alkyl), --NHC(.dbd.O)N(C.sub.1-6
alkyl).sub.2, --NHC(.dbd.O)NH(C.sub.1-6 alkyl),
--NHC(.dbd.O)NH.sub.2, --C(.dbd.NH)O(C.sub.1-6 alkyl),
--OC(.dbd.NH)(C.sub.1-6 alkyl), --OC(.dbd.NH)OC.sub.1-6 alkyl,
--C(.dbd.NH)N(C.sub.1-6 alkyl).sub.2, --C(.dbd.NH)NH(C.sub.1-6
alkyl), --C(.dbd.NH)NH.sub.2, --OC(.dbd.NH)N(C.sub.1-6
alkyl).sub.2, --OC(NH)NH(C.sub.1-6 alkyl), --OC(NH)NH.sub.2,
--NHC(NH)N(C.sub.1-6 alkyl).sub.2, --NHC(.dbd.NH)NH.sub.2,
--NHSO.sub.2(C.sub.1-6 alkyl), --SO.sub.2N(C.sub.1-6 alkyl).sub.2,
--SO.sub.2NH(C.sub.1-6 alkyl), --SO.sub.2NH.sub.2,
--SO.sub.2C.sub.1-6 alkyl, --SO.sub.2OC.sub.1-6 alkyl,
--OSO.sub.2C.sub.1-6 alkyl, --SOC.sub.1-6 alkyl, --Si(C.sub.1-6
alkyl).sub.3, --OSi(C.sub.1-6 alkyl).sub.3--C(.dbd.S)N(C.sub.1-6
alkyl).sub.2, C(.dbd.S)NH(C.sub.1-6 alkyl), C(.dbd.S)NH.sub.2,
--C(.dbd.O)S(C.sub.1-6 alkyl), --C(.dbd.S)SC.sub.1-6 alkyl,
--SC(.dbd.S)SC.sub.1-6 alkyl, --P(.dbd.O)(OC.sub.1-6 alkyl).sub.2,
--P(.dbd.O)(C.sub.1-6 alkyl).sub.2, --OP(.dbd.O)(C.sub.1-6
alkyl).sub.2, --OP(.dbd.O)(OC.sub.1-6 alkyl).sub.2, C.sub.1-6
alkyl, C.sub.1-6 perhaloalkyl, C.sub.2-6 alkenyl, C.sub.2-6
alkynyl, heteroC.sub.1-6alkyl, heteroC.sub.2-6alkenyl,
heteroC.sub.2-6alkynyl, C.sub.3-10 carbocyclyl, C.sub.6-10 aryl,
3-10 membered heterocyclyl, 5-10 membered heteroaryl; or two
geminal R.sup.gg substituents can be joined to form .dbd.O or
.dbd.S; wherein X.sup.- is a counterion. Alternatively, two geminal
hydrogens on a carbon atom are replaced with the group .dbd.O,
.dbd.S, .dbd.NN(R.sup.bb).sub.2, --NNR.sup.bbC(.dbd.O)R.sup.aa,
--NNR.sup.bbC(.dbd.O)OR.sup.aa,
--NNR.sup.bbS(.dbd.O).sub.2R.sup.aa, --NR.sup.bb, or
.dbd.NOR.sup.cc; wherein each alkyl, alkenyl, alkynyl, heteroalkyl,
heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and
heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5
R.sup.dd groups; wherein X.sup.- is a counterion;
wherein: [0114] each instance of R.sup.aa is, independently,
selected from C.sub.1-10 alkyl, C.sub.1-10 perhaloalkyl, C.sub.2-10
alkenyl, C.sub.2-10 alkynyl, heteroC.sub.1-10 alkyl,
heteroC.sub.2-10 alkenyl, heteroC.sub.2-10 alkynyl, C.sub.3-10
carbocyclyl, 3-14 membered heterocyclyl, C.sub.6-14 aryl, and 5-14
membered heteroaryl, or two R.sup.aa groups are joined to form a
3-14 membered heterocyclyl or 5-14 membered heteroaryl ring,
wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,
heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is
independently substituted with 0, 1, 2, 3, 4, or 5 R.sup.dd
groups;
[0115] each instance of R.sup.bb is, independently, selected from
hydrogen, --OH, --OR.sup.aa, --N(R.sup.cc).sub.2, --CN,
--C(.dbd.O)R.sup.aa, --C(.dbd.O)N(R.sup.cc), --CO.sub.2R.sup.aa,
--SO.sub.2R.sup.aa, --C(.dbd.NR.sup.cc)OR.sup.aa,
--C(.dbd.NR.sup.cc)N(R.sup.cc).sub.2, --SO.sub.2N(R.sup.cc).sub.2,
--SO.sub.2R.sup.cc, --SO.sub.2OR.sup.cc, --SOR.sup.aa,
--C(.dbd.S)N(R.sup.cc).sub.2, --C(.dbd.O)SR.sup.cc,
--C(.dbd.S)SR.sup.cc, --P(.dbd.O)(R.sup.aa).sub.2,
--P(.dbd.O)(OR.sup.cc).sub.2, --P(.dbd.O)(N(R.sup.cc).sub.2).sub.2,
C.sub.1-10 alkyl, C.sub.1-10 perhaloalkyl, C.sub.2-10 alkenyl,
C.sub.2-10 alkynyl, heteroC.sub.1-10 alkyl, heteroC.sub.2-10
alkenyl, heteroC.sub.2-10 alkynyl, C.sub.3-10 carbocyclyl, 3-14
membered heterocyclyl, C.sub.6-14 aryl, and 5-14 membered
heteroaryl, or two R.sup.bb groups are joined to form a 3-14
membered heterocyclyl or 5-14 membered heteroaryl ring, wherein
each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,
heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is
independently substituted with 0, 1, 2, 3, 4, or 5 R.sup.dd groups;
wherein X is a counterion;
[0116] each instance of R.sup.cc is, independently, selected from
hydrogen, C.sub.1-10 alkyl, C.sub.1-10 perhaloalkyl, C.sub.2-10
alkenyl, C.sub.2-10 alkynyl, heteroC.sub.1-10 alkyl,
heteroC.sub.2-10 alkenyl, heteroC.sub.2-10 alkynyl, C.sub.3-10
carbocyclyl, 3-14 membered heterocyclyl, C.sub.6-14 aryl, and 5-14
membered heteroaryl, or two R.sup.cc groups are joined to form a
3-14 membered heterocyclyl or 5-14 membered heteroaryl ring,
wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,
heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is
independently substituted with 0, 1, 2, 3, 4, or 5 R.sup.dd
groups;
[0117] each instance of R.sup.dd is, independently, selected from
halogen, --CN, --NO.sub.2, --N.sub.3, --SO.sub.2H, --SO.sub.3H,
--OH, --OR.sup.ee, --ON(R.sup.ff).sub.2, --N(R.sup.ff).sub.2,
--N(R).sub.3.sup.+X.sup.-, --N(OR.sup.ee)R.sup.ff, --SH,
--SR.sup.ee, --SSR.sup.ee, --C(.dbd.O)R.sup.ee, --CO.sub.2H,
--CO.sub.2R.sup.ee, --OC(.dbd.O)R.sup.ee, --OCO.sub.2R.sup.ee,
--C(.dbd.O)N(R.sup.ff).sub.2, --OC(.dbd.O)N(R.sup.ff).sub.2,
--NR.sup.ffC(.dbd.O)R.sup.ee, --NR.sup.ffCO.sub.2R.sup.ee,
--NR.sup.ffC(.dbd.O)N(RE).sub.2, --C(.dbd.NR.sup.ff)OR.sup.ee,
--OC(.dbd.NR.sup.ff)R.sup.ee, --OC(.dbd.NR.sup.ff)OR.sup.ee,
--C(.dbd.NR.sup.ff)N(R.sup.ee).sub.2,
--OC(.dbd.NR.sup.ff)N(R.sup.ff).sub.2,
--NR.sup.ffC(.dbd.NR.sup.ff)N(R.sup.ff).sub.2,
--NR.sup.ffSO.sub.2R.sup.ee, --SO.sub.2N(R.sup.ff).sub.2,
--SO.sub.2R.sup.ee, --SO.sub.2OR.sup.ee, --OSO.sub.2R.sup.ee,
--S(.dbd.O)R.sup.ee, --Si(R.sup.ee).sub.3, --OSi(R.sup.ee).sub.3,
--C(.dbd.S)N(R.sup.ff).sub.2, --C(.dbd.O)SR.sup.ee,
--C(.dbd.S)SR.sup.ee, --SC(.dbd.S)SR.sup.ee,
--P(.dbd.O)(OR.sup.ee).sub.2, --P(.dbd.O)(R.sup.ee).sub.2,
--OP(.dbd.O)(R.sup.ee), --OP(.dbd.O)(OR.sup.ee).sub.2, C.sub.1-6
alkyl, C.sub.1-6 perhaloalkyl, C.sub.2-6 alkenyl, C.sub.2-6
alkynyl, heteroC.sub.1-6alkyl, heteroC.sub.2-6alkenyl,
heteroC.sub.2-6alkynyl, C.sub.3-10 carbocyclyl, 3-10 membered
heterocyclyl, C.sub.6-10 aryl, 5-10 membered heteroaryl, wherein
each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,
heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is
independently substituted with 0, 1, 2, 3, 4, or 5 R.sup.gg groups,
or two geminal R.sup.dd substituents can be joined to form .dbd.O
or .dbd.S; wherein X.sup.- is a counterion;
[0118] each instance of R.sup.cc is, independently, selected from
C.sub.1-6 alkyl, C.sub.1-6 perhaloalkyl, C.sub.2-6 alkenyl,
C.sub.2-6 alkynyl, heteroC.sub.1-6 alkyl, heteroC.sub.2-6alkenyl,
heteroC.sub.2-6 alkynyl, C.sub.3-10 carbocyclyl, C.sub.6-10 aryl,
3-10 membered heterocyclyl, and 3-10 membered heteroaryl, wherein
each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,
heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is
independently substituted with 0, 1, 2, 3, 4, or 5 R.sup.gg
groups;
[0119] each instance of R.sup.ff is, independently, selected from
hydrogen, C.sub.1-6 alkyl, C.sub.1-6 perhaloalkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, heteroC.sub.1-6alkyl,
heteroC.sub.2-6alkenyl, heteroC.sub.2-6alkynyl, C.sub.3-10
carbocyclyl, 3-10 membered heterocyclyl, C.sub.6-10 aryl and 5-10
membered heteroaryl, or two R.sup.ff groups are joined to form a
3-10 membered heterocyclyl or 5-10 membered heteroaryl ring,
wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,
heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is
independently substituted with 0, 1, 2, 3, 4, or 5 R.sup.gg groups;
and
[0120] each instance of R.sup.gg is, independently, halogen, --CN,
--NO.sub.2, --N.sub.3, --SO.sub.2H, --SO.sub.3H, --OH, --OC.sub.1-6
alkyl, --ON(C.sub.1-6 alkyl).sub.2, --N(C.sub.1-6 alkyl).sub.2,
--N(C.sub.1-6 alkyl).sub.3.sup.+X.sup.-, --NH(C.sub.1-6
alkyl).sub.2.sup.+X.sup.-, --NH.sub.2(C.sub.1-6
alkyl).sup.+X.sup.-, --NH.sub.3.sup.+X.sup.-, --N(OC.sub.1-6
alkyl)(C.sub.1-6 alky, --NN(OH)(C.sub.1-6 alkyl), --NH(OH), --SH,
--SC.sub.1-6 alkyl, --SS(C.sub.1-6 alkyl), --C(.dbd.O)(C.sub.1-6
alkyl), CO.sub.2H, --CO.sub.2(C.sub.1-6 alkyl),
--OC(.dbd.O)(C.sub.1-6 alkyl), --OCO.sub.2(C.sub.1-6 alkyl),
--C(.dbd.O)NH.sub.2, --C(.dbd.O)N(C.sub.1-6 alkyl).sub.2,
--OC(.dbd.C)N(C.sub.1-6 alkyl)), --NHC(.dbd.O)(C.sub.1-6 alkyl),
--N(C.sub.1-6 alkyl)C(.dbd.O)(C.sub.1-6 alkyl),
--NHCO.sub.2(C.sub.1-6 alkyl), --NHC(.dbd.O)N(C.sub.1-6
alkyl).sub.2, --NHC(.dbd.O)NH(C.sub.1-6 alkyl),
--NC(.dbd.O)NH.sub.2, --C(.dbd.NH)O(C.sub.1-6 alkyl),
--OC(.dbd.NH)(C.sub.1-6 alkyl), --OC(.dbd.NH)OC.sub.1-6 alkyl,
--C(.dbd.NH)N(C.sub.1-6 alkyl).sub.2, --C(.dbd.NH)NH(C.sub.1-6
alkyl), --C(.dbd.NH)NH.sub.2, --OC(.dbd.NH)N(C.sub.1-6
alkyl).sub.2, --OC(NH)NH(C.sub.1-6 alkyl), --OC(NH)NH.sub.2,
--NHC(NH)N(C.sub.1-6 alkyl).sub.2, --NHC(.dbd.NH)NH.sub.2,
--NHSO.sub.2(C.sub.1-6 alkyl), --SO.sub.2N(C.sub.1-6 alkyl).sub.2,
--SO.sub.2NH(C.sub.1-6 alkyl), --SO.sub.2NH.sub.2,
--SO.sub.2C.sub.1-6 alkyl, --SO.sub.2OC.sub.1-6 alkyl,
--OSO.sub.2C.sub.1-6 alkyl, --SOC.sub.1-6 alkyl, --Si(C.sub.1-6
alkyl).sub.3, --OSi(C.sub.1-6 alkyl).sub.3--C(.dbd.S)N(C.sub.1-6
alkyl).sub.2, C(.dbd.S)NH(C.sub.1-6 alkyl), C(.dbd.S)NH.sub.2,
--C(.dbd.O)S(C.sub.1-6 alkyl), --C(.dbd.S)SC.sub.1-6 alkyl,
--SC(.dbd.S)SC.sub.1-6 alkyl, --P(.dbd.O)(OC.sub.1-6 alkyl).sub.2,
--P(.dbd.O)(C.sub.1-6 alkyl).sub.2, --OP(.dbd.O)(C.sub.1-6
alkyl).sub.2, --OP(.dbd.O)(OC.sub.1-6 alkyl).sub.2, C.sub.1-6
alkyl, C.sub.1-6 perhaloalkyl, C.sub.2-6 alkenyl, C.sub.2-6
alkynyl, heteroC.sub.1-6alkyl, heteroC.sub.2-6alkenyl,
heteroC.sub.2-6alkynyl, C.sub.3-10 carbocyclyl, C.sub.6-10 aryl,
3-10 membered heterocyclyl, 5-10 membered heteroaryl; or two
geminal R.sup.gg substituents can be joined to form .dbd.O or
.dbd.S; wherein X.sup.- is a counterion.
[0121] A "counterion" or "anionic counterion" is a negatively
charged group associated with a positively charged group in order
to maintain electronic neutrality. An anionic counterion may be
monovalent (i.e., including one formal negative charge). An anionic
counterion may also be multivalent (i.e., including more than one
formal negative charge), such as divalent or trivalent. Exemplary
counterions include halide ions (e.g., F.sup.-, Cl.sup.-, Br.sup.-,
I.sup.-), NO.sub.3.sup.-, ClO.sub.4.sup.-, OH.sup.-,
H.sub.2PO.sub.4.sup.-, HCO.sub.3.sup.-, HSO.sub.4.sup.-, sulfonate
ions (e.g., methansulfonate, trifluoromethanesulfonate,
p-toluenesulfonate, benzenesulfonate, 10-camphor sulfonate,
naphthalene-2-sulfonate, naphthalene-1-sulfonic acid-5-sulfonate,
ethan-1-sulfonic acid-2-sulfonate, and the like), carboxylate ions
(e.g., acetate, propanoate, benzoate, glycerate, lactate, tartrate,
glycolate, gluconate, and the like), BF.sub.4.sup.-,
PF.sub.4.sup.-, PF.sub.6.sup.-, AsF6.sup.-, SbF.sub.6.sup.-,
B[3,5-(CF.sub.3).sub.2C.sub.6H.sub.3].sub.4].sup.-,
B(C.sub.6F.sub.5).sub.4.sup.-, BPh.sub.4.sup.-,
Al(OC(CF.sub.3).sub.3).sub.4.sup.-, and carborane anions (e.g.,
CB.sub.11H.sub.12.sup.- or (HCB.sub.11Me.sub.5Br.sub.6).sup.-).
Exemplary counterions which may be multivalent include
CO.sub.3.sup.2-, HPO.sub.4.sup.2-, PO.sub.4.sup.3-,
B.sub.4O.sub.7.sup.2-, SO.sub.4.sup.2-, S.sub.2O.sub.3.sup.2-,
carboxylate anions (e.g., tartrate, citrate, fumarate, maleate,
malate, malonate, gluconate, succinate, glutarate, adipate,
pimelate, suberate, azelate, sebacate, salicylate, phthalates,
aspartate, glutamate, and the like), and carboranes.
[0122] The term "pharmaceutically acceptable salt" refers to those
salts which are, within the scope of sound medical judgment,
suitable for use in contact with the tissues of humans and lower
animals without undue toxicity, irritation, allergic response and
the like, and are commensurate with a reasonable benefit/risk
ratio. Pharmaceutically acceptable salts are well known in the art.
For example, Berge et al., describe pharmaceutically acceptable
salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19,
incorporated by reference. Pharmaceutically acceptable salts of the
compounds disclosed in this application include those derived from
suitable inorganic and organic acids and bases. Examples of
pharmaceutically acceptable, nontoxic acid addition salts are salts
of an amino group formed with inorganic acids such as hydrochloric
acid, hydrobromic acid, phosphoric acid, sulfuric acid, and
perchloric acid or with organic acids such as acetic acid, oxalic
acid, maleic acid, tartaric acid, citric acid, succinic acid, or
malonic acid or by using other methods known in the art such as ion
exchange. Other pharmaceutically acceptable salts include adipate,
alginate, ascorbate, aspartate, benzenesulfonate, benzoate,
bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate,
cyclopentanepropionate, digluconate, dodecylsulfate,
ethanesulfonate, formate, fumarate, glucoheptonate,
glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate,
hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate,
laurate, lauryl sulfate, malate, maleate, malonate,
methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate,
oleate, oxalate, palmitate, pamoate, pectinate, persulfate,
3-phenylpropionate, phosphate, picrate, pivalate, propionate,
stearate, succinate, sulfate, tartrate, thiocyanate,
p-toluenesulfonate, undecanoate, valerate salts, and the like.
Salts derived from appropriate bases include alkali metal, alkaline
earth metal, ammonium and N.sup.+(C.sub.1-4 alkyl).sub.4 salts.
Representative alkali or alkaline earth metal salts include sodium,
lithium, potassium, calcium, magnesium, and the like. Further
pharmaceutically acceptable salts include, when appropriate,
nontoxic ammonium, quaternary ammonium, and amine cations formed
using counterions such as halide, hydroxide, carboxylate, sulfate,
phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate.
[0123] The term "solvate" refers to forms of a compound that are
associated with a solvent, usually by a solvolysis reaction. This
physical association may include hydrogen bonding. Conventional
solvents include water, methanol, ethanol, acetic acid, DMSO, THF,
diethyl ether, and the like. The compounds of Formula (1), (9),
(10), and (11) may be prepared, e.g., in crystalline form, and may
be solvated. Suitable solvates include pharmaceutically acceptable
solvates and further include both stoichiometric solvates and
non-stoichiometric solvates. In certain instances, the solvate will
be capable of isolation, for example, when one or more solvent
molecules are incorporated in the crystal lattice of a crystalline
solid. "Solvate" encompasses both solution-phase and isolable
solvates. Representative solvates include hydrates, ethanolates,
and methanolates.
[0124] The term "hydrate" refers to a compound that is associated
with water. Typically, the number of the water molecules contained
in a hydrate of a compound is in a definite ratio to the number of
the compound molecules in the hydrate. Therefore, a hydrate of a
compound may be represented, for example, by the general formula
R.x H.sub.2O, wherein R is the compound and wherein x is a number
greater than 0. A given compound may form more than one type of
hydrates, including, e.g., monohydrates (x is 1), lower hydrates (x
is a number greater than 0 and smaller than 1, e.g., hemihydrates
(R.0.5 H.sub.2O)), and polyhydrates (x is a number greater than 1,
e.g., dihydrates (R.2 H.sub.2O) and hexahydrates (R.6
H.sub.2O)).
[0125] The term "tautomers" refer to compounds that are
interchangeable forms of a particular compound structure, and that
vary in the displacement of hydrogen atoms and electrons. Thus, two
structures may be in equilibrium through the movement of .pi.
electrons and an atom (usually H). For example, enols and ketones
are tautomers because they are rapidly interconverted by treatment
with either acid or base. Another example of tautomerism is the
aci- and nitro-forms of phenylnitromethane, which are likewise
formed by treatment with acid or base. Tautomeric forms may be
relevant to the attainment of the optimal chemical reactivity and
biological activity of a compound of interest.
[0126] It is also to be understood that compounds that have the
same molecular formula but differ in the nature or sequence of
bonding of their atoms or the arrangement of their atoms in space
are termed "isomers." Isomers that differ in the arrangement of
their atoms in space are termed "stereoisomers."
[0127] Stereoisomers that are not mirror images of one another are
termed "diastereomers" and those that are non-superimposable mirror
images of each other are termed "enantiomers." When a compound has
an asymmetric center, for example, it is bonded to four different
groups, a pair of enantiomers is possible. An enantiomer can be
characterized by the absolute configuration of its asymmetric
center and described by the R- and S-sequencing rules of Cahn and
Prelog. An enantiomer can also be characterized by the manner in
which the molecule rotates the plane of polarized light, and
designated as dextrorotatory or levorotatory (i.e., as (+) or
(-)-isomers respectively). A chiral compound can exist as either an
individual enantiomer or as a mixture of enantiomers. A mixture
containing equal proportions of the enantiomers is called a
"racemic mixture."
[0128] The term "co-crystal" refers to a crystalline structure
comprising at least two different components (e.g., a compound
described in this application and an acid), wherein each of the
components is independently an atom, ion, or molecule. In certain
embodiments, none of the components is a solvent. In certain
embodiments, at least one of the components is a solvent. A
co-crystal of a compound and an acid is different from a salt
formed from a compound and the acid. In the salt, a compound
described in this application is complexed with the acid in a way
that proton transfer (e.g., a complete proton transfer) from the
acid to a compound described in this application easily occurs at
room temperature. In the co-crystal, however, a compound described
in this application is complexed with the acid in a way that proton
transfer from the acid to a compound described in this application
does not easily occur at room temperature. In certain embodiments,
in the co-crystal, there is no proton transfer from the acid to a
compound described in this application. In certain embodiments, in
the co-crystal, there is partial proton transfer from the acid to a
compound described in this application. Co-crystals may be useful
to improve the properties (e.g., solubility, stability, and ease of
formulation) of a compound described in this application.
[0129] The term "polymorphs" refers to a crystalline form of a
compound (or a salt, hydrate, or solvate thereof) in a particular
crystal packing arrangement. All polymorphs of the same compound
have the same elemental composition. Different crystalline forms
usually have different X-ray diffraction patterns, infrared
spectra, melting points, density, hardness, crystal shape, optical
and electrical properties, stability, and solubility.
Recrystallization solvent, rate of crystallization, storage
temperature, and other factors may cause one crystal form to
dominate. Various polymorphs of a compound can be prepared by
crystallization under different conditions.
[0130] The term "prodrug" refers to compounds, including
derivatives of the compounds of Formula (X), (8), (9), (10), or
(11), that have cleavable groups and become by solvolysis or under
physiological conditions the compounds of Formula (X), (8), (9),
(10), or (11) and that are pharmaceutically active in vivo. The
prodrugs may have attributes such as, without limitation,
solubility, bioavailability, tissue compatibility, or delayed
release in a mammalian organism. Examples include, but are not
limited to, derivatives of compounds described in this application,
including derivatives formed from glycosylation of the compounds
described in this application (e.g., glycoside derivatives),
carrier-linked prodrugs (e.g., ester derivatives), bioprecursor
prodrugs (a prodrug metabolized by molecular modification into the
active compound), and the like. Non-limiting examples of glycoside
derivatives are disclosed in and incorporated by reference from
WO2018208875 and US20190078168. Non-limiting examples of ester
derivatives are disclosed in and incorporated by reference from
US20170362195.
[0131] Other derivatives of the compounds of this invention have
activity in both their acid and acid derivative forms, but the acid
sensitive form often offers advantages of solubility,
bioavailability, tissue compatibility, or delayed release in a
mammalian organism (see, Bundgard, H., Design of Prodrugs, pp. 7-9,
21-24, Elsevier, Amsterdam 1985). Prodrugs include acid derivatives
well known to practitioners of the art, such as, for example,
esters prepared by reaction of the parent acid with a suitable
alcohol, or amides prepared by reaction of the parent acid compound
with a substituted or unsubstituted amine, or acid anhydrides, or
mixed anhydrides. Simple aliphatic or aromatic esters, amides, and
anhydrides derived from acidic groups pendant on the compounds of
this invention are particular prodrugs. In some cases it is
desirable to prepare double ester type prodrugs such as
(acyloxy)alkyl esters or ((alkoxycarbonyl)oxy)alkylesters.
C.sub.1-C.sub.8 alkyl, C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.8
alkynyl, aryl, C.sub.7-C.sub.12 substituted aryl, and
C.sub.7-C.sub.12 arylalkyl esters of the compounds of Formula (X),
(8), (9), (10), or (11) may be preferred.
Cannabinoids
[0132] As used in this application, the term "cannabinoid" includes
compounds of Formula (X):
##STR00026##
or a pharmaceutically acceptable salt, co-crystal, tautomer,
stereoisomer, solvate, hydrate, polymorph, isotopically enriched
derivative, or prodrug thereof, wherein R1 is hydrogen, optionally
substituted acyl, optionally substituted alkyl, optionally
substituted alkenyl, optionally substituted alkynyl, optionally
substituted carbocyclyl, or optionally substituted aryl; R2 and R6
are, independently, hydrogen or carboxyl; R3 and R5 are,
independently, hydroxyl, halogen, or alkoxy; and R4 is a hydrogen
or an optionally substituted prenyl moiety; or optionally R4 and R3
are taken together with their intervening atoms to form a cyclic
moiety, or optionally R4 and R5 are taken together with their
intervening atoms to form a cyclic moiety, or optionally both 1) R4
and R3 are taken together with their intervening atoms to form a
cyclic moiety and 2) R4 and R5 are taken together with their
intervening atoms to form a cyclic moiety. In certain embodiments,
R4 and R3 are taken together with their intervening atoms to form a
cyclic moiety. In certain embodiments, R4 and R5 are taken together
with their intervening atoms to form a cyclic moiety. In certain
embodiments, "cannabinoid" refers to a compound of Formula (X), or
a pharmaceutically acceptable salt thereof. In certain embodiments,
both 1) R4 and R3 are taken together with their intervening atoms
to form a cyclic moiety and 2) R4 and R5 are taken together with
their intervening atoms to form a cyclic moiety.
[0133] In some embodiments, cannabinoids may be synthesized via the
following steps: a) one or more reactions to incorporate three
additional ketone moieties onto an acyl-CoA scaffold, where the
acyl moiety in the acyl-CoA scaffold comprises between four and
fourteen carbons; b) a reaction cyclizing the product of step (a);
and c) a reaction to incorporate a prenyl moiety to the product of
step (b) or a derivative of the product of step (b). In some
embodiments, non-limiting examples of the acyl-CoA scaffold
described in step (a) include hexanoyl-CoA and butyryl-CoA. In some
embodiments, non-limiting examples of the product of step (b) or a
derivative of the product of step (b) include olivetolic acid and
divarinic acid.
[0134] In some embodiments, a cannabinoid compound of Formula (X)
is of Formula (X-A), (X-B), or (X-C):
##STR00027##
or a pharmaceutically acceptable salt, solvate, hydrate, polymorph,
co-crystal, tautomer, stereoisomer, isotopically labeled
derivative, or prodrug thereof, wherein is a double bond or a
single bond, as valency permits;
[0135] R is hydrogen, optionally substituted acyl, optionally
substituted alkyl, optionally substituted alkenyl, optionally
substituted alkynyl, optionally substituted carbocyclyl, or
optionally substituted aryl;
[0136] R.sup.Z1 is hydrogen, optionally substituted acyl,
optionally substituted alkyl, optionally substituted alkenyl,
optionally substituted alkynyl, optionally substituted carbocyclyl,
or optionally substituted aryl;
[0137] R.sup.Z2 is hydrogen, optionally substituted acyl,
optionally substituted alkyl, optionally substituted alkenyl,
optionally substituted alkynyl, optionally substituted carbocyclyl,
or optionally substituted aryl;
[0138] or optionally, R.sup.Z1 and R.sup.Z2 are taken together with
their intervening atoms to form an optionally substituted
carbocyclic ring;
[0139] R.sup.3A is hydrogen, optionally substituted acyl,
optionally substituted alkyl, optionally substituted alkenyl, or
optionally substituted alkynyl;
[0140] R.sup.3B is hydrogen, optionally substituted acyl,
optionally substituted alkyl, optionally substituted alkenyl, or
optionally substituted alkynyl;
[0141] R.sup.Y is hydrogen, optionally substituted acyl, optionally
substituted alkyl, optionally substituted alkenyl, or optionally
substituted alkynyl;
[0142] R.sup.Z is hydrogen, optionally substituted acyl, optionally
substituted alkyl, optionally substituted alkenyl, or optionally
substituted alkynyl.
[0143] In certain embodiments, a cannabinoid compound is of Formula
(X-A):
##STR00028##
wherein is a double bond, and each of R.sup.Z1 and R.sup.Z2 is
hydrogen, one of R.sup.3A and R.sup.3B is optionally substituted
C.sub.2-6 alkenyl, and the other one of R.sup.3A and R.sup.3B is
optionally substituted C.sub.2-6 alkyl. In some embodiments, a
cannabinoid compound of Formula (X) is of Formula (X-A), wherein
each of R.sup.Z1 and R.sup.Z2 is hydrogen, one of R.sup.3A and
R.sup.3B is a prenyl group, and the other one of R.sup.3A and
R.sup.3B is optionally substituted methyl.
[0144] In certain embodiments, a cannabinoid compound of Formula
(X) of Formula (X-A) is of Formula (11-z):
##STR00029##
wherein is a double bond or single bond, as valency permits; one of
R.sup.3A and R.sup.3B is C.sub.1-6 alkyl optionally substituted
with alkenyl, and the other of R.sup.3A and R.sup.3B is optionally
substituted C.sub.1-6 alkyl. In certain embodiments, in a compound
of Formula (11-z), is a single bond; one of R.sup.3A and R.sup.3B
is C.sub.1-6 alkyl optionally substituted with prenyl; and the
other of one of R.sup.3A and R.sup.3B is unsubstituted methyl; and
R is as described in this application. In certain embodiments, in a
compound of Formula (11-z), is a single bond; one of R.sup.3A and
R.sup.3B is
##STR00030##
and the other of one of R.sup.3A and R.sup.3B is unsubstituted
methyl; and R is as described in this application. In certain
embodiments, a cannabinoid compound of Formula (11-z) is of Formula
(11a):
##STR00031##
[0145] In certain embodiments, a cannabinoid compound of Formula
(X-A) is of Formula (10-z):
##STR00032##
wherein is a double bond or single bond, as valency permits;
R.sup.Y is hydrogen, optionally substituted acyl, optionally
substituted alkyl, optionally substituted alkenyl, or optionally
substituted alkynyl; and each of R.sup.3A and R.sup.3B is
independently optionally substituted C.sub.1-6 alkyl. In certain
embodiments, in a compound of Formula (10-z), is a single bond;
each of R.sup.3A and R.sup.3B is unsubstituted methyl, and R is as
described in this application. In certain embodiments, a
cannabinoid compound of Formula (10-z) is of Formula (10a):
##STR00033##
In certain embodiments, a compound of Formula (10a)
##STR00034##
has a chiral atom labeled with * at carbon 10 and a chiral atom
labeled with ** at carbon 6. In certain embodiments, in a compound
of Formula (10a)
##STR00035##
the chiral atom labeled with * at carbon 10 is of the
R-configuration or S-configuration; and a chiral atom labeled with
** at carbon 6 is of the R-configuration. In certain embodiments,
in a compound of Formula (10a)
##STR00036##
the chiral atom labeled with * at carbon 10 is of the
S-configuration; and a chiral atom labeled with ** at carbon 6 is
of the R-configuration or S-configuration. In certain embodiments,
in a compound of Formula (10a)
##STR00037##
the chiral atom labeled with * at carbon 10 is of the
R-configuration and a chiral atom labeled with ** at carbon 6 is of
the R-configuration. In certain embodiments, a compound of Formula
(10a)
##STR00038##
is of the formula:
##STR00039##
In certain embodiments, in a compound of Formula (10a)
##STR00040##
the chiral atom labeled with * at carbon 10 is of the
S-configuration and a chiral atom labeled with ** at carbon 6 is of
the S-configuration. In certain embodiments, a compound of Formula
(10a)
##STR00041##
is of the formula:
##STR00042##
[0146] In certain embodiments, a cannabinoid compound is of Formula
(X-B):
##STR00043##
wherein is a double bond; R.sup.Y is hydrogen, optionally
substituted acyl, optionally substituted alkyl, optionally
substituted alkenyl, or optionally substituted alkynyl; and each of
R.sup.3A and R.sup.3B is independently optionally substituted
C.sub.1-6 alkyl. In certain embodiments, in a compound of Formula
(X-B), R.sup.Y is optionally substituted C.sub.1-6 alkyl; one of
R.sup.3A and R.sup.3B is
##STR00044##
and the other one of R.sup.3A and R.sup.3B is unsubstituted methyl,
and R is as described in this application. In certain embodiments,
a compound of Formula (X-B) is of Formula (9a):
##STR00045##
In certain embodiments, a compound of Formula (9a)
##STR00046##
has a chiral atom labeled with * at carbon 3 and a chiral atom
labeled with ** at carbon 4. In certain embodiments, in a compound
of Formula (9a)
##STR00047##
the chiral atom labeled with * at carbon 3 is of the
R-configuration or S-configuration; and a chiral atom labeled with
** at carbon 4 is of the R-configuration. In certain embodiments,
in a compound of Formula (9a)
##STR00048##
the chiral atom labeled with * at carbon 3 is of the
S-configuration; and a chiral atom labeled with ** at carbon 4 is
of the R-configuration or S-configuration. In certain embodiments,
in a compound of Formula (9a)
##STR00049##
the chiral atom labeled with * at carbon 3 is of the
R-configuration and a chiral atom labeled with ** at carbon 4 is of
the R-configuration. In certain embodiments, a compound of Formula
(9a)
##STR00050##
is of the formula:
##STR00051##
In certain embodiments, in a compound of Formula (9a)
##STR00052##
the chiral atom labeled with * at carbon 3 is of the
S-configuration and a chiral atom labeled with ** at carbon 4 is of
the S-configuration. In certain embodiments, a compound of Formula
(9a)
##STR00053##
is of the formula:
##STR00054##
[0147] In certain embodiments, a cannabinoid compound is of Formula
(X-C):
##STR00055##
wherein R.sup.Z is optionally substituted alkyl or optionally
substituted alkenyl. In certain embodiments, a compound of Formula
(X-C) is of formula:
##STR00056##
wherein a is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In certain
embodiments, a is 1. In certain embodiments, a is 2. In certain
embodiments, a is 3. In certain embodiments, a is 1, 2, or 3 for a
compound of Formula (X-C). In certain embodiments, a cannabinoid
compound is of Formula (X-C), and a is 1, 2, 3, 4, or 5. In certain
embodiments, a compound of Formula (X-C) is of Formula (8a):
##STR00057##
[0148] In some embodiments, cannabinoids of the present disclosure
comprise cannabinoid receptor ligands. Cannabinoid receptors are a
class of cell membrane receptors in the G protein-coupled receptor
superfamily. Cannabinoid receptors include the CB.sub.1 receptor
and the CB.sub.2 receptor. In some embodiments, cannabinoid
receptors comprise GPR18, GPR55, and PPAR. (See Bram et al.
"Activation of GPR18 by cannabinoid compounds: a tale of biased
agonism" Br J Pharmcol v171 (16) (2014); Shi et al. "The novel
cannabinoid receptor GPR55 mediates anxiolytic-like effects in the
medial orbital cortex of mice with acute stress" Molecular Brain
10, No. 38 (2017); and O'Sullvan, Elizabeth. "An update on PPAR
activation by cannabinoids" Br J Pharmcol v. 173(12) (2016)).
[0149] In some embodiments, cannabinoids comprise endocannabinoids,
which are substances produced within the body, and
phytocannabinoids, which are cannabinoids that are naturally
produced by plants of genus Cannabis. In some embodiments,
phytocannabinoids comprise the acidic and decarboxylated acid forms
of the naturally-occurring plant-derived cannabinoids, and their
synthetic and biosynthetic equivalents.
[0150] Over 94 phytocannabinoids have been identified to date
(Berman, Paula, et al. "A new ESI-LC/MS approach for comprehensive
metabolic profiling of phytocannabinoids in Cannabis." Scientific
reports 8.1 (2018): 14280; El-Alfy et al., 2010,
"Antidepressant-like effect of delta-9-tetrahydrocannabinol and
other cannabinoids isolated from Cannabis sativa L", Pharmacology
Biochemistry and Behavior 95 (4): 434-42; Rudolf Brenneisen, 2007,
Chemistry and Analysis of Phytocannabinoids, each of which is
incorporated by reference in this application in its entirety). In
some embodiments, cannabinoids comprise
.DELTA..sup.9-tetrahydrocannabinol (THC) type (e.g.,
(-)-trans-delta-9-tetrahydrocannabinol or dronabinol,
(+)-trans-delta-9-tetrahydrocannabinol,
(-)-cis-delta-9-tetrahydrocannabinol, or
(+)-cis-delta-9-tetrahydrocannabinol), cannabidiol (CBD) type,
cannabigerol (CBG) type, cannabichromene (CBC) type, cannabicyclol
(CBL) type, cannabinodiol (CBND) type, or cannabitriol (CBT) type
cannabinoids, or any combination thereof (see, e.g., R Pertwee, ed,
Handbook of Cannabis (Oxford, UK: Oxford University Press, 2014)),
which is incorporated by reference in this application in its
entirety). A non-limiting list of cannabinoids comprises:
cannabiorcol-C1 (CBNO), CBND-C1 (CBNDO),
.DELTA..sup.9-trans-Tetrahydrocannabiorcolic acid-C1
(.DELTA..sup.9-THCO), Cannabidiorcol-C1 (CBDO),
Cannabiorchromene-C1 (CBCO),
(-)-.DELTA..sup.8-trans-(6aR,10aR)-Tetrahydrocannabiorcol-C1
(.DELTA..sup.8-THCO), Cannabiorcyclol C1 (CBLO), CBG-C1 (CBGO),
Cannabinol-C2 (CBN-C2), CBND-C2, .DELTA..sup.9-THC-C2, CBD-C2,
CBC-C2, .DELTA..sup.8-THC-C2, CBL-C2, Bisnor-cannabielsoin-C1
(CBEO), CBG-C2, Cannabivarin-C3 (CBNV), Cannabinodivarin-C3
(CBNDV), (-)-.DELTA..sup.9-trans-Tetrahydrocannabivarin-C3
(.DELTA..sup.9-THCV), (-)-Cannabidivarin-C3 (CBDV),
(.+-.)-Cannabichromevarin-C3 (CBCV), (-)-.DELTA..sup.8-trans-THC-C3
(.DELTA..sup.8-THCV), (.+-.)-(1aS,3aR,8bR,8cR)-Cannabicyclovarin-C3
(CBLV),
2-Methyl-2-(4-methyl-2-pentenyl)-7-propyl-2H-1-benzopyran-5-ol,
.DELTA..sup.7-tetrahydrocannabivarin-C3 (.DELTA..sup.7-THCV),
CBE-C2, Cannabigerovarin-C3 (CBGV), Cannabitriol-C1 (CBTO),
Cannabinol-C4 (CBN-C4), CBND-C4,
(-)-.DELTA..sup.9-trans-Tetrahydrocannabinol-C4
(.DELTA..sup.9-THC-C4), Cannabidiol-C4 (CBD-C4), CBC-C4,
(-)-trans-.DELTA..sup.8-THC-C4, CBL-C4, Cannabielsoin-C3 (CBEV),
CBG-C4, CBT-C2, Cannabichromanone-C3, Cannabiglendol-C3
(OH-iso-HHCV-C3), Cannabioxepane-C5 (CBX), Dehydrocannabifuran-C5
(DCBF), Cannabinol-C5 (CBN), Cannabinodiol-C5 (CBND),
(-)-.DELTA..sup.9-trans-Tetrahydrocannabinol-C5
(.DELTA..sup.9-THC),
(-)-.DELTA..sup.8-trans-(6aR,10aR)-Tetrahydrocannabinol-C5
(.DELTA..sup.8-THC), (.+-.)-Cannabichromene-C5 (CBC),
(-)-Cannabidiol-C5 (CBD), (.+-.)-(1aS,3aR,8bR,8cR)-CannabicyclolC5
(CBL), Cannabicitran-C5 (CBR),
(-)-.DELTA..sup.9-(6aS,10aR-cis)-Tetrahydrocannabinol-C5
((-)-cis-.DELTA..sup.9-THC),
(-)-.DELTA..sup.7-trans-(1R,3R,6R)-Isotetrahydrocannabinol-C5
(trans-iso.DELTA..sup.7-THC), CBE-C4, Cannabigerol-C5 (CBG),
Cannabitriol-C3 (CBTV), Cannabinol methyl ether-C5 (CBNM),
CBNDM-C5, 8-OH--CBN-C5 (OH-CBN), OH-CBND-C5 (OH-CBND),
10-Oxo-.DELTA..sup.6a(10a)-Tetrahydrocannabinol-C5 (OTHC),
Cannabichromanone D-C5, Cannabicoumaronone-C5 (CBCON-C5),
Cannabidiol monomethyl ether-C5 (CBDM), .DELTA..sup.9-THCM-C5,
(.+-.)-3''-hydroxy-.DELTA..sup.4''-cannabichromene-C5,
(5aS,6S,9R,9aR)-Cannabielsoin-C5 (CBE),
2-geranyl-5-hydroxy-3-n-pentyl-1,4-benzoquinone-C5, 5-geranyl
olivetolic acid, 5-geranyl olivetolate,
8.alpha.-Hydroxy-.DELTA..sup.9-Tetrahydrocannabinol-C5
(8.alpha.-OH-.DELTA..sup.9-THC),
8.beta.-Hydroxy-.DELTA..sup.9-Tetrahydrocannabinol-C5
(8.beta.-OH-.DELTA..sup.9-THC),
10.alpha.-Hydroxy-.DELTA..sup.8-Tetrahydrocannabinol-C5
(10.alpha.-OH-.DELTA..sup.8-THC),
10.beta.-Hydroxy-.DELTA..sup.8-Tetrahydrocannabinol-C5
(10.beta.-OH-.DELTA..sup.8-THC),
10.alpha.-hydroxy-.DELTA..sup.9,11-hexahydrocannabinol-C5,
9.beta.,10.beta.-Epoxyhexahydrocannabinol-C5, OH-CBD-C5 (OH-CBD),
Cannabigerol monomethyl ether-C5 (CBGM), Cannabichromanone-C5,
CBT-C4, (.+-.)-6,7-cis-epoxycannabigerol-C5,
(.+-.)-6,7-trans-epoxycannabigerol-C5,
(-)-7-hydroxycannabichromane-C5, Cannabimovone-C5,
(-)-trans-Cannabitriol-C5 ((-)-trans-CBT),
(+)-trans-Cannabitriol-C5 ((+)-trans-CBT),
(.+-.)-cis-Cannabitriol-C5 ((.+-.)-cis-CBT),
(-)-trans-1-Ethoxy-9-hydroxy-.DELTA..sup.6a(10a)-tetrahydrocannabivarin-C-
3 [(-)-trans-CBT-OEt],
(-)-(6aR,9S,10S,10aR)-9,10-Dihydroxyhexahydrocannabinol-C5
[(-)-Cannabiripsol] (CBR), Cannabichromanone C-C5,
(-)-6a,7,10a-Trihydroxy-.DELTA..sup.9-tetrahydrocannabinol-C5
[(-)-Cannabitetrol] (CBTT), Cannabichromanone B-C5,
8,9-Dihydroxy-.DELTA..sup.6a(10a)-tetrahydrocannabinol-C5
(8,9-Di-OHCBT), (.+-.)-4-acetoxycannabichromene-C5,
2-acetoxy-6-geranyl-3-n-pentyl-1,4-benzoquinone-C5,
11-Acetoxy-.DELTA. 9-TetrahydrocannabinolC5 (11-OAc-.DELTA. 9-THC),
5-acetyl-4-hydroxycannabigerol-C5,
4-acetoxy-2-geranyl-5-hydroxy-3-npentylphenol-C5,
(-)-trans-10-Ethoxy-9-hydroxy-.DELTA..sup.6a(10a)-tetrahydrocannabinol-C5
((-)-trans-CBTOEt), sesquicannabigerol-C5 (SesquiCBG),
carmagerol-C5, 4-terpenyl cannabinolate-C5,
.beta.-fenchyl-.DELTA..sup.9-tetrahydrocannabinolate-C5,
.alpha.-fenchyl-.DELTA..sup.9-tetrahydrocannabinolate-C5,
epi-bornyl-.DELTA..sup.9-tetrahydrocannabinolate-C5,
bornyl-.DELTA..sup.9-tetrahydrocannabinolate-C5,
.alpha.-terpenyl-.DELTA..sup.9-tetrahydrocannabinolate-C5,
4-terpenyl-.DELTA..sup.9-tetrahydrocannabinolate-C5,
6,6,9-trimethyl-3-pentyl-6H-dibenzo[b,d]pyran-1-ol,
3-(1,1-dimethylheptyl)-6,6a,7,8,10,10a-hexahydro-1-hydroxy-6,6-dimethyl-9-
H-dibenzo[b,d]pyran-9-one, (-)-(3
S4S)-7-hydroxy-.DELTA..sup.6-tetrahydrocannabinol-1,1-dimethylheptyl,
(+)-(3S,4S)-7-hydroxy-A-tetrahydrocannabinol-1,1-dimethylheptyl,
11-hydroxy-.DELTA..sup.9-tetrahydrocannabinol, and
.DELTA..sup.8-tetrahydrocannabinol-11-oic acid)); certain
piperidine analogs (e g.,
(-)-(6S,6aR,9R,10aR)-5,6,6a,7,8,9,10,10a-octahydro-6-methyl-3-[(R)-1-meth-
yl-4-phenylbutoxy]-1,9-phenanthridinediol 1-acetate)), certain
aminoalkylindole analogs (e.g.,
(R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)-pyrrolo[1,2,3-de]-1-
,4-benzoxazin-6-yl]-1-naphthalenyl-methanone), certain open pyran
ring analogs (e.g.,
2-[3-methyl-6-(1-methylethenyl)-2-cyclohexen-1-yl]-5-pentyl-1,3-benzenedi-
ol and
4-(1,1-dimethylheptyl)-2,3'-dihydroxy-6'alpha-(3-hydroxypropyl)-1',-
2',3',4',5',6'-hexahydrobiphenyl, tetrahydrocannabiphorol (THCP),
cannabidiphorol (CBDP), CBGP, CBCP, their acidic forms, salts of
the acidic forms, or any combination thereof.
[0151] A cannabinoid described in this application can be a rare
cannabinoid. For example, in some embodiments, a cannabinoid
described in this application corresponds to a cannabinoid that is
naturally produced in conventional Cannabis varieties at
concentrations of less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%,
1%, 0.9%, 0.8%, 0.7%, 0.6% 0.5%, 0.25%, or 0.1% by dry weight of
the female flower. In some embodiments, rare cannabinoids include
CBGA, CBGVA, THCVA, CBDVA, CBCVA, and CBCA. In some embodiments,
rare cannabinoids are cannabinoids that are not THCA, THC, CBDA or
CBD.
[0152] A cannabinoid described in this application can also be a
non-rare cannabinoid.
[0153] In some embodiments, the cannabinoid is selected from the
cannabinoids listed in Table 1.
TABLE-US-00001 TABLE 1 Non-limiting examples of cannabinoids
according to the present disclosure. ##STR00058## ##STR00059##
##STR00060## ##STR00061## ##STR00062## ##STR00063## ##STR00064##
##STR00065## ##STR00066## ##STR00067## ##STR00068## ##STR00069##
##STR00070## ##STR00071## ##STR00072## ##STR00073## ##STR00074##
##STR00075## ##STR00076## ##STR00077## ##STR00078## ##STR00079##
##STR00080## ##STR00081## ##STR00082## ##STR00083## ##STR00084##
##STR00085## ##STR00086## ##STR00087## ##STR00088## ##STR00089##
##STR00090## ##STR00091## ##STR00092## ##STR00093## ##STR00094##
##STR00095## ##STR00096## ##STR00097## ##STR00098## ##STR00099##
##STR00100## ##STR00101## ##STR00102## ##STR00103## ##STR00104##
##STR00105## ##STR00106## ##STR00107## ##STR00108## ##STR00109##
##STR00110## ##STR00111## ##STR00112## ##STR00113## ##STR00114##
##STR00115## ##STR00116## ##STR00117## ##STR00118## ##STR00119##
##STR00120## ##STR00121##
Biosynthesis of Cannabinoids and Cannabinoid Precursors
[0154] Aspects of the present disclosure provide tools, sequences,
and methods for the biosynthetic production of cannabinoids in host
cells. In some embodiments, the present disclosure teaches
expression of enzymes that are capable of producing cannabinoids by
biosynthesis.
[0155] As a non-limiting example, one or more of the enzymes
depicted in FIG. 2 may be used to produce a cannabinoid or
cannabinoid precursor of interest. FIG. 1 shows a cannabinoid
biosynthesis pathway for the most abundant phytocannabinoids found
in Cannabis. See also, de Meijer et al. I, II, III, and IV (I:
2003, Genetics, 163:335-346; II: 2005, Euphytica, 145:189-198; III:
2009, Euphytica, 165:293-311; and IV: 2009, Euphytica, 168:95-112),
and Carvalho et al. "Designing Microorganisms for Heterologous
Biosynthesis of Cannabinoids" (2017) FEMS Yeast Research June 1;
17(4), each of which is in this application incorporated by
reference in its entirety for all purposes.
[0156] It should be appreciated that a precursor substrate for use
in cannabinoid biosynthesis is generally selected based on the
cannabinoid of interest. Non-limiting examples of cannabinoid
precursors include compounds of Formulae 1-8 in FIG. 2. In some
embodiments, polyketides, including compounds of Formula 5, could
be prenylated. In certain embodiments, the precursor is a precursor
compound shown in FIG. 1, 2, or 3. Substrates containing 1-40
carbon atoms are preferred. In some embodiments, substrates
containing 3-8 carbon atoms are most preferred.
[0157] As used in this application, a cannabinoid or a cannabinoid
precursor may comprise an R group. See, e.g., FIG. 2. In some
embodiments, R may be a hydrogen. In certain embodiments, R is
optionally substituted alkyl. In certain embodiments, R is
optionally substituted C1-40 alkyl. In certain embodiments, R is
optionally substituted C2-40 alkyl. In certain embodiments, R is
optionally substituted C2-40 alkyl, which is straight chain or
branched alkyl. In certain embodiments, R is optionally substituted
C3-8 alkyl. In certain embodiments, R is optionally substituted
C1-C40 alkyl, C1-C20 alkyl, C1-C10 alkyl, C1-C8 alkyl, C1-C5 alkyl,
C3-C5 alkyl, C3 alkyl, or C5 alkyl. In certain embodiments, R is
optionally substituted C1-C20 alkyl. In certain embodiments, R is
optionally substituted C1-C10 alkyl. In certain embodiments, R is
optionally substituted C1-C8 alkyl. In certain embodiments, R is
optionally substituted C1-C5 alkyl. In certain embodiments, R is
optionally substituted C1-C7 alkyl. In certain embodiments, R is
optionally substituted C3-C5 alkyl. In certain embodiments, R is
optionally substituted C3 alkyl. In certain embodiments, R is
unsubstituted C3 alkyl. In certain embodiments, R is n-C3 alkyl. In
certain embodiments, R is n-propyl. In certain embodiments, R is
n-butyl. In certain embodiments, R is n-pentyl. In certain
embodiments, R is n-hexyl. In certain embodiments, R is n-heptyl.
In certain embodiments, R is of formula:
##STR00122##
In certain embodiments, R is optionally substituted C4 alkyl. In
certain embodiments, R is unsubstituted C4 alkyl. In certain
embodiments, R is optionally substituted C5 alkyl. In certain
embodiments, R is unsubstituted C5 alkyl. In certain embodiments, R
is optionally substituted C6 alkyl. In certain embodiments, R is
unsubstituted C6 alkyl. In certain embodiments, R is optionally
substituted C7 alkyl. In certain embodiments, R is unsubstituted C7
alkyl. In certain embodiments, R is of formula:
##STR00123##
In certain embodiments, R is of formula:
##STR00124##
In certain embodiments, R is of formula:
##STR00125##
In certain embodiments, R is of formula:
##STR00126##
In certain embodiments, R is of formula:
##STR00127##
In certain embodiments, R is optionally substituted n-propyl. In
certain embodiments, R is n-propyl optionally substituted with
optionally substituted aryl. In certain embodiments, R is n-propyl
optionally substituted with optionally substituted phenyl. In
certain embodiments, R is n-propyl substituted with unsubstituted
phenyl. In certain embodiments, R is optionally substituted butyl.
In certain embodiments, R is optionally substituted n-butyl. In
certain embodiments, R is n-butyl optionally substituted with
optionally substituted aryl. In certain embodiments, R is n-butyl
optionally substituted with optionally substituted phenyl. In
certain embodiments, R is n-butyl substituted with unsubstituted
phenyl. In certain embodiments, R is optionally substituted pentyl.
In certain embodiments, R is optionally substituted n-pentyl. In
certain embodiments, R is n-pentyl optionally substituted with
optionally substituted aryl. In certain embodiments, R is n-pentyl
optionally substituted with optionally substituted phenyl. In
certain embodiments, R is n-pentyl substituted with unsubstituted
phenyl. In certain embodiments, R is optionally substituted hexyl.
In certain embodiments, R is optionally substituted n-hexyl. In
certain embodiments, R is optionally substituted n-heptyl. In
certain embodiments, R is optionally substituted n-octyl. In
certain embodiments, R is alkyl optionally substituted with aryl
(e.g., phenyl). In certain embodiments, R is optionally substituted
acyl (e.g., --C(.dbd.O)Me).
[0158] In certain embodiments, R is optionally substituted alkenyl
(e.g., substituted or unsubstituted C.sub.2-6 alkenyl). In certain
embodiments, R is substituted or unsubstituted C.sub.2-6 alkenyl.
In certain embodiments, R is substituted or unsubstituted C.sub.2-5
alkenyl. In certain embodiments, R is of formula:
##STR00128##
In certain embodiments, R is optionally substituted alkynyl (e.g.,
substituted orunsubstituted C.sub.2-6 alkynyl). In certain
embodiments, R is substituted or unsubstituted C.sub.2-6 alkynyl.
In certain embodiments, R is of formula:
##STR00129##
In certain embodiments, R is optionally substituted carbocyclyl. In
certain embodiments, R is optionally substituted aryl (e.g., phenyl
or napthyl).
[0159] The chain length of a precursor substrate can be from
C1-C40. Those substrates can have any degree and any kind of
branching or saturation or chain structure, including, without
limitation, aliphatic, alicyclic, and aromatic. In addition, they
may include any functional groups including hydroxy, halogens,
carbohydrates, phosphates, methyl-containing or nitrogen-containing
functional groups.
[0160] For example, FIG. 3 shows a non-exclusive set of putative
precursors for the cannabinoid pathway. Aliphatic carboxylic acids
including four to eight total carbons ("C4"-"C8" in FIG. 3) and up
to 10-12 total carbons with either linear or branched chains may be
used as precursors for the heterologous pathway. Non-limiting
examples include methanoic acid, butyric acid, pentanoic acid,
hexanoic acid, heptanoic acid, isovaleric acid, octanoic acid, and
decanoic acid. Additional precursors may include ethanoic acid and
propanoic acid. In some embodiments, in addition to acids, the
ester, salt, and acid forms may all be used as substrates.
Substrates may have any degree and any kind of branching,
saturation, and chain structure, including, without limitation,
aliphatic, alicyclic, and aromatic. In addition, they may include
any functional modifications or combination of modifications
including, without limitation, halogenation, hydroxylation,
amination, acylation, alkylation, phenylation, and/or installation
of pendant carbohydrates, phosphates, sulfates, heterocycles, or
lipids, or any other functional groups.
[0161] Substrates for any of the enzymes disclosed in this
application may be provided exogenously or may be produced
endogenously by a host cell. In some embodiments, the cannabinoids
are produced from a glucose substrate, so that compounds of Formula
1 shown in FIG. 2 and CoA precursors are synthesized by the cell.
In other embodiments, a precursor is fed into the reaction. In some
embodiments, a precursor is a compound selected from Formulae 1-8
in FIG. 2.
[0162] Cannabinoids produced by methods disclosed in this
application include rare cannabinoids. Due to the low
concentrations at which rare cannabinoids occur in nature,
producing industrially significant amounts of isolated or purified
rare cannabinoids from the Cannabis plant may become prohibitive
due to, e.g., the large volumes of Cannabis plants, and the large
amounts of space, labor, time, and capital requirements to grow,
harvest, and/or process the plant materials. The disclosure
provided in this application represents a potentially efficient
method for producing high yields of cannabinoids, including rare
cannabinoids.
[0163] Cannabinoids produced by the disclosed methods also include
non-rare cannabinoids. Without being bound by a particular theory,
the methods described in this application may be advantageous
compared with traditional plant-based methods for producing
non-rare cannabinoids. For example, methods provided in this
application represent potentially efficient means for producing
consistent and high yields of non-rare cannabinoids. With
traditional methods of cannabinoid production, in which
cannabinoids are harvested from plants, maintaining consistent and
uniform conditions, including airflow, nutrients, lighting,
temperature, and humidity, can be difficult. For example, with
plant-based methods, there can be microclimates created by
branching, which can lead to inconsistent yields and by-product
formation. In some embodiments, the methods described in this
application are more efficient at producing a cannabinoid of
interest as compared to harvesting cannabinoids from plants. For
example, with plant-based methods, seed-to-harvest can take up to
half a year, while cutting-to-harvest usually takes about 4 months.
Additional steps including drying, curing, and extraction are also
usually needed with plant-based methods. In contrast, in some
embodiments, the fermentation-based methods described in this
application only take about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days.
In some embodiments, the fermentation-based methods described in
this application only take about 3-5 days. In some embodiments, the
fermentation-based methods described in this application only take
about 5 days. In some embodiments, the methods provided in this
application reduce the amount of security needed to comply with
regulatory standards. For example, a smaller secured area may be
needed to be monitored and secured to practice the methods
described in this application as compared to the cultivation of
plants. In some embodiments, the methods described in this
application are advantageous over plant-sourced cannabinoids.
Cannabinoid Pathway Enzymes
[0164] Methods for production of cannabinoids and cannabinoid
precursors can include expression of one or more of: an acyl
activating anzyme (AAE); a polyketide synthase (PKS) (e.g., OLS); a
polyketide cyclase (PKC); a prenyltransferase (PT) and a terminal
synthase (TS).
Acyl Activating Enzyme (AAE)
[0165] A host cell described in this disclosure may comprise an
AAE. As used in this disclosure, an AAE refers to an enzyme that is
capable of catalyzing the esterification between a thiol and a
substrate (e.g., optionally substituted aliphatic or aryl group)
that has a carboxylic acid moiety. In some embodiments, an AAE is
capable of using Formula (1):
##STR00130##
or a salt, solvate, hydrate, polymorph, co-crystal, tautomer,
stereoisomer, isotopically labeled derivative thereof to produce a
product of Formula (2):
##STR00131##
[0166] R is as defined in this application. In certain embodiments,
R is hydrogen. In certain embodiments, R is optionally substituted
alkyl. In certain embodiments, R is optionally substituted C1-40
alkyl. In certain embodiments, R is optionally substituted C2-40
alkyl. In certain embodiments, R is optionally substituted C2-40
alkyl, which is straight chain or branched alkyl. In certain
embodiments, R is optionally substituted C2-10 alkyl, optionally
substituted C10-C20 alkyl, optionally substituted C20-C30 alkyl,
optionally substituted C30-C40 alkyl, or optionally substituted
C40-C50 alkyl, which is straight chain or branched alkyl. In
certain embodiments, R is optionally substituted C3-8 alkyl. In
certain embodiments, R is optionally substituted C1-C40 alkyl,
C1-C20 alkyl, C1-C10 alkyl, C1-C8 alkyl, C1-C5 alkyl, C3-C5 alkyl,
C3 alkyl, or C.sub.5 alkyl. In certain embodiments, R is optionally
substituted C.sub.1-C20 alkyl. In certain embodiments, R is
optionally substituted C1-C20 branched alkyl. In certain
embodiments, R is optionally substituted C1-C20 alkyl, optionally
substituted C1-C10 alkyl, optionally substituted C10-C20 alkyl,
optionally substituted C20-C30 alkyl, optionally substituted
C30-C40 alkyl, or optionally substituted C40-C50 alkyl. In certain
embodiments, R is optionally substituted C1-C10 alkyl. In certain
embodiments, R is optionally substituted C3 alkyl. In certain
embodiments, R is optionally substituted n-propyl. In certain
embodiments, R is unsubstituted n-propyl. In certain embodiments, R
is optionally substituted C.sub.1-C8 alkyl. In some embodiments, R
is a C.sub.2-C6 alkyl. In certain embodiments, R is optionally
substituted C1-C5 alkyl. In certain embodiments, R is optionally
substituted C3-C5 alkyl. In certain embodiments, R is optionally
substituted C3 alkyl. In certain embodiments, R is optionally
substituted C5 alkyl. In certain embodiments, R is of formula:
##STR00132##
In certain embodiments, R is of formula:
##STR00133##
In certain embodiments, R is of formula:
##STR00134##
In certain embodiments, R is of formula:
##STR00135##
In certain embodiments, R is optionally substituted propyl. In
certain embodiments, R is optionally substituted n-propyl. In
certain embodiments, R is n-propyl optionally substituted with
optionally substituted aryl. In certain embodiments, R is n-propyl
optionally substituted with optionally substituted phenyl. In
certain embodiments, R is n-propyl substituted with unsubstituted
phenyl. In certain embodiments, R is optionally substituted butyl.
In certain embodiments, R is optionally substituted n-butyl. In
certain embodiments, R is n-butyl optionally substituted with
optionally substituted aryl. In certain embodiments, R is n-butyl
optionally substituted with optionally substituted phenyl. In
certain embodiments, R is n-butyl substituted with unsubstituted
phenyl. In certain embodiments, R is optionally substituted pentyl.
In certain embodiments, R is optionally substituted n-pentyl. In
certain embodiments, R is n-pentyl optionally substituted with
optionally substituted aryl. In certain embodiments, R is n-pentyl
optionally substituted with optionally substituted phenyl. In
certain embodiments, R is n-pentyl substituted with unsubstituted
phenyl. In certain embodiments, R is optionally substituted hexyl.
In certain embodiments, R is optionally substituted n-hexyl. In
certain embodiments, R is optionally substituted n-heptyl. In
certain embodiments, R is optionally substituted n-octyl. In
certain embodiments, R is alkyl optionally substituted with aryl
(e.g., phenyl). In certain embodiments, R is optionally substituted
acyl (e.g., --C(.dbd.O)Me).
[0167] In certain embodiments, R is optionally substituted alkenyl
(e.g., substituted or unsubstituted C.sub.2-6 alkenyl). In certain
embodiments, R is substituted or unsubstituted C.sub.2-6 alkenyl.
In certain embodiments, R is substituted or unsubstituted C.sub.2-5
alkenyl. In certain embodiments, R is of formula:
##STR00136##
In certain embodiments, R is optionally substituted alkynyl (e.g.,
substituted orunsubstituted C.sub.2-6 alkynyl). In certain
embodiments, R is substituted orunsubstituted C.sub.2-6 alkynyl. In
certain embodiments, R is of formula:
##STR00137##
In certain embodiments, R is optionally substituted carbocyclyl. In
certain embodiments, R is optionally substituted aryl (e.g., phenyl
or napthyl).
[0168] In some embodiments, a substrate for an AAE is produced by
fatty acid metabolism within a host cell. In some embodiments, a
substrate for an AAE is provided exogenously.
[0169] In some embodiments, an AAE is capable of catalyzing the
formation of hexanoyl-coenzyme A (hexanoyl-CoA) from hexanoic acid
and coenzyme A (CoA). In some embodiments, an AAE is capable of
catalyzing the formation of butanoyl-coenzyme A (butanoyl-CoA) from
butanoic acid and coenzyme A (CoA).
[0170] As one of ordinary skill in the art would appreciate, an AAE
could be obtained from any source, including naturally occurring
sources and synthetic sources (e.g., a non-naturally occurring
AAE). In some embodiments, an AAE is a Cannabis enzyme.
Non-limiting examples of AAEs include C. sativa hexanoyl-CoA
synthetase 1 (CsHCS1) and C. sativa hexanoyl-CoA synthetase 2
(CsHCS2) as disclosed in U.S. Pat. No. 9,546,362, which is
incorporated by reference in this application in its entirety.
TABLE-US-00002 CsHCS1 has the sequence: (SEQ ID NO: 109)
MGKNYKSLDSVVASDFIALGITSEVAETLHGRLAEIVCNYGAATPQTWI
NIANHILSPDLPFSLHQMLFYGCYKDFGPAPPAWIPDPEKVKSTNLGAL
LEKRGKEFLGVKYKDPISSFSHFQEFSVRNPEVYWRTVLMDEMKISFSK
DPECILRRDDINNPGGSEWLPGGYLNSAKNCLNVNSNKKLNDTMIVWRD
EGNDDLPLNKLTLDQLRKRVWLVGYALEEMGLEKGCAIAIDMPMHVDAV
VIYLAIVLAGYVVVSIADSFSAPEISTRLRLSKAKAIFTQDHIIRGKKR
IPLYSRVVEAKSPMAIVIPCSGSNIGAELRDGDISWDYFLERAKEFKNC
EFTAREQPVDAYTNILFSSGTTGEPKAIPWTQATPLKAAADGWSHLDIR
KGDVIVWPTNLGWMMGPWLVYASLLNGASIALYNGSPLVSGFAKFVQDA
KVTMLGVVPSIVRSWKSTNCVSGYDWSTIRCFSSSGEASNVDEYLWLMG
RANYKPVIEMCGGTEIGGAFSAGSFLQAQSLSSFSSQCMGCTLYILDKN
GYPMPKNKPGIGELALGPVMFGASKTLLNGNHHDVYFKGMPTLNGEVLR
RHGDIFELTSNGYYHAHGRADDTMNIGGIKISSIEIERVCNEVDDRVFE
TTAIGVPPLGGGPEQLVIFFVLKDSNDTTIDLNQLRLSFNLGLQKKLNP
LFKVTRVVPLSSLPRTATNKIMRRVLRQFSHFE. CsHCS2 has the sequence: (SEQ ID
NO: 129) MEKSGYGRDGIYRSLRPPLHLPNNNNLSMVSFLFRNSSSYPQKPALIDS
ETNQILSFSHFKSTVIKVSHGFLNLGIKKNDVVLIYAPNSIHFPVCFLG
IIASGAIATTSNPLYTVSELSKQVKDSNPKLIITVPQLLEKVKGFNLPT
ILIGPDSEQESSSDKVMTFNDLVNLGGSSGSEFPIVDDFKQSDTAALLY
SSGTTGMSKGVVLTHKNFIASSLMVTMEQDLVGEMDNVFLCFLPMFHVF
GLAIITYAQLQRGNTVISMARFDLEKMLKDVEKYKVTHLWVVPPVILAL
SKNSMVKKFNLSSIKYIGSGAAPLGKDLMEECSKVVPYGIVAQGYGMTE
TCGIVSMEDIRGGKRNSGSAGMLASGVEAQIVSVDTLKPLPPNQLGEIW
VKGPNMMQGYFNNPQATKLTIDKKGWVHTGDLGYFDEDGHLYVVDRIKE
LIKYKGFQVAPAELEGLLVSHPEILDAVVIPFPDAEAGEVPVAYVVRSP
NSSLTENDVKKFIAGQVASFKRLRKVTFINSVPKSASGKILRRELIQKV RSNM.
[0171] In some embodiments, an AAE comprises a sequence that is at
least 5%, at least 10%, at least 15%, at least 20%, at least 25%,
at least 30%, at least 35%, at least 40%, at least 45%, at least
50%, at least 55%, at least 60%, at least 65%, at least 70%, at
least 71%, at least 72%, at least 73%, at least 74%, at least 75%,
at least 76%, at least 77%, at least 78%, at least 79%, at least
80%, at least 81%, at least 82%, at least 83%, at least 84%, at
least 85%, at least 86%, at least 87%, at least 88%, at least 89%,
at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99%, or is 100% identical, including all values in between,
to a sequence (e.g., nucleic acid or amino acid sequence) set forth
in SEQ ID NOs:63-69, 141-142, or 707-708.
[0172] In some embodiments, an AAE acts on multiple substrates,
while in other embodiments, it exhibits substrate specificity. For
example, in some embodiments, an AAE exhibits substrate specificity
for one or more of hexanoic acid, butyric acid, isovaleric acid,
octanoic acid, or decanoic acid. In other embodiments, an AAE
exhibits activity on at least two of hexanoic acid, butyric acid,
isovaleric acid, octanoic acid, and decanoic acid. AAE enzymes were
identified herein that exhibited activity on butyrate and/or
hexanoate (FIGS. 5 and 6). Activity on butyrate was unexpected in
view of disclosure in Carvalho et al. "Designing Microorganisms for
Heterologous Biosynthesis of Cannabinoids" (2017) FEMS Yeast
Research June 1; 17(4).
[0173] In some embodiments, an AAE described herein comprises: N at
a residue corresponding to position 90 in UniProtKB--Q6C577 (SEQ ID
NO:64); A at a residue corresponding to position 100 in
UniProtKB--Q6C577 (SEQ ID NO:64); G at a residue corresponding to
position 105 in UniProtKB--Q6C577 (SEQ ID NO:64); E at a residue
corresponding to position 162 in UniProtKB--Q6C577 (SEQ ID NO:64);
Y at a residue corresponding to position 195 in UniProtKB--Q6C577
(SEQ ID NO:64); G at a residue corresponding to position 205 in
UniProtKB--Q6C577 (SEQ ID NO:64); K at a residue corresponding to
position 208 in UniProtKB--Q6C577 (SEQ ID NO:64); P at a residue
corresponding to position 243 in UniProtKB--Q6C577 (SEQ ID NO:64);
H at a residue corresponding to position 246 in UniProtKB--Q6C577
(SEQ ID NO:64); G at a residue corresponding to position 261 in
UniProtKB--Q6C577 (SEQ ID NO:64); F at a residue corresponding to
position 270 in UniProtKB--Q6C577 (SEQ ID NO:64); V at a residue
corresponding to position 284 in UniProtKB--Q6C577 (SEQ ID NO:64);
L at a residue corresponding to position 289 in UniProtKB--Q6C577
(SEQ ID NO:64); V at a residue corresponding to position 290 in
UniProtKB--Q6C577 (SEQ ID NO:64); P at a residue corresponding to
position 291 in UniProtKB--Q6C577 (SEQ ID NO:64); P at a residue
corresponding to position 301 in UniProtKB--Q6C577 (SEQ ID NO:64);
A at a residue corresponding to position 321 in UniProtKB--Q6C577
(SEQ ID NO:64); V at a residue corresponding to position 328 in
UniProtKB--Q6C577 (SEQ ID NO:64); Y at a residue corresponding to
position 356 in UniProtKB--Q6C577 (SEQ ID NO:64); G at a residue
corresponding to position 381 in UniProtKB--Q6C577 (SEQ ID NO:64);
I at a residue corresponding to position 391 in UniProtKB--Q6C577
(SEQ ID NO:64); P at a residue corresponding to position 400 in
UniProtKB--Q6C577 (SEQ ID NO:64); G at a residue corresponding to
position 423 in UniProtKB--Q6C577 (SEQ ID NO:64); Y at a residue
corresponding to position 436 in UniProtKB--Q6C577 (SEQ ID NO:64);
P at a residue corresponding to position 440 in UniProtKB--Q6C577
(SEQ ID NO:64); W at a residue corresponding to position 464 in
UniProtKB--Q6C577 (SEQ ID NO:64); G at a residue corresponding to
position 468 in UniProtKB--Q6C577 (SEQ ID NO:64); D at a residue
corresponding to position 469 in UniProtKB--Q6C577 (SEQ ID NO:64);
D at a residue corresponding to position 474 in UniProtKB--Q6C577
(SEQ ID NO:64); G at a residue corresponding to position 477 in
UniProtKB--Q6C577 (SEQ ID NO:64); D at a residue corresponding to
position 483 in UniProtKB--Q6C577 (SEQ ID NO:64); R at a residue
corresponding to position 484 in UniProtKB--Q6C577 (SEQ ID NO:64);
I at a residue corresponding to position 489 in UniProtKB--Q6C577
(SEQ ID NO:64); S at a residue corresponding to position 491 in
UniProtKB--Q6C577 (SEQ ID NO:64); E at a residue corresponding to
position 500 in UniProtKB--Q6C577 (SEQ ID NO:64); E at a residue
corresponding to position 502 in UniProtKB--Q6C577 (SEQ ID NO:64);
H at a residue corresponding to position 508 in UniProtKB--Q6C577
(SEQ ID NO:64); V at a residue corresponding to position 511 in
UniProtKB--Q6C577 (SEQ ID NO:64); A at a residue corresponding to
position 515 in UniProtKB--Q6C577 (SEQ ID NO:64); V at a residue
corresponding to position 516 in UniProtKB--Q6C577 (SEQ ID NO:64);
G at a residue corresponding to position 518 in UniProtKB--Q6C577
(SEQ ID NO:64); A at a residue corresponding to position 531 in
UniProtKB--Q6C577 (SEQ ID NO:64); K at a residue corresponding to
position 557 in UniProtKB--Q6C577 (SEQ ID NO:64); P at a residue
corresponding to position 570 in UniProtKB--Q6C577 (SEQ ID NO:64);
G at a residue corresponding to position 575 in UniProtKB--Q6C577
(SEQ ID NO:64); K at a residue corresponding to position 576 in
UniProtKB--Q6C577 (SEQ ID NO:64); R at a residue corresponding to
position 580 in UniProtKB--Q6C577 (SEQ ID NO:64); and/or L at a
residue corresponding to position 582 in UniProtKB--Q6C577 (SEQ ID
NO:64).
[0174] In some embodiments an AAE described herein comprises one or
more of: an amino acid sequence set forth as SGAAPLG (SEQ ID NO:
114); an amino acid sequence set forth as AYLGMSSGTSGG (SEQ ID NO:
115); an amino acid sequence set forth as DQPA (SEQ ID NO: 116); an
amino acid sequence set forth as QVAPAELE (SEQ ID NO: 117); an
amino acid sequence set forth as VVID (SEQ ID NO: 118); and/or an
amino acid sequence set forth as SGKILRRLLR (SEQ ID NO: 119).
[0175] In some embodiments an AAE described herein comprises: the
amino acid sequence set forth as SGAAPLG (SEQ ID NO: 114) at
residues corresponding to positions 319-325 in UniProtKB--Q6C577
(SEQ ID NO:64); the amino acid sequence set forth as AYLGMSSGTSGG
(SEQ ID NO: 115) at residues corresponding to positions 194-205 in
UniProtKB--Q6C577 (SEQ ID NO:64); the amino acid sequence set forth
as DQPA (SEQ ID NO: 116) at residues corresponding to positions
398-401 in UniProtKB--Q6C577 (SEQ ID NO:64); the amino acid
sequence set forth as QVAPAELE (SEQ ID NO: 117) at residues
corresponding to positions 495-502 in UniProtKB--Q6C577 (SEQ ID
NO:64); the amino acid sequence set forth as VVID (SEQ ID NO: 118)
at residues corresponding to positions 564-567 in UniProtKB--Q6C577
(SEQ ID NO:64); and/or the amino acid sequence set forth as
SGKILRRLLR (SEQ ID NO: 119) at residues corresponding to positions
574-583 in UniProtKB--Q6C577 (SEQ ID NO:64).
[0176] In some embodiments an AAE described herein comprises: an
amino acid sequence with no more than three amino acid
substitutions at residues corresponding to positions 428-440 in
UniProtKB--Q6C577 (SEQ ID NO:64); or an amino acid sequence with no
more than one amino acid substitution at residues corresponding to
positions 482-491 in UniProtKB--Q6C577 (SEQ ID NO:64).
[0177] In some embodiments an AAE described herein comprises: I or
V at a residue corresponding to position 432 in UniProtKB--Q6C577
(SEQ ID NO:64); S or D at a residue corresponding to position 434
in UniProtKB--Q6C577 (SEQ ID NO:64); K or N at a residue
corresponding to position 438 in UniProtKB--Q6C577 (SEQ ID NO:64);
and/or L or M at a residue corresponding to position 488 in
UniProtKB--Q6C577 (SEQ ID NO:64).
[0178] In some embodiments an AAE described herein comprises: an
amino acid sequence set forth as RGPQIMSGYHKNP (SEQ ID NO: 120); an
amino acid sequence set forth as RGPQVMDGYHNNP (SEQ ID NO: 121); an
amino acid sequence set forth as RGPQIMDGYHKNP (SEQ ID NO: 122); an
amino acid sequence set forth as VDRTKELIKS (SEQ ID NO: 123);
and/or an amino acid sequence set forth as VDRTKEMIKS (SEQ ID NO:
124).
[0179] A recombinant host cell that expresses a heterologous gene
encoding an AAE described herein may be capable of producing at
least 1% (e.g., at least 5%, at least 10%, at least 15%, at least
20%, at least 25%, at least 30%, at least 35%, at least 40%, at
least 45%, at least 50%, at least 55%, at least 60%, at least 65%,
at least 70%, at least 75%, at least 80%, at least 85%, at least
90%, at least 95%, at least 100%, at least 125%, at least 150%, at
least 175%, at least 200%, at least 300%, at least 400%, at least
500%, at least 600%, at least 700%, at least 800%, at least 900%,
or at least 1,000%) more hexanoyl-CoA and/or more butanoyl-coenzyme
A relative to a control. In some embodiments, a control is a host
cell that does not express a heterologous gene encoding an AAE.
Polyketide Synthases (PKS)
[0180] A host cell described in this application may comprise a
PKS. As used in this application, a "PKS" refers to an enzyme that
is capable of producing a polyketide. In certain embodiments, a PKS
converts a compound of Formula (2) to a compound of Formula (4),
(5), and/or (6). In certain embodiments, a PKS converts a compound
of Formula (2) to a compound of Formula (4). In certain
embodiments, a PKS converts a compound of Formula (2) to a compound
of Formula (5). In certain embodiments, a PKS converts a compound
of Formula (2) to a compound of Formula (4) and/or (5). In certain
embodiments, a PKS converts a compound of Formula (2) to a compound
of Formula (5) and/or (6).
[0181] In some embodiments, a PKS is a tetraketide synthase (TKS).
In certain embodiments, a PKS is an olivetol synthase (OLS). As
used in this application, an "OLS" refers to an enzyme that is
capable of using a substrate of Formula (2a) to form a compound of
Formula (4a), (5a) and/or (6a) as shown in FIG. 1. In some
embodiments, an OLS catalyzes the formation of olivetol (Formula
(5a)). In some embodiments, an olivetol synthase (OLS) catalyzes
the formation of olivetol with minimal production of
3,5,7-trioxoalkanoyl-CoA and/or olivetolic acid. In some instances,
an OLS that is capable of catalyzing the formation of olivetol may
be useful in providing olivetol as a substrate for a
prenyltransferase. As a non-limiting example, NphB can use olivetol
as reactant. See, e.g., Kumano et al., Bioorg Med Chem. 2008 Sep.
1; 16(17): 8117-8126.
[0182] In certain embodiments, a PKS is a divarinic acid synthase
(DVS).
[0183] A non-limiting example of an OLS is provided by
UniProtKB--B1Q2B6 from C. sativa. In C. sativa, this OLS uses
hexanoyl-CoA and malonyl-CoA as substrates to form
3,5,7-trioxododecanoyl-CoA. OLS (e.g., UniProtKB--B1Q2B6) in
combination with olivetolic acid cyclase (OAC) produces olivetolic
acid (OA) in C. sativa.
[0184] The amino acid sequence of UniProtKB--B1Q2B6 is:
TABLE-US-00003 (SEQ ID NO: 5)
MNHLRAEGPASVLAIGTANPENILLQDEFPDYYFRVTKSEHMTQLKEKF
RKICDKSMIRKRNCFLNEEHLKQNPRLVEHEMQTLDARQDMLVVEVPKL
GKDACAKAIKEWGQPKSKITHLIFTSASTTDMPGADYHCAKLLGLSPSV
KRVMMYQLGCYGGGTVLRIAKDIAENNKGARVLAVCCDIMACLFRGPSE
SDLELLVGQAIFGDGAAAVIVGAEPDESVGERPIFELVSTGQTILPNSE
GTIGGHIREAGLIFDLHKDVPMLISNNIEKCLIEAFTPIGISDWNSIFW
ITHPGGKAILDKVEEKLHLKSDKFVDSRHVLSEHGNIVISSSTVLFVMD
ELRKRSLEEGKSTTGDGFEWGVLFGFGPGLTVERVVVRSVPIKY.
[0185] Structurally, an OLS comprises a triad of conserved
residues, which have been implicated as catalytic residues. This
triad of conserved residues may be referred to as a catalytic
triad. See, e.g., Taura et al., FEBS Letters 583 (2009) 2061-2066.
The catalytic triad of UniProtKB--B1Q2B6 (SEQ ID NO: 5) comprises
C157, H297, and N330. One of ordinary skill in the art would be
able to identify corresponding catalytic residues in other PKSs,
including OLSs, by aligning the amino acid sequence of interest
with UniProtKB--B1Q2B6. A PKS, including an OLS, may comprise the
amino acid C at a residue corresponding to position 157 in SEQ ID
NO: 5, the amino acid H at a residue corresponding to position 297
in SEQ ID NO: 5, and the amino acid N at a residue corresponding to
residue 330 in SEQ ID NO: 5. As a non-limiting example, the
residues corresponding to positions 157, 297, and 330 in SEQ ID NO:
5 are C164, H304, and N337, respectively in SEQ ID NO: 6.
Similarly, the residues corresponding to positions 157, 297, and
330 in SEQ ID NO: 5 are C164, H304, and N337, respectively, in SEQ
ID NO: 7.
[0186] The active site of a PKS may be defined by generating the
three-dimensional structure of the PKS and identifying the residues
within a particular distance of any of the residues within the
catalytic triad and/or within a particular distance of a docked
substrate within the PKS (e.g., a compound of Formula (2)). A
substrate docks or binds in the substrate binding pocket of a PKS.
The substrate binding pocket may comprise the active site of the
PKS. As a non-limiting example, the structure of a PKS may be
generated using ROSETTA software. See, e.g., Kaufmann et al.,
Biochemistry 2010, 49, 2987-2998.
[0187] As used herein, a residue is within the active site of an
OLS enzyme if it is within about 12 angstroms of any of the
residues within the catalytic triad of the OLS enzyme and/or within
about 12 angstroms of a docked substrate within the OLS enzyme.
[0188] In some embodiments, a residue is within 12 angstroms
(.ANG.), within 11 .ANG., within 10 .ANG., within 9 .ANG., within 8
.ANG., within 7 .ANG., within 6 .ANG., within 5 .ANG., within 4
.ANG., within 3 .ANG., within 2 .ANG., or within 1 .ANG. from any
of the residues within the catalytic triad (i.e., 157, 297, and 330
in SEQ ID NO: 5) and/or from a docked substrate (e.g.,
hexanoyl-CoA).
[0189] In some embodiments, a residue in a PKS is within 20 .ANG.,
within 19 .ANG., within 18 .ANG., within 17 .ANG., within 16 .ANG.,
within 15 .ANG., within 14 .ANG., within 13 .ANG., within 12 .ANG.,
within 11 .ANG., within 10 .ANG., within 9 .ANG., within 8 .ANG.,
within 7 .ANG., within 6 .ANG., within 5 .ANG., within 4 .ANG.,
within 3 .ANG., within 2 .ANG., and/or within 1 .ANG. from any of
the residues within the catalytic triad (i.e., residues in the PKS
corresponding to positions 157, 297, and 330 in SEQ ID NO: 5)
and/or a docked substrate.
[0190] As a non-limiting example, positions 17, 23, 25, 51, 54, 64,
95, 123, 125, 153, 196, 201, 207, 241, 247, 267, 273, 277, 296,
307, 320, 324, 326, 328, 334, 335, and 375 in SEQ ID NO: 5 may be
located within the active site of a PKS comprising SEQ ID NO: 5.
Positions 51, 54, 123, 125, 201, 207, 241, 247, 267, 273, 296, 307,
324, 326, 328, 334, 335, and 375 in SEQ ID NO: 5 may be located
within about 8 .ANG. from any of the residues within the catalytic
triad and/or a docked substrate of the PKS comprising SEQ ID NO:
5.
[0191] In some embodiments, a PKS comprises an amino acid
substitution, insertion, or deletion at a residue that is within
the active site of the PKS. In some embodiments, a PKS comprises an
amino acid substitution, insertion, or deletion at a residue that
is within 12 angstroms (.ANG.), within 11 .ANG., within 10 .ANG.,
within 9 .ANG., within 8 .ANG., within 7 .ANG., within 6 .ANG.,
within 5 .ANG., within 4 .ANG., within 3 .ANG., within 2 .ANG., or
within 1 .ANG. away from any one of the catalytic triad residues
(i.e., positions 157, 297, and 330 in SEQ ID NO: 5) and/or from a
docked substrate. In some embodiments, the amino acid substitution,
insertion, or deletion is at a residue corresponding to position
17, 23, 25, 51, 54, 64, 95, 123, 125, 153, 196, 201, 207, 241, 247,
267, 273, 277, 296, 307, 320, 324, 326, 328, 334, 335, and/or 375
in SEQ ID NO: 5. In some embodiments, a residue in a PKS
corresponding to position 17, 23, 25, 51, 54, 64, 95, 123, 125,
153, 196, 201, 207, 241, 247, 267, 273, 277, 296, 307, 320, 324,
326, 328, 334, 335, and/or 375 in SEQ ID NO: 5 is located within 12
.ANG. from the active site of the PKS. In some embodiments, a
residue in a PKS corresponding to position 51, 54, 123, 125, 201,
207, 241, 247, 267, 273, 296, 307, 324, 326, 328, 334, 335, and/or
375 in SEQ ID NO: 5 is located within 8 .ANG. from the active site
of the PKS. In some embodiments, the PKS comprises one or more of:
T17K, I23C, L25R, K51R, D54R, F64Y, V95A, T123C, A125S, Y153G,
E196K, L201C, 1207L, L241I, T247A, M267K, M267G, 1273V, L277M,
T296A, V307I, D320A, V324I, S326R, H328Y, S334P, S334A, T335C,
and/or R375T relative to SEQ ID NO: 5. In some embodiments, a host
cell comprising one or more of these amino acid substitutions
relative to SEQ ID NO: 5 is capable of producing at least 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more than 15 mg/L
Olivetol, including all values in between.
[0192] In some embodiments, a PKS comprises: an amino acid
substitution, insertion, or deletion at a residue that is more than
12 angstroms (.ANG.), more than 11 .ANG., more than 10 .ANG., more
than 9 .ANG., more than 8 .ANG., more than 7 .ANG., more than 6
.ANG., more than 5 .ANG., more than 4 .ANG., more than 3 .ANG.,
more than 2 .ANG., or more than 1 .ANG. away from the catalytic
triad (i.e., 157, 297, and 330 in SEQ ID NO: 5) and/or from a
docked substrate. In some embodiments, the residue corresponds to
position 71, 92, 100, 108, 116, 128, 135, 229, 278, 284, and/or 348
in SEQ ID NO: 5. In some embodiments, the residue in a PKS
corresponding to position 71, 92, 100, 108, 116, 128, 135, 229,
278, 284, and/or 348 in SEQ ID NO: 5 is more than 12 .ANG. from the
active site of the PKS. In some embodiments, the PKS comprises one
or more of: I284Y, K100L, K116R, I278E, K108D, L348S, K71R, V92G,
T128V, K100M, Y135V, P229A, T128A, and/or T128I. In some
embodiments, a host cell comprising one or more of these mutations
is capable of producing at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15 or more than 15 mg/L Olivetol, including all values
in between.
[0193] In some embodiments, a PKS comprises the amino acid C at a
residue corresponding to position 335 of SEQ ID NO: 5. In some
embodiments, a PKS comprises the amino acid substitution T335C
relative to a control. In some embodiments, the control is a PKS
comprising SEQ ID NO: 5. In some embodiments, a PKS comprises a
sequence at least 5%, at least 10%, at least 15%, at least 20%, at
least 25%, at least 30%, at least 35%, at least 40%, at least 45%,
at least 50%, at least 55%, at least 60%, at least 65%, at least
70%, at least 71%, at least 72%, at least 73%, at least 74%, at
least 75%, at least 76%, at least 77%, at least 78%, at least 79%,
at least 80%, at least 81%, at least 82%, at least 83%, at least
84%, at least 85%, at least 86%, at least 87%, at least 88%, at
least 89%, at least 90%, at least 91%, at least 92%, at least 93%,
at least 94%, at least 95%, at least 96%, at least 97%, at least
98%, at least 99%, or is 100% identical, including all values in
between, to a sequence (e.g., amino acid or nucleic sequence) set
forth in SEQ ID NOs: 38, 172, 175, 176, 196, 204, 205, 7, 17, 145,
13, 8, and 15. In some embodiments, a PKS comprises a sequence at
most 5%, at most 10%, at most 15%, at most 20%, at most 25%, at
most 30%, at most 35%, at most 40%, at most 45%, at most 50%, at
most 55%, at most 60%, at most 65%, at most 70%, at most 71%, at
most 72%, at most 73%, at most 74%, at most 75%, at most 76%, at
most 77%, at most 78%, at most 79%, at most 80%, at most 81%, at
most 82%, at most 83%, at most 84%, at most 85%, at most 86%, at
most 87%, at most 88%, at most 89%, at most 90%, at most 91%, at
most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at
most 97%, at most 98%, at most 99%, or is 100% identical, including
all values in between, to a sequence (e.g., amino acid or nucleic
sequence) set forth in SEQ ID NOs: 38, 172, 175, 176, 196, 204,
205, 7, 17, 145, 13, 8, and 15.
[0194] In some embodiments, a PKS described herein comprises a
sequence that is at least 5%, at least 10%, at least 15%, at least
20%, at least 25%, at least 30%, at least 35%, at least 40%, at
least 45%, at least 50%, at least 55%, at least 60%, at least 65%,
at least 70%, at least 71%, at least 72%, at least 73%, at least
74%, at least 75%, at least 76%, at least 77%, at least 78%, at
least 79%, at least 80%, at least 81%, at least 82%, at least 83%,
at least 84%, at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, at least 99%, or is 100% identical, including all
values in between, to a sequence (e.g., nucleic acid or amino acid
sequence) set forth in UniProtKB--A0A088G5Z5 (SEQ ID NO: 7), SEQ ID
NO: 714, SEQ ID NO: 715, or SEQ ID NO: 38.
[0195] In some embodiments, relative to the sequence of SEQ ID NO:
7, the PKS comprises an amino acid substitution at a residue
corresponding to position 28, 34, 50, 70, 71, 76, 88, 100, 151,
203, 219, 285, 359, and/or 385 in SEQ ID NO: 7. In some
embodiments, the PKS comprises: the amino acid P at a residue
corresponding to position 28 in SEQ ID NO: 7; the amino acid Q at a
residue corresponding to position 34 in SEQ ID NO: 7; the amino
acid N at a residue corresponding to position 50 in SEQ ID NO: 7;
the amino acid M at a residue corresponding to position 70 in SEQ
ID NO: 7; the amino acid Y at a residue corresponding to position
71 in SEQ ID NO: 7; the amino acid I at a residue corresponding to
position 76 in SEQ ID NO: 7; the amino acid A at a residue
corresponding to position 88 in SEQ ID NO: 7; the amino acid P or T
at a residue corresponding to position 100 in SEQ ID NO: 7; the
amino acid P at a residue corresponding to position 151 in SEQ ID
NO: 7; the amino acid K at a residue corresponding to position 203
in SEQ ID NO: 7; the amino acid C at a residue corresponding to
position 219 in SEQ ID NO: 7; the amino acid A at a residue
corresponding to position 285 in SEQ ID NO: 7; the amino acid M at
a residue corresponding to position 359 in SEQ ID NO: 7; and/or the
amino acid M at a residue corresponding to position 385 in SEQ
IDNO: 7. In some embodiments, the PKS comprises one or more of the
following amino acid substitutions relative to SEQ ID NO: 7: E28P,
S34Q, V50N, F70M, V71Y, L76I, D88A, R100P, R100T, N151P, E203K,
A219C, E285A, K359M, and/or L385M. In some embodiments, the PKS
comprises V71Y and/or F70M. In some embodiments, the PKS comprises
C at a residue corresponding to position 164 in SEQ ID NO: 7; H at
a residue corresponding to position 304 in SEQ ID NO: 7; and/or N
at a residue corresponding to position 337 in SEQ ID NO: 7.
[0196] In some embodiments, a host cell with a PKS that comprises
an amino acid substitution at a residue corresponding to position
to position 28, 34, 50, 70, 71, 76, 88, 100, 151, 203, 219, 285,
359, and/or 385 in SEQ ID NO: 7 produces at least 1% (e.g., at
least 5%, at least 10%, at least 15%, at least 20%, at least 25%,
at least 30%, at least 35%, at least 40%, at least 45%, at least
50%, at least 55%, at least 60%, at least 65%, at least 70%, at
least 75%, at least 80%, at least 85%, at least 90%, at least 95%,
at least 100%, at least 125%, at least 150%, at least 175%, at
least 200%, at least 300%, at least 400%, at least 500%, at least
600%, at least 700%, at least 800%, at least 900%, or at least
1,000%) more of a product (e.g., a compound of Formula (4), (5),
and/or (6)) relative to a host cell comprising SEQ ID NO: 7.
[0197] In some embodiments, a PKS described herein comprises: A at
a residue corresponding to position 17 in UniProtKB--A0A1R3HSU5
(SEQ ID NO:6); A at a residue corresponding to position 21 in
UniProtKB--A0A1R3HSU5 (SEQ ID NO:6); I at a residue corresponding
to position 22 in UniProtKB--A0A1R3HSU5 (SEQ ID NO:6); G at a
residue corresponding to position 23 in UniProtKB--A0A1R3HSU5 (SEQ
ID NO: 6); Q at a residue corresponding to position 33 in
UniProtKB--A0A1R3HSU5 (SEQ ID NO: 6); D at a residue corresponding
to position 38 in UniProtKB--A0A1R3HSU5 (SEQ ID NO: 6); F at a
residue corresponding to position 41 in UniProtKB--A0A1R3HSU5 (SEQ
ID NO:6); L at a residue corresponding to position 52 in
UniProtKB--A0A1R3HSU5 (SEQ ID NO:6); K at a residue corresponding
to position 55 in UniProtKB--A0A1R3HSU5 (SEQ ID NO:6); C at a
residue corresponding to position 60 in UniProtKB--A0A1R3HSU5 (SEQ
ID NO:6); R at a residue corresponding to position 68 in
UniProtKB--A0A1R3HSU5 (SEQ ID NO:6); R at a residue corresponding
to position 94 in UniProtKB--A0A1R3HSU5 (SEQ ID NO:6); A at a
residue corresponding to position 109 in UniProtKB--A0A1R3HSU5 (SEQ
ID NO:6); A at a residue corresponding to position 113 in
UniProtKB--A0A1R3HSU5 (SEQ ID NO:6); W at a residue corresponding
to position 117 in UniProtKB--A0A1R3HSU5 (SEQ ID NO:6); G at a
residue corresponding to position 118 in UniProtKB--A0A1R3HSU5 (SEQ
ID NO:6); S at a residue corresponding to position 122 in
UniProtKB--A0A1R3HSU5 (SEQ ID NO:6); I at a residue corresponding
to position 124 in UniProtKB--A0A1R3HSU5 (SEQ ID NO:6); T at a
residue corresponding to position 125 in UniProtKB--A0A1R3HSU5 (SEQ
ID NO:6); H at a residue corresponding to position 126 in
UniProtKB--A0A1R3HSU5 (SEQ ID NO:6); P at a residue corresponding
to position 138 in UniProtKB--A0A1R3HSU5 (SEQ ID NO:6); D at a
residue corresponding to position 141 in UniProtKB--A0A1R3HSU5 (SEQ
ID NO:6); L at a residue corresponding to position 150 in
UniProtKB--A0A1R3HSU5 (SEQ ID NO:6); G at a residue corresponding
to position 163 in UniProtKB--A0A1R3HSU5 (SEQ ID NO:6); C at a
residue corresponding to position 164 in UniProtKB--A0A1R3HSU5 (SEQ
ID NO:6); G at a residue corresponding to position 168 in
UniProtKB--A0A1R3HSU5 (SEQ ID NO:6); R at a residue corresponding
to position 172 in UniProtKB--A0A1R3HSU5 (SEQ ID NO:6); K at a
residue corresponding to position 175 in UniProtKB--A0A1R3HSU5 (SEQ
ID NO:6); E at a residue corresponding to position 179 in
UniProtKB--A0A1R3HSU5 (SEQ ID NO:6); R at a residue corresponding
to position 185 in UniProtKB--A0A1R3HSU5 (SEQ ID NO:6); L at a
residue corresponding to position 187 in UniProtKB--A0A1R3HSU5 (SEQ
ID NO:6); V at a residue corresponding to position 189 in
UniProtKB--A0A1R3HSU5 (SEQ ID NO:6); C at a residue corresponding
to position 190 in UniProtKB--A0A1R3HSU5 (SEQ ID NO:6); P at a
residue corresponding to position 201 in UniProtKB--A0A1R3HSU5 (SEQ
ID NO:6); F at a residue corresponding to position 215 in
UniProtKB--A0A1R3HSU5 (SEQ ID NO:6); D at a residue corresponding
to position 217 in UniProtKB--A0A1R3HSU5 (SEQ ID NO:6); G at a
residue corresponding to position 218 in UniProtKB--A0A1R3HSU5 (SEQ
ID NO:6); G at a residue corresponding to position 225 in
UniProtKB--A0A1R3HSU5 (SEQ ID NO:6); E at a residue corresponding
to position 234 in UniProtKB--A0A1R3HSU5 (SEQ ID NO:6); G at a
residue corresponding to position 263 in UniProtKB--A0A1R3HSU5 (SEQ
ID NO:6); P at a residue corresponding to position 273 in
UniProtKB--A0A1R3HSU5 (SEQ ID NO:6); F at a residue corresponding
to position 288 in UniProtKB--A0A1R3HSU5 (SEQ ID NO:6); D at a
residue corresponding to position 295 in UniProtKB--A0A1R3HSU5 (SEQ
ID NO:6); N at a residue corresponding to position 297 in
UniProtKB--A0A1R3HSU5 (SEQ ID NO:6); F at a residue corresponding
to position 300 in UniProtKB--A0A1R3HSU5 (SEQ ID NO:6); H at a
residue corresponding to position 304 in UniProtKB--A0A1R3HSU5 (SEQ
ID NO:6); G at a residue corresponding to position 306 in
UniProtKB--A0A1R3HSU5 (SEQ ID NO:6); G at a residue corresponding
to position 307 in UniProtKB--A0A1R3HSU5 (SEQ ID NO:6); L at a
residue corresponding to position 311 in UniProtKB--A0A1R3HSU5 (SEQ
ID NO:6); G at a residue corresponding to position 336 in
UniProtKB--A0A1R3HSU5 (SEQ ID NO:6); N at a residue corresponding
to position 337 in UniProtKB--A0A1R3HSU5 (SEQ ID NO:6); M at a
residue corresponding to position 338 in UniProtKB--A0A1R3HSU5 (SEQ
ID NO:6); V at a residue corresponding to position 343 in
UniProtKB--A0A1R3HSU5 (SEQ ID NO:6); D at a residue corresponding
to position 348 in UniProtKB--A0A1R3HSU5 (SEQ ID NO:6); R at a
residue corresponding to position 351 in UniProtKB--A0A1R3HSU5 (SEQ
ID NO:6); G at a residue corresponding to position 363 in
UniProtKB--A0A1R3HSU5 (SEQ ID NO:6); G at a residue corresponding
to position 365 in UniProtKB--A0A1R3HSU5 (SEQ ID NO:6); G at a
residue corresponding to position 369 in UniProtKB--A0A1R3HSU5 (SEQ
ID NO:6); G at a residue corresponding to position 375 in
UniProtKB--A0A1R3HSU5 (SEQ ID NO: 6); P at a residue corresponding
to position 376 in UniProtKB--A0A1R3HSU5 (SEQ ID NO:6); G at a
residue corresponding to position 377 in UniProtKB--A0A1R3HSU5 (SEQ
ID NO:6); E at a residue corresponding to position 381 in
UniProtKB--A0A1R3HSU5 (SEQ ID NO:6); and/or S at a residue
corresponding to position 387 in UniProtKB--A0A1R3HSU5 (SEQ ID
NO:6).
[0198] In some embodiments, a PKS described herein comprises: S, T,
or G at a residue corresponding to position 18 in
UniProtKB--A0A1R3HSU5 (SEQ ID NO:6); V or I at a residue
corresponding to position 19 in UniProtKB--A0A1R3HSU5 (SEQ ID
NO:6); E, P, S, A, or D at a residue corresponding to position 28
in UniProtKB--A0A1R3HSU5 (SEQ ID NO:6); I, C, I, S, F, Y, Q, H, A,
or V at a residue corresponding to position 30 in
UniProtKB--A0A1R3HSU5 (SEQ ID NO:6); D, S, C, I, A, or D at a
residue corresponding to position 34 in UniProtKB--A0A1R3HSU5 (SEQ
ID NO:6); F or Y at a residue corresponding to position 36 in
UniProtKB--A0A1R3HSU5 (SEQ ID NO:6); Y, F, or V at a residue
corresponding to position 39 in UniProtKB--A0A1R3HSU5 (SEQ ID
NO:6); K, N, D, or S at a residue corresponding to position 45 in
UniProtKB--A0A1R3HSU5 (SEQ ID NO:6); K, R, or H at a residue
corresponding to position 58 in UniProtKB--A0A1R3HSU5 (SEQ ID
NO:6); F, H, V, Y, or N at a residue corresponding to position 71
in UniProtKB--A0A1R3HSU5 (SEQ ID NO:6); R, N, C, E, S, or H at a
residue corresponding to position 82 in UniProtKB--A0A1R3HSU5 (SEQ
ID NO:6); M, A, D, S, E, or V at a residue corresponding to
position 88 in UniProtKB--A0A1R3HSU5 (SEQ ID NO:6); Q, P, S, N, L,
or K at a residue corresponding to position 89 in
UniProtKB--A0A1R3HSU5 (SEQ ID NO:6); T or S at a residue
corresponding to position 90 in UniProtKB--A0A1R3HSU5 (SEQ ID
NO:6); M, I, F, L, or V at a residue corresponding to position 97
in UniProtKB--A0A1R3HSU5 (SEQ ID NO:6); D, E, K, or A at a residue
corresponding to position 108 in UniProtKB--A0A1R3HSU5 (SEQ ID
NO:6); C, A, or S at a residue corresponding to position 110 in
UniProtKB--A0A1R3HSU5 (SEQ ID NO:6); T, C, or Y at a residue
corresponding to position 130 in UniProtKB--A0A1R3HSU5 (SEQ ID
NO:6); S or T at a residue corresponding to position 131 in
UniProtKB--A0A1R3HSU5 (SEQ ID NO:6); A, S, T, or I at a residue
corresponding to position 132 in UniProtKB--A0A1R3HSU5 (SEQ ID
NO:6); L, Q, or H at a residue corresponding to position 162 in
UniProtKB--A0A1R3HSU5 (SEQ ID NO:6); G, A, or S at a residue
corresponding to position 166 in UniProtKB--A0A1R3HSU5 (SEQ ID
NO:6); I, L, V, T, M, or Y at a residue corresponding to position
173 in UniProtKB--A0A1R3HSU5 (SEQ ID NO:6); I, L, F, or V at a
residue corresponding to position 177 in UniProtKB--A0A1R3HSU5 (SEQ
ID NO:6); C, S, or A at a residue corresponding to position 191 in
UniProtKB--A0A1R3HSU5 (SEQ ID NO:6); D or E at a residue
corresponding to position 192 in UniProtKB--A0A1R3HSU5 (SEQ ID
NO:6); M or T at a residue corresponding to position 194 in
UniProtKB--A0A1R3HSU5 (SEQ ID NO:6); L, C, T, S, M, or N at a
residue corresponding to position 197 in UniProtKB--A0A1R3HSU5 (SEQ
ID NO:6); E or D at a residue corresponding to position 207 in
UniProtKB--A0A1R3HSU5 (SEQ ID NO:6); V, L, M, or I at a residue
corresponding to position 222 in UniProtKB--A0A1R3HSU5 (SEQ ID
NO:6); I, L, C, S, V, or M at a residue corresponding to position
237 in UniProtKB--A0A1R3HSU5 (SEQ ID NO:6); T, A, N, or S at a
residue corresponding to position 243 in UniProtKB--A0A1R3HSU5 (SEQ
ID NO:6); N, D, E, or G at a residue corresponding to position 250
in UniProtKB--A0A1R3HSU5 (SEQ ID NO:6); I or L at a residue
corresponding to position 299 in UniProtKB--A0A1R3HSU5 (SEQ ID
NO:6); K, P, or R at a residue corresponding to position 308 in
UniProtKB--A0A1R3HSU5 (SEQ ID NO:6); F, L, or M at a residue
corresponding to position 325 in UniProtKB--A0A1R3HSU5 (SEQ ID
NO:6); H or Y at a residue corresponding to position 335 in
UniProtKB--A0A1R3HSU5; M or L at a residue corresponding to
position 347 in UniProtKB--A0A1R3HSU5 (SEQ ID NO:6); L, M, I, or T
at a residue corresponding to position 350 in UniProtKB--A0A1R3HSU5
(SEQ ID NO:6); F, L, or M at a residue corresponding to position
366 in UniProtKB--A0A1R3HSU5 (SEQ ID NO:6); and/or R or T at a
residue corresponding to position 382 in UniProtKB--A0A1R3HSU5 (SEQ
ID NO:6).
[0199] In some embodiments, a PKS comprises a sequence that is at
least 5%, at least 10%, at least 15%, at least 20%, at least 25%,
at least 30%, at least 35%, at least 40%, at least 45%, at least
50%, at least 55%, at least 60%, at least 65%, at least 70%, at
least 71%, at least 72%, at least 73%, at least 74%, at least 75%,
at least 76%, at least 77%, at least 78%, at least 79%, at least
80%, at least 81%, at least 82%, at least 83%, at least 84%, at
least 85%, at least 86%, at least 87%, at least 88%, at least 89%,
at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99%, or is 100% identical, including all values in between,
to a sequence (e.g., nucleic acid or amino acid sequence) set forth
in SEQ ID NOs: 1-31, 77-92, 143-171, 207-249, 293-420, 549-627,
32-62, 93-108, 172-206, 250-292, 421-548, 628-705, and 706 or to a
sequence selected from Tables 5-6 and 13-16.
[0200] In some embodiments, a PKS comprises at least 1, at least 2,
at least 3, at least 4, at least 5, at least 6, at least 7, at
least 8, at least 9, at least 10, at least 11, at least 12, at
least 13, at least 14, at least 15, at least 16, at least 17, at
least 18, at least 19, at least 20, at least 21, at least 22, at
least 23, at least 24, at least 25, at least 26, at least 27, at
least 28, at least 29, at least 30, at least 31, at least 32, at
least 33, at least 34, at least 35, at least 36, at least 37, at
least 38, at least 39, at least 40, at least 41, at least 42, at
least 43, at least 44, at least 45, at least 46, at least 47, at
least 48, at least 49, at least 50, at least 51, at least 52, at
least 53, at least 54, at least 55, at least 56, at least 57, at
least 58, at least 59, at least 60, at least 61, at least 62, at
least 63, at least 64, at least 65, at least 66, at least 67, at
least 68, at least 69, at least 70, at least 71, at least 72, at
least 73, at least 74, at least 75, at least 76, at least 77, at
least 78, at least 79, at least 80, at least 81, at least 82, at
least 83, at least 84, at least 85, at least 86, at least 87, at
least 88, at least 89, at least 90, at least 91, at least 92, at
least 93, at least 94, at least 95, at least 96, at least 97, at
least 98, at least 99, at least 100, at least 101, at least 102, at
least 103, at least 104, at least 105, at least 106, at least 107,
at least 108, at least 109, at least 110, at least 111, at least
112, at least 113, at least 114, at least 115, at least 116, at
least 117, at least 118, at least 119, at least 120, at least 121,
at least 122, at least 123, at least 124, at least 125, at least
126, at least 127, at least 128, at least 129, at least 130, at
least 131, at least 132, at least 133, at least 134, at least 135,
at least 136, at least 137, at least 138, at least 139, at least
140, at least 141, at least 142, at least 143, at least 144, at
least 145, at least 146, at least 147, at least 148, at least 149,
at least 150, at least 151, at least 152, at least 153, at least
154, at least 155, at least 156, at least 157, at least 158, at
least 159, at least 160, at least 161, at least 162, at least 163,
at least 164, at least 165, at least 166, at least 167, at least
168, at least 169, at least 170, at least 171, at least 172, at
least 173, at least 174, at least 175, at least 176, at least 177,
at least 178, at least 179, at least 180, at least 181, at least
182, at least 183, at least 184, at least 185, at least 186, at
least 187, at least 188, at least 189, at least 190, at least 191,
at least 192, at least 193, at least 194, at least 195, at least
196, at least 197, at least 198, at least 199, at least 200, at
least 201, at least 202, at least 203, at least 204, at least 205,
at least 206, at least 207, at least 208, at least 209, at least
210, at least 211, at least 212, at least 213, at least 214, at
least 215, at least 216, at least 217, at least 218, at least 219,
at least 220, at least 221, at least 222, at least 223, at least
224, at least 225, at least 226, at least 227, at least 228, at
least 229, at least 230, at least 231, at least 232, at least 233,
at least 234, at least 235, at least 236, at least 237, at least
238, at least 239, at least 240, at least 241, at least 242, at
least 243, at least 244, at least 245, at least 246, at least 247,
at least 248, at least 249, at least 250, at least 251, at least
252, at least 253, at least 254, at least 255, at least 256, at
least 257, at least 258, at least 259, at least 260, at least 261,
at least 262, at least 263, at least 264, at least 265, at least
266, at least 267, at least 268, at least 269, at least 270, at
least 271, at least 272, at least 273, at least 274, at least 275,
at least 276, at least 277, at least 278, at least 279, at least
280, at least 281, at least 282, at least 283, at least 284, at
least 285, at least 286, at least 287, at least 288, at least 289,
at least 290, at least 291, at least 292, at least 293, at least
294, at least 295, at least 296, at least 297, at least 298, at
least 299, at least 300, at least 301, at least 302, at least 303,
at least 304, at least 305, at least 306, at least 307, at least
308, at least 309, at least 310, at least 311, at least 312, at
least 313, at least 314, at least 315, at least 316, at least 317,
at least 318, at least 319, at least 320, at least 321, at least
322, at least 323, at least 324, at least 325, at least 326, at
least 327, at least 328, at least 329, at least 330, at least 331,
at least 332, at least 333, at least 334, at least 335, at least
336, at least 337, at least 338, at least 339, at least 340, at
least 341, at least 342, at least 343, at least 344, at least 345,
at least 346, at least 347, at least 348, at least 349, at least
350, at least 351, at least 352, at least 353, at least 354, at
least 355, at least 356, at least 357, at least 358, at least 359,
at least 360, at least 361, at least 362, at least 363, at least
364, at least 365, at least 366, at least 367, at least 368, at
least 369, at least 370, at least 371, at least 372, at least 373,
at least 374, at least 375, at least 376, at least 377, at least
378, at least 379, or at least 380 amino acid substitutions,
deletions, or insertions relative to SEQ ID NOs: 1-31, 77-92,
143-171, 207-249, 293-420, and 549-627 or to an amino acid sequence
selected from Tables 5-6 and 13-16.
[0201] In some embodiments, a PKS comprises at most 1, at most 2,
at most 3, at most 4, at most 5, at most 6, at most 7, at most 8,
at most 9, at most 10, at most 11, at most 12, at most 13, at most
14, at most 15, at most 16, at most 17, at most 18, at most 19, at
most 20, at most 21, at most 22, at most 23, at most 24, at most
25, at most 26, at most 27, at most 28, at most 29, at most 30, at
most 31, at most 32, at most 33, at most 34, at most 35, at most
36, at most 37, at most 38, at most 39, at most 40, at most 41, at
most 42, at most 43, at most 44, at most 45, at most 46, at most
47, at most 48, at most 49, at most 50, at most 51, at most 52, at
most 53, at most 54, at most 55, at most 56, at most 57, at most
58, at most 59, at most 60, at most 61, at most 62, at most 63, at
most 64, at most 65, at most 66, at most 67, at most 68, at most
69, at most 70, at most 71, at most 72, at most 73, at most 74, at
most 75, at most 76, at most 77, at most 78, at most 79, at most
80, at most 81, at most 82, at most 83, at most 84, at most 85, at
most 86, at most 87, at most 88, at most 89, at most 90, at most
91, at most 92, at most 93, at most 94, at most 95, at most 96, at
most 97, at most 98, at most 99, at most 100, at most 101, at most
102, at most 103, at most 104, at most 105, at most 106, at most
107, at most 108, at most 109, at most 110, at most 111, at most
112, at most 113, at most 114, at most 115, at most 116, at most
117, at most 118, at most 119, at most 120, at most 121, at most
122, at most 123, at most 124, at most 125, at most 126, at most
127, at most 128, at most 129, at most 130, at most 131, at most
132, at most 133, at most 134, at most 135, at most 136, at most
137, at most 138, at most 139, at most 140, at most 141, at most
142, at most 143, at most 144, at most 145, at most 146, at most
147, at most 148, at most 149, at most 150, at most 151, at most
152, at most 153, at most 154, at most 155, at most 156, at most
157, at most 158, at most 159, at most 160, at most 161, at most
162, at most 163, at most 164, at most 165, at most 166, at most
167, at most 168, at most 169, at most 170, at most 171, at most
172, at most 173, at most 174, at most 175, at most 176, at most
177, at most 178, at most 179, at most 180, at most 181, at most
182, at most 183, at most 184, at most 185, at most 186, at most
187, at most 188, at most 189, at most 190, at most 191, at most
192, at most 193, at most 194, at most 195, at most 196, at most
197, at most 198, at most 199, at most 200, at most 201, at most
202, at most 203, at most 204, at most 205, at most 206, at most
207, at most 208, at most 209, at most 210, at most 211, at most
212, at most 213, at most 214, at most 215, at most 216, at most
217, at most 218, at most 219, at most 220, at most 221, at most
222, at most 223, at most 224, at most 225, at most 226, at most
227, at most 228, at most 229, at most 230, at most 231, at most
232, at most 233, at most 234, at most 235, at most 236, at most
237, at most 238, at most 239, at most 240, at most 241, at most
242, at most 243, at most 244, at most 245, at most 246, at most
247, at most 248, at most 249, at most 250, at most 251, at most
252, at most 253, at most 254, at most 255, at most 256, at most
257, at most 258, at most 259, at most 260, at most 261, at most
262, at most 263, at most 264, at most 265, at most 266, at most
267, at most 268, at most 269, at most 270, at most 271, at most
272, at most 273, at most 274, at most 275, at most 276, at most
277, at most 278, at most 279, at most 280, at most 281, at most
282, at most 283, at most 284, at most 285, at most 286, at most
287, at most 288, at most 289, at most 290, at most 291, at most
292, at most 293, at most 294, at most 295, at most 296, at most
297, at most 298, at most 299, at most 300, at most 301, at most
302, at most 303, at most 304, at most 305, at most 306, at most
307, at most 308, at most 309, at most 310, at most 311, at most
312, at most 313, at most 314, at most 315, at most 316, at most
317, at most 318, at most 319, at most 320, at most 321, at most
322, at most 323, at most 324, at most 325, at most 326, at most
327, at most 328, at most 329, at most 330, at most 331, at most
332, at most 333, at most 334, at most 335, at most 336, at most
337, at most 338, at most 339, at most 340, at most 341, at most
342, at most 343, at most 344, at most 345, at most 346, at most
347, at most 348, at most 349, at most 350, at most 351, at most
352, at most 353, at most 354, at most 355, at most 356, at most
357, at most 358, at most 359, at most 360, at most 361, at most
362, at most 363, at most 364, at most 365, at most 366, at most
367, at most 368, at most 369, at most 370, at most 371, at most
372, at most 373, at most 374, at most 375, at most 376, at most
377, at most 378, at most 379, or at most 380 amino acid
substitutions, deletions, or insertions relative to 1-31, 77-92,
143-171, 207-249, 293-420, and 549-627 or to an amino acid sequence
selected from Tables 5-6 and 13-16.
[0202] As one of ordinary skill in the art would appreciate a PKS,
such as an OLS, could be obtained from any source, including
naturally occurring sources and synthetic sources (e.g., a
non-naturally occurring PKS). In some embodiments a PKS is from
Cannabis. In some embodiments a PKS is from Dictyostelium.
Non-limiting examples of PKS enzymes may be found in U.S. Pat. No.
6,265,633; WO2019/202510; WO 2018/148848 A1; WO 2018/148849 A1; and
US 2018/155748 (granted as U.S. Pat. No. 10,435,727), which are
incorporated by reference in this application in their entireties.
For example, PKSs include SEQ ID NO: 2 from WO2019/202510, SEQ ID
NO: 9 from WO2019/202510, SEQ ID NO: 37 from WO 2018/148848 A1, SEQ
ID NO: 38 from WO 2018/148848 A1, SEQ ID NO: 9 from WO 2018/148849
A1, SEQ ID NO: 10 from WO 2018/148849 A1, SEQ ID NO: 13 from WO
2018/148849 A1; and SEQ ID NO: 35 from U.S. Pat. No.
10,435,727.
[0203] In certain embodiments, polyketide synthases can use
hexanoyl-CoA or any acyl-CoA (or a product of Formula (2)):
##STR00138##
and three malonyl-CoAs as substrates to form
3,5,7-trioxododecanoyl-CoA or other 3,5,7-trioxo-acyl-CoA
derivatives; or to form a compound of Formula (4):
##STR00139##
wherein R is hydrogen, optionally substituted acyl, optionally
substituted alkyl, optionally substituted alkenyl, optionally
substituted alkynyl, optionally substituted carbocyclyl, or
optionally substituted aryl; depending on substrate. R is as
defined in this application. In some embodiments, R is a C2-C6
optionally substituted alkyl. In some embodiments, R is a propyl or
pentyl. In some embodiments, R is pentyl. In some embodiments, R is
propyl. A PKS may also bind isovaleryl-CoA, octanoyl-CoA,
hexanoyl-CoA, and butyryl-CoA. In some embodiments, a PKS is
capable of catalyzing the formation of a 3,5,7-trioxoalkanoyl-CoA
(e.g. 3,5,7-trioxododecanoyl-CoA). In some embodiments, an OLS is
capable of catalyzing the formation of a 3,5,7-trioxoalkanoyl-CoA
(e.g. 3,5,7-trioxododecanoyl-CoA).
[0204] In some embodiments, a PKS uses a substrate of Formula (2)
to form a compound of Formula (4):
##STR00140##
wherein R is unsubstituted pentyl.
[0205] A recombinant host cell that expresses a heterologous gene
encoding an PKS described herein may be capable of producing at
least 1% (e.g., at least 5%, at least 10%, at least 15%, at least
20%, at least 25%, at least 30%, at least 35%, at least 40%, at
least 45%, at least 50%, at least 55%, at least 60%, at least 65%,
at least 70%, at least 75%, at least 80%, at least 85%, at least
90%, at least 95%, at least 100%, at least 125%, at least 150%, at
least 175%, at least 200%, at least 300%, at least 400%, at least
500%, at least 600%, at least 700%, at least 800%, at least 900%,
or at least 1,000%) more of a product (e.g., a compound of Formula
(4), (5), and/or (6)) relative to a control. In some embodiments, a
compound of Formula (4) is a compound of Formula (4a), a compound
of Formula (5) is a compound of Formula (5a), and a compound of
Formula (6) is a compound of Formula (6a). In some embodiments, a
control is a recombinant host cell that expresses a heterologous
gene encoding UniProtKB--B1Q2B6. In some embodiments, a control is
a recombinant host cell that expresses a heterologous gene encoding
a wild-type PKS.
[0206] A recombinant host cell that expresses a heterologous gene
encoding an PKS described herein may be capable of producing at
least 0.5 mg/L, at least 1 mg/L, at least 1.5 mg/L, at least 2
mg/L, at least 2.5 mg/L, at least 3 mg/L, at least 3.5 mg/L, at
least 4 mg/L, at least 4.5 mg/L, at least 5 mg/L, at least 5.5
mg/L, at least 6 mg/L, at least 6.5 mg/L, at least 7 mg/L, at least
7.5 mg/L, at least 8 mg/L, at least 8.5 mg/L, at least 9 mg/L, at
least 9.5 mg/L, at least 10 mg/L, at least 10.5 mg/L, at least 11
mg/L, at least 11.5 mg/L, at least 12 mg/L, at least 12.5 mg/L, at
least 13 mg/L, at least 13.5 mg/L, at least 14 mg/L, at least 14.5
mg/L, at least 15 mg/L, at least 15.5 mg/L, at least 16 mg/L, at
least 16.5 mg/L, at least 17 mg/L, at least 17.5 mg/L, at least 18
mg/L, at least 18.5 mg/L, at least 19 mg/L, at least 19.5 mg/L, at
least 20 mg/L, at least 20.5 mg/L, at least 21 mg/L, at least 21.5
mg/L, at least 22 mg/L, at least 22.5 mg/L, at least 23 mg/L, at
least 23.5 mg/L, at least 24 mg/L, at least 24.5 mg/L, at least 25
mg/L, at least 25.5 mg/L, at least 26 mg/L, at least 26.5 mg/L, at
least 27 mg/L, at least 27.5 mg/L, at least 28 mg/L, at least 28.5
mg/L, at least 29 mg/L, at least 29.5 mg/L, at least 30 mg/L, at
least 30.5 mg/L, at least 31 mg/L, at least 31.5 mg/L, at least 32
mg/L, at least 32.5 mg/L, at least 33 mg/L, at least 33.5 mg/L, at
least 34 mg/L, at least 34.5 mg/L, at least 35 mg/L, at least 35.5
mg/L, at least 36 mg/L, at least 36.5 mg/L, at least 37 mg/L, at
least 37.5 mg/L, at least 38 mg/L, at least 38.5 mg/L, at least 39
mg/L, at least 39.5 mg/L, at least 40 mg/L, at least 40.5 mg/L, at
least 41 mg/L, at least 41.5 mg/L, at least 42 mg/L, at least 42.5
mg/L, at least 43 mg/L, at least 43.5 mg/L, at least 44 mg/L, at
least 44.5 mg/L, at least 45 mg/L, at least 45.5 mg/L, at least 46
mg/L, at least 46.5 mg/L, at least 47 mg/L, at least 47.5 mg/L, at
least 48 mg/L, at least 48.5 mg/L, at least 49 mg/L, at least 49.5
mg/L, at least 50 mg/L, at least 50.5 mg/L, at least 51 mg/L, at
least 51.5 mg/L, at least 52 mg/L, at least 52.5 mg/L, at least 53
mg/L, at least 53.5 mg/L, at least 54 mg/L, at least 54.5 mg/L, at
least 55 mg/L, at least 55.5 mg/L, at least 56 mg/L, at least 56.5
mg/L, at least 57 mg/L, at least 57.5 mg/L, at least 58 mg/L, at
least 58.5 mg/L, at least 59 mg/L, at least 59.5 mg/L, at least 60
mg/L, at least 60.5 mg/L, at least 61 mg/L, at least 61.5 mg/L, at
least 62 mg/L, at least 62.5 mg/L, at least 63 mg/L, at least 63.5
mg/L, at least 64 mg/L, at least 64.5 mg/L, at least 65 mg/L, at
least 65.5 mg/L, at least 66 mg/L, at least 66.5 mg/L, at least 67
mg/L, at least 67.5 mg/L, at least 68 mg/L, at least 68.5 mg/L, at
least 69 mg/L, at least 69.5 mg/L, at least 70 mg/L, at least 70.5
mg/L, at least 71 mg/L, at least 71.5 mg/L, at least 72 mg/L, at
least 72.5 mg/L, at least 73 mg/L, at least 73.5 mg/L, at least 74
mg/L, at least 74.5 mg/L, at least 75 mg/L, at least 75.5 mg/L, at
least 76 mg/L, at least 76.5 mg/L, at least 77 mg/L, at least 77.5
mg/L, at least 78 mg/L, at least 78.5 mg/L, at least 79 mg/L, at
least 79.5 mg/L, at least 80 mg/L, at least 80.5 mg/L, at least 81
mg/L, at least 81.5 mg/L, at least 82 mg/L, at least 82.5 mg/L, at
least 83 mg/L, at least 83.5 mg/L, at least 84 mg/L, at least 84.5
mg/L, at least 85 mg/L, at least 85.5 mg/L, at least 86 mg/L, at
least 86.5 mg/L, at least 87 mg/L, at least 87.5 mg/L, at least 88
mg/L, at least 88.5 mg/L, at least 89 mg/L, at least 89.5 mg/L, at
least 90 mg/L, at least 90.5 mg/L, at least 91 mg/L, at least 91.5
mg/L, at least 92 mg/L, at least 92.5 mg/L, at least 93 mg/L, at
least 93.5 mg/L, at least 94 mg/L, at least 94.5 mg/L, at least 95
mg/L, at least 95.5 mg/L, at least 96 mg/L, at least 96.5 mg/L, at
least 97 mg/L, at least 97.5 mg/L, at least 98 mg/L, at least 98.5
mg/L, at least 99 mg/L, at least 99.5 mg/L, or at least 100 mg/L of
a product (e.g., a compound of Formula (4), (5), and/or (6). In
some instances, OLSs may form triketide (PDAL) and/or tetraketide
(HTAL and olivetol) by-products. Triketides convert to PDAL, and
tetraketides convert to HTAL and olivetol, not to olivetolic acid.
In some embodiments, production of by-products is undesirable. In
some embodiments, OLS enzymes described herein do not produce
by-products or produce minimal by-products relative to a control.
In some embodiments, OLS enzymes are selected, at least in part,
based on the ratio of olivetolic acid produced relative to
olivetol.
[0207] It was surprisingly discovered herein that OLSs can exhibit
both OLS and OAC activity. PKS enzymes described in this
application may or may not have cyclase activity. In some
embodiments where the PKS enzyme does not have cyclase activity,
one or more exogenous polynucleotides that encode a polyketide
cyclase (PKC) enzyme may also be co-expressed in the same host
cells to enable conversion of hexanoic acid or butyric acid or
other fatty acid conversion into olivetolic acid or divarinolic
acid or other precursors of cannabinoids. In some embodiments, the
PKS enzyme and a PKC enzyme are expressed as separate and distinct
enzymes. In some embodiments, a PKS enzyme that lacks cyclase
activity and a PKC are linked as part of a fusion polypeptide that
is a bifunctional PKS. In some embodiments, a bifunctional PKC is
referred to
[0208] As used in this application, a bifunctional PKS is an enzyme
that is capable of producing a compound of Formula (6):
##STR00141##
from a compound of Formula (2):
##STR00142##
and a compound of Formula (3):
##STR00143##
In some embodiments, a PKS produces more of a compound of Formula
(6):
##STR00144##
as compared to a compound of Formula (5):
##STR00145##
As a non-limiting example, a compound of Formula (6):
##STR00146##
is olivetolic acid (Formula (6a)):
##STR00147##
As a non-limiting example, a compound of Formula (5):
##STR00148##
is olivetol (Formula (5a)):
##STR00149##
[0209] In some embodiments, a polyketide synthase of the present
disclosure is capable of catalyzing a compound of Formula (2):
##STR00150##
and a compound of Formula (3):
##STR00151##
to produce a compound of Formula (4):
##STR00152##
and also further catalyzes a compound of Formula (4):
##STR00153##
to produce a compound of Formula (6):
##STR00154##
In some embodiments, the PKS is not a fusion protein. In some
embodiments, a PKS that is capable of catalyzing a compound of
Formula (2):
##STR00155##
and a compound of Formula (3):
##STR00156##
to produce a compound of Formula (4):
##STR00157##
and is also capable of further catalyzing the production of a
compound of Formula (6):
##STR00158##
from the compound of Formula (4):
##STR00159##
is preferred because it avoids the need for an additional
polyketide cyclase to produce a compound of Formula (6):
##STR00160##
In some embodiments, such an enzyme that is a bifunctional PKS
eliminates the transport considerations needed with addition of a
polyketide cyclase, whereby the compound of Formula (4), being the
product of the PKS, must be transported to the PKS for use as a
substrate to be converted into the compound of Formula (6).
[0210] In some embodiments, a PKS is capable of producing
olivetolic acid in the presence of a compound of Formula (2a):
##STR00161##
and Formula (3a):
##STR00162##
[0212] In some embodiments, an OLS is capable of producing
olivetolic acid in the presence of a compound of Formula (2a):
##STR00163##
and Formula (3a):
##STR00164##
[0214] Without being bound by a particular theory, the presence of
the amino acid W at a residue in a PKS corresponding to position
339 of SEQ ID NO: 6 may render or enhance bifunctionality of a PKS.
In some embodiments, a bifunctional PKS comprises the amino acid W
at a residue corresponding to position 339 of SEQ ID NO: 6. In some
embodiments, a bifunctional PKS does not comprise the amino acid S
at a residue corresponding to position 339 of SEQ ID NO: 6. As a
non-limiting example, a PKS may comprise the amino acid
substitution S332W relative to SEQ ID NO: 5 (see, e.g., t606899,
SEQ ID NO: 298). In some embodiments, a PKS may comprise the amino
acid substitution S339W relative to SEQ ID NO: 7 (see, e.g.,
t607377, SEQ ID NO: 409) In some embodiments, the PKS comprises a
sequence that is at least 5%, at least 10%, at least 15%, at least
20%, at least 25%, at least 30%, at least 35%, at least 40%, at
least 45%, at least 50%, at least 55%, at least 60%, at least 65%,
at least 70%, at least 71%, at least 72%, at least 73%, at least
74%, at least 75%, at least 76%, at least 77%, at least 78%, at
least 79%, at least 80%, at least 81%, at least 82%, at least 83%,
at least 84%, at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, at least 99%, or is 100% identical, including all
values in between, to a sequence (e.g., nucleic acid or amino acid
sequence) set forth in SEQ ID NO: 6.
[0215] In some embodiments, an OLS is capable of producing
olivetolic acid in the presence of a compound of Formula (2a) and
Formula (3a). In some embodiments, the OLS produces more olivetolic
acid (OA) than olivetol. In some embodiments, the OLS produces at
least 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6
times, 1.7 times, 1.8 times, 1.9 times, 2 times, 2.1 times, 2.2
times, 2.3 times, 2.4 times, 2.5 times, 2.6 times, 2.7 times, 2.8
times, 2.9 times, 3 times, 3.1 times, 3.2 times, 3.3 times, 3.4
times, 3.5 times, 3.6 times, 3.7 times, 3.8 times, 3.9 times, 4
times, 5 times, 6 times, 8 times, 9 times, 10 times, 20 times, 30
times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times,
100 times, 200 times, 300 times, 400 times, 500 times, 600 times,
700 times, 800 times or 1,000 times more olivetolic acid (OA) than
olivetol.
[0216] Without wishing to be bound by any theory, in some
embodiments, bifunctional OLSs differ from other OLSs in the
geometry of the substrate binding pocket, an internal substrate
holding cavity, and/or a substrate exit tunnel. For example, the
substrate binding pocket of the bifunctional OLSs may be wider as
compared to the substrate binding pocket of Cannabis sativa OLS
(SEQ ID NO: 5). Without wishing to be bound by any theory, this
extra space may alleviate steric clashes between the protein and
substrate and permit the pro-cyclization configuration.
Polyketide Cyclase (PKC)
[0217] A host cell described in this application may comprise a
PKC. As used in this application, a "PKC" refers to an enzyme that
is capable of cyclizing a polyketide.
[0218] In certain embodiments, a polyketide cyclase (PKC) catalyzes
the cyclization of an oxo fatty acyl-CoA (e.g., a compound of
Formula (4):
##STR00165##
[0219] or 3,5,7-trioxododecanoyl-COA, 3,5,7-trioxodecanoyl-COA) to
the corresponding intramolecular cyclization product (e.g.,
compound of Formula (6), including olivetolic acid and divarinic
acid). In some embodiments, a PKC catalyzes the formation of a
compound which occurs in the presence of a PKS. PKC substrates
include trioxoalkanol-CoA, such as 3,5,7-Trioxododecanoyl-CoA, or a
compound of Formula (4):
##STR00166##
wherein R is hydrogen, optionally substituted acyl, optionally
substituted alkyl, optionally substituted alkenyl, optionally
substituted alkynyl, optionally substituted carbocyclyl, or
optionally substituted aryl. In certain embodiments, a PKC
catalyzes a compound of Formula (4):
##STR00167##
wherein R is hydrogen, optionally substituted acyl, optionally
substituted alkyl, optionally substituted alkenyl, optionally
substituted alkynyl, optionally substituted carbocyclyl, or
optionally substituted aryl; to form a compound of Formula (6):
##STR00168##
wherein R is hydrogen, optionally substituted acyl, optionally
substituted alkyl, optionally substituted alkenyl, optionally
substituted alkynyl, optionally substituted carbocyclyl, or
optionally substituted aryl; as substrates. R is as defined in this
application. In some embodiments, R is a C2-C6 optionally
substituted alkyl. In some embodiments, R is a propyl or pentyl. In
some embodiments, R is pentyl. In some embodiments, R is propyl. In
certain embodiments, a PKC is an olivetolic acid cyclase (OAC). In
certain embodiments, a PKC is a divarinic acid cyclase (DAC).
[0220] As one of ordinary skill in the art would appreciate a PKC
could be obtained from any source, including naturally occurring
sources and synthetic sources (e.g., a non-naturally occurring
PKC). In some embodiments, a PKC is from Cannabis. Non-limiting
examples of PKCs include those disclosed in U.S. Pat. Nos.
9,611,460; 10,059,971; and US Pub 2019/0169661, which are
incorporated by reference in this application in their
entireties.
[0221] In some embodiments, a PKC is an OAC. As used in this
application, an "OAC" refers to an enzyme that is capable of
catalyzing the formation of olivetolic acid (OA). In some
embodiments, an OAC is an enzyme that is capable of using a
substrate of Formula (4a) (3,5,7-trioxododecanoyl-CoA):
##STR00169##
to form a compound of Formula (6a) (olivetolic acid):
##STR00170##
[0222] Olivetolic acid cyclase from C. sativa (CsOAC) is a 101
amino acid enzyme that performs non-decaboxylative cyclization of
the tetraketide product of olivetol synthase (FIG. 4 Structure 4a)
via aldol condensation to form olivetolic acid (FIG. 4 Structure
6a). CsOAC was identified and characterized by Gagne et al. (PNAS
2012) via transcriptome mining, and its cyclization function was
recapitulated in vitro to demonstrate that CsOAC is required for
formation of olivetolic acid in C. sativa. A crystal structure of
the enzyme was published by Yang et al. (FEBS J. 2016 March;
283(6):1088-106), which revealed that the enzyme is a homodimer and
belongs to the .alpha.+.beta. barrel (DABB) superfamily of protein
folds. CsOAC is the only known plant polyketide cyclase. Multiple
fungal Type III polyketide synthases have been identified that
perform both polyketide synthase and cyclization functions (Funa et
al., J Biol Chem. 2007 May 11; 282(19):14476-81); however, in
plants such a dual function enzyme has not yet been discovered.
[0223] A non-limiting example of an amino acid sequence encoding
OAC in C. sativa is provided by UniProtKB--I6WU39 (SEQ ID NO: 125),
which catalyzes the formation of olivetolic acid (OA) from
3,5,7-Trioxododecanoyl-CoA.
[0224] The sequence of UniProtKB--I6WU39 (SEQ ID NO: 125) is:
TABLE-US-00004 MAVKHLIVLKFKDEITEAQKEEFFKTYVNLVNIIPAMKDVYWGKDVTQK
NKEEGYTHIVEVTFESVETIQDYIIHPAHVGFGDVYRSFWEKLLIFDYT PRK.
[0225] A non-limiting example of a nucleic acid sequence encoding
C. sativa OAC is:
TABLE-US-00005 (SEQ ID NO: 130)
atggcagtgaagcatttgattgtattgaagttcaaagatgaaatcacag
aagcccaaaaggaagaatttttcaagacgtatgtgaatcttgtgaatat
catcccagccatgaaagatgtatactggggtaaagatgtgactcaaaag
aataaggaagaagggtacactcacatagttgaggtaacatttgagagtg
tggagactattcaggactacattattcatcctgcccatgttggatttgg
agatgtctatcgttctttctgggaaaaacttctcatttttgactacaca ccacgaaag.
Prenyltransferase (PT)
[0226] A host cell described in this application may comprise a
prenyltransferase (PT). As used in this application, a "PT" refers
to an enzyme that is capable of transferring prenyl groups to
acceptor molecule substrates. Non-limiting examples of
prenyltransferases are described in WO2018200888 (e.g., CsPT4),
U.S. Pat. No. 8,884,100 (e.g., CsPT1); CA2718469; Valliere et al.,
Nat Commun. 2019 Feb. 4; 10(1):565; and Luo et al., Nature 2019
March; 567(7746):123-126, which are incorporated by reference in
their entireties. In some embodiments, a PT is capable of producing
cannabigerolic acid (CBGA), cannabigerovarinic acid (CBGVA), or
other cannabinoids or cannabinoid-like substances. In some
embodiments, a PT is cannabigerolic acid synthase (CBGAS). In some
embodiments, a PT is cannabigerovarinic acid synthase (CBGVAS).
[0227] In some embodiments, the PT is an NphB prenyltransferase.
See, e.g., U.S. Pat. No. 7,544,498; and Kumano et al., Bioorg Med
Chem. 2008 Sep. 1; 16(17): 8117-8126, which are incorporated by
reference in this application in their entireties. In some
embodiments, a PT corresponds to NphB from Streptomyces sp. (see,
e.g., UniprotKB Accession No. Q4R2T2; see also SEQ ID NO: 2 of U.S.
Pat. No. 7,361,483). The protein sequence corresponding to
UniprotKB Accession No. Q4R2T2 is provided by SEQ ID NO: 131:
TABLE-US-00006 (SEQ ID NO: 131)
MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLSTFQDTLVEGGSVVV
FSMASGRHSTELDFSISVPTSHGDPYATVVEKGLFPATGHPVDDLLADT
QKHLPVSMFAIDGEVTGGFKKTYAFFPTDNMPGVAELSAIPSMPPAVAE
NAELFARYGLDKVQMTSMDYKKRQVNLYFSELSAQTLEAESVLALVREL
GLHVPNELGLKFCKRSFSVYPTLNWETGKIDRLCFAVISNDPTLVPSSD
EGDIEKFHNYATKAPYAYVGEKRTLVYGLTLSPKEEYYKLGAYYHITDV
QRGLLKAFDSLED.
[0228] A non-limiting example of a nucleic acid sequence encoding
NphB is:
TABLE-US-00007 (SEQ ID NO: 132)
atgtcagaagccgcagatgtcgaaagagtttacgccgctatggaagaag
ccgccggtttgttaggtgttgcctgtgccagagataagatctacccatt
gttgtctacttttcaagatacattagttgaaggtggttcagttgttgtt
ttctctatggcttcaggtagacattctacagaattggatttctctatct
cagttccaacatcacatggtgatccatacgctactgttgttgaaaaagg
tttatttccagcaacaggtcatccagttgatgatttgttggctgatact
caaaagcatttgccagtttctatgtttgcaattgatggtgaagttactg
gtggtttcaagaaaacttacgctttetttccaactgataacatgccagg
tgttgcagaattatctgctattccatcaatgccaccagctgttgcagaa
aatgcagaattatttgctagatacggtttggataaggttcaaatgacat
ctatggattacaagaaaagacaagttaatttgtacttttctgaattatc
agcacaaactttggaagctgaatcagttttggcattagttagagaattg
ggtttacatgttccaaacgaattgggtttgaagttttgtaaaagatctt
tctcagtttatccaactttaaactgggaaacaggcaagatcgatagatt
atgtttcgcagttatctctaacgatccaacattggttccatcttcagat
gaaggtgatatcgaaaagtttcataactacgctactaaagcaccatatg
cttacgttggtgaaaagagaacattagtttatggtttgactttatcacc
aaaggaagaatactacaagttgggtgcttactaccacattaccgacgta
caaagaggtttattgaaagcattcgatagtttagaagactaa.
[0229] In other embodiments, a PT corresponds to CsPT1, which is
disclosed as SEQ ID NO:2 in U.S. Pat. No. 8,884,100 (C. sativa;
corresponding to SEQ ID NO: 110 in this application):
TABLE-US-00008 (SEQ ID NO: 110)
MGLSSVCTFSFQTNYHTLLNPHNNNPKTSLLCYRHPKTPIKYSYNNFPS
KHCSTKSFHLQNKCSESLSIAKNSIRAATTNQTEPPESDNHSVATKILN
FGKACWKLQRPYTIIAFTSCACGLFGKELLHNTNLISWSLMFKAFFFLV
AILCIASFTTTINQIYDLHIDRINKPDLPLASGEISVNTAWEVISIIVA
LFGLIITIKMKGGPLYIFGYCFGIFGGIVYSVPPFRWKQNPSTAFLLNF
LAHIITNFTFYYASRAALGLPFELRPSFTFLLAFMKSMGSALALIKDAS
DVEGDTKFGISTLASKYGSRNLTLFCSGIVLLSYVAAILAGIIWPQAFN
SNVMLLSHAILAFWLILQTRDFALTNYDPEAGRRFYEFMWKLYYAEYLV YVFI.
[0230] In some embodiments, a PT corresponds to CsPT4, which is
disclosed as SEQ ID NO:1 in WO2019071000, corresponding to SEQ ID
NO: 133 in this application:
TABLE-US-00009 (SEQ ID NO: 133)
MGLSLVCTFSFQTNYHTLLNPHNKNPKNSLLSYQHPKTPIIKSSYDNFP
SKYCLTKNFHLLGLNSHNRISSQSRSIRAGSDQIEGSPHHESDNSIATK
ILNFGHTCWKLQRPYVVKGMISIACGLFGRELFNNRHLFSWGLMWKAFF
ALVPILSFNFFAAIMNQIYDVDIDRINKPDLPLVSGEMSIETAWILSII
VALTGLIVTIKLKSAPLFVFIYIFGIFAGFAYSVPPIRWKQYPFTNFLI
TISSHVGLAFTSYSATTSALGLPFVWRPAFSFITAFMTVMGMTIAFAKD
ISDIEGDAKYGVSTVATKLGARNIVITFVVSGVLLLNYLVSISIGIIWP
QVFKSNIMILSHAILAFCLIFQTRELALANYASAPSRQFFEFIWLLYYA EYFVYVFI.
[0231] In some embodiments, a PT corresponds to a truncated CsPT4,
which is provided as SEQ ID NO: 134 herein:
TABLE-US-00010 MSAGSDQIEGSPHHESDNSIATKILNFGHTCWKLQRPYVVKGMISIACG
LFGRELFNNRHLFSWGLMWKAFFALVPILSFNFFAAIMNQIYDVDIDRI
NKPDLPLVSGEMSIETAWILSIIVALTGLIVTIKLKSAPLFVFIYIFGI
FAGFAYSVPPIRWKQYPFTNFLITISSHVGLAFTSYSATTSALGLPFVW
RPAFSFITAFMTVMGMTIAFAKDISDIEGDAKYGVSTVATKLGARNMTF
VVSGVLLLNYLVSISIGIIWPQVFKSNIMILSHAILAFCLIFQTRELAL
ANYASAPSRQFFEFIWLLYYAEYFVYVFI.
[0232] Functional expression of paralog C. sativa CBGAS enzymes in
S. cerevisiae and production of the major cannabinoid CBGA has been
reported (Page and Boubakir US 20120144523, 2012, and Luo et al.
Nature, 2019). Luo et al. reported the production of CBGA in S.
cerevisiae by expressing a truncated version of a C. sativa CBGAS,
CsPT4, with its native signal peptide removed (Luo et al. Nature,
2019). Without being bound by a particular theory, the
integral-membrane nature of C. sativa CBGAS enzymes may render
functional expression of C. sativa CBGAS enzymes in heterologous
hosts challenging. Removal of transmembrane domain(s) or signal
sequences or use of prenyltransferases that are not associated with
the membrane and are not integral membrane proteins may facilitate
increased interaction between the enzyme and available substrate,
for example in the cellular cytosol and/or in organelles that may
be targeted using peptides that confer localization.
[0233] In some embodiments, the PT is a soluble PT. In some
embodiments, the PT is a cytosolic PT. In some embodiments, the PT
is a secreted protein. In some embodiments, the PT is not a
membrane-associated protein. In some embodiments, the PT is not an
integral membrane protein. In some embodiments, the PT does not
comprise a transmembrane domain or a predicted transmembrane. In
some embodiments, the PT may be primarily detected in the cytosol
(e.g., detected in the cytosol to a greater extent than detected
associated with the cell membrane). In some embodiments, the PT is
a protein from which one or more transmembrane domains have been
removed and/or mutated (e.g., by truncation, deletions,
substitutions, insertions, and/or additions) so that the PT
localizes or is predicted to localize in the cytosol of the host
cell, or to cytosolic organelles within the host cell, or, in the
case of bacterial hosts, in the periplasm. In some embodiments, the
PT is a protein from which one or more transmembrane domains have
been removed or mutated (e.g., by truncation, deletions,
substitutions, insertions, and/or additions) so that the PT has
increased localization to the cytosol, organelles, or periplasm of
the host cell, as compared to membrane localization.
[0234] Within the scope of the term "transmembrane domains" are
predicted or putative transmembrane domains in addition to
transmembrane domains that have been empirically determined. In
general, transmembrane domains are characterized by a region of
hydrophobicity that facilitates integration into the cell membrane.
Methods of predicting whether a protein is a membrane protein or a
membrane-associated protein are known in the art and may include,
for example, amino acid sequence analysis, hydropathy plots, and/or
protein localization assays.
[0235] In some embodiments, the PT is a protein from which a signal
sequence has been removed and/or mutated so that the PT is not
directed to the cellular secretory pathway. In some embodiments,
the PT is a protein from which a signal sequence has been removed
and/or mutated so that the PT is localized to the cytosol or has
increased localization to the cytosol (e.g., as compared to the
secretory pathway).
[0236] In some embodiments, the PT is a secreted protein. In some
embodiments, the PT contains a signal sequence.
[0237] In some embodiments, a PT is a fusion protein. For example,
a PT may be fused to one or more genes in the metabolic pathway of
a host cell. In certain embodiments, a PT may be fused to mutant
forms of one or more genes in the metabolic pathway of a host
cell.
[0238] In some embodiments, a PT described in this application
transfers one or more prenyl groups to any of positions 1, 2, 3, 4,
or 5 in a compound of Formula (6), shown below:
##STR00171##
[0239] In some embodiments, the PT transfers a prenyl group to any
of positions 1, 2, 3, 4, or 5 in a compound of Formula (6), shown
below:
##STR00172##
to form a compound of one or more of Formula (8w), Formula (8x),
Formula (8'), Formula (8y), Formula (8z):
##STR00173##
or a pharmaceutically acceptable salt, solvate, hydrate, polymorph,
co-crystal, tautomer, stereoisomer, isotopically labeled
derivative, or prodrug thereof, wherein a is 1, 2, 3, 4, 5, 6, 7,
8, 9, or 10.
Terminal Synthases (TS)
[0240] A host cell described in this application may comprise a
terminal synthase (TS). As used in this application, a "TS" refers
to an enzyme that is capable of catalyzing oxidative cyclization of
a prenyl moiety (e.g., terpene) to produce a ring-containing
product (e.g., heterocyclic ring-containing product). In certain
embodiments, a TS is capable of catalyzing oxidative cyclization of
a prenyl moiety (e.g., terpene) to produce a carbocyclic-ring
containing product (e.g., cannabinoid). In certain embodiments, a
TS is capable of catalyzing oxidative cyclization of a prenyl
moiety (e.g., terpene) to produce a heterocyclic-ring containing
product (e.g., cannabinoid). In certain embodiments, a TS is
capable of catalyzing oxidative cyclization of a prenyl moiety
(e.g., terpene) to produce a cannabinoid.
[0241] In some embodiments, a TS is a tetrahydrocannabinolic acid
synthase (THCAS), a cannabidiolic acid synthase (CBDAS), and/or a
cannabichromenic acid synthase (CBCAS). As one of ordinary skill in
the art would appreciate a TS could be obtained from any source,
including naturally occurring sources and synthetic sources (e.g.,
a non-naturally occurring TS).
[0242] a. Substrates
[0243] A TS may be capable of using one or more substrates. In some
instances, the location of the prenyl group and/or the R group
differs between TS substrates. For example, a TS may be capable of
using as a substrate one or more compounds of Formula (8w), Formula
(8x), Formula (8'), Formula (8y), and/or Formula (8z):
##STR00174##
or a pharmaceutically acceptable salt, solvate, hydrate, polymorph,
co-crystal, tautomer, stereoisomer, isotopically labeled
derivative, or prodrug thereof, wherein a is 1, 2, 3, 4, 5, 6, 7,
8, 9, or 10.
[0244] In certain embodiments, a compound of Formula (8') is a
compound of Formula (8):
##STR00175##
[0245] In some embodiments, a TS catalyzes oxidative cyclization of
the prenyl moiety (e.g., terpene) of a compound of Formula (8)
described in this application and shown in FIG. 2. In certain
embodiments, a compound of Formula (8) is a compound of Formula
(8.alpha.):
##STR00176##
[0246] b. Products
[0247] In embodiments wherein CBGA is the substrate, the TS enzymes
CBDAS, THCAS and CBCAS would generally catalyze the formation of
cannabidiolic acid (CBDA), .DELTA.9-tetrahydrocannabinolic acid
(THCA) and cannabichromenic acid (CBCA), respectively. However, in
some embodiments, a TS can produce more than one different product
depending on reaction conditions. For example, the pH of the
reaction environment may cause a THCAS or a CBDAS to produce CBCA
in greater proportions than THCA or CBDAS, respectively (see, for
example, U.S. Pat. No. 9,359,625 to Winnicki and Donsky,
incorporated by reference in its entirety).
[0248] A TS may be capable of using one or more substrates
described in this application to produce one or more products.
Non-limiting example of TS products are shown in Table 1. In some
instances, a TS is capable of using one substrate to produce 1, 2,
3, 4, 5, 6, 7, 8, 9, or 10 different products. In some embodiments,
a TS is capable of using more than one substrate to produce 1, 2,
3, 4, 5, 6, 7, 8, 9, or 10 different products.
[0249] In some embodiments, a TS is capable of producing a compound
of Formula (X-A) and/or a compound of Formula (X-B):
##STR00177##
or a pharmaceutically acceptable salt, solvate, hydrate, polymorph,
co-crystal, tautomer, stereoisomer, isotopically labeled
derivative, or prodrug thereof, wherein is a double bond or a
single bond, as valency permits;
[0250] R is hydrogen, optionally substituted acyl, optionally
substituted alkyl, optionally substituted alkenyl, optionally
substituted alkynyl, optionally substituted carbocyclyl, or
optionally substituted aryl;
[0251] R.sup.Z1 is hydrogen, optionally substituted acyl,
optionally substituted alkyl, optionally substituted alkenyl,
optionally substituted alkynyl, optionally substituted carbocyclyl,
or optionally substituted aryl;
[0252] R.sup.Z2 is hydrogen, optionally substituted acyl,
optionally substituted alkyl, optionally substituted alkenyl,
optionally substituted alkynyl, optionally substituted carbocyclyl,
or optionally substituted aryl;
[0253] or optionally, R.sup.Z1 and R.sup.Z2 are taken together with
their intervening atoms to form an optionally substituted
carbocyclic ring;
[0254] R.sup.3A is hydrogen, optionally substituted acyl,
optionally substituted alkyl, optionally substituted alkenyl, or
optionally substituted alkynyl;
[0255] R.sup.3B is hydrogen, optionally substituted acyl,
optionally substituted alkyl, optionally substituted alkenyl, or
optionally substituted alkynyl; and/or
[0256] R.sup.Y is hydrogen, optionally substituted acyl, optionally
substituted alkyl, optionally substituted alkenyl, or optionally
substituted alkynyl.
[0257] In some embodiments, a compound of Formula (X-A) is:
##STR00178##
[0258] In certain embodiments, a compound of Formula (10)
##STR00179##
has a chiral atom labeled with * at carbon 10 and a chiral atom
labeled with ** at carbon 6. In certain embodiments, in a compound
of Formula (10)
##STR00180##
the chiral atom labeled with * at carbon 10 is of the
R-configuration or S-configuration; and a chiral atom labeled with
** at carbon 6 is of the R-configuration. In certain embodiments,
in a compound of Formula (10)
##STR00181##
the chiral atom labeled with * at carbon 10 is of the
S-configuration; and a chiral atom labeled with ** at carbon 6 is
of the R-configuration or S-configuration. In certain embodiments,
in a compound of Formula (10)
##STR00182##
the chiral atom labeled with * at carbon 10 is of the
R-configuration and a chiral atom labeled with ** at carbon 6 is of
the R-configuration. In certain embodiments, a compound of Formula
(10)
##STR00183##
is of the formula:
##STR00184##
In certain embodiments, in a compound of Formula (10)
##STR00185##
the chiral atom labeled with * at carbon 10 is of the
S-configuration and a chiral atom labeled with ** at carbon 6 is of
the S-configuration. In certain embodiments, a compound of Formula
(10)
##STR00186##
is of the formula:
##STR00187##
[0259] In certain embodiments, a compound of Formula
(10.alpha.)
##STR00188##
has a chiral atom labeled with * at carbon 10 and a chiral atom
labeled with ** at carbon 6. In certain embodiments, in a compound
of Formula (10.alpha.)
##STR00189##
the chiral atom labeled with * at carbon 10 is of the
R-configuration or S-configuration; and a chiral atom labeled with
** at carbon 6 is of the R-configuration. In certain embodiments,
in a compound of Formula (10.alpha.)
##STR00190##
the chiral atom labeled with * at carbon 10 is of the
S-configuration; and a chiral atom labeled with ** at carbon 6 is
of the R-configuration or S-configuration. In certain embodiments,
in a compound of Formula (10.alpha.)
##STR00191##
the chiral atom labeled with * at carbon 10 is of the
R-configuration and a chiral atom labeled with ** at carbon 6 is of
the R-configuration. In certain embodiments, a compound of Formula
(10.alpha.)
##STR00192##
is of the formula:
##STR00193##
In certain embodiments, in a compound of Formula (10.alpha.)
##STR00194##
the chiral atom labeled with * at carbon 10 is of the
S-configuration and a chiral atom labeled with ** at carbon 6 is of
the S-configuration. In certain embodiments, a compound of Formula
(10.alpha.)
##STR00195##
is of the formula:
##STR00196##
[0260] In some embodiments, a compound of Formula (X-A) is:
##STR00197##
[0261] In some embodiments, a compound of Formula (X-A) is:
##STR00198##
[0262] In some embodiments, a compound of Formula (X-B) is:
##STR00199##
[0263] In certain embodiments, a compound of Formula (9)
##STR00200##
has a chiral atom labeled with * at carbon 3 and a chiral atom
labeled with ** at carbon 4. In certain embodiments, in a compound
of Formula (9)
##STR00201##
the chiral atom labeled with * at carbon 3 is of the
R-configuration or S-configuration; and a chiral atom labeled with
** at carbon 4 is of the R-configuration. In certain embodiments,
in a compound of Formula (9)
##STR00202##
the chiral atom labeled with * at carbon 3 is of the
S-configuration; and a chiral atom labeled with ** at carbon 4 is
of the R-configuration or S-configuration. In certain embodiments,
in a compound of Formula (9)
##STR00203##
the chiral atom labeled with * at carbon 3 is of the
R-configuration and a chiral atom labeled with ** at carbon 4 is of
the R-configuration. In certain embodiments, a compound of Formula
(9)
##STR00204##
is of the formula:
##STR00205##
In certain embodiments, in a compound of Formula (9)
##STR00206##
the chiral atom labeled with * at carbon 3 is of the
S-configuration and a chiral atom labeled with ** at carbon 4 is of
the S-configuration. In certain embodiments, a compound of Formula
(9)
##STR00207##
is of the formula:
##STR00208##
[0264] In certain embodiments, a compound of Formula (9a)
(CBDA)
##STR00209##
has a chiral atom labeled with * at carbon 3 and a chiral atom
labeled with ** at carbon 4. In certain embodiments, in a compound
of Formula (9a)
##STR00210##
the chiral atom labeled with * at carbon 3 is of the
R-configuration or S-configuration; and a chiral atom labeled with
** at carbon 4 is of the R-configuration. In certain embodiments,
in a compound of Formula (9a)
##STR00211##
the chiral atom labeled with * at carbon 3 is of the
S-configuration; and a chiral atom labeled with ** at carbon 4 is
of the R-configuration or S-configuration. In certain embodiments,
in a compound of Formula (9a)
##STR00212##
the chiral atom labeled with * at carbon 3 is of the
R-configuration and a chiral atom labeled with ** at carbon 4 is of
the R-configuration. In certain embodiments, a compound of Formula
(9a)
##STR00213##
is of the formula:
##STR00214##
In certain embodiments, in a compound of Formula (9a)
##STR00215##
the chiral atom labeled with * at carbon 3 is of the
S-configuration and a chiral atom labeled with ** at carbon 4 is of
the S-configuration. In certain embodiments, a compound of Formula
(9a)
##STR00216##
is of the formula:
##STR00217##
[0265] In some embodiments, as shown in FIG. 2, a TS is capable of
producing a cannabinoid from the product of a PT, including,
without limitation, an enzyme capable of producing a compound of
Formula (9), (10), or (11):
##STR00218##
or a pharmaceutically acceptable salt, solvate, hydrate, polymorph,
co-crystal, tautomer, stereoisomer, isotopically labeled
derivative, or prodrug thereof, wherein R is hydrogen, optionally
substituted acyl, optionally substituted alkyl, optionally
substituted alkenyl, optionally substituted alkynyl, optionally
substituted carbocyclyl, or optionally substituted aryl; produced
from a compound of Formula (8'):
##STR00219##
wherein a is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and R is hydrogen,
optionally substituted acyl, optionally substituted alkyl,
optionally substituted alkenyl, optionally substituted alkynyl,
optionally substituted carbocyclyl, or optionally substituted aryl;
or using any other substrate. In certain embodiments, a compound of
Formula (8') is a compound of Formula (8):
##STR00220##
[0266] In certain embodiments, a compound of Formula (9), (10), or
(11) is produced using a TS from a substrate compound of Formula
(8') (e.g., compound of Formula (8)), for example. Non-limiting
examples of substrate compounds of Formula (8') include but are not
limited to cannabigerolic acid (CBGA), cannabigerovarinic acid
(CBGVA), or cannabinerolic acid. In certain embodiments, at least
one of the hydroxyl groups of the product compounds of Formula (9),
(10), or (11) is further methylated. In certain embodiments, a
compound of Formula (9) is methylated to form a compound of Formula
(12):
##STR00221##
or a pharmaceutically acceptable salt, solvate, hydrate, polymorph,
co-crystal, tautomer, stereoisomer, isotopically labeled
derivative, or prodrug thereof.
Tetrahydrocannabinolic Acid Synthase (THCAS)
[0267] A host cell described in this application may comprise a TS
that is a tetrahydrocannabinolic acid synthase (THCAS). As used in
this application "tetrahydrocannabinolic acid synthase (THCAS)" or
".DELTA..sup.1-tetrahydrocannabinolic acid (THCA) synthase" refers
to an enzyme that is capable of catalyzing oxidative cyclization of
a prenyl moiety (e.g., terpene) of a compound of Formula (8) to
produce a ring-containing product (e.g., heterocyclic
ring-containing product, carbocyclic-ring containing product) of
Formula (10). In certain embodiments, a THCAS refers to an enzyme
that is capable of producing .DELTA.9-tetrahydrocannabinolic acid
(.DELTA.9-THCA, THCA, .DELTA.9-Tetrahydro-cannabivarinic acid A
(.DELTA.9-THCVA-C3 A), THCVA, THCP, or a compound of Formula 10(a),
from a compound of Formula (8). In certain embodiments, a THCAS is
capable of producing .DELTA..sup.9-tetrahydrocannabinolic acid
(.DELTA..sup.9-THCA, THCA, or a compound of Formula 10(a)).
[0268] A THCAS may use cannabigerolic acid (CBGA) as a substrate.
In some embodiments, the THCAS produces .DELTA..sup.9-THCA from
CBGA. In some embodiments, a THCAS may catalyze the oxidative
cyclization of other substrates, such as
3-geranyl-2,4-dihydro-6-alkylbenzoic acids. In some embodiments, a
THCAS may catalyze the oxidative cyclization of other substrates,
such as 3-geranyl-2,4-dihydro-6-alkylbenzoic acids like
cannabigerovarinic acid (CBGVA)). In some embodiments, a THCAS
exhibits specificity for CBGA substrates. In some embodiments, a
THCAS may use cannabivarinic acid (CBDVA) as a substrate. In some
embodiments, the THCAS exhibits specificity for CBDVA substrates.
In some embodiments, a THCAS may use cannabiphorol acid (CBDP) as a
substrate. In some embodiments, the THCAS exhibits specificity for
CBDP substrates.
[0269] In some embodiments, a THCAS is from C. sativa. C. sativa
THCAS performs the oxidative cyclization of the geranyl moiety of
Cannabigerolic Acid (CBGA) (FIG. 4 Structure 8.alpha.) to form
Tetrahydrocannabinolic Acid (FIG. 4 Structure 10.alpha.) using
covalently bound flavin adenine dinucleotide (FAD) as a cofactor
and molecular oxygen as the final electron acceptor. THCAS was
first discovered and characterized by Taura et al. (JACS. 1995)
following extraction of the enzyme from the leaf buds of C. sativa
and confirmation of its THCA synthase activity in vitro upon the
addition of CBGA as a substrate. Additional analysis indicated that
the enzyme is a monomer and possesses FAD binding and Berberine
Bridge Enzyme (BBE) sequence motifs. A crystal structure of the
enzyme published by Shoyama et al. (J Mol Biol. 2012 Oct. 12;
423(1):96-105) revealed that the enzyme covalently binds to a
molecule of the cofactor FAD. See also, e.g., Sirikantarams et al.,
J. Biol. Chem. 2004 Sep. 17; 279(38):39767-39774. There are several
THCAS isozymes in Cannabis sativa.
[0270] In some embodiments, a C. sativa THCAS (Uniprot KB Accession
No.: I1V0C5) comprises the amino acid sequence shown below:
TABLE-US-00011 (SEQ ID NO: 135)
MNCSAFSFWFVCKIIFFFLSFNIQISIANPQENFLKCFSEYIPNNPANP
KFIYTQHDQLYMSVLNSTIQNLRFTSDTTPKPLVIVTPSNVSHIQASIL
CSKKVGLQIRTRSGGHDAEGMSYISQVPFVVVDLRNMHSIKIDVHSQTA
WVEAGATLGEVYYWINEKNENFSFPGGYCPTVGVGGHFSGGGYGALMRN
YGLAADNIIDAHLVNVDGKVLDRKSMGEDLFWAIRGGGGENFGIIAAWK
IKLVAVPSKSTIFSVKKNMEIHGLVKLFNKWQNIAYKYDKDLVLMTHFI
TKNITDNHGKNKTTVHGYFSSIFHGGVDSLVDLMNKSFPELGIKKTDCK
EFSWIDTTIFYSGVVNFNTANFKKEILLDRSAGKKTAFSIKLDYVKKPI
PETAMVKILEKLYEEDVGVGMYVLYPYGGIMEEISESAIPFPHRAGIMY
ELWYTASWEKQEDNEKHINWVRSVYNFTTPYVSQNPRLAYLNYRDLDLG
KTNPESPNNYTQARIWGEKYFGKNFNRLVKVKTKADPNNFFRNEQSIPP LPPHHH.
[0271] In some embodiments, a THCAS comprises the sequence shown
below:
TABLE-US-00012 (SEQ ID NO: 136)
NPQENFLKCFSEYIPNNPANPKFIYTQHDQLYMSVLNSTIQNLRFTSDT
TPKPLVIVTPSNVSHIQASILCSKKVGLQIRTRSGGHDAEGMSYISQVP
FVVVDLRNMHSIKIDVHSQTAWVEAGATLGEVYYWINEKNENFSFPGGY
CPTVGVGGHFSGGGYGALMRNYGLAADNIIDAHLVNVDGKVLDRKSMGE
DLFWAIRGGGGENFGIIAAWKIKLVAVPSKSTIFSVKKNMEIHGLVKLF
NKWQNIAYKYDKDLVLMTHFITKNITDNHGKNKTTVHGYFSSIFHGGVD
SLVDLMNKSFPELGIKKTDCKEFSWIDTTIFYSGVVNFNTANFKKEILL
DRSAGKKTAFSIKLDYVKKPIPETAMVKILEKLYEEDVGVGMYVLYPYG
GEVIEEISESAIPFPHRAGEVIYELWYTASWEKQEDNEKHINWVRSVYN
FTTPYVSQNPRLAYLNYRDLDLGKTNPESPNNYTQARIWGEKYFGKNFN
RLVKVKTKADPNNFFRNEQSIPPLPPHHH.
[0272] A non-limiting example of a nucleotide sequence encoding SEQ
ID NO: 136 is:
TABLE-US-00013 (SEQ ID NO: 137)
aacccgcaagaaaactttctaaaatgcttttctgaatacattcctaaca
accctgccaacccgaagtttatctacacacaacacgatcaattgtatat
gagcgtgttgaatagtacaatacagaacctgaggtttacatccgacaca
acgccgaaaccgctagtgatcgtcacaccctccaacgtaagccacattc
aggcaagcattttatgcagcaagaaagtcggactgcagataaggacgag
gtccggaggacacgacgccgaagggatgagctatatctcccaggtacct
tttgtggtggtagacttgagaaatatgcactctatcaagatagacgttc
actcccaaaccgcttgggttgaggcgggagccacccttggtgaggtcta
ctactggatcaacgaaaagaatgaaaattttagctttcctgggggatat
tgcccaactgtaggtgttggcggccacttctcaggaggcggttatgggg
ccttgatgcgtaactacggacttgcggccgacaacattatagacgcaca
tctagtgaatgtagacggcaaagttttagacaggaagagcatgggtgag
gatcttttttgggcaattagaggcggagggggagaaaattttggaatta
tcgctgcttggaaaattaagctagttgcggtaccgagcaaaagcactat
attctctgtaaaaaagaacatggagatacatggtttggtgaagcttttt
aataagtggcaaaacatcgcgtacaagtacgacaaagatctggttctga
tgacgcattttataacgaaaaatatcaccgacaaccacggaaaaaacaa
aaccacagtacatggctacttctctagtatatttcatgggggagtcgat
tctctggttgatttaatgaacaaatcattcccagagttgggtataaaga
agacagactgtaaggagttctcttggattgacacaactatattctattc
aggcgtagtcaactttaacacggcgaatttcaaaaaagagatccttctg
gacagatccgcaggtaagaaaactgcgttctctatcaaattggactatg
tgaagaagcctattcccgaaaccgcgatggtcaagatacttgagaaatt
atacgaggaagatgtgggagttggaatgtacgtactttatccctatggt
gggataatggaagaaatcagcgagagcgccattccatttccccatcgtg
ccggcatcatgtacgagctgtggtatactgcgagttgggagaagcaaga
agacaacgaaaagcacattaactgggtcagatcagtttacaatttcacc
accccatacgtgtcccagaatccgcgtctggcttacttgaactaccgtg
atcttgacctgggtaaaacgaacccggagtcacccaacaattacactca
agctagaatctggggagagaaatactttgggaagaacttcaacaggtta
gtaaaggttaaaaccaaggcagatccaaacaacttttttagaaatgaac
aatccattcccccgctacccccgcaccatcac.
[0273] In some embodiments, a C. sativa THCAS comprises the amino
acid sequence set forth in UniProtKB--Q8GTB6 (SEQ ID NO: 112):
TABLE-US-00014 MNCSAFSFWFVCKIIFFFLSFHIQISIANPRENFLKCFSKHIPNNVANP
KLVYTQHDQLYMSILNSTIQNLRFISDTTPKPLVIVTPSNNSHIQATIL
CSKKVGLQIRTRSGGHDAEGMSYISQVPFVVVDLRNMHSIKIDVHSQTA
WVEAGATLGEVYYWINEKNENLSFPGGYCPTVGVGGHFSGGGYGALMRN
YGLAADNIIDAHLVNVDGKVLDRKSMGEDLFWAIRGGGGENFGIIAAWK
IKLVAVPSKSTIFSVKKNMEIHGLVKLFNKWQNIAYKYDKDLVLMTHFI
TKNITDNHGKNKTTVHGYFSSIFHGGVDSLVDLMNKSFPELGIKKTDCK
EFSWIDTTIFYSGVVNFNTANFKKEILLDRSAGKKTAFSIKLDYVKKPI
PETAMVKILEKLYEEDVGAGMYVLYPYGGEVIEEISESAIPFPHRAGEV
IYELWYTASWEKQEDNEKHINWVRSVYNFTTPYVSQNPRLAYLNYRDLD
LGKTNHASPNNYTQARIWGEKYFGKNFNRLVKVKTKVDPNNFFRNEQSI PPLPPHHH.
[0274] Additional non-limiting examples of THCAS enzymes may also
be found in U.S. Pat. No. 9,512,391 and US Publication No.
2018/0179564, which are incorporated by reference in this
application in their entireties.
Cannabidiolic Acid Synthase (CBDAS)
[0275] A host cell described in this application may comprise a TS
that is a cannabidiolic acid synthase (CBDAS). As used in this
application, a "CBDAS" refers to an enzyme that is capable of
catalyzing oxidative cyclization of a prenyl moiety (e.g., terpene)
of a compound of Formula (8) to produce a compound of Formula 9. In
some embodiments, a compound of Formula 9 is a compound of Formula
(9a) (cannabidiolic acid (CBDA)), CBDVA, or CBDP. A CBDAS may use
cannabigerolic acid (CBGA) or cannabinerolic acid as a substrate.
In some embodiments, a cannabidiolic acid synthase is capable of
oxidative cyclization of cannabigerolic acid (CBGA) to produce
cannabidiolic acid (CBDA). In some embodiments, the CBDAS may
catalyze the oxidative cyclization of other substrates, such as
3-geranyl-2,4-dihydro-6-alkylbenzoic acids like cannabigerovarinic
acid (CBVGA). In some embodiments, the CBDAS exhibits specificity
for CBGA substrates.
[0276] In some embodiments, a CBDAS is from Cannabis. In C. sativa,
CBDAS is encoded by the CBDAS gene and is a flavoenzyme. A
non-limiting example of an amino acid sequence encoding CBDAS is
provided by UniProtKB--A6P6V9 (SEQ ID NO: 111) from C. sativa:
TABLE-US-00015 MKCSTFSFWFVCKIIFFFFSFNIQTSIANPRENFLKCFSQYIPNNATNL
KLVYTQNNPLYMSVLNSTIHNLRFTSDTTPKPLVIVTPSHVSHIQGTIL
CSKKVGLQIRTRSGGHDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTA
WVEAGATLGEVYYWVNEKNENLSLAAGYCPTVCAGGHFGGGGYGPLMRN
YGLAADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIVAWK
IRLVAVPKSTMFSVKKEVIEIHELVKLVNKWQNIAYKYDKDLLLMTHFI
TRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNKSFPELGIKKTDCR
QLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAGQNGAFKIKLDYVKKPI
PESVFVQILEKLYEEDIGAGMYALYPYGGIMDEISESAIPFPHRAGILY
ELWYICSWEKQEDNEKHLNWIRNIYNFMTPYVSKNPRLAYLNYRDLDIG
INDPKNPNNYTQARIWGEKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPP LPRHRH.
[0277] Additional non-limiting examples of CBDAS enzymes may also
be found in U.S. Pat. No. 9,512,391 and US Publication No.
2018/0179564, which are incorporated by reference in this
application in their entireties.
Cannabichromenic Acid Synthase (CBCAS)
[0278] A host cell described in this application may comprise a TS
that is a cannabichromenic acid synthase (CBCAS). As used in this
application, a "CBCAS" refers to an enzyme that is capable of
catalyzing oxidative cyclization of a prenyl moiety (e.g., terpene)
of a compound of Formula (8) to produce a compound of Formula (11).
In some embodiments, a compound of Formula (11) is a compound of
Formula (IIa) (cannabichromenic acid (CBCA)), CBCVA, or CBCPA. A
CBCAS may use cannabigerolic acid (CBGA) as a substrate. In some
embodiments, a CBCAS produces cannabichromenic acid (CBCA) from
cannabigerolic acid (CBGA). In some embodiments, the CBCAS may
catalyze the oxidative cyclization of other substrates, such as
3-geranyl-2,4-dihydro-6-alkylbenzoic acids like cannabigerovarinic
acid (CBVGA), or CBCPA. In some embodiments, the CBCAS exhibits
specificity for CBGA substrates.
[0279] In some embodiments, a CBCAS is from Cannabis. In C. sativa,
an amino acid sequence encoding CBCAS is provided by, and
incorporated by reference from, SEQ ID NO:2 disclosed in U.S.
Patent Publication No. 20170211049. In other embodiments, a CBCAS
may be a THCAS described in and incorporated by reference from U.S.
Pat. No. 9,359,625. SEQ ID NO:2 disclosed in U.S. Patent
Publication No. 20170211049 (corresponding to SEQ ID NO: 113 in
this application) has the amino acid sequence:
TABLE-US-00016 MNCSTFSFWFVCKIIFFFLSFNIQISIANPQENFLKCFSEYIPNNPANP
KFIYTQHDQLYMSVLNSTIQNLRFTSDTTPKPLVIVTPSNVSHIQASIL
CSKKVGLQIRTRSGGHDAEGLSYISQVPFAIVDLRNMHTVKVDIHSQTA
WVEAGATLGEVYYWINEMNENFSFPGGYCPTVGVGGHFSGGGYGALMRN
YGLAADNIIDAHLVNVDGKVLDRKSMGEDLFWAIRGGGGENFGIIAACK
IKLVVVPSKATIFSVKKNMEIHGLVKLFNKWQNIAYKYDKDLMLTTHFR
TRNITDNHGKNKTTVHGYFSSIFLGGVDSLVDLMNKSFPELGIKKTDCK
ELSWIDTTIFYSGVVNYNTANFKKEILLDRSAGKKTAFSIKLDYVKKLI
PETAMVKILEKLYEEEVGVGMYVLYPYGGIMDEISESAIPFPHRAGEVI
YELWYTATWEKQEDNEKHINWVRSVYNFTTPYVSQNPRLAYLNYRDLDL
GKTNPESPNNYTQARIWGEKYFGKNFNRLVKVKTKADPNNFFRNEQSIP PLPPRHH.
[0280] Any of the enzymes, host cells, and methods described in
this application may be used for the production of cannabinoids and
cannabinoid precursors, such as those provided in Table 1. In
general, the term "production" is used to refer to the generation
of one or more products (e.g., products of interest and/or
by-products/off-products), for example, from a particular substrate
or reactant. The amount of production may be evaluated at any one
or more steps of a pathway, such as a final product or an
intermediate product, using metrics familiar to one of ordinary
skill in the art. For example, the amount of production may be
assessed for a single enzymatic reaction (e.g., conversion of a
compound of Formula (8) to a compound of Formula (10) by a TS).
Alternatively or in addition, the amount of production may be
assessed for a series of enzymatic reactions (e.g., the
biosynthetic pathway shown in FIG. 1 and/or FIG. 2). Production may
be assessed by any metrics known in the art, for example, by
assessing volumetric productivity, enzyme kinetics/reaction rate,
specific productivity biomass-specific productivity, titer, yield,
and total titer of one or more products (e.g., products of interest
and/or by-products/off-products).
[0281] In some embodiments, the metric used to measure production
may depend on whether a continuous process is being monitored
(e.g., several cannabinoid biosynthesis steps are used in
combination) or whether a particular end product is being measured.
For example, in some embodiments, metrics used to monitor
production by a continuous process may include volumetric
productivity, enzyme kinetics and reaction rate. In some
embodiments, metrics used to monitor production of a particular
product may include specific productivity, biomass-specific
productivity, titer, yield, and/or total titer of one or more
products (e.g., products of interest and/or
by-products/off-products).
[0282] Production of one or more products (e.g., products of
interest and/or by-products/off-products) may be assessed
indirectly, for example by determining the amount of a substrate
remaining following termination of the reaction/fermentation. For
example, for a TS that catalyzes the formation of products (e.g., a
compound of Formula (10), including tetrahydrocannabinolic acid
(THCA) (Formula (10.alpha.)) from a compound of Formula (8),
including CBGA (Formula 8(a))), production of the products may be
assessed by quantifying the compound of Formula (10) directly or by
quantifying the amount of substrate remaining following the
reaction (e.g., amount of the compound of Formula (8)).
Variants
[0283] Aspects of the disclosure relate to nucleic acids encoding
any of the polypeptides (e.g., AAE, PKS, PKC, PT, or TS) described
in this application. In some embodiments, a nucleic acid
encompassed by the disclosure is a nucleic acid that hybridizes
under high or medium stringency conditions to a nucleic acid
encoding an AAE, PKS, PKC, PT, or TS and is biologically active.
For example, high stringency conditions of 0.2 to 1.times.SSC at
65.degree. C. followed by a wash at 0.2.times.SSC at 65.degree. C.
can be used. In some embodiments, a nucleic acid encompassed by the
disclosure is a nucleic acid that hybridizes under low stringency
conditions to a nucleic acid encoding an AAE, PKS, PKC, PT, or TS
and is biologically active. For example, low stringency conditions
of 6.times.SSC at room temperature followed by a wash at
2.times.SSC at room temperature can be used. Other hybridization
conditions include 3.times.SSC at 40 or 50.degree. C., followed by
a wash in 1 or 2.times.SSC at 20, 30, 40, 50, 60, or 65.degree.
C.
[0284] Hybridizations can be conducted in the presence of
formaldehyde, e.g., 10%, 20%, 30% 40% or 50%, which further
increases the stringency of hybridization. Theory and practice of
nucleic acid hybridization is described, e.g., in S. Agrawal (ed.)
Methods in Molecular Biology, volume 20; and Tijssen (1993)
Laboratory Techniques in biochemistry and molecular
biology-hybridization with nucleic acid probes, e.g., part I
chapter 2 "Overview of principles of hybridization and the strategy
of nucleic acid probe assays," Elsevier, New York provide a basic
guide to nucleic acid hybridization.
[0285] Variants of enzyme sequences described in this application
(e.g., AAE, PKS, PKC, PT, or TS, including nucleic acid or amino
acid sequences) are also encompassed by the present disclosure. A
variant may share at least 5%, at least 10%, at least 15%, at least
20%, at least 25%, at least 30%, at least 35%, at least 40%, at
least 45%, at least 50%, at least 55%, at least 60%, at least 65%,
at least 70%, at least 71%, at least 72%, at least 73%, at least
74%, at least 75%, at least 76%, at least 77%, at least 78%, at
least 79%, at least 80%, at least 81%, at least 82%, at least 83%,
at least 84%, at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, at least 99%, or 100% sequence identity with a
reference sequence, including all values in between.
[0286] Unless otherwise noted, the term "sequence identity," as
known in the art, refers to a relationship between the sequences of
two polypeptides or polynucleotides, as determined by sequence
comparison (alignment). In some embodiments, sequence identity is
determined across the entire length of a sequence (e.g., AAE, PKS,
PKC, PT, or TS sequence). In some embodiments, sequence identity is
determined over a region (e.g., a stretch of amino acids or nucleic
acids, e.g., the sequence spanning an active site) of a sequence
(e.g., AAE, PKS, PKC, PT, or TS sequence).
[0287] Identity can also refer to the degree of sequence
relatedness between two sequences as determined by the number of
matches between strings of two or more residues (e.g., nucleic acid
or amino acid residues). Identity measures the percent of identical
matches between the smaller of two or more sequences with gap
alignments (if any) addressed by a particular mathematical model,
algorithms, or computer program.
[0288] Identity of related polypeptides or nucleic acid sequences
can be readily calculated by any of the methods known to one of
ordinary skill in the art. The "percent identity" of two sequences
(e.g., nucleic acid or amino acid sequences) may, for example, be
determined using the algorithm of Karlin and Altschul Proc. Natl.
Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul
Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is
incorporated into the NBLAST.RTM. and XBLAST.RTM. programs (version
2.0) of Altschul et al., J. Mol. Biol. 215:403-10, 1990. BLAST.RTM.
protein searches can be performed, for example, with the XBLAST
program, score=50, wordlength=3 to obtain amino acid sequences
homologous to the proteins described in this application. Where
gaps exist between two sequences, Gapped BLAST.RTM. can be
utilized, for example, as described in Altschul et al., Nucleic
Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST.RTM. and
Gapped BLAST.RTM. programs, the default parameters of the
respective programs (e.g., XBLAST.RTM. and NBLAST.RTM.) can be
used, or the parameters can be adjusted appropriately as would be
understood by one of ordinary skill in the art.
[0289] Another local alignment technique which may be used, for
example, is based on the Smith-Waterman algorithm (Smith, T. F.
& Waterman, M. S. (1981) "Identification of common molecular
subsequences." J. Mol. Biol. 147:195-197). A general global
alignment technique which may be used, for example, is the
Needleman-Wunsch algorithm (Needleman, S. B. & Wunsch, C. D.
(1970) "A general method applicable to the search for similarities
in the amino acid sequences of two proteins." J. Mol. Biol.
48:443-453), which is based on dynamic programming.
[0290] More recently, a Fast Optimal Global Sequence Alignment
Algorithm (FOGSAA) was developed that purportedly produces global
alignment of nucleic acid and amino acid sequences faster than
other optimal global alignment methods, including the
Needleman-Wunsch algorithm. In some embodiments, the identity of
two polypeptides is determined by aligning the two amino acid
sequences, calculating the number of identical amino acids, and
dividing by the length of one of the amino acid sequences. In some
embodiments, the identity of two nucleic acids is determined by
aligning the two nucleotide sequences and calculating the number of
identical nucleotide and dividing by the length of one of the
nucleic acids.
[0291] For multiple sequence alignments, computer programs
including Clustal Omega (Sievers et al., Mol Syst Biol. 2011 Oct.
11; 7:539) may be used.
[0292] It should be appreciated that a sequence, including a
nucleic acid or amino acid sequence, may be found to have a
specified percent identity to a reference sequence, such as a
sequence disclosed in this application and/or recited in the
claims, using any method known to one of ordinary skill in the art.
Different algorithms may yield different percent identity values
for a given set of sequences. The claims of this application should
be understood to encompass sequences for which percent identity to
a reference sequence is calculated using default parameters and/or
parameters typically used by the skilled artisan for a given
algorithm.
[0293] In some embodiments, a sequence, including a nucleic acid or
amino acid sequence, is found to have a specified percent identity
to a reference sequence, such as a sequence disclosed in this
application and/or recited in the claims when sequence identity is
determined using the algorithm of Karlin and Altschul Proc. Natl.
Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul
Proc. Natl. Acad. Sci. USA 90:5873-77, 1993 (e.g., BLAST.RTM.,
NBLAST.RTM., XBLAST.RTM. or Gapped BLAST.RTM. programs, using
default parameters of the respective programs).
[0294] In some embodiments, a sequence, including a nucleic acid or
amino acid sequence, is found to have a specified percent identity
to a reference sequence, such as a sequence disclosed in this
application and/or recited in the claims when sequence identity is
determined using the Smith-Waterman algorithm (Smith, T. F. &
Waterman, M. S. (1981) "Identification of common molecular
subsequences." J. Mol. Biol. 147:195-197) or the Needleman-Wunsch
algorithm (Needleman, S. B. & Wunsch, C. D. (1970) "A general
method applicable to the search for similarities in the amino acid
sequences of two proteins." J. Mol. Biol. 48:443-453).
[0295] In some embodiments, a sequence, including a nucleic acid or
amino acid sequence, is found to have a specified percent identity
to a reference sequence, such as a sequence disclosed in this
application and/or recited in the claims when sequence identity is
determined using a Fast Optimal Global Sequence Alignment Algorithm
(FOGSAA).
[0296] In some embodiments, a sequence, including a nucleic acid or
amino acid sequence, is found to have a specified percent identity
to a reference sequence, such as a sequence disclosed in this
application and/or recited in the claims when sequence identity is
determined using Clustal Omega (Sievers et al., Mol Syst Biol. 2011
Oct. 11; 7:539).
[0297] As used in this application, a residue (such as a nucleic
acid residue or an amino acid residue) in sequence "X" is referred
to as corresponding to a position or residue (such as a nucleic
acid residue or an amino acid residue) "Z" in a different sequence
"Y" when the residue in sequence "X" is at the counterpart position
of "Z" in sequence "Y" when sequences X and Y are aligned using
amino acid sequence alignment tools known in the art.
[0298] As used in this application, variant sequences may be
homologous sequences. As used in this application, homologous
sequences are sequences (e.g., nucleic acid or amino acid
sequences) that share a certain percent identity (e.g., at least
5%, at least 10%, at least 15%, at least 20%, at least 25%, at
least 30%, at least 35%, at least 40%, at least 45%, at least 50%,
at least 55%, at least 60%, at least 65%, at least 70%, at least
71%, at least 72%, at least 73%, at least 74%, at least 75%, at
least 76%, at least 77%, at least 78%, at least 79%, at least 80%,
at least 81%, at least 82%, at least 83%, at least 84%, at least
85%, at least 86%, at least 87%, at least 88%, at least 89%, at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, at least
99%, or 100% percent identity, including all values in between).
Homologous sequences include but are not limited to paralogous or
orthologous sequences. Paralogous sequences arise from duplication
of a gene within a genome of a species, while orthologous sequences
diverge after a speciation event.
[0299] In some embodiments, a polypeptide variant (e.g., AAE, PKS,
PKC, PT, or TS enzyme variant) comprises a domain that shares a
secondary structure (e.g., alpha helix, beta sheet) with a
reference polypeptide (e.g., a reference AAE, PKS, PKC, PT, or TS
enzyme). In some embodiments, a polypeptide variant (e.g., AAE,
PKS, PKC, PT, or TS enzyme variant) shares a tertiary structure
with a reference polypeptide (e.g., a reference AAE, PKS, PKC, PT,
or TS enzyme). As a non-limiting example, a polypeptide variant
(e.g., AAE, PKS, PKC, PT, or TS enzyme) may have low primary
sequence identity (e.g., less than 80%, less than 75%, less than
70%, less than 65%, less than 60%, less than 55%, less than 50%,
less than 45%, less than 40%, less than 35%, less than 30%, less
than 25%, less than 20%, less than 15%, less than 10%, or less than
5% sequence identity) compared to a reference polypeptide, but
share one or more secondary structures (e.g., including but not
limited to loops, alpha helices, or beta sheets), or have the same
tertiary structure as a reference polypeptide. For example, a loop
may be located between a beta sheet and an alpha helix, between two
alpha helices, or between two beta sheets. Homology modeling may be
used to compare two or more tertiary structures.
[0300] Functional variants of the recombinant AAE, PKS, PKC, PT, or
TS enzyme disclosed in this application are encompassed by the
present disclosure. For example, functional variants may bind one
or more of the same substrates or produce one or more of the same
products. Functional variants may be identified using any method
known in the art. For example, the algorithm of Karlin and Altschul
Proc. Natl. Acad. Sci. USA 87:2264-68, 1990 described above may be
used to identify homologous proteins with known functions.
[0301] Putative functional variants may also be identified by
searching for polypeptides with functionally annotated domains.
Databases including Pfam (Sonnhammer et al., Proteins. 1997 July;
28(3):405-20) may be used to identify polypeptides with a
particular domain.
[0302] Homology modeling may also be used to identify amino acid
residues that are amenable to mutation (e.g., substitution,
deletion, and/or insertion) without affecting function. A
non-limiting example of such a method may include use of
position-specific scoring matrix (PSSM) and an energy minimization
protocol.
[0303] Position-specific scoring matrix (PSSM) uses a position
weight matrix to identify consensus sequences (e.g., motifs). PSSM
can be conducted on nucleic acid or amino acid sequences. Sequences
are aligned and the method takes into account the observed
frequency of a particular residue (e.g., an amino acid or a
nucleotide) at a particular position and the number of sequences
analyzed. See, e.g., Stormo et al., Nucleic Acids Res. 1982 May 11;
10(9):2997-3011. The likelihood of observing a particular residue
at a given position can be calculated. Without being bound by a
particular theory, positions in sequences with high variability may
be amenable to mutation (e.g., substitution, deletion, and/or
insertion; e.g., PSSM score .gtoreq.0) to produce functional
homologs.
[0304] PSSM may be paired with calculation of a Rosetta energy
function, which determines the difference between the wild-type and
the single-point mutant. The Rosetta energy function calculates
this difference as (.DELTA..DELTA.G.sub.calc). With the Rosetta
function, the bonding interactions between a mutated residue and
the surrounding atoms are used to determine whether an amino acid
substitution, deletion, or insertion increases or decreases protein
stability. For example, an amino acid substitution, deletion, or
insertion that is designated as favorable by the PSSM score (e.g.
PSSM score .gtoreq.0), can then be analyzed using the Rosetta
energy function to determine the potential impact of the amino acid
substitution, deletion, or insertion on protein stability. Without
being bound by a particular theory, potentially stabilizing amino
acid substitutions, deletions, or insertions are desirable for
protein engineering (e.g., production of functional homologs). In
some embodiments, a potentially stabilizing amino acid
substitution, deletion, or insertion has a .DELTA..DELTA.G.sub.calc
value of less than -0.1 (e.g., less than -0.2, less than -0.3, less
than -0.35, less than -0.4, less than -0.45, less than -0.5, less
than -0.55, less than -0.6, less than -0.65, less than -0.7, less
than -0.75, less than -0.8, less than -0.85, less than -0.9, less
than -0.95, or less than -1.0) Rosetta energy units (R.e.u.). See,
e.g., Goldenzweig et al., Mol Cell. 2016 Jul. 21; 63(2):337-346.
Doi: 10.1016/j.molcel.2016.06.012.
[0305] In some embodiments, an AAE, PKS, PKC, PT, or TS enzyme
coding sequence comprises an amino acid substitution, deletion,
and/or insertion at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,
49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,
66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,
83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
100 or more than 100 positions corresponding to a reference (e.g.,
AAE, PKS, PKC, PT, or TS enzyme) coding sequence. In some
embodiments, the AAE, PKS, PKC, PT, or TS enzyme coding sequence
comprises an amino acid substitution, deletion, and/or insertion in
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,
37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,
54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,
71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,
88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more codons
of the coding sequence relative to a reference (e.g., AAE, PKS,
PKC, PT, or TS enzyme) coding sequence. As will be understood by
one of ordinary skill in the art, a substitution, insertion, or
deletion within a codon may or may not change the amino acid that
is encoded by the codon due to degeneracy of the genetic code. In
some embodiments, the one or more substitutions, insertions, or
deletions in the coding sequence do not alter the amino acid
sequence of the coding sequence (e.g., AAE, PKS, PKC, PT, or TS
enzyme) relative to the amino acid sequence of a reference
polypeptide (e.g., AAE, PKS, PKC, PT, or TS enzyme).
[0306] In some embodiments, the one or more substitutions,
deletions, and/or insertions in a recombinant AAE, PKS, PKC, PT, or
TS enzyme sequence alters the amino acid sequence of the
polypeptide (e.g., AAE, PKS, PKC, PT, or TS enzyme) relative to the
amino acid sequence of a reference polypeptide (e.g., AAE, PKS,
PKC, PT, or TS enzyme). In some embodiments, the one or more
substitutions, insertions, or deletions alters the amino acid
sequence of the recombinant polypeptide (e.g., AAE, PKS, PKC, PT,
or TS enzyme) relative to the amino acid sequence of a reference
polypeptide (e.g., AAE, PKS, PKC, PT, or TS enzyme) and alters
(enhances or reduces) an activity of the polypeptide relative to
the reference polypeptide.
[0307] The activity (e.g., specific activity) of any of the
recombinant polypeptides described in this application (e.g., AAE,
PKS, PKC, PT, or TS enzyme) may be measured using routine methods.
As a non-limiting example, a recombinant polypeptide's activity may
be determined by measuring its substrate specificity, product(s)
produced, the concentration of product(s) produced, or any
combination thereof. As used in this application, "specific
activity" of a recombinant polypeptide refers to the amount (e.g.,
concentration) of a particular product produced for a given amount
(e.g., concentration) of the recombinant polypeptide per unit
time.
[0308] The skilled artisan will also realize that insertions,
substitutions, or deletions in a recombinant polypeptide (e.g.,
AAE, PKS, PKC, PT, or TS enzyme) coding sequence may result in
conservative amino acid substitutions to provide functionally
equivalent variants of the foregoing polypeptides, e.g., variants
that retain the activities of the polypeptides. As used in this
application, a "conservative amino acid substitution" refers to an
amino acid substitution that does not alter the relative charge or
size characteristics or functional activity of the protein in which
the amino acid substitution is made.
[0309] In some instances, an amino acid is characterized by its R
group (see, e.g., Table 3). For example, an amino acid may comprise
a nonpolar aliphatic R group, a positively charged R group, a
negatively charged R group, a nonpolar aromatic R group, or a polar
uncharged R group. Non-limiting examples of an amino acid
comprising a nonpolar aliphatic R group include alanine, glycine,
valine, leucine, methionine, and isoleucine. Non-limiting examples
of an amino acid comprising a positively charged R group includes
lysine, arginine, and histidine. Non-limiting examples of an amino
acid comprising a negatively charged R group include aspartate and
glutamate. Non-limiting examples of an amino acid comprising a
nonpolar, aromatic R group include phenylalanine, tyrosine, and
tryptophan. Non-limiting examples of an amino acid comprising a
polar uncharged R group include serine, threonine, cysteine,
proline, asparagine, and glutamine.
[0310] Non-limiting examples of functionally equivalent variants of
polypeptides may include conservative amino acid substitutions in
the amino acid sequences of proteins disclosed in this application.
As used in this application "conservative substitution" is used
interchangeably with "conservative amino acid substitution" and
refers to any one of the amino acid substitutions provided in Table
2.
[0311] In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20 or more than 20 residues can be
changed when preparing variant polypeptides. In some embodiments,
amino acids are replaced by conservative amino acid
substitutions.
TABLE-US-00017 TABLE 2 Conservative Amino Acid Substitutions
Conservative Original Amino Acid Residue R Group Type Substitutions
Ala nonpolar aliphatic R group Cys, Gly, Ser Arg positively charged
R group His, Lys Asn polar uncharged R group Asp, Gln, Glu Asp
negatively charged R group Asn, Gln, Glu Cys polar uncharged R
group Ala, Ser Gln polar uncharged R group Asn, Asp, Glu Glu
negatively charged R group Asn, Asp, Gln Gly nonpolar aliphatic R
group Ala, Ser His positively charged R group Arg, Tyr, Trp Ile
nonpolar aliphatic R group Leu, Met, Val Leu nonpolar aliphatic R
group Ile, Met, Val Lys positively charged R group Arg, His Met
nonpolar aliphatic R group Ile, Leu, Phe, Val Pro polar uncharged R
group Phe nonpolar aromatic R group Met, Trp, Tyr Ser polar
uncharged R group Ala, Gly, Thr Thr polar uncharged R group Ala,
Asn, Ser Trp nonpolar aromatic R group His, Phe, Tyr, Met Tyr
nonpolar aromatic R group His, Phe, Trp Val nonpolar aliphatic R
group Ile, Leu, Met, Thr
[0312] Amino acid substitutions in the amino acid sequence of a
polypeptide to produce a recombinant polypeptide (e.g., AAE, PKS,
PKC, PT, or TS enzyme) variant having a desired property and/or
activity can be made by alteration of the coding sequence of the
polypeptide (e.g., AAE, PKS, PKC, PT, or TS enzyme). Similarly,
conservative amino acid substitutions in the amino acid sequence of
a polypeptide to produce functionally equivalent variants of the
polypeptide typically are made by alteration of the coding sequence
of the recombinant polypeptide (e.g., AAE, PKS, PKC, PT, or TS
enzyme).
[0313] Mutations (e.g., substitutions, insertions, additions, or
deletions) can be made in a nucleic acid sequence by a variety of
methods known to one of ordinary skill in the art. For example,
mutations (e.g., substitutions, insertions, additions, or
deletions) can be made by PCR-directed mutation, site-directed
mutagenesis according to the method of Kunkel (Kunkel, Proc. Nat.
Acad. Sci. U.S.A. 82: 488-492, 1985), by chemical synthesis of a
gene encoding a polypeptide, by CRISPR, or by insertions, such as
insertion of a tag (e.g., a HIS tag or a GFP tag). Mutations can
include, for example, substitutions, insertions, additions,
deletions, and translocations, generated by any method known in the
art. Methods for producing mutations may be found in in references
such as Molecular Cloning: A Laboratory Manual, J. Sambrook, et
al., eds., Fourth Edition, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y., 2012, or Current Protocols in Molecular
Biology, F. M. Ausubel, et al., eds., John Wiley & Sons, Inc.,
New York, 2010.
[0314] In some embodiments, methods for producing variants include
circular permutation (Yu and Lutz, Trends Biotechnol. 2011 January;
29(1):18-25). In circular permutation, the linear primary sequence
of a polypeptide can be circularized (e.g., by joining the
N-terminal and C-terminal ends of the sequence) and the polypeptide
can be severed ("broken") at a different location. Thus, the linear
primary sequence of the new polypeptide may have low sequence
identity (e.g., less than 80%, less than 75%, less than 70%, less
than 65%, less than 60%, less than 55%, less than 50%, less than
45%, less than 40%, less than 35%, less than 30%, less than 25%,
less than 20%, less than 15%, less than 10%, less or less than 5%,
including all values in between) as determined by linear sequence
alignment methods (e.g., Clustal Omega or BLAST). Topological
analysis of the two proteins, however, may reveal that the tertiary
structure of the two polypeptides is similar or dissimilar. Without
being bound by a particular theory, a variant polypeptide created
through circular permutation of a reference polypeptide and with a
similar tertiary structure as the reference polypeptide can share
similar functional characteristics (e.g., enzymatic activity,
enzyme kinetics, substrate specificity or product specificity). In
some instances, circular permutation may alter the secondary
structure, tertiary structure or quaternary structure and produce
an enzyme with different functional characteristics (e.g.,
increased or decreased enzymatic activity, different substrate
specificity, or different product specificity). See, e.g., Yu and
Lutz, Trends Biotechnol. 2011 January; 29(1):18-25.
[0315] It should be appreciated that in a protein that has
undergone circular permutation, the linear amino acid sequence of
the protein would differ from a reference protein that has not
undergone circular permutation. However, one of ordinary skill in
the art would be able to determine which residues in the protein
that has undergone circular permutation correspond to residues in
the reference protein that has not undergone circular permutation
by, for example, aligning the sequences and detecting conserved
motifs, and/or by comparing the structures or predicted structures
of the proteins, e.g., by homology modeling.
[0316] In some embodiments, an algorithm that determines the
percent identity between a sequence of interest and a reference
sequence described in this application accounts for the presence of
circular permutation between the sequences. The presence of
circular permutation may be detected using any method known in the
art, including, for example, RASPODOM (Weiner et al.,
Bioinformatics. 2005 Apr. 1; 21(7):932-7). In some embodiments, the
presence of circulation permutation is corrected for (e.g., the
domains in at least one sequence are rearranged) prior to
calculation of the percent identity between a sequence of interest
and a sequence described in this application. The claims of this
application should be understood to encompass sequences for which
percent identity to a reference sequence is calculated after taking
into account potential circular permutation of the sequence.
Expression of Nucleic Acids in Host Cells
[0317] Aspects of the present disclosure relate to recombinant
enzymes, functional modifications and variants thereof, as well as
their uses. For example, the methods described in this application
may be used to produce cannabinoids and/or cannabinoid precursors.
The methods may comprise using a host cell comprising an enzyme
disclosed in this application, cell lysate, isolated enzymes, or
any combination thereof. Methods comprising recombinant expression
of genes encoding an enzyme disclosed in this application in a host
cell are encompassed by the present disclosure. In vitro methods
comprising reacting one or more cannabinoid precursors or
cannabinoids in a reaction mixture with an enzyme disclosed in this
application are also encompassed by the present disclosure. In some
embodiments, the enzyme is a TS.
[0318] A nucleic acid encoding any of the recombinant polypeptides
(e.g., AAE, PKS, PKC, PT, or TS enzyme) described in this
application may be incorporated into any appropriate vector through
any method known in the art. For example, the vector may be an
expression vector, including but not limited to a viral vector
(e.g., a lentiviral, retroviral, adenoviral, or adeno-associated
viral vector), any vector suitable for transient expression, any
vector suitable for constitutive expression, or any vector suitable
for inducible expression (e.g., a galactose-inducible or
doxycycline-inducible vector).
[0319] A vector encoding any of the recombinant polypeptides (e.g.,
AAE, PKS, PKC, PT, or TS enzyme) described in this application may
be introduced into a suitable host cell using any method known in
the art. Non-limiting examples of yeast transformation protocols
are described in Gietz et al., Yeast transformation can be
conducted by the LiAc/SS Carrier DNA/PEG method. Methods Mol Biol.
2006; 313:107-20, which is hereby incorporated by reference in its
entirety. Host cells may be cultured under any conditions suitable
as would be understood by one of ordinary skill in the art. For
example, any media, temperature, and incubation conditions known in
the art may be used. For host cells carrying an inducible vector,
cells may be cultured with an appropriate inducible agent to
promote expression.
[0320] In some embodiments, a vector replicates autonomously in the
cell. In some embodiments, a vector integrates into a chromosome
within a cell. A vector can contain one or more endonuclease
restriction sites that are cut by a restriction endonuclease to
insert and ligate a nucleic acid containing a gene described in
this application to produce a recombinant vector that is able to
replicate in a cell. Vectors are typically composed of DNA,
although RNA vectors are also available. Cloning vectors include,
but are not limited to: plasmids, fosmids, phagemids, virus genomes
and artificial chromosomes. As used in this application, the terms
"expression vector" or "expression construct" refer to a nucleic
acid construct, generated recombinantly or synthetically, with a
series of specified nucleic acid elements that permit transcription
of a particular nucleic acid in a host cell (e.g., microbe), such
as a yeast cell. In some embodiments, the nucleic acid sequence of
a gene described in this application is inserted into a cloning
vector so that it is operably joined to regulatory sequences and,
in some embodiments, expressed as an RNA transcript. In some
embodiments, the vector contains one or more markers, such as a
selectable marker as described in this application, to identify
cells transformed or transfected with the recombinant vector. In
some embodiments, a host cell has already been transformed with one
or more vectors. In some embodiments, a host cell that has been
transformed with one or more vectors is subsequently transformed
with one or more vectors. In some embodiments, a host cell is
transformed simultaneously with more than one vector. In some
embodiments, the nucleic acid sequence of a gene described in this
application is recoded. Recoding may increase production of the
gene product by at least 10%, at least 15%, at least 20%, at least
25%, at least 30%, at least 35%, at least 40%, at least 45%, at
least 50%, at least 55%, at least 60%, at least 65%, at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, at least
95%, or 100%, including all values in between) relative to a
reference sequence that is not recoded.
[0321] In some embodiments, the nucleic acid encoding any of the
proteins described in this application is under the control of
regulatory sequences (e.g., enhancer sequences). In some
embodiments, a nucleic acid is expressed under the control of a
promoter. The promoter can be a native promoter, e.g., the promoter
of the gene in its endogenous context, which provides normal
regulation of expression of the gene. Alternatively, a promoter can
be a promoter that is different from the native promoter of the
gene, e.g., the promoter is different from the promoter of the gene
in its endogenous context.
[0322] In some embodiments, the promoter is a eukaryotic promoter.
Non-limiting examples of eukaryotic promoters include TDH3, PGK1,
PKC1, PDC1, TEF1, TEF2, RPL18B, SSA1, TDH2, PYK1, TPI1, GAL1,
GAL10, GAL7, GAL3, GAL2, MET3, MET25, HXT3, HXT7, ACT1, ADH1, ADH2,
CUP1-1, ENO2, and SOD1, as would be known to one of ordinary skill
in the art (see, e.g., Addgene website:
blog.addgene.org/plasmids-101-the-promoter-region). In some
embodiments, the promoter is a prokaryotic promoter (e.g.,
bacteriophage or bacterial promoter). Non-limiting examples of
bacteriophage promoters include Plslcon, T3, T7, SP6, and PL.
Non-limiting examples of bacterial promoters include Pbad, PmgrB,
Ptrc2, Plac/ara, Ptac, and Pm.
[0323] In some embodiments, the promoter is an inducible promoter.
As used in this application, an "inducible promoter" is a promoter
controlled by the presence or absence of a molecule. This may be
used, for example, to controllably induce the expression of an
enzyme. In some embodiments, an inducible promoter linked to a PT
and/or a TS may be used to regulate expression of the enzyme(s),
for example to reduce cannabinoid production in certain scenarios
(e.g., during transport of the genetically modified organism to
satisfy regulatory restrictions in certain jurisdictions, or
between jurisdictions where cannabinoids may not be shipped). In
some embodiments, an inducible promoter linked to a CBGAS and/or a
TS, the CBGAS and/or TS may be used to regulate expression of the
enzyme(s), for example to reduce cannabinoid production in certain
scenarios (e.g., during transport of the genetically modified
organism to satisfy regulatory restrictions in certain
jurisdictions, or between jurisdictions where cannabinoids may not
be shipped). Non-limiting examples of inducible promoters include
chemically regulated promoters and physically regulated promoters.
For chemically regulated promoters, the transcriptional activity
can be regulated by one or more compounds, such as alcohol,
tetracycline, galactose, a steroid, a metal, an amino acid, or
other compounds. For physically regulated promoters,
transcriptional activity can be regulated by a phenomenon such as
light or temperature. Non-limiting examples of
tetracycline-regulated promoters include anhydrotetracycline
(aTc)-responsive promoters and other tetracycline-responsive
promoter systems (e.g., a tetracycline repressor protein (tetR), a
tetracycline operator sequence (tetO) and a tetracycline
transactivator fusion protein (tTA)). Non-limiting examples of
steroid-regulated promoters include promoters based on the rat
glucocorticoid receptor, human estrogen receptor, moth ecdysone
receptors, and promoters from the steroid/retinoid/thyroid receptor
superfamily. Non-limiting examples of metal-regulated promoters
include promoters derived from metallothionein (proteins that bind
and sequester metal ions) genes. Non-limiting examples of
pathogenesis-regulated promoters include promoters induced by
salicylic acid, ethylene or benzothiadiazole (BTH). Non-limiting
examples of temperature/heat-inducible promoters include heat shock
promoters. Non-limiting examples of light-regulated promoters
include light responsive promoters from plant cells. In certain
embodiments, the inducible promoter is a galactose-inducible
promoter. In some embodiments, the inducible promoter is induced by
one or more physiological conditions (e.g., pH, temperature,
radiation, osmotic pressure, saline gradients, cell surface
binding, or concentration of one or more extrinsic or intrinsic
inducing agents). Non-limiting examples of an extrinsic inducer or
inducing agent include amino acids and amino acid analogs,
saccharides and polysaccharides, nucleic acids, protein
transcriptional activators and repressors, cytokines, toxins,
petroleum-based compounds, metal containing compounds, salts, ions,
enzyme substrate analogs, hormones or any combination.
[0324] In some embodiments, the promoter is a constitutive
promoter. As used in this application, a "constitutive promoter"
refers to an unregulated promoter that allows continuous
transcription of a gene. Non-limiting examples of a constitutive
promoter include TDH3, PGK1, PKC1, PDC1, TEF1, TEF2, RPL18B, SSA1,
TDH2, PYK1, TPI1, HXT3, HXT7, ACT1, ADH1, ADH2, ENO2, and SOD1.
[0325] Other inducible promoters or constitutive promoters,
including synthetic promoters, that may be known to one of ordinary
skill in the art are also contemplated.
[0326] The precise nature of the regulatory sequences needed for
gene expression may vary between species or cell types, but
generally include, as necessary, 5' non-transcribed and 5'
non-translated sequences involved with the initiation of
transcription and translation respectively, such as a TATA box,
capping sequence, CAAT sequence, and the like. In particular, such
5' non-transcribed regulatory sequences will include a promoter
region which includes a promoter sequence for transcriptional
control of the operably joined gene. Regulatory sequences may also
include enhancer sequences or upstream activator sequences. The
vectors disclosed may include 5' leader or signal sequences. The
regulatory sequence may also include a terminator sequence. In some
embodiments, a terminator sequence marks the end of a gene in DNA
during transcription. The choice and design of one or more
appropriate vectors suitable for inducing expression of one or more
genes described in this application in a heterologous organism is
within the ability and discretion of one of ordinary skill in the
art.
[0327] Expression vectors containing the necessary elements for
expression are commercially available and known to one of ordinary
skill in the art (see, e.g., Sambrook et al., Molecular Cloning: A
Laboratory Manual, Fourth Edition, Cold Spring Harbor Laboratory
Press, 2012).
Host Cells
[0328] The disclosed cannabinoid biosynthetic methods and host
cells are exemplified with S. cerevisiae, but are also applicable
to other host cells, as would be understood by one of ordinary
skill in the art.
[0329] Suitable host cells include, but are not limited to: yeast
cells, bacterial cells, algal cells, plant cells, fungal cells,
insect cells, and animal cells, including mammalian cells. In one
illustrative embodiment, suitable host cells include E. coli (e.g.,
Shuffle.TM. competent E. coli available from New England BioLabs in
Ipswich, Mass.).
[0330] Other suitable host cells of the present disclosure include
microorganisms of the genus Corynebacterium. In some embodiments,
preferred Corynebacterium strains/species include: C. efficiens,
with the deposited type strain being DSM44549, C. glutamicum, with
the deposited type strain being ATCC13032, and C. ammoniagenes,
with the deposited type strain being ATCC6871. In some embodiments
the preferred host cell of the present disclosure is C.
glutamicum.
[0331] Suitable host cells of the genus Corynebacterium, in
particular of the species Corynebacterium glutamicum, are in
particular the known wild-type strains: Corynebacterium glutamicum
ATCC13032, Corynebacterium acetoglutamicum ATCC15806,
Corynebacterium acetoacidophilum ATCC13870, Corynebacterium
melassecola ATCC17965, Corynebacterium thermoaminogenes FERM
BP-1539, Brevibacterium flavum ATCC14067, Brevibacterium
lactofermentum ATCC13869, and Brevibacterium divaricatum ATCC14020;
and L-amino acid-producing mutants, or strains, prepared therefrom,
such as, for example, the L-lysine-producing strains:
Corynebacterium glutamicum FERM-P 1709, Brevibacterium flavum
FERM-P 1708, Brevibacterium lactofermentum FERM-P 1712,
Corynebacterium glutamicum FERM-P 6463, Corynebacterium glutamicum
FERM-P 6464, Corynebacterium glutamicum DM58-1, Corynebacterium
glutamicum DG52-5, Corynebacterium glutamicum DSM5714, and
Corynebacterium glutamicum DSM12866.
[0332] Suitable yeast host cells include, but are not limited to:
Candida, Hansenula, Saccharomyces, Schizosaccharomyces, Pichia,
Kluyveromyces, and Yarrowia. In some embodiments, the yeast cell is
Hansenula polymorpha, Saccharomyces cerevisiae, Saccaromyces
carlsbergensis, Saccharomyces diastaticus, Saccharomyces norbensis,
Saccharomyces kluyveri, Schizosaccharomyces pombe, Komagataella
phaffii, formerly known as Pichia pastoris, Pichia finlandica,
Pichia trehalophila, Pichia kodamae, Pichia membranaefaciens,
Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia
quercuum, Pichia pijperi, Pichia stipitis, Pichia methanolica,
Pichia angusta, Kluyveromyces lactis, Candida albicans, or Yarrowia
lipolytica.
[0333] In some embodiments, the yeast strain is an industrial
polyploid yeast strain. Other non-limiting examples of fungal cells
include cells obtained from Aspergillus spp., Penicillium spp.,
Fusarium spp., Rhizopus spp., Acremonium spp., Neurospora spp.,
Sordaria spp., Magnaporthe spp., Allomyces spp., Ustilago spp.,
Botrytis spp., and Trichoderma spp.
[0334] In certain embodiments, the host cell is an algal cell such
as, Chlamydomonas (e.g., C. Reinhardtii) and Phormidium (P. sp.
ATCC29409).
[0335] In other embodiments, the host cell is a prokaryotic cell.
Suitable prokaryotic cells include gram positive, gram negative,
and gram-variable bacterial cells. The host cell may be a species
of, but not limited to: Agrobacterium, Alicyclobacillus, Anabaena,
Anacystis, Acinetobacter, Acidothermus, Arthrobacter, Azobacter,
Bacillus, Bifidobacterium, Brevibacterium, Butyrivibrio, Buchnera,
Campestris, Camplyobacter, Clostridium, Corynebacterium,
Chromatium, Coprococcus, Escherichia, Enterococcus, Enterobacter,
Erwinia, Fusobacterium, Faecalibacterium, Francisella,
Flavobacterium, Geobacillus, Haemophilus, Helicobacter, Klebsiella,
Lactobacillus, Lactococcus, Ilyobacter, Micrococcus,
Microbacterium, Mesorhizobium, Methylobacterium, Methylobacterium,
Mycobacterium, Neisseria, Pantoea, Pseudomonas, Prochlorococcus,
Rhodobacter, Rhodopseudomonas, Rhodopseudomonas, Roseburia,
Rhodospirillum, Rhodococcus, Scenedesmus, Streptomyces,
Streptococcus, Synecoccus, Saccharomonospora, Saccharopolyspora,
Staphylococcus, Serratia, Salmonella, Shigella,
Thermoanaerobacterium, Tropheryma, Tularensis, Temecula,
Thermosynechococcus, Thermococcus, Ureaplasma, Xanthomonas,
Xylella, Yersinia, and Zymomonas.
[0336] In some embodiments, the bacterial host strain is an
industrial strain. Numerous bacterial industrial strains are known
and suitable for the methods and compositions described in this
application.
[0337] In some embodiments, the bacterial host cell is of the
Agrobacterium species (e.g., A. radiobacter, A. rhizogenes, A.
rubi), the Arthrobacter species (e.g., A. aurescens, A. citreus, A.
globformis, A. hydrocarboglutamicus, A. mysorens, A. nicotianae, A.
paraffineus, A. protophonniae, A. roseoparaffinus, A. sulfureus, A.
ureafaciens), the Bacillus species (e.g., B. thuringiensis, B.
anthracis, B. megaterium, B. subtilis, B. lentus, B. circulars, B.
pumilus, B. lautus, B. coagulans, B. brevis, B. firmus, B.
alkaophius, B. licheniformis, B. clausii, B. stearothermophilus, B.
halodurans and B. amyloliquefaciens. In particular embodiments, the
host cell will be an industrial Bacillus strain including but not
limited to B. subtilis, B. pumilus, B. licheniformis, B.
megaterium, B. clausii, B. stearothermophilus and B.
amyloliquefaciens. In some embodiments, the host cell will be an
industrial Clostridium species (e.g., C. acetobutylicum, C. tetani
E88, C. lituseburense, C. saccharobutylicum, C. perfringens, C.
beijerinckii). In some embodiments, the host cell will be an
industrial Corynebacterium species (e.g., C. glutamicum, C.
acetoacidophilum). In some embodiments, the host cell will be an
industrial Escherichia species (e.g., E. coli). In some
embodiments, the host cell will be an industrial Erwinia species
(e.g., E. uredovora, E. carotovora, E. ananas, E. herbicola, E.
punctata, E. terreus). In some embodiments, the host cell will be
an industrial Pantoea species (e.g., P. citrea, P. agglomerans). In
some embodiments, the host cell will be an industrial Pseudomonas
species, (e.g., P. putida, P. aeruginosa, P. mevalonii). In some
embodiments, the host cell will be an industrial Streptococcus
species (e.g., S. equisimiles, S. pyogenes, S. uberis). In some
embodiments, the host cell will be an industrial Streptomyces
species (e.g., S. ambofaciens, S. achromogenes, S. avermitilis, S.
coelicolor, S. aureofaciens, S. aureus, S. fungicidicus, S.
griseus, S. lividans). In some embodiments, the host cell will be
an industrial Zymomonas species (e.g., Z. mobilis, Z. lipolytica),
and the like.
[0338] The present disclosure is also suitable for use with a
variety of animal cell types, including mammalian cells, for
example, human (including 293, HeLa, WI38, PER.C6 and Bowes
melanoma cells), mouse (including 3T3, NS0, NS1, Sp2/0), hamster
(CHO, BHK), monkey (COS, FRhL, Vero), insect cells, for example
fall armyworm (including Sf9 and Sf21), silkmoth (including BmN),
cabbage looper (including BTI-Tn-5B1-4) and common fruit fly
(including Schneider 2), and hybridoma cell lines.
[0339] In various embodiments, strains that may be used in the
practice of the disclosure including both prokaryotic and
eukaryotic strains, and are readily accessible to the public from a
number of culture collections such as American Type Culture
Collection (ATCC), Deutsche Sammlung von Mikroorganismen and
Zellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures
(CBS), and Agricultural Research Service Patent Culture Collection,
Northern Regional Research Center (NRRL). The present disclosure is
also suitable for use with a variety of plant cell types. In some
embodiments, the plant is of the Cannabis genus in the family
Cannabaceae. In certain embodiments, the plant is of the species
Cannabis sativa, Cannabis indica, or Cannabis ruderalis. In other
embodiments, the plant is of the genus Nicotiana in the family
Solanaceae. In certain embodiments, the plant is of the species
Nicotiana rustica.
[0340] The term "cell," as used in this application, may refer to a
single cell or a population of cells, such as a population of cells
belonging to the same cell line or strain. Use of the singular term
"cell" should not be construed to refer explicitly to a single cell
rather than a population of cells. The host cell may comprise
genetic modifications relative to a wild-type counterpart.
Reduction of gene expression and/or gene inactivation in a host
cell may be achieved through any suitable method, including but not
limited to, deletion of the gene, introduction of a point mutation
into the gene, selective editing of the gene and/or truncation of
the gene. For example, polymerase chain reaction (PCR)-based
methods may be used (see, e.g., Gardner et al., Methods Mol Biol.
2014; 1205:45-78). As a non-limiting example, genes may be deleted
through gene replacement (e.g., with a marker, including a
selection marker). A gene may also be truncated through the use of
a transposon system (see, e.g., Poussu et al., Nucleic Acids Res.
2005; 33(12): e104). A gene may also be edited through of the use
of gene editing technologies known in the art, such as CRISPR-based
technologies.
Culturing of Host Cells
[0341] Any of the cells disclosed in this application can be
cultured in media of any type (rich or minimal) and any composition
prior to, during, and/or after contact and/or integration of a
nucleic acid. The conditions of the culture or culturing process
can be optimized through routine experimentation as would be
understood by one of ordinary skill in the art. In some
embodiments, the selected media is supplemented with various
components. In some embodiments, the concentration and amount of a
supplemental component is optimized. In some embodiments, other
aspects of the media and growth conditions (e.g., pH, temperature,
etc.) are optimized through routine experimentation. In some
embodiments, the frequency that the media is supplemented with one
or more supplemental components, and the amount of time that the
cell is cultured, is optimized.
[0342] Culturing of the cells described in this application can be
performed in culture vessels known and used in the art. In some
embodiments, an aerated reaction vessel (e.g., a stirred tank
reactor) is used to culture the cells. In some embodiments, a
bioreactor or fermentor is used to culture the cell. Thus, in some
embodiments, the cells are used in fermentation. As used in this
application, the terms "bioreactor" and "fermentor" are
interchangeably used and refer to an enclosure, or partial
enclosure, in which a biological, biochemical and/or chemical
reaction takes place that involves a living organism or part of a
living organism. A "large-scale bioreactor" or "industrial-scale
bioreactor" is a bioreactor that is used to generate a product on a
commercial or quasi-commercial scale. Large scale bioreactors
typically have volumes in the range of liters, hundreds of liters,
thousands of liters, or more.
[0343] Non-limiting examples of bioreactors include: stirred tank
fermentors, bioreactors agitated by rotating mixing devices,
chemostats, bioreactors agitated by shaking devices, airlift
fermentors, packed-bed reactors, fixed-bed reactors, fluidized bed
bioreactors, bioreactors employing wave induced agitation,
centrifugal bioreactors, roller bottles, and hollow fiber
bioreactors, roller apparatuses (for example benchtop,
cart-mounted, and/or automated varieties), vertically-stacked
plates, spinner flasks, stirring or rocking flasks, shaken
multi-well plates, MD bottles, T-flasks, Roux bottles,
multiple-surface tissue culture propagators, modified fermentors,
and coated beads (e.g., beads coated with serum proteins,
nitrocellulose, or carboxymethyl cellulose to prevent cell
attachment).
[0344] In some embodiments, the bioreactor includes a cell culture
system where the cell (e.g., yeast cell) is in contact with moving
liquids and/or gas bubbles. In some embodiments, the cell or cell
culture is grown in suspension. In other embodiments, the cell or
cell culture is attached to a solid phase carrier. Non-limiting
examples of a carrier system includes microcarriers (e.g., polymer
spheres, microbeads, and microdisks that can be porous or
non-porous), cross-linked beads (e.g., dextran) charged with
specific chemical groups (e.g., tertiary amine groups), 2D
microcarriers including cells trapped in nonporous polymer fibers,
3D carriers (e.g., carrier fibers, hollow fibers, multicartridge
reactors, and semi-permeable membranes that can comprising porous
fibers), microcarriers having reduced ion exchange capacity,
encapsulation cells, capillaries, and aggregates. In some
embodiments, carriers are fabricated from materials such as
dextran, gelatin, glass, or cellulose.
[0345] In some embodiments, industrial-scale processes are operated
in continuous, semi-continuous or non-continuous modes.
Non-limiting examples of operation modes are batch, fed batch,
extended batch, repetitive batch, draw/fill, rotating-wall,
spinning flask, and/or perfusion mode of operation. In some
embodiments, a bioreactor allows continuous or semi-continuous
replenishment of the substrate stock, for example a carbohydrate
source and/or continuous or semi-continuous separation of the
product, from the bioreactor.
[0346] In some embodiments, the bioreactor or fermentor includes a
sensor and/or a control system to measure and/or adjust reaction
parameters. Non-limiting examples of reaction parameters include
biological parameters (e.g., growth rate, cell size, cell number,
cell density, cell type, or cell state, etc.), chemical parameters
(e.g., pH, redox-potential, concentration of reaction substrate
and/or product, concentration of dissolved gases, such as oxygen
concentration and CO.sub.2 concentration, nutrient concentrations,
metabolite concentrations, concentration of an oligopeptide,
concentration of an amino acid, concentration of a vitamin,
concentration of a hormone, concentration of an additive, serum
concentration, ionic strength, concentration of an ion, relative
humidity, molarity, osmolarity, concentration of other chemicals,
for example buffering agents, adjuvants, or reaction by-products),
physical/mechanical parameters (e.g., density, conductivity, degree
of agitation, pressure, and flow rate, shear stress, shear rate,
viscosity, color, turbidity, light absorption, mixing rate,
conversion rate, as well as thermodynamic parameters, such as
temperature, light intensity/quality, etc.). Sensors to measure the
parameters described in this application are well known to one of
ordinary skill in the relevant mechanical and electronic arts.
Control systems to adjust the parameters in a bioreactor based on
the inputs from a sensor described in this application are well
known to one of ordinary skill in the art in bioreactor
engineering.
[0347] In some embodiments, the method involves batch fermentation
(e.g., shake flask fermentation). General considerations for batch
fermentation (e.g., shake flask fermentation) include the level of
oxygen and glucose. For example, batch fermentation (e.g., shake
flask fermentation) may be oxygen and glucose limited, so in some
embodiments, the capability of a strain to perform in a
well-designed fed-batch fermentation is underestimated. Also, the
final product (e.g., cannabinoid or cannabinoid precursor) may
display some differences from the substrate in terms of solubility,
toxicity, cellular accumulation and secretion and in some
embodiments can have different fermentation kinetics.
[0348] In some embodiments, the cells of the present disclosure are
adapted to produce cannabinoids or cannabinoid precursors in vivo.
In some embodiments, the cells are adapted to secrete one or more
enzymes for cannabinoid synthesis (e.g., AAE, PKS, PKC, PT, or TS).
In some embodiments, the cells of the present disclosure are lysed,
and the remaining lysates are recovered for subsequent use. In such
embodiments, the secreted or lysed enzyme can catalyze reactions
for the production of a cannabinoid or precursor by bioconversion
in an in vitro or ex vivo process. In some embodiments, any and all
conversions described in this application can be conducted
chemically or enzymatically, in vitro or in vivo.
Purification and Further Processing
[0349] In some embodiments, any of the methods described in this
application may include isolation and/or purification of the
cannabinoids and/or cannabinoid precursors produced (e.g., produced
in a bioreactor). For example, the isolation and/or purification
can involve one or more of cell lysis, centrifugation, extraction,
column chromatography, distillation, crystallization, and
lyophilization.
[0350] The methods described in this application encompass
production of any cannabinoid or cannabinoid precursor known in the
art. Cannabinoids or cannabinoid precursors produced by any of the
recombinant cells disclosed in this application or any of the in
vitro methods described in this application may be identified and
extracted using any method known in the art. Mass spectrometry
(e.g., LC-MS, GC-MS) is a non-limiting example of a method for
identification and may be used to extract a compound of
interest.
[0351] In some embodiments, any of the methods described in this
application further comprise decarboxylation of a cannabinoid or
cannabinoid precursor. As a non-limiting example, the acid form of
a cannabinoid or cannabinoid precursor may be heated (e.g., at
least 90.degree. C.) to decarboxylate the cannabinoid or
cannabinoid precursor. See, e.g., U.S. Pat. Nos. 10,159,908,
10,143,706, 9,908,832 and 7,344,736. See also, e.g., Wang et al.,
Cannabis Cannabinoid Res. 2016; 1(1): 262-271.
Compositions, Kits, and Administration
[0352] The present disclosure provides compositions, including
pharmaceutical compositions, comprising a cannabinoid or a
cannabinoid precursor, or pharmaceutically acceptable salt thereof,
produced by any of the methods described in this application, and
optionally a pharmaceutically acceptable excipient.
[0353] In certain embodiments, a cannabinoid or cannabinoid
precursor described in this application is provided in an effective
amount in a composition, such as a pharmaceutical composition. In
certain embodiments, the effective amount is a therapeutically
effective amount. In certain embodiments, the effective amount is a
prophylactically effective amount.
[0354] Compositions, such as pharmaceutical compositions, described
in this application can be prepared by any method known in the art.
In general, such preparatory methods include bringing a compound
described in this application (i.e., the "active ingredient") into
association with a carrier or excipient, and/or one or more other
accessory ingredients, and then, if necessary and/or desirable,
shaping, and/or packaging the product into a desired single- or
multi-dose unit.
[0355] Pharmaceutical compositions can be prepared, packaged,
and/or sold in bulk, as a single unit dose, and/or as a plurality
of single unit doses. A "unit dose" is a discrete amount of the
pharmaceutical composition comprising a predetermined amount of the
active ingredient. The amount of the active ingredient is generally
equal to the dosage of the active ingredient which would be
administered to a subject and/or a convenient fraction of such a
dosage, such as one-half or one-third of such a dosage.
[0356] Relative amounts of the active ingredient, the
pharmaceutically acceptable excipient, and/or any additional
ingredients in a pharmaceutical composition described in this
application will vary, depending upon the identity, size, and/or
condition of the subject treated and further depending upon the
route by which the composition is to be administered. The
composition may comprise between 0.1% and 100% (w/w) active
ingredient.
[0357] Pharmaceutically acceptable excipients used in the
manufacture of pharmaceutical compositions include inert diluents,
dispersing and/or granulating agents, surface active agents and/or
emulsifiers, disintegrating agents, binding agents, preservatives,
buffering agents, lubricating agents, and/or oils. Excipients such
as cocoa butter and suppository waxes, coloring agents, coating
agents, sweetening, flavoring, and perfuming agents may also be
present in the composition. Exemplary excipients include diluents,
dispersing and/or granulating agents, surface active agents and/or
emulsifiers, disintegrating agents, binding agents, preservatives,
buffering agents, lubricating agents, and/or oils (e.g., synthetic
oils, semi-synthetic oils) as disclosed in this application.
[0358] Exemplary diluents include calcium carbonate, sodium
carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate,
calcium hydrogen phosphate, sodium phosphate lactose, sucrose,
cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol,
inositol, sodium chloride, dry starch, cornstarch, powdered sugar,
and mixtures thereof.
[0359] Exemplary granulating and/or dispersing agents include
potato starch, corn starch, tapioca starch, sodium starch
glycolate, clays, alginic acid, guar gum, citrus pulp, agar,
bentonite, cellulose, and wood products, natural sponge,
cation-exchange resins, calcium carbonate, silicates, sodium
carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone),
sodium carboxymethyl starch (sodium starch glycolate),
carboxymethyl cellulose, cross-linked sodium carboxymethyl
cellulose (croscarmellose), methylcellulose, pregelatinized starch
(starch 1500), microcrystalline starch, water insoluble starch,
calcium carboxymethyl cellulose, magnesium aluminum silicate
(Veegum), sodium lauryl sulfate, quaternary ammonium compounds, and
mixtures thereof.
[0360] Exemplary surface active agents and/or emulsifiers include
natural emulsifiers (e.g., acacia, agar, alginic acid, sodium
alginate, tragacanth, chondrux, cholesterol, xanthan, pectin,
gelatin, egg yolk, casein, wool fat, cholesterol, wax, and
lecithin), colloidal clays (e.g., bentonite (aluminum silicate) and
Veegum (magnesium aluminum silicate)), long chain amino acid
derivatives, high molecular weight alcohols (e.g., stearyl alcohol,
cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene
glycol distearate, glyceryl monostearate, and propylene glycol
monostearate, polyvinyl alcohol), carbomers (e.g., carboxy
polymethylene, polyacrylic acid, acrylic acid polymer, and
carboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g.,
carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl
cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose,
methylcellulose), sorbitan fatty acid esters (e.g., polyoxyethylene
sorbitan monolaurate (Tween.RTM. 20), polyoxyethylene sorbitan
(Tween.RTM. 60), polyoxyethylene sorbitan monooleate (Tween.RTM.
80), sorbitan monopalmitate (Span.RTM. 40), sorbitan monostearate
(Span.RTM. 60), sorbitan tristearate (Span.RTM. 65), glyceryl
monooleate, sorbitan monooleate (Span.RTM. 80), polyoxyethylene
esters (e.g., polyoxyethylene monostearate (Myrj.RTM. 45),
polyoxyethylene hydrogenated castor oil, polyethoxylated castor
oil, polyoxymethylene stearate, and Solutol.RTM.), sucrose fatty
acid esters, polyethylene glycol fatty acid esters (e.g.,
Cremophor.RTM.), polyoxyethylene ethers, (e.g., polyoxyethylene
lauryl ether (Brij.RTM. 30)), poly(vinyl-pyrrolidone), diethylene
glycol monolaurate, triethanolamine oleate, sodium oleate,
potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium
lauryl sulfate, Pluronic.RTM. F-68, poloxamer P-188, cetrimonium
bromide, cetylpyridinium chloride, benzalkonium chloride, docusate
sodium, and/or mixtures thereof.
[0361] Exemplary binding agents include starch (e.g., cornstarch
and starch paste), gelatin, sugars (e.g., sucrose, glucose,
dextrose, dextrin, molasses, lactose, lactitol, mannitol, etc.),
natural and synthetic gums (e.g., acacia, sodium alginate, extract
of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks,
carboxymethylcellulose, methylcellulose, ethylcellulose,
hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl
methylcellulose, microcrystalline cellulose, cellulose acetate,
poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum.RTM.),
and larch arabogalactan), alginates, polyethylene oxide,
polyethylene glycol, inorganic calcium salts, silicic acid,
polymethacrylates, waxes, water, alcohol, and/or mixtures
thereof.
[0362] Exemplary preservatives include antioxidants, chelating
agents, antimicrobial preservatives, antifungal preservatives,
antiprotozoan preservatives, alcohol preservatives, acidic
preservatives, and other preservatives. In certain embodiments, the
preservative is an antioxidant. In other embodiments, the
preservative is a chelating agent.
[0363] Exemplary antioxidants include alpha tocopherol, ascorbic
acid, acorbyl palmitate, butylated hydroxyanisole, butylated
hydroxytoluene, monothioglycerol, potassium metabisulfite,
propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite,
sodium metabisulfite, and sodium sulfite.
[0364] Exemplary chelating agents include
ethylenediaminetetraacetic acid (EDTA) and salts and hydrates
thereof (e.g., sodium edetate, disodium edetate, trisodium edetate,
calcium disodium edetate, dipotassium edetate, and the like),
citric acid and salts and hydrates thereof (e.g., citric acid
monohydrate), fumaric acid and salts and hydrates thereof, malic
acid and salts and hydrates thereof, phosphoric acid and salts and
hydrates thereof, and tartaric acid and salts and hydrates thereof.
Exemplary antimicrobial preservatives include benzalkonium
chloride, benzethonium chloride, benzyl alcohol, bronopol,
cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol,
chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin,
hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol,
phenylmercuric nitrate, propylene glycol, and thimerosal.
[0365] Exemplary antifungal preservatives include butyl paraben,
methyl paraben, ethyl paraben, propyl paraben, benzoic acid,
hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium
benzoate, sodium propionate, and sorbic acid.
[0366] Exemplary alcohol preservatives include ethanol,
polyethylene glycol, phenol, phenolic compounds, bisphenol,
chlorobutanol, hydroxybenzoate, and phenylethyl alcohol.
[0367] Exemplary acidic preservatives include vitamin A, vitamin C,
vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic
acid, ascorbic acid, sorbic acid, and phytic acid.
[0368] Other preservatives include tocopherol, tocopherol acetate,
deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA),
butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl
sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium
bisulfite, sodium metabisulfite, potassium sulfite, potassium
metabisulfite, Glydant.RTM. Plus, Phenonip.RTM., methylparaben,
Germall.RTM. 115, Germaben.RTM. II, Neolone.RTM., Kathon.RTM., and
Euxyl.RTM..
[0369] Exemplary buffering agents include citrate buffer solutions,
acetate buffer solutions, phosphate buffer solutions, ammonium
chloride, calcium carbonate, calcium chloride, calcium citrate,
calcium glubionate, calcium gluceptate, calcium gluconate,
D-gluconic acid, calcium glycerophosphate, calcium lactate,
propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium
phosphate, phosphoric acid, tribasic calcium phosphate, calcium
hydroxide phosphate, potassium acetate, potassium chloride,
potassium gluconate, potassium mixtures, dibasic potassium
phosphate, monobasic potassium phosphate, potassium phosphate
mixtures, sodium acetate, sodium bicarbonate, sodium chloride,
sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic
sodium phosphate, sodium phosphate mixtures, tromethamine,
magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free
water, isotonic saline, Ringer's solution, ethyl alcohol, and
mixtures thereof.
[0370] Exemplary lubricating agents include magnesium stearate,
calcium stearate, stearic acid, silica, talc, malt, glyceryl
behanate, hydrogenated vegetable oils, polyethylene glycol, sodium
benzoate, sodium acetate, sodium chloride, leucine, magnesium
lauryl sulfate, sodium lauryl sulfate, and mixtures thereof.
[0371] Exemplary natural oils include almond, apricot kernel,
avocado, babassu, bergamot, black current seed, borage, cade,
camomile, canola, caraway, carnauba, castor, cinnamon, cocoa
butter, coconut, cod liver, coffee, corn, cotton seed, emu,
eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd,
grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui
nut, lavandin, lavender, lemon, Litsea cubeba, macademia nut,
mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange,
orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed,
pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood,
sasquana, savoury, sea buckthorn, sesame, shea butter, silicone,
soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut,
and wheat germ oils. Exemplary synthetic or semi-synthetic oils
include, but are not limited to, butyl stearate, medium chain
triglycerides (such as caprylic triglyceride and capric
triglyceride), cyclomethicone, diethyl sebacate, dimethicone 360,
isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol,
silicone oil, and mixtures thereof. In certain embodiments,
exemplary synthetic oils comprise medium chain triglycerides (such
as caprylic triglyceride and capric triglyceride).
[0372] Liquid dosage forms for oral and parenteral administration
include pharmaceutically acceptable emulsions, microemulsions,
solutions, suspensions, syrups and elixirs. In addition to the
active ingredients, the liquid dosage forms may comprise inert
diluents commonly used in the art such as, for example, water or
other solvents, solubilizing agents and emulsifiers such as ethyl
alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl
alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,
dimethylformamide, oils (e.g., cottonseed, groundnut, corn, germ,
olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl
alcohol, polyethylene glycols and fatty acid esters of sorbitan,
and mixtures thereof. Besides inert diluents, the oral compositions
can include adjuvants such as wetting agents, emulsifying and
suspending agents, sweetening, flavoring, and perfuming agents. In
certain embodiments for parenteral administration, the conjugates
described in this application are mixed with solubilizing agents
such as Cremophor.RTM., alcohols, oils, modified oils, glycols,
polysorbates, cyclodextrins, polymers, and mixtures thereof.
[0373] Injectable preparations, for example, sterile injectable
aqueous or oleaginous suspensions can be formulated according to
the known art using suitable dispersing or wetting agents and
suspending agents. The sterile injectable preparation can be a
sterile injectable solution, suspension, or emulsion in a nontoxic
parenterally acceptable diluent or solvent, for example, as a
solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that can be employed are water, Ringer's solution, U.S.P.,
and isotonic sodium chloride solution. In addition, sterile, fixed
oils are conventionally employed as a solvent or suspending medium.
For this purpose, any bland fixed oil can be employed including
synthetic mono- or di-glycerides. In addition, fatty acids such as
oleic acid are used in the preparation of injectables.
[0374] The injectable formulations can be sterilized, for example,
by filtration through a bacterial-retaining filter, or by
incorporating sterilizing agents in the form of sterile solid
compositions which can be dissolved or dispersed in sterile water
or other sterile injectable medium prior to use.
[0375] In order to prolong the effect of a drug, it is often
desirable to slow the absorption of the drug from subcutaneous or
intramuscular injection. This can be accomplished by the use of a
liquid suspension of crystalline or amorphous material with poor
water solubility. The rate of absorption of the drug then depends
upon its rate of dissolution, which, in turn, may depend upon
crystal size and crystalline form. Alternatively, delayed
absorption of a parenterally administered drug form may be
accomplished by dissolving or suspending the drug in an oil
vehicle.
[0376] Compositions for rectal or vaginal administration are
typically suppositories which can be prepared by mixing the
conjugates described in this application with suitable
non-irritating excipients or carriers such as cocoa butter,
polyethylene glycol, or a suppository wax which are solid at
ambient temperature but liquid at body temperature and therefore
melt in the rectum or vaginal cavity and release the active
ingredient.
[0377] Solid dosage forms for oral administration include capsules,
tablets, pills, powders, and granules. In such solid dosage forms,
the active ingredient is mixed with at least one inert,
pharmaceutically acceptable excipient or carrier such as sodium
citrate or dicalcium phosphate and/or (a) fillers or extenders such
as starches, lactose, sucrose, glucose, mannitol, and silicic acid,
(b) binders such as, for example, carboxymethylcellulose,
alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia,
(c) humectants such as glycerol, (d) disintegrating agents such as
agar, calcium carbonate, potato or tapioca starch, alginic acid,
certain silicates, and sodium carbonate, (e) solution retarding
agents such as paraffin, (f) absorption accelerators such as
quaternary ammonium compounds, (g) wetting agents such as, for
example, cetyl alcohol and glycerol monostearate, (h) absorbents
such as kaolin and bentonite clay, and (i) lubricants such as talc,
calcium stearate, magnesium stearate, solid polyethylene glycols,
sodium lauryl sulfate, and mixtures thereof. In the case of
capsules, tablets, and pills, the dosage form may include a
buffering agent.
[0378] Solid compositions of a similar type can be employed as
fillers in soft and hard-filled gelatin capsules using such
excipients as lactose or milk sugar as well as high molecular
weight polyethylene glycols and the like. The solid dosage forms of
tablets, dragees, capsules, pills, and granules can be prepared
with coatings and shells such as enteric coatings and other
coatings well known in the art of pharmacology. They may optionally
comprise opacifying agents and can be of a composition that they
release the active ingredient(s) only, or preferentially, in a
certain part of the intestinal tract, optionally, in a delayed
manner. Examples of encapsulating compositions which can be used
include polymeric substances and waxes. Solid compositions of a
similar type can be employed as fillers in soft and hard-filled
gelatin capsules using such excipients as lactose or milk sugar as
well as high molecular weight polethylene glycols and the like.
[0379] The active ingredient can be in a micro-encapsulated form
with one or more excipients as noted above. The solid dosage forms
of tablets, dragees, capsules, pills, and granules can be prepared
with coatings and shells such as enteric coatings, release
controlling coatings, and other coatings well known in the
pharmaceutical formulating art. In such solid dosage forms the
active ingredient can be admixed with at least one inert diluent
such as sucrose, lactose, or starch. Such dosage forms may
comprise, as is normal practice, additional substances other than
inert diluents, e.g., tableting lubricants and other tableting aids
such a magnesium stearate and microcrystalline cellulose. In the
case of capsules, tablets and pills, the dosage forms may comprise
buffering agents. They may optionally comprise opacifying agents
and can be of a composition that they release the active
ingredient(s) only, or preferentially, in a certain part of the
intestinal tract, optionally, in a delayed manner. Examples of
encapsulating agents which can be used include polymeric substances
and waxes.
[0380] Dosage forms for topical and/or transdermal administration
of a compound described in this application may include ointments,
pastes, creams, lotions, gels, powders, solutions, sprays,
inhalants, and/or patches. Generally, the active ingredient is
admixed under sterile conditions with a pharmaceutically acceptable
carrier or excipient and/or any needed preservatives and/or buffers
as can be required. Additionally, the present disclosure
contemplates the use of transdermal patches, which often have the
added advantage of providing controlled delivery of an active
ingredient to the body. Such dosage forms can be prepared, for
example, by dissolving and/or dispensing the active ingredient in
the proper medium. Alternatively or additionally, the rate can be
controlled by either providing a rate controlling membrane and/or
by dispersing the active ingredient in a polymer matrix and/or
gel.
[0381] Suitable devices for use in delivering intradermal
pharmaceutical compositions described in this application include
short needle devices. Intradermal compositions can be administered
by devices which limit the effective penetration length of a needle
into the skin. Alternatively or additionally, conventional syringes
can be used in the classical mantoux method of intradermal
administration. Jet injection devices which deliver liquid
formulations to the dermis via a liquid jet injector and/or via a
needle which pierces the stratum corneum and produces a jet which
reaches the dermis are suitable. Ballistic powder/particle delivery
devices which use compressed gas to accelerate the compound in
powder form through the outer layers of the skin to the dermis are
suitable.
[0382] Formulations suitable for topical administration include,
but are not limited to, liquid and/or semi-liquid preparations such
as liniments, lotions, oil-in-water and/or water-in-oil emulsions
such as creams, ointments, and/or pastes, and/or solutions and/or
suspensions. Topically administrable formulations may, for example,
comprise from about 1% to about 10% (w/w) active ingredient,
although the concentration of the active ingredient can be as high
as the solubility limit of the active ingredient in the solvent.
Formulations for topical administration may further comprise one or
more of the additional ingredients described in this
application.
[0383] A pharmaceutical composition described in this application
can be prepared, packaged, and/or sold in a formulation suitable
for pulmonary administration via the buccal cavity. Such a
formulation may comprise dry particles which comprise the active
ingredient and which have a diameter in the range from about 0.5 to
about 7 nanometers, or from about 1 to about 6 nanometers. Such
compositions are conveniently in the form of dry powders for
administration using a device comprising a dry powder reservoir to
which a stream of propellant can be directed to disperse the powder
and/or using a self-propelling solvent/powder dispensing container
such as a device comprising the active ingredient dissolved and/or
suspended in a low-boiling propellant in a sealed container. Such
powders comprise particles wherein at least 98% of the particles by
weight have a diameter greater than 0.5 nanometers and at least 95%
of the particles by number have a diameter less than 7 nanometers.
Alternatively, at least 95% of the particles by weight have a
diameter greater than 1 nanometer and at least 90% of the particles
by number have a diameter less than 6 nanometers. Dry powder
compositions may include a solid fine powder diluent such as sugar
and are conveniently provided in a unit dose form.
[0384] Low boiling propellants generally include liquid propellants
having a boiling point of below 65.degree. F. at atmospheric
pressure. Generally, the propellant may constitute 50 to 99.9%
(w/w) of the composition, and the active ingredient may constitute
0.1 to 20% (w/w) of the composition. The propellant may further
comprise additional ingredients such as a liquid non-ionic and/or
solid anionic surfactant and/or a solid diluent (which may have a
particle size of the same order as particles comprising the active
ingredient).
[0385] Although the descriptions of pharmaceutical compositions
provided in this application are principally directed to
pharmaceutical compositions which are suitable for administration
to humans, it will be understood by the skilled artisan that such
compositions are generally suitable for administration to animals
of all sorts. Modification of pharmaceutical compositions suitable
for administration to humans in order to render the compositions
suitable for administration to various animals is well understood,
and the ordinarily skilled veterinary pharmacologist can design
and/or perform such modification with ordinary experimentation.
[0386] Compounds provided in this application are typically
formulated in dosage unit form for ease of administration and
uniformity of dosage. It will be understood, however, that the
total daily usage of the compositions described in this application
will be decided by a physician within the scope of sound medical
judgment. The specific therapeutically effective dose level for any
particular subject or organism will depend upon a variety of
factors including the disease being treated and the severity of the
disorder; the activity of the specific active ingredient employed;
the specific composition employed; the age, body weight, general
health, sex, and diet of the subject; the time of administration,
route of administration, and rate of excretion of the specific
active ingredient employed; the duration of the treatment; drugs
used in combination or coincidental with the specific active
ingredient employed; and like factors well known in the medical
arts.
[0387] The compounds and compositions provided in this application
can be administered by any route, including enteral (e.g., oral),
parenteral, intravenous, intramuscular, intra-arterial,
intramedullary, intrathecal, subcutaneous, intraventricular,
transdermal, interdermal, rectal, intravaginal, intraperitoneal,
topical (as by powders, ointments, creams, and/or drops), mucosal,
nasal, bucal, sublingual; by intratracheal instillation, bronchial
instillation, and/or inhalation; and/or as an oral spray, nasal
spray, and/or aerosol. Specifically contemplated routes are oral
administration, intravenous administration (e.g., systemic
intravenous injection), regional administration via blood and/or
lymph supply, and/or direct administration to an affected site. In
general, the most appropriate route of administration will depend
upon a variety of factors including the nature of the agent (e.g.,
its stability in the environment of the gastrointestinal tract),
and/or the condition of the subject (e.g., whether the subject is
able to tolerate oral administration).
[0388] In some embodiments, compounds or compositions disclosed in
this application are formulated and/or administered in
nanoparticles. Nanoparticles are particles in the nanoscale. In
some embodiments, nanoparticles are less than 1 .mu.m in diameter.
In some embodiments, nanoparticles are between about 1 and 100 nm
in diameter. Nanoparticles include organic nanoparticles, such as
dendrimers, liposomes, or polymeric nanoparticles. Nanoparticles
also include inorganic nanoparticles, such as fullerenes, quantum
dots, and gold nanoparticles. Compositions may comprise an
aggregate of nanoparticles. In some embodiments, the aggregate of
nanoparticles is homogeneous, while in other embodiments the
aggregate of nanoparticles is heterogeneous.
[0389] The exact amount of a compound required to achieve an
effective amount will vary from subject to subject, depending, for
example, on species, age, and general condition of a subject,
severity of the side effects or disorder, identity of the
particular compound, mode of administration, and the like. An
effective amount may be included in a single dose (e.g., single
oral dose) or multiple doses (e.g., multiple oral doses). In
certain embodiments, when multiple doses are administered to a
subject or applied to a tissue or cell, any two doses of the
multiple doses include different or substantially the same amounts
of a compound described in this application. In certain
embodiments, when multiple doses are administered to a subject or
applied to a tissue or cell, the frequency of administering the
multiple doses to the subject or applying the multiple doses to the
tissue or cell is three doses a day, two doses a day, one dose a
day, one dose every other day, one dose every third day, one dose
every week, one dose every two weeks, one dose every three weeks,
or one dose every four weeks. In certain embodiments, the frequency
of administering the multiple doses to the subject or applying the
multiple doses to the tissue or cell is one dose per day. In
certain embodiments, the frequency of administering the multiple
doses to the subject or applying the multiple doses to the tissue
or cell is two doses per day. In certain embodiments, the frequency
of administering the multiple doses to the subject or applying the
multiple doses to the tissue or cell is three doses per day. In
certain embodiments, when multiple doses are administered to a
subject or applied to a tissue or cell, the duration between the
first dose and last dose of the multiple doses is one day, two
days, four days, one week, two weeks, three weeks, one month, two
months, three months, four months, six months, nine months, one
year, two years, three years, four years, five years, seven years,
ten years, fifteen years, twenty years, or the lifetime of the
subject, tissue, or cell. In certain embodiments, the duration
between the first dose and last dose of the multiple doses is three
months, six months, or one year. In certain embodiments, the
duration between the first dose and last dose of the multiple doses
is the lifetime of the subject, tissue, or cell. In certain
embodiments, a dose (e.g., a single dose, or any dose of multiple
doses) described in this application includes independently between
0.1 .mu.g and 1 .mu.g, between 0.001 mg and 0.01 mg, between 0.01
mg and 0.1 mg, between 0.1 mg and 1 mg, between 1 mg and 3 mg,
between 3 mg and 10 mg, between 10 mg and 30 mg, between 30 mg and
100 mg, between 100 mg and 300 mg, between 300 mg and 1,000 mg, or
between 1 g and 10 g, inclusive, of a compound described in this
application. In certain embodiments, a dose described in this
application includes independently between 1 mg and 3 mg,
inclusive, of a compound described in this application. In certain
embodiments, a dose described in this application includes
independently between 3 mg and 10 mg, inclusive, of a compound
described in this application. In certain embodiments, a dose
described in this application includes independently between 10 mg
and 30 mg, inclusive, of a compound described in this application.
In certain embodiments, a dose described in this application
includes independently between 30 mg and 100 mg, inclusive, of a
compound described in this application.
[0390] Dose ranges as described in this application provide
guidance for the administration of provided pharmaceutical
compositions to an adult. The amount to be administered to, for
example, a child or an adolescent can be determined by a medical
practitioner or person skilled in the art and can be lower or the
same as that administered to an adult.
[0391] A compound or composition, as described in this application,
can be administered in combination with one or more additional
pharmaceutical agents (e.g., therapeutically and/or
prophylactically active agents). The compounds or compositions can
be administered in combination with additional pharmaceutical
agents that improve their activity, improve bioavailability,
improve safety, reduce drug resistance, reduce and/or modify
metabolism, inhibit excretion, and/or modify distribution in a
subject or cell. It will also be appreciated that the therapy
employed may achieve a desired effect for the same disorder, and/or
it may achieve different effects. In certain embodiments, a
pharmaceutical composition described in this application including
a compound described in this application and an additional
pharmaceutical agent shows a synergistic effect that is absent in a
pharmaceutical composition including one of the compound and the
additional pharmaceutical agent, but not both.
[0392] The compound or composition can be administered concurrently
with, prior to, or subsequent to one or more additional
pharmaceutical agents, which may be useful as, e.g., combination
therapies. Pharmaceutical agents include therapeutically active
agents. Pharmaceutical agents also include prophylactically active
agents. Pharmaceutical agents include small organic molecules such
as drug compounds (e.g., compounds approved for human or veterinary
use by the U.S. Food and Drug Administration as provided in the
Code of Federal Regulations (CFR)), peptides, proteins,
carbohydrates, monosaccharides, oligosaccharides, polysaccharides,
nucleoproteins, mucoproteins, lipoproteins, synthetic polypeptides
or proteins, small molecules linked to proteins, glycoproteins,
steroids, nucleic acids, DNAs, RNAs, nucleotides, nucleosides,
oligonucleotides, antisense oligonucleotides, lipids, hormones,
vitamins, and cells. In certain embodiments, the additional
pharmaceutical agent is a pharmaceutical agent useful for treating
and/or preventing a disease (e.g., proliferative disease,
neurological disease, painful condition, psychiatric disorder, or
metabolic disorder). Each additional pharmaceutical agent may be
administered at a dose and/or on a time schedule determined for
that pharmaceutical agent. The additional pharmaceutical agents may
also be administered together with each other and/or with the
compound or composition described in this application in a single
dose or administered separately in different doses. The particular
combination to employ in a regimen will take into account
compatibility of the compound described in this application with
the additional pharmaceutical agent(s) and/or the desired
therapeutic and/or prophylactic effect to be achieved. In general,
it is expected that the additional pharmaceutical agent(s) in
combination be utilized at levels that do not exceed the levels at
which they are utilized individually. In some embodiments, the
levels utilized in combination will be lower than those utilized
individually.
[0393] In some embodiments, one or more of the compositions
described in this application are administered to a subject. In
certain embodiments, the subject is an animal. The animal may be of
either sex and may be at any stage of development. In certain
embodiments, the subject is a human. In other embodiments, the
subject is a non-human animal. In certain embodiments, the subject
is a mammal. In certain embodiments, the subject is a non-human
mammal. In certain embodiments, the subject is a domesticated
animal, such as a dog, cat, cow, pig, horse, sheep, or goat. In
certain embodiments, the subject is a companion animal, such as a
dog or cat. In certain embodiments, the subject is a livestock
animal, such as a cow, pig, horse, sheep, or goat. In certain
embodiments, the subject is a zoo animal. In another embodiment,
the subject is a research animal, such as a rodent (e.g., mouse,
rat), dog, pig, or non-human primate.
[0394] Also encompassed by the disclosure are kits (e.g.,
pharmaceutical packs). The kits provided may comprise a
composition, such as a pharmaceutical composition, or a compound
described in this application and a container (e.g., a vial,
ampule, bottle, syringe, and/or dispenser package, or other
suitable container). In some embodiments, provided kits may
optionally further include a second container comprising a
pharmaceutical excipient for dilution or suspension of a
pharmaceutical composition or compound described in this
application. In some embodiments, the pharmaceutical composition or
compound described in this application provided in the first
container and the second container a combined to form one unit
dosage form.
[0395] Thus, in one aspect, provided are kits including a first
container comprising a compound or composition described in this
application. In certain embodiments, the kits are useful for
treating a disease in a subject in need thereof. In certain
embodiments, the kits are useful for preventing a disease in a
subject in need thereof. In certain embodiments, the kits are
useful for reducing the risk of developing a disease in a subject
in need thereof.
[0396] In certain embodiments, a kit described in this application
further includes instructions for using the kit. A kit described in
this application may also include information as required by a
regulatory agency such as the U.S. Food and Drug Administration
(FDA). In certain embodiments, the information included in the kits
is prescribing information. In certain embodiments, the kits and
instructions provide for treating a disease in a subject in need
thereof. In certain embodiments, the kits and instructions provide
for preventing a disease in a subject in need thereof. In certain
embodiments, the kits and instructions provide for reducing the
risk of developing a disease in a subject in need thereof. A kit
described in this application may include one or more additional
pharmaceutical agents described in this application as a separate
composition.
[0397] The present invention is further illustrated by the
following Examples, which in no way should be construed as
limiting. The entire contents of all of the references (including
literature references, issued patents, published patent
applications, and co-pending patent applications) cited throughout
this application are hereby expressly incorporated by reference. If
a reference incorporated in this application contains a term whose
definition is incongruous or incompatible with the definition of
same term as defined in the present disclosure, the meaning
ascribed to the term in this disclosure shall govern. However,
mention of any reference, article, publication, patent, patent
publication, and patent application cited in this application is
not, and should not be taken as an acknowledgment or any form of
suggestion that they constitute valid prior art or form part of the
common general knowledge in any country in the world.
EXAMPLES
Example 1: Functional Expression of AAE Genes in E. coli and S.
cerevisiae
[0398] It was reported previously that S. cerevisiae has endogenous
AAE activity that allows conversion of hexanoate to hexanoyl-CoA
(Gagne et al. 2012). However, in some embodiments, the endogenous
AAE activity of S. cerevisiae may be insufficient for
industrial-scale synthesis of downstream products. This example
validates novel genes with AAE activity that can be used in the
cells, reactions, and methods of the present disclosure.
[0399] Several DNA sequences with predicted AAE functionality were
identified from the genomes of the yeast Yarrowia lipolytica (Y.
lipolytica) and the bacterium Rhodopseudomonas palustris (R.
palustris). The predicted AAE genes were first codon-optimized in
silico for expression in E. coli. The codon-optimized gene
sequences were synthesized via standard DNA synthesis techniques
and were expressed in recombinant E. coli host cells (FIG. 4).
Lysates from the recombinant E. coli host cells were then tested
for AAE activity using an assay described below.
[0400] FIG. 4 shows the results from the AAE activity assay in E.
coli host cells. 3 out of 4 predicted Y. lipolytica AAEs (strains
t49578, t49594, and t51477) and both of the predicted R. palustris
AAEs (strains t55127 and t55128) exhibited activity on a hexanoate
substrate. Strains t49594 and t51477 expressed the candidate AAE
enzyme as a fusion protein with an N-terminal MYC tag. In addition,
the assays also showed that 2 out of 4 predicted Y. lipolytica AAEs
(strains t49594 and t51477) and both of the predicted R. palustris
AAEs (strains t55127 and t55128) also demonstrated activity on a
butyrate substrate.
[0401] The newly described AAEs were also found to be capable of
exhibiting AAE activity in eukaryotes. Briefly, the Y. lipolytica
AAE that produced the best results in E. coli host cells was
selected. This corresponded to the AAE expressed by strain t49594
(which encodes a protein corresponding to the protein provided by
Uniprot Accession No. Q6C577 with a N-terminal MYC tag). The gene
encoding this AAE was codon-optimized for expression in S.
cerevisiae, and the last three residues (peroxisomal targeting
signal 1) were removed. Two different codon-optimized versions of
this AAE were synthesized in the replicative yeast expression
vector shown in FIGS. 5A-5B. The recoded sequences only shared
81.66% sequence identity at the DNA level, while encoding for the
same polypeptide. Both AAE expression constructs were then
transformed into a CEN.PK S. cerevisiae strain, and transformants
were selected based on ability to grow on media lacking uracil. The
transformants were tested for AAE activity with a colorimetric AAE
assay (described below). An S. cerevisiae strain expressing GFP was
used as a negative control (strain t390338).
[0402] The results from the colorimetric AAE assay are shown in
FIG. 6. Both of the codon-optimized versions of the Y. lipolytica
AAE (strains t392878 and t392879) exhibited AAE activity on a
hexanoate substrate, demonstrating that the newly disclosed AAEs
could also be used in eukaryotic hosts. These enzymes were thus
demonstrated to be capable of catalyzing the first enzymatic step
in microbial production of cannabinoids from carboxylic acids.
[0403] This Example demonstrates identification of AAEs that are
capable of using hexanoate and butyrate as substrates to produce
cannabinoid precursors. Detailed results for the AAE activity
experiments in E. coli host cells are provided below in Table 3.
Sequence information for strains described in this Example are
provided in Table 4 at the end of the Examples section.
TABLE-US-00018 TABLE 3 Activity of AAE Enzymes on Hexanoate and
Butyrate in E. Coli Standard Standard Average Deviation Average
Deviation Strain Activity on Activity on Activity on Activity on
(E. coli) Hexanoate Hexanoate Butyrate Butyrate t49568 -0.0495
0.014849 -0.0105 0.04879 (Negative control) t49578 0.222 0.016971
-0.0195 0.010607 t49580 -0.065 0.019799 -0.055 0.007071 t49594
0.458 0.005657 0.3895 0.000707 t51477 0.347 0.005657 0.046 0.005657
t55127 0.395 0.011314 0.1835 0.024749 t55128 0.2495 0.012021 0.2205
0.012021
Materials and Methods
[0404] AAE Assay for E. coli
[0405] E. coli BL21 strains harboring a plasmid that contained AAE
genes driven by a T7 promoter were inoculated from glycerol stocks
into shake flasks with 25 mL LB and grown overnight at 37.degree.
C. with shaking at 250 RPM. The next day, strains were inoculated
1% (v/v) into LB and grown for 3-6 hours until an OD600 of -0.6 was
attained. They were then induced with 1 mM IPTG and incubated
overnight at 23.degree. C. with shaking at 250 RPM. The next day,
the cultures were harvested and pelleted. Cell pellets were lysed
with BugBuster.TM. reagent (5 mL per g wet pellet) at 18.degree. C.
and shaken at 250 RPM for 20 min. Lysates were centrifuged at
4.degree. C. and 4000 RPM for 20 min. The soluble fractions of the
lysates were taken for the enzyme assay. The enzyme assay mixture
contained 5 mM substrate (sodium hexanoate or sodium butyrate), 3
mM ATP, 1 mM CoA, 5 mM MgCl.sub.2, and 100 mM HEPES (pH 7.5). 25
.mu.L of E. coli lysates were added to 500 .mu.L of assay mixture
and allowed to react at 30.degree. C., 250 RPM for 20 min. Assays
were then quenched by adding 50 .mu.L of the reaction to 50 .mu.L
of 2 mM DTNB. Absorbance was measured at 412 nm to quantify the
decrease in free CoA.
AAE Assay for S. cerevisiae
[0406] 5 .mu.L/well of thawed glycerol stocks were stamped into 300
.mu.L/well of SC-URA+4% dextrose in half-height deepwell plates,
which were sealed with AeraSeal.TM. film. Samples were incubated at
30.degree. C. and shaken at 1000 RPM in 80% humidity for 2 days. 10
.mu.L/well of resulting precultures were stamped into 300
.mu.L/well of SC-URA+4% dextrose in half-height deepwell plates,
which were sealed with AeraSeal.TM. film. Samples were incubated at
30.degree. C. and shaken at 1000 RPM in 80% humidity for 3 days. 10
.mu.L of resulting production cultures were stamped into 140
.mu.L/well PBS in flat bottom plates. Optical measurements were
taken on a plate reader, with absorbance measured at 600 nm and
fluorescence at 528 nm with 485 mn excitation.
[0407] Production culture plates were centrifuged at 4000 RPM for
10 min. Supernatant was removed, and the plates of pellets were
heat-sealed and frozen at -80.degree. C.
[0408] Pellets were thawed and 200 .mu.L Y-PER per well was added.
Samples were agitated at room temperature for 20 minutes and then
pelleted at 3500 RPM for 10 minutes. 50 .mu.L of the clarified
lysate was combined with 50 .mu.L of feed buffer or CoA standard in
clear bottom plates. Plates were then incubated at 30.degree. C.
and shaken at 1000 RPM in 80% humidity for 60 min. 1 .mu.L of DTNB
buffer was added to each well to a final concentration of 100 .mu.M
DTNB, and samples were agitated at room temperature for 15 minutes.
Absorbance was measured at 412 nm to quantify the decrease in free
CoA.
[0409] Materials included: [0410] Feed Buffers: [0411] 10 mM
MgCl.sub.2 [0412] 1 mM sodium hexanoate [0413] 0.5 mM CoA [0414] 1
mM ATP [0415] 100 mM Tris HCl pH 7.6 [0416] DTNB Buffer: [0417] 10
mM DTNB (Sigma D8130) (stock of DTNB in DMSO) [0418] 100 mM
Tris-HCl pH7.6 (Teknova) [0419] Y-PER Yeast Protein Extraction
Reagent (Thermo 78990): [0420] +1 tablet/50 mL complete, EDTA-free,
protease inhibitors (Sigma, 11873580001) [0421] Coenzyme A
trilithium salt (Sigma C3019)
Example 2: Functional Expression of OLS Genes in S. cerevisiae
[0422] Functional expression of C. sativa olivetol synthase (OLS)
and olivetolic acid cyclase (OAC) enzymes in S. cerevisiae was
previously reported (Gagne et al. 2012). To identify other OLS
genes that can be functionally expressed, a library of
approximately 2000 OLS candidate genes was designed. The genes
within the library were codon-optimized for expression in S.
cerevisiae and synthesized in the replicative yeast expression
vector shown in FIGS. 5A-5B. Each candidate OLS was transformed
into an auxotrophic S. cerevisiae CEN.PK GAL80 knockout strain, and
transformants were selected based on ability to grow on media
lacking uracil. The transformants were tested for olivetol and
olivetolic acid production from sodium hexanoate in vivo in a
high-throughput primary screen, as described in the materials and
methods section below. Top olivetol and/or olivetolic
acid-producing strains that were identified in the primary screen
were subsequently tested in a secondary screen to verify and
further quantify olivetol and olivetolic acid production.
[0423] Numerous yeast transformants were observed to be capable of
producing olivetol in the primary screen (FIG. 8). In particular,
two of the top olivetol-producing strains were strain t395094 and
strain t393991 (FIG. 8). These two strains were also found to be
among the top olivetol-producing strains in the secondary
screen.
[0424] When the OLS library described in this Example was designed
and screened in the primary and secondary screens, it was expected
that the strains expressed full-length candidate OLS enzymes.
Specifically, strain t395094 was believed to express a full-length
OLS protein from Araucaria cunninghamii (Hoop pine) (corresponding
to Uniprot Accession No. A0A0D6QTX3) and strain t393991 was
believed to express a full-length OLS protein from Cymbidium hybrid
cultivar (corresponding to Uniprot Accession No. A0A088G5Z5).
However, as explained further in Table 5 at the end of the Examples
section, sequencing analysis of strains from the OLS library used
for these screens later revealed that there was a 6-nucleotide
deletion in the sequences of many of the genes encoding the OLS
enzymes in the library. Specifically, this deletion affected all of
the candidate OLSs expressed by the strains identified in FIGS.
8-10, including the candidate OLSs expressed by strains t395094 and
t393991 (Table 5).
[0425] The 6-nucleotide deletion included the first two nucleotides
within the start codon of the genes encoding the OLS enzymes. As
one of ordinary skill in the art would appreciate, such a deletion
may result in the truncation of one or more amino acids from the
N-terminus of the proteins encoded by the affected genes, and such
a deletion could potentially extend to the next in-frame methionine
residue in the intended protein sequence. For example, strain
t393991 expressed a truncated version of a codon-optimized nucleic
acid encoding an OLS protein from Cymbidium hybrid cultivar (Table
5). The full-length Cymbidium hybrid cultivar protein corresponds
to SEQ ID NO: 7. If the deletion in the nucleic acid encoding this
OLS protein were to result in translation commencing from the next
start codon within the same reading frame, this would result in an
N-terminally truncated version of the full-length OLS protein from
Cymbidium hybrid cultivar. A protein sequence for a truncated
protein that commences from the next start codon within the same
reading frame is provided by SEQ ID NO: 714. SEQ ID NO: 714 has a
truncation of the first 86 amino acids of SEQ ID NO: 7 and is
approximately 77.9% identical to SEQ ID NO: 7.
[0426] Due to the truncation of OLS candidate genes within the
library, candidate OLS genes screened in this Example were
independently screened again using a new library that expressed
only full-length OLS genes (Example 3). As discussed in Example 3,
screening with a full-length OLS library independently identified
both the OLS protein from Araucaria cunninghamii (Hoop pine)
(corresponding to Uniprot Accession No. A0A0D6QTX3) and the OLS
protein from Cymbidium hybrid cultivar (corresponding to Uniprot
Accession No. A0A088G5Z5), discussed above, verifying the
identification of these candidate OLSs as being highly effective
for olivetol production in recombinant host cells.
[0427] It was determined that the OLS enzymes expressed by positive
control strains t339579 and t339582, depicted in FIGS. 8-10, were
also affected by the 6-nucleotide deletion discussed above.
Accordingly, the low amounts of olivetol and olivetolic acid
produced by the strains labelled as positive controls in FIGS. 8-10
may have been caused by disrupted expression of these proteins due
to truncation.
Identification of Bifunctional PKS-PKC Enzymes
[0428] It was previously observed that S. cerevisiae possesses
native OAC activity which enables some amount of the OLS product,
3,5,7-trioxododecanoyl-CoA, to be converted to olivetolic acid
instead of undergoing a spontaneous decarboxylative cyclization to
olivetol in the absence of OAC activity (FIG. 1). Most strains
tested in the primary screen were observed to produce a constant
(i.e., fixed) ratio of olivetolic acid to olivetol (FIG. 10).
Without wishing to be bound by any theory, the accumulation of
olivetolic acid and olivetol in these strains may be due to the
reported endogenous S. cerevisiae OAC activity competing with
spontaneous conversion of 3,5,7-trioxododecanoyl-CoA to olivetol.
Both products, olivetol and olivetolic acid, increase
proportionally with their shared precursor.
[0429] However, multiple strains were identified in the primary
screen that demonstrated olivetolic acid production outside of the
constant olivetolic acid to olivetol ratio discussed above. Strain
t393974 demonstrated the highest olivetolic acid production (FIG.
9). In particular, strain t393974 was observed to produce
substantially more olivetolic acid than olivetol in the primary
screen a quantity of olivetolic acid that was outside of the fixed
ratio exhibited by other tested strains (FIG. 10). These data
suggested that the OLS enzyme expressed by strain t393974 may be a
bifunctional enzyme possessing both polyketide synthase and
polyketide cyclase catalytic functions and may be capable of
catalyzing both reactions R2 and R3 in FIG. 2, and, at least, both
reactions R2a and R3a in FIG. 1 ("Bifunctional PKS-PKC").
[0430] As discussed above, when the OLS library described in this
Example was designed and screened in the primary and secondary
screens, it was expected that the strains expressed full-length
candidate OLS enzymes. Specifically, strain t393974 was believed to
express a full-length OLS protein from Corchorus olitorius (Jute)
(corresponding to UniProt Accession No. A0A1R3HSU5). However, as
discussed above and explained further in Table 5 at the end of the
Examples section, sequencing analysis of strains from the OLS
library used for these screens later revealed that there was a
6-nucleotide deletion in the sequences of many of the genes
encoding the OLS enzymes in the library, which affected all of the
candidate OLSs expressed by the strains identified in FIGS. 8-10,
including the candidate OLS expressed by strain t393974 (Table 5).
Accordingly, strain t393974 expressed a truncated version of a
codon-optimized nucleic acid encoding an OLS protein from Corchorus
olitorius (Jute) (Table 5). The full-length Corchorus olitorius
(Jute) protein corresponds to SEQ ID NO: 6.
[0431] Due to the sequence truncation of OLS candidate genes within
the library, candidate OLS genes screened in this Example were
independently screened again using a new library that expressed
only full-length OLS genes (Example 3). As discussed in Example 3,
screening with a full-length OLS library also independently
identified the candidate OLS protein from Corchorus olitorius
(Jute) (corresponding to UniProt Accession No. A0A1R3HSU5; SEQ ID
NO: 6) as an OLS that produced both olivetol and olivetolic
acid.
Materials and Methods
OLS Assay
[0432] A library of approximately 2000 OLS enzymes was transformed
into S. cerevisiae. 5 .mu.L/well of thawed glycerol stocks were
stamped into 300 .mu.L/well of SC-URA+4% dextrose in half-height
deepwell plates, which were sealed with AeraSeal.TM. films. Samples
were incubated at 30.degree. C. and shaken at 1000 RPM in 80%
humidity for 2 days. 10 .mu.L/well of the resulting precultures
were stamped into 300 .mu.L/well of SC-URA+4% Dextrose+1 mM sodium
hexanoate in half-height deepwell plates, which were sealed with
AeraSeal.TM. films. Samples were incubated at 30.degree. C. and
shaken at 1000 RPM in 80% humidity for 4 days. 10 .mu.L/well of the
resulting production cultures were stamped into 140 .mu.L/well PBS
in flat bottom plates. Optical measurements were taken on a plate
reader, with absorbance measured at 600 nm and fluorescence at 528
nm with 485 mn excitation.
[0433] 30 .mu.L/well of production cultures were stamped into 270
.mu.L/well of 100% methanol containing 300 .mu.g/L
3-(3-Hydroxypropyl)phenol (3HPP) in half-height deepwell plates.
Plates were heat sealed and frozen at -80.degree. C. for two hours.
Plates were then thawed for 30 minutes and spun down at 4.degree.
C. at 4000 rpm for 10 min. 75 .mu.L of supernatant from each well
of each plate was stamped into Corning 3694 (half area) plates,
which were then submitted for LC-MS quantification of olivetol and
olivetolic acid.
[0434] The experimental protocol for the secondary screen was the
same as described above, except that four replicates per strain
were tested and standard curves of both olivetol and olivetolic
acid were prepared so that both products could be quantified.
Example 3: Generation and Screening of Full-Length OLS Library
[0435] As discussed in Example 2, sequencing analysis of strains
from the OLS library used for the screening described in Example 2
revealed that many of the genes encoding the OLS enzymes in the
library were inadvertently truncated N-terminally. This truncation
affected all of the strains identified in FIGS. 8-10, including the
positive control strains.
[0436] Accordingly, a new OLS library was generated to contain only
OLS genes that produce full-length OLS enzymes. The full-length OLS
library contained full-length versions of the approximately 2000
OLS enzymes from the original library described in Example 2 and
also included approximately 900 additional candidate OLS enzymes.
All candidate OLS enzymes were codon optimized for expression in S.
cerevisiae. Strain t527340, comprising an OLS from C. sativa, was
included in the library as a positive control, and strain t527338,
comprising GFP, was included in the library as a negative control.
A high-throughput primary screen was conducted with the full-length
OLS library using the same OLS assay described in Example 2 with
the following exceptions: all library members and controls were
transformed into an auxotrophic CEN.PK strain that comprised a
chromosomally integrated heterologous gene encoding AAE VcsA
(Uniprot Accession Q6N4N8) from R. palustris; and neither sodium
hexanoate nor sodium butyrate were included in the production
cultures.
[0437] Top olivetol and/or olivetolic acid-producing strains from
the high-throughput primary screen were carried over to a secondary
screen to verify and further quantify olivetol production. The
experimental protocol for the secondary screen was the same as the
primary screen except that: four replicates per strain were tested;
and one set of cultures was supplemented with 1 mM sodium
hexanoate, while the second set of cultures was not supplemented
with sodium hexanoate. Olivetol production was normalized to a
positive control strain expressing a C. Sativa OLS. Table 7
provides results for strains that exhibited average normalized
olivetol >1 and/or that produced higher amounts of olivetolic
acid than the positive control in samples that were supplemented
with sodium hexanoate. FIG. 12A depicts olivetol production in
library strains supplemented with sodium hexanoate. FIG. 12B
depicts olivetolic acid production in library strains supplemented
with sodium hexanoate. Table 8 provides results for strains that
exhibited average normalized olivetol >1 and/or that produced
higher amounts of olivetolic acid than the positive control in
samples that were not supplemented with sodium hexanoate. Table 6
provides sequence information for strains described in Tables 7 and
8. The Average Normalized Olivetol for each strain was calculated
by taking the mean of the following ratio for each replicate of
that strain: the ratio of olivetol to absorbance measured at 600 nm
to average olivetol produced by the C. sativa OLS positive control
in the same plate.
[0438] One of the top olivetol-producing strains identified in this
Example was strain t527346, comprising an OLS enzyme from Cymbidium
hybrid cultivar (Accession ID: A0A088G5Z5; SEQ ID NO: 7; Tables
6-8). As discussed above, this candidate OLS was also identified in
the screening conducted in Example 2. Protein alignments conducted
with BLASTP using default parameters identified two other OLS
candidates that shared at least 90% identity with the OLS enzyme
from Cymbidium hybrid cultivar (Accession ID: A0A088G5Z5; SEQ ID
NO: 7). These strains were: strain t598916 (expressing an OLS
corresponding to SEQ ID NO: 145) and strain t599231 (expressing an
OLS corresponding to SEQ ID NO: 15) (Tables 6-8), which were 93.07%
and 91.79%, identical to SEQ ID NO: 7, respectively.
[0439] Another notable olivetol-producing strain identified in this
Example was strain t599285, comprising an OLS enzyme from Araucaria
cunninghamii (Accession A0A0D6QTX3; SEQ ID NO: 17). As discussed
above, this candidate OLS was also identified in the screening
conducted in Example 2.
[0440] Consistent with the identification in Example 2 of a
candidate OLS from Corchorus olitorius (Jute) as a potentially
bifunctional OLS, strain t598084, comprising a full-length version
of the Corchorus olitorius (Jute) OLS (corresponding to UniProt
Accession No. A0A1R3HSU5; SEQ ID NO: 6) was independently
identified in this Example as a candidate OLS that produced more
olivetolic acid than a Cannabis OLS positive control, and produced
more olivetolic acid than olivetol, based on average olivetol and
olivetolic acid produced (Tables 6-8).
Example 4: Generation and Screening of C. sativa OLS Protein
Engineering Library in S. cerevisiae
[0441] To identify C. sativa OLS (CsOLS) enzyme variants with
improved olivetol production, a library comprised of approximately
1300 members was designed. The library included CsOLS enzymes
containing single or multiple amino acid substitutions or
deletions. Nucleotide sequences were codon-optimized for expression
in S. cerevisiae and synthesized in the replicative yeast
expression vector shown in FIGS. 5A-5B. Each candidate enzyme
expression construct was transformed into an auxotrophic S.
cerevisiae CEN.PK GAL80 knockout strain. Transformants were
selected based on ability to grow on media lacking uracil. Strain
t346317, carrying GFP, was included in the library as a negative
control.
[0442] The library of candidate CsOLS enzyme variants was assayed
for activity in a high-throughput primary screen using the OLS
assay described in Example 2 (FIG. 13). LC-MS analysis revealed
that approximately 95% of library members produced measurable
amounts of olivetol.
[0443] The top olivetol and/or olivetolic acid-producing strains
from the primary screen were carried over to a secondary screen to
verify the results. The experimental protocol for the secondary
screen was the same as the primary screen, except that four
replicates per strain were tested and a standard curve for olivetol
and olivetolic acid was generated so that the amount of olivetol
and olivetolic acid could be quantified via LC-MS (FIG. 13.)
[0444] Multiple OLS variants were identified that were capable of
producing olivetol (Table 9). In order to investigate where the
point mutations in the OLS variants were located relative to the
OLS enzyme structure, a 3D model of the wildtype C. sativa OLS
protein (corresponding to SEQ ID NO: 5) was generated using Rosetta
protein modeling software. The active site of the C. sativa OLS
enzyme was identified based on the catalytic triad of residues
described in Taura et al. (2009) FEBS Letters for OLS enzymes,
consisting of residues H297, C157, and N330 in the C. sativa OLS
enzyme. The active site was also defined to include a docked
molecule of hexanoyl-CoA (OLS substrate). Residues were considered
to be within the active site if they were within about 12 angstroms
of any of the residues within the catalytic triad of the OLS enzyme
and/or within about 12 angstroms of a docked substrate within the
OLS enzyme.
[0445] A subset of OLS point mutations was identified that included
strains that produced at least 10 mg/L olivetol and mapped to
within the active site. This group of point mutations included:
T17K, I23C, L25R, K51R, D54R, F64Y, V95A, T123C, A125S, Y153G,
E196K, L201C, I207L, L241I, T247A, M267K, M267G, I273V, L277M,
T296A, V307I, D320A, V324I, S326R, H328Y, S334P, S334A, T335C,
R375T (Table 9). FIG. 17 provides a schematic of the 3D structure
of the C. Sativa OLS protein (corresponding to SEQ ID NO: 5),
showing the catalytic triad, the bound hexanoyl-CoA substrate, and
the cluster of point mutations identified within the active
site.
[0446] OLS point mutations from strains that produced at least 10
mg/L olivetol and mapped to within about 8 angstroms of any of the
residues within the catalytic triad of the OLS enzyme and/or within
about 8 angstroms of a docked substrate within the OLS included:
K51R, D54R, T123C, A125S, L201C, I207L, L241I, T247A, M267K, M267G,
I273V, T296A, V307I, V324I, S326R, H328Y, S334P, T335C, and R375T.
FIG. 16 provides a schematic of the 3D structure of the C. Sativa
OLS protein (corresponding to SEQ ID NO: 5), showing the catalytic
triad, the bound hexanoyl-CoA substrate, and the cluster of point
mutations identified within about 8 angstroms of any of the
residues within the catalytic triad of the OLS enzyme and/or within
about 8 angstroms of a docked substrate within the PKS.
[0447] The point mutation that was found to be associated with the
most olivetol production was a T335C mutation in the C. sativa OLS
sequence (Table 9). This residue maps to the active site of the OLS
enzyme (FIGS. 16-17). In further support of the importance of this
residue for olivetol production, at least 5 of the high-producing
olivetol candidate OLSs identified in Example 3 contain a C residue
at this position (strain IDs t527346 (SEQ ID NO: 7), t598265 (SEQ
ID NO: 13), t598301 (SEQ ID NO: 7), t598916 (SEQ ID NO: 145),
t598976 (SEQ ID NO: 8), t599231 (SEQ ID NO: 15)). Strains t527346
and t598301 comprise an OLS that has the same amino acid sequence
but the OLS is encoded by different nucleic acid sequences.
[0448] Additional C. sativa OLS variants were identified that did
not map within the active site, but which were observed to produce
more than approximately 13 mg/L olivetol (Table 9). This group of
point mutations included: I284Y, K100L, K116R, 1278E, K108D, L348S,
K71R, V92G, T128V, K100M, Y135V, P229A, T128A, T128I (Table 9).
[0449] Table 13 provides sequence information for strains described
in Table 9.
[0450] Thus, novel variants of the C. sativa OLS protein that may
be useful for olivetol production in recombinant host cells were
identified.
Example 5. Generation and Screening of C. sativa OLS (CsOLS),
Cymbidium OLS (ChOLS), and Corchorus OLS (CoOLS) Protein
Engineering Libraries in S. cerevisiae
[0451] An additional OLS protein engineering library was generated
that included OLS variants based on three different OLS templates:
C. sativa OLS (CsOLS); Cymbidium hybrid cultivar OLS (ChOLS) and
Corchorus olitorius OLS (CoOLS), which were among the candidate
OLSs identified in the screens described in Examples 2 and 3. As
discussed above, ChOLS was identified as being one of the strongest
olivetol-producing candidate OLS enzymes, while CoOLS was
identified as being a potential bifunctional enzyme possessing both
polyketide synthase and polyketide cyclase catalytic functions.
[0452] The library included approximately 300 variants of ChOLS and
approximately 200 variants of CoOLS. Variants of ChOLS and CoOLS
included both single and multiple amino acid substitutions. For
ChOLS, some of the variants were designed by taking beneficial
mutations discovered from screening of CsOLS variants described in
Example 4 and mapping the corresponding mutations onto the ChOLS
template. Corresponding positions in ChOLS were identified and
mutated.
[0453] For CoOLS, some of the variants were designed to investigate
whether there were any specific residues that may contribute to
conferring or enhancing bifunctionality. The sequence of the
bifunctional CoOLS enzyme (SEQ ID NO: 6) was aligned with the
sequence of the CsOLS enzyme (SEQ ID NO: 5), which is not
bifunctional, and residues that are different between the sequences
were considered for mutagenesis in both the CsOLS and CoOLS
sequences. The impact of these mutations on bifunctionality was
investigated by measuring the ratio of production of olivetolic
acid to olivetol. A specific residue that was investigated with
respect to bifunctionality was residue W339 in CoOLS, which
corresponds to residue S332 in CsOLS.
[0454] Nucleotide sequences of the genes within the library were
codon-optimized for expression in S. cerevisiae and synthesized in
the replicative yeast expression vector shown in FIGS. 5A-5B. Each
candidate OLS expression construct was transformed into a S.
cerevisiae CEN.PK strain expressing a heterologous AAE VcsA-Q6N4N8
from R. palustris. The library was screened in a high-throughput
primary screen in which the OLS assay was conducted as described in
Example 2, except that production cultures were not supplemented
with either sodium hexanoate or sodium butyrate. Instead the
strains' natural pools of hexanoyl-CoA and butyryl-CoA were used as
substrates. Top olivetol and/or olivetolic acid producing strains
were carried over to a secondary screen to verify production of
olivetol and/or olivetolic acid. The experimental protocol for the
secondary screen was identical to the primary screen, except that
four replicates per strain were tested; and olivetol production was
assessed both in the context of production cultures being
supplemented with sodium hexanoate and in the context of production
cultures that were not being supplemented with sodium
hexanoate.
[0455] Strain t527338, expressing a fluorescent protein, was
included in the library as a negative control for enzyme activity.
Strain t527340, expressing wild-type CsOLS, was included in the
library as a positive control. Strain t527346, expressing wild-type
ChOLS, was included in the library as a positive control and was
used to establish hit ranking for variants designed using ChOLS as
a template. Similarly, strain t606797, expressing wild-type CoOLS,
was included in the library as a positive control and was used to
establish hit ranking for variants designed using CoOLS as a
template. Olivetol was normalized to the mean production of its
wild-type template (e.g., olivetol produced by a variant of ChOLS
was normalized to the mean olivetol titer produced by strain
t527346) except that for variants made to the CoOLS template,
olivetol was normalized against each of a CsOLS template and a
CoOLS template due to inconsistent activity of the CoOLS wild type
control (Tables 10A-B and 11A-B). The Average Normalized Olivetol
value for wild-type templates was not necessarily 1.0. For example,
for the wild-type C. sativa strain t527340 in Table 10B, the
Average Normalized Olivetol value was 1.02159. This was because the
mean by which values were normalized was based on library controls
that were included on each plate within a screen (e.g., strain
t527340). The library further contained additional in-library
controls of the same strain (e.g., strain t527340). Those
additional in-library controls were not used to calculate the mean.
In instances where the average normalized olivetol values for all
samples of strain t527340 were calculated, if the in-library
controls produced slightly more olivetol than the mean olivetol
produced by the library controls that were included on each plate,
then the Average Normalized Olivetol value was slightly above
1.0.
[0456] Results from the secondary screen are provided in Tables
10-11. Table 10 provides results for samples that were supplemented
with sodium hexanoate, while Table 11 provides results for samples
that were not supplemented with sodium hexanoate. In Table 10,
strains comprising ChOLS mutants that produced an average
normalized olivetol level of at least 0.5 are shown. The
performance of multi-mutation ChOLS enzymes are also shown.
[0457] For ChOLS, the approach of mapping equivalent variants from
the CsOLS sequence led to the identification of multiple variants
that exhibited improved olivetol production. These variants
included the following point mutations: V71Y, F70M, L385M, E285A,
L76I, N151P, E203K, V50N, S34Q, R100P, A219C, K359M, and R100T
(Table 11). Several additional variants exhibited improved olivetol
production in samples that were supplemented with sodium hexanoate.
These variants included the following point mutations: V71Y and
F70M (Table 10).
[0458] For CoOLS, when the mutation W339S was made to the CoOLS
template (strain t607112), the ratio of olivetolic acid to olivetol
decreased, from approximately 1.5 to approximately 0.157 (based on
517.075 ug/L olivetol and 81.225 ug/L olivetolic acid, as shown in
Table 10A). However, olivetol levels reported in Table 10A for
strain t607112 were within the standard deviation. Accordingly,
while the mutation may have had an impact on bifunctionality, it
also appears to have more generally affected overall functionality
of the enzyme. The reverse mutation was also tested in CsOLS. For
CsOLS, S332W (strain t606899) had a significantly negative impact
on the function of the enzyme (Table 10A). Similarly, mutation
S339W in ChOLS (strain t607377) had a significantly negative impact
on the overall function of the enzyme (Table 10A).
Example 6. Functional Expression of OLS Enzymes in a Prototrophic
S. cerevisiae Strain
[0459] Examples 2-5 utilized an auxotrophic S. cerevisiae CEN.PK
strain as a host chassis for the expression of OLS enzyme
candidates from a replicative plasmid. OLS candidate enzymes
determined to be active in Examples 2-5 were also assessed in a
prototrophic S. cerevisiae CEN.PK strain.
[0460] A library of approximately 58 OLS genes under the control of
the same genetic regulatory elements shown in FIGS. 5A-5B (GAL1
promoter and CYC1 terminator) were integrated into the genome of a
prototrophic S. cerevisiae CEN.PK strain. The parental chassis
strain t473139, not expressing a heterologous OLS enzyme, was
included as a negative control for enzyme activity. Strain t496084,
expressing the CsOLS T335C point-mutant, which was the highest
ranking CsOLS point mutant identified in Example 5 based on
production of olivetol, was also included. The OLS assay was
conducted as described in Example 2 with the following exceptions:
glycerol stocks were stamped into YEP+4% glucose; a portion of the
resulting cultures were then stamped into production cultures
containing YEP+4% glucose+1 mM sodium hexanoate; and three
bio-replicates were used instead of two.
[0461] Despite differences between auxotrophic and prototrophic
strains that may impact production of olivetol, candidate OLS
enzymes identified in Examples 2-5 through screening in auxotrophic
strains were also found to be effective in production of olivetol
in a prototrophic strain (Table 12 and FIG. 15). As shown in Table
12, strain t496073, corresponding to a prototrophic S. cerevisiae
strain comprising a chromosomally integrated, codon-optimized
nucleotide sequence encoding the OLS candidate from Cymbidium
hybrid cultivar (Accession ID: A0A088G5Z5), which was identified in
Examples 2 and 3, produced the highest olivetol titer of any
library member and significantly more olivetol than the C. sativa
control (FIG. 15 and Table 12).
[0462] Thus, novel candidate OLS enzymes identified in Examples 2-5
were found to be effective for olivetol production when expressed
in prototrophic strains as well as auxotrophic strains.
Example 7. Biosynthesis of Cannabinoids in Engineered S. cerevisiae
Host Cells
[0463] The activation of an organic acid to its CoA-thioester and
the subsequent condensation of this thioester with a number of
malonyl-CoA molecules, or other similar polyketide extender units,
represent the first two steps in the biosynthesis of all known
cannabinoids. To demonstrate the biosynthesis of CBGA (FIG. 1,
Formula (8.alpha.)), CBDA (FIG. 1, Formula (9a)), THCA (FIG. 1,
Formula (10.alpha.)), and CBCA (FIG. 1, Formula (IIa)) the
cannabinoid biosynthetic pathway shown in FIG. 1 is assembled in
the genome of a prototrophic S. cerevisiae CEN.PK host cell wherein
each enzyme (R1a-R5a) may be present in one or more copies. For
example, the S. cerevisiae host cell may express one or more copies
of one or more of: an AAE, an OLS, an OAC, a CBGAS, and a TS.
[0464] An AAE enzyme expressed heterologously in a host cell may be
one or more of the AAE candidates from Y. lipolytica or R.
palustris that are shown in Example 1 to be functionally expressed
in S. cerevisiae. An OLS enzyme expressed heterologously in a host
cell may be an OLS identified and characterized in Examples 2-8,
such as a Cymbidium hybrid cultivar OLS (SEQ ID NO: 7) or a
Phalaenopsis.times.Doritaenopsis hybrid cultivar OLS (SEQ ID NO:
15), or an OLS corresponding to SEQ ID NO: 145. The OLS enzyme may
also be an engineered OLS such as CsOLS T335C (SEQ ID NO: 207) or
an engineered version of any other OLS enzyme described in this
disclosure. An OAC enzyme expressed heterologously in a host cell
may be a naturally occurring or synthetic OAC that is functionally
expressed in S. cerevisiae, or a variant thereof, including an OAC
from C. sativa or a variant of an OAC from C. sativa. In instances
where a bifunctional OLS, such as Corchorus olitorius OLS (SEQ ID
NO: 6), is used, a separate OAC enzyme may be omitted.
[0465] A CBGAS enzyme, such as a PT enzyme, expressed
heterologously in a host cell may be a naturally occurring or
synthetic PT that is functionally expressed in S. cerevisiae, or a
variant thereof, including a PT from C. sativa or a variant of a PT
from C. sativa. For example, a PT may comprise CsPT4 from C.
sativa, or a variant thereof, or NphB from Streptomyces sp. Strain
CL190, or a variant thereof.
[0466] A TS enzyme expressed heterologously in a host cell may be a
naturally occurring or synthetic TS that is functionally expressed
in S. cerevisiae, or a variant thereof, including a TS from C.
sativa or a variant of a TS from C. sativa. The TS enzyme may be a
TS that produces one or more of CBDA, THCA, and CBCA as a majority
product.
[0467] The cannabinoid fermentation procedure may be similar to the
OLS assay described in the Examples above with the following
exceptions: the incubation of production cultures may last from,
for example, 48-144 hours, and production cultures may be
supplemented with, for example, 4% galactose and 1 mM sodium
hexanoate approximately every 24 hours. Titers of CBGA, CBDA, THCA,
and CBCA may be quantified via LC-MS.
[0468] It should be appreciated that sequences disclosed herein may
or may not contain signal sequences. The sequences disclosed herein
encompass versions with or without signal sequences. It should also
be understood that protein sequences disclosed herein may be
depicted with or without a start codon (M). Accordingly, in some
instances amino acid numbering may correspond to protein sequences
containing a start codon, while in other instances, amino acid
numbering may correspond to protein sequences that do not contain a
start codon. Aspects of the disclosure encompass host cells
comprising any of the sequences described herein, including the
sequences within Tables 4-6, and 13-16 and fragments thereof.
TABLE-US-00019 TABLE 4 Sequence Information For Strains Described
in Example 1 Strain ID AAE Sequence Information t49578 This strain
comprises a codon-optimized nucleic acid (SEQ ID NO: 70), which
encodes a (E. coli) Yarrowia lipolytica protein (SEQ ID NO: 63).
The protein sequence of SEQ ID NO: 63 corresponds to the protein
sequence provided by UniProt Accession No. Q6CFE4. t49594 This
strain comprises a codon-optimized nucleic acid (SEQ ID NO: 71),
which encodes a (E. coli) Yarrowia lipolytica protein (SEQ ID NO:
64). The protein sequence of SEQ ID NO: 64 corresponds to the
protein sequence provided by UniProt Accession No. Q6C577. This
protein was expressed as a fusion protein with an N-terminal MYC
tag (SEQ ID NO: 140). SEQ ID NO: 707 corresponds to the fusion
protein. SEQ ID NO: 712 is a codon-optimized nucleic acid encoding
SEQ ID NO: 707. t51477 This strain comprises a codon-optimized
nucleic acid (SEQ ID NO: 72), which encodes a (E. coli) Yarrowia
lipolytica protein (SEQ ID NO: 65). The protein sequence of SEQ ID
NO: 65 corresponds to the protein sequence provided by UniProt
Accession No. Q6C650. This protein was expressed as a fusion
protein with an N-terminal MYC tag (SEQ ID NO: 140). SEQ ID NO: 708
corresponds to the fusion protein). SEQ ID NO: 713 is a codon-
optimized nucleic acid encoding SEQ ID NO: 708. t392878 This strain
comprises a codon-optimized nucleic acid (SEQ ID NO: 75), which
encodes a (S. cerevisiae) Yarrowia lipolytica protein (SEQ ID NO:
141). SEQ ID NO: 141 corresponds to residues 1-595 of SEQ ID NO:
68. The protein sequence of (SEQ ID NO: 141) corresponds to the
protein sequence provided by UniProt Accession No. Q6C577 except
that the last three residues (peroxisomal targeting signal 1) were
removed. t392879 This strain comprises a codon-optimized nucleic
acid (SEQ ID NO: 76), which encodes a (S. cerevisiae) Yarrowia
lipolytica protein (SEQ ID NO: 142). SEQ ID NO: 142 corresponds to
residues 1-595 of SEQ ID NO: 69. The protein sequence of (SEQ ID
NO: 142) corresponds to the protein sequence provided by UniProt
Accession No. Q6C577 except that the last three residues
(peroxisomal targeting signal 1) were removed. t55127 This strain
comprises a codon-optimized nucleic acid (SEQ ID NO: 73), which
encodes a (E. coli) Rhodopseudomonas palustris protein (SEQ ID NO:
66). The protein sequence of SEQ ID NO: 66 corresponds to the
protein sequence provided by UniProt Accession No. Q6N948. t55128
This strain comprises a codon-optimized nucleic acid (SEQ ID NO:
74), which encodes a (E. coli) Rhodopseudomonas palustris protein
(SEQ ID NO: 67). The protein sequence of SEQ ID NO: 67 corresponds
to the protein sequence provided by UniProt Accession No. Q6N4N8.
t49580 This strain comprises a codon-optimized nucleic acid (SEQ ID
NO:72), which encodes a (E. coli) Yarrowia lipolytica protein (SEQ
ID NO:65). The protein sequence of SEQ ID NO: 65 corresponds to the
protein sequence provided by UniProt Accession No. Q6C650.
TABLE-US-00020 TABLE 5 Sequence Information for Strains Described
in Example 2 and FIGs. 8-10 Strain ID OLS Sequence Information
t394087 This strain comprises a codon-optimized nucleic acid that
corresponds to nucleotides 3-1182 of SEQ ID NO: 32 (due to a
truncation of nucleotides 1-2 of SEQ ID NO: 32). Translation of
this sequence is expected to produce a truncated version of a
protein corresponding to SEQ ID NO. 1. The protein sequence of SEQ
ID NO: 1 corresponds to the protein sequence provided by UniProt
Accession No. A0A2G5F4L7, from Aquilegia coerulea (Rocky mountain
columbine) t394687 This strain comprises a codon-optimized nucleic
acid that corresponds to nucleotides 3-1179 of SEQ ID NO: 33 (due
to a truncation of nucleotides 1-2 of SEQ ID NO: 33). Translation
of this sequence is expected to produce a truncated version of a
protein corresponding to SEQ ID NO. 2. The protein sequence of SEQ
ID NO: 2 corresponds to the protein sequence provided by UniProt
Accession No. I6VW41, from Vitis pseudoreticulata (Chinese wild
grapevine) t393495 This strain comprises a codon-optimized nucleic
acid that corresponds to nucleotides 3-1185 of SEQ ID NO: 34 (due
to a truncation of nucleotides 1-2 of SEQ ID NO: 34). Translation
of this sequence is expected to produce a truncated version of a
protein corresponding to SEQ ID NO. 3. The protein sequence of SEQ
ID NO: 3 corresponds to the protein sequence provided by UniProt
Accession No. M4DVZ4, from Brassica rapa subsp. pekinensis (Chinese
cabbage) (Brassica pekinensis) t393563 This strain comprises a
codon-optimized nucleic acid that corresponds to nucleotides 3-1197
of SEQ ID NO: 35 (due to a truncation of nucleotides 1-2 of SEQ ID
NO: 35). Translation of this sequence is expected to produce a
truncated version of a protein corresponding to SEQ ID NO. 4. The
protein sequence of SEQ ID NO: 4 corresponds to the protein
sequence provided by UniProt Accession No. Q8VWQ7, from Sorghum
bicolor (Sorghum) (Sorghum vulgare) t339568 This strain comprises a
codon-optimized nucleic acid that corresponds to nucleotides 3-1158
of SEQ ID NO: 36 (due to a truncation of nucleotides 1-2 of SEQ ID
NO: 36). Translation of this sequence is expected to produce a
truncated version of a protein corresponding to SEQ ID NO. 5. The
protein sequence of SEQ ID NO: 5 corresponds to the protein
sequence provided by UniProt Accession No. B1Q2B6, from Cannabis
sativa t393974 This strain comprises a codon-optimized nucleic acid
that corresponds to nucleotides 3-1191 of SEQ ID NO: 37 (due to a
truncation of nucleotides 1-2 of SEQ ID NO: 37). Translation of
this sequence is expected to produce a truncated version of a
protein corresponding to SEQ ID NO. 6. The protein sequence of SEQ
ID NO: 6 corresponds to the protein sequence provided by UniProt
Accession No. A0A1R3HSU5, from Corchorus olitorius t393991 This
strain comprises a codon-optimized nucleic acid that corresponds to
nucleotides 3-1173 of SEQ ID NO: 38 (due to a truncation of
nucleotides 1-2 of SEQ ID NO: 38). Translation of this sequence is
expected to produce a truncated version of a protein corresponding
to SEQ ID NO. 7. The protein sequence of SEQ ID NO: 7 corresponds
to the protein sequence provided by UniProt Accession No.
A0A088G5Z5, from Cymbidium hybrid cultivar t394336 This strain
comprises a codon-optimized nucleic acid that corresponds to
nucleotides 3-1185 of SEQ ID NO: 39 (due to a truncation of
nucleotides 1-2 of SEQ ID NO: 39). Translation of this sequence is
expected to produce a truncated version of a protein corresponding
to SEQ ID NO. 8. The protein sequence of SEQ ID NO: 8 corresponds
to the protein sequence provided by UniProt Accession No.
A0A0A6Z8B1, from Paphiopedilum helenae t394547 This strain
comprises a codon-optimized nucleic acid that corresponds to
nucleotides 3-1185 of SEQ ID NO: 40 (due to a truncation of
nucleotides 1-2 of SEQ ID NO: 40). Translation of this sequence is
expected to produce a truncated version of a protein corresponding
to SEQ ID NO. 9. The protein sequence of SEQ ID NO: 9 corresponds
to the protein sequence provided by UniProt Accession No.
A0A078IM49, from Brassica napus (Rape) t394457 This strain
comprises a codon-optimized nucleic acid that corresponds to
nucleotides 3-1191 of SEQ ID NO: 41 (due to a truncation of
nucleotides 1-2 of SEQ ID NO: 41). Translation of this sequence is
expected to produce a truncated version of a protein corresponding
to SEQ ID NO. 10. The protein sequence of SEQ ID NO: 10 corresponds
to the protein sequence provided by UniProt Accession No.
A0A140KXU1, from Picea jezoensis t394521 This strain comprises a
codon-optimized nucleic acid that corresponds to nucleotides 3-1191
of SEQ ID NO: 42 (due to a truncation of nucleotides 1-2 of SEQ ID
NO: 42). Translation of this sequence is expected to produce a
truncated version of a protein corresponding to SEQ ID NO. 11. The
protein sequence of SEQ ID NO: 11 corresponds to the protein
sequence provided by UniProt Accession No. P48408, from Pinus
strobus (Eastern white pine) t394790 This strain comprises a
codon-optimized nucleic acid that corresponds to nucleotides 3-1170
of SEQ ID NO: 43 (due to a truncation of nucleotides 1-2 of SEQ ID
NO: 43). Translation of this sequence is expected to produce a
truncated version of a protein corresponding to SEQ ID NO. 12. The
protein sequence of SEQ ID NO: 12 corresponds to the protein
sequence provided by UniProt Accession No. I3QQ50, from Arachis
hypogaea (Peanut) t394905 This strain comprises a codon-optimized
nucleic acid that corresponds to nucleotides 3-1296 of SEQ ID NO:
44 (due to a truncation of nucleotides 1-2 of SEQ ID NO: 44).
Translation of this sequence is expected to produce a truncated
version of a protein corresponding to SEQ ID NO. 13. The protein
sequence of SEQ ID NO: 13 corresponds to the protein sequence
provided by UniProt Accession No. A0A1S4ATN2, from Nicotiana
tabacum (Common tobacco) t394981 This strain comprises a
codon-optimized nucleic acid that corresponds to nucleotides 3-1197
of SEQ ID NO: 45 (due to a truncation of nucleotides 1-2 of SEQ ID
NO: 45). Translation of this sequence is expected to produce a
truncated version of a protein corresponding to SEQ ID NO. 14. The
protein sequence of SEQ ID NO: 14 corresponds to the protein
sequence provided by UniProt Accession No. K3Y7T4, from Setaria
italica (Foxtail millet) (Panicum italicum) t395011 This strain
comprises a codon-optimized nucleic acid that corresponds to
nucleotides 3-1173 of SEQ ID NO: 46 (due to a truncation of
nucleotides 1-2 of SEQ ID NO: 46). Translation of this sequence is
expected to produce a truncated version of a protein corresponding
to SEQ ID NO. 15. The protein sequence of SEQ ID NO: 15 corresponds
to the protein sequence provided by UniProt Accession No. Q6WJD6,
from Phalaenopsis x Doritaenopsis hybrid cultivar t394797 This
strain comprises a codon-optimized nucleic acid that corresponds to
nucleotides 3-1170 of SEQ ID NO: 47 (due to a truncation of
nucleotides 1-2 of SEQ ID NO: 47). Translation of this sequence is
expected to produce a truncated version of a protein corresponding
to SEQ ID NO. 16. The protein sequence of SEQ ID NO: 16 corresponds
to the protein sequence provided by UniProt Accession No. K7XD27,
from Arachis hypogaea (Peanut) t395094 This strain comprises a
codon-optimized nucleic acid that corresponds to nucleotides 3-1179
of SEQ ID NO: 48 (due to a truncation of nucleotides 1-2 of SEQ ID
NO: 48). Translation of this sequence is expected to produce a
truncated version of a protein corresponding to SEQ ID NO. 17. The
protein sequence of SEQ ID NO: 17 corresponds to the protein
sequence provided by UniProt Accession No. A0A0D6QTX3, from
Araucaria cunninghamii (Hoop pine) (Moreton Bay pine) t395103 This
strain comprises a codon-optimized nucleic acid that corresponds to
nucleotides 3-1182 of SEQ ID NO: 49 (due to a truncation of
nucleotides 1-2 of SEQ ID NO: 49). Translation of this sequence is
expected to produce a truncated version of a protein corresponding
to SEQ ID NO. 18. The protein sequence of SEQ ID NO: 18 corresponds
to the protein sequence provided by UniProt Accession No. V7AZ15,
from Phaseolus vulgaris (common bean) t393835 This strain comprises
a codon-optimized nucleic acid that corresponds to nucleotides
3-1179 of SEQ ID NO: 50 (due to a truncation of nucleotides 1-2 of
SEQ ID NO: 50). Translation of this sequence is expected to produce
a truncated version of a protein corresponding to SEQ ID NO. 19.
The protein sequence of SEQ ID NO: 19 corresponds to the protein
sequence provided by UniProt Accession No. I6S977, from Vitis
quinquangularis t394115 This strain comprises a codon-optimized
nucleic acid that corresponds to nucleotides 3-1188 of SEQ ID NO:
51 (due to a truncation of nucleotides 1-2 of SEQ ID NO: 51).
Translation of this sequence is expected to produce a truncated
version of a protein corresponding to SEQ ID NO: 20. The protein
sequence of SEQ ID NO: 20 corresponds to the protein sequence
provided by UniProt Accession No. Q9FR69, from Cardamine penzesii
t394091 This strain comprises a codon-optimized nucleic acid that
corresponds to nucleotides 3-1170 of SEQ ID NO: 52 (due to a
truncation of nucleotides 1-2 of SEQ ID NO: 52). Translation of
this sequence is expected to produce a truncated version of a
protein corresponding to SEQ ID NO. 21. The protein sequence of SEQ
ID NO: 21 corresponds to the protein sequence provided by UniProt
Accession No. G7IQL2, from Medicago truncatula (Barrel medic)
(Medicago tribuloides) t394037 This strain comprises a
codon-optimized nucleic acid that corresponds to nucleotides 3-1179
of SEQ ID NO: 53 (due to a truncation of nucleotides 1-2 of SEQ ID
NO: 53). Translation of this sequence is expected to produce a
truncated version of a protein corresponding to SEQ ID NO. 22. The
protein sequence of SEQ ID NO: 22 corresponds to the protein
sequence provided by UniProt Accession No. I6W888, from Vitis
pseudoreticulata (Chinese wild grapevine) t394279 This strain
comprises a codon-optimized nucleic acid that corresponds to
nucleotides 3-1188 of SEQ ID NO: 54 (due to a truncation of
nucleotides 1-2 of SEQ ID NO: 54). Translation of this sequence is
expected to produce a truncated version of a protein corresponding
to SEQ ID NO. 23. The protein sequence of SEQ ID NO: 23 corresponds
to the protein sequence provided by UniProt Accession No. P13114,
from Arabidopsis thaliana (Mouse-ear cress) t394043 This strain
comprises a codon-optimized nucleic acid that corresponds to
nucleotides 3-1344 of SEQ ID NO: 55 (due to a truncation of
nucleotides 1-2 of SEQ ID NO: 55). Translation of this sequence is
expected to produce a truncated version of a protein corresponding
to SEQ ID NO. 24. The protein sequence of SEQ ID NO: 24 corresponds
to the protein sequence provided by UniProt Accession No.
A0A251SHA8, from Helianthus annuus (Common sunflower) t394404 This
strain comprises a codon-optimized nucleic acid that corresponds to
nucleotides 3-1170 of SEQ ID NO: 56 (due to a truncation of
nucleotides 1-2 of SEQ ID NO: 56). Translation of this sequence is
expected to produce a truncated version of a protein corresponding
to SEQ ID NO. 25. The protein sequence of SEQ ID NO: 25 corresponds
to the protein sequence provided by UniProt Accession No. X5I326,
from Vaccinium ashei t394436 This strain comprises a
codon-optimized nucleic acid that corresponds to nucleotides 3-1197
of SEQ ID NO: 57 (due to a truncation of nucleotides 1-2 of SEQ ID
NO: 57). Translation of this sequence is expected to produce a
truncated version of a protein corresponding to SEQ ID NO. 26. The
protein sequence of SEQ ID NO: 26 corresponds to the protein
sequence provided by UniProt Accession No. A0A164ZDA1, from Daucus
carota subsp. Sativus t393720 This strain comprises a
codon-optimized nucleic acid that corresponds to nucleotides 3-1212
of SEQ ID NO: 58 (due to a truncation of nucleotides 1-2 of SEQ ID
NO: 58). Translation of this sequence is expected to produce a
truncated version of a protein corresponding to SEQ ID NO. 27. The
protein sequence of SEQ ID NO: 27 corresponds to the protein
sequence provided by UniProt Accession No. Q58VP7, from Aloe
alborescens (Kidachi aloe) t394911 This strain comprises a
codon-optimized nucleic acid that
corresponds to nucleotides 3-1203 of SEQ ID NO: 59 (due to a
truncation of nucleotides 1-2 of SEQ ID NO: 59). Translation of
this sequence is expected to produce a truncated version of a
protein corresponding to SEQ ID NO. 28. The protein sequence of SEQ
ID NO: 28 corresponds to the protein sequence provided by UniProt
Accession No. A0A2K3P0B5, from Trifolium pratense (Red clover)
t395023 This strain comprises a codon-optimized nucleic acid that
corresponds to nucleotides 3-1200 of SEQ ID NO: 60 (due to a
truncation of nucleotides 1-2 of SEQ ID NO: 60). Translation of
this sequence is expected to produce a truncated version of a
protein corresponding to SEQ ID NO. 29. The protein sequence of SEQ
ID NO: 29 corresponds to the protein sequence provided by UniProt
Accession No. Q8GZP4, from Hydrangea macrophylla (Bigleaf
hydrangea) (Viburnum macrophyllum) t339579 This strain comprises a
codon-optimized nucleic acid that corresponds to nucleotides 3-1158
of SEQ ID NO: 61 (due to a truncation of nucleotides 1-2 of SEQ ID
NO: 61). Translation of this sequence is expected to produce a
truncated version of a protein corresponding to SEQ ID NO. 30. The
protein sequence of SEQ ID NO: 30 corresponds to the protein
sequence provided by UniProt Accession No. B1Q2B6, from C. sativa
t339582 This strain comprises a codon-optimized nucleic acid that
corresponds to nucleotides 3-1158 of SEQ ID NO: 62 (due to a
truncation of nucleotides 1-2 of SEQ ID NO: 62). Translation of
this sequence is expected to produce a truncated version of a
protein corresponding to SEQ ID NO. 31. The protein sequence of SEQ
ID NO: 31 corresponds to the protein sequence provided by UniProt
Accession No. B1Q2B6, from C. sativa t394396 This strain comprises
a codon-optimized nucleic acid that corresponds to nucleotides
3-1158 of SEQ ID NO: 93 (due to a truncation of nucleotides 1-2 of
SEQ ID NO: 93). Translation of this sequence is expected to produce
a truncated version of a protein corresponding to SEQ ID NO. 77.
The protein sequence of SEQ ID NO: 77 corresponds to the protein
sequence provided by UniProt Accession No. B1Q2B6, from C. sativa
t339546 This strain comprises a codon-optimized nucleic acid that
corresponds to nucleotides 3-1158 of SEQ ID NO: 94 (due to a
truncation of nucleotides 1-2 of SEQ ID NO: 94). Translation of
this sequence is expected to produce a truncated version of a
protein corresponding to SEQ ID NO: 78. The protein sequence of SEQ
ID NO: 78 corresponds to the protein sequence provided by UniProt
Accession No. B1Q2B6, from C. sativa t339549 This strain comprises
a codon-optimized nucleic acid that corresponds to nucleotides
3-1158 of SEQ ID NO: 95 (due to a truncation of nucleotides 1-2 of
SEQ ID NO: 95). Translation of this sequence is expected to produce
a truncated version of a protein corresponding to SEQ ID NO. 79.
The protein sequence of SEQ ID NO: 79 corresponds to the protein
sequence provided by UniProt Accession No. B1Q2B6, from C. sativa
t393360 This strain comprises a codon-optimized nucleic acid that
corresponds to nucleotides 3-1158 of SEQ ID NO: 96 (due to a
truncation of nucleotides 1-2 of SEQ ID NO: 96). Translation of
this sequence is expected to produce a truncated version of a
protein corresponding to SEQ ID NO. 80. The protein sequence of SEQ
ID NO: 80 corresponds to the protein sequence provided by UniProt
Accession No. F1LKH5, from C. sativa t393555 This strain comprises
a codon-optimized nucleic acid that corresponds to nucleotides
3-1188 of SEQ ID NO: 97 (due to a truncation of nucleotides 1-2 of
SEQ ID NO: 97). Translation of this sequence is expected to produce
a truncated version of a protein corresponding to SEQ ID NO. 81.
The protein sequence of SEQ ID NO: 81 corresponds to the protein
sequence provided by UniProt Accession No. Q9SENO, from Fourraea
alpina (Rock-cress) (Arabis pauciflora) t394593 This strain
comprises a codon-optimized nucleic acid that corresponds to
nucleotides 3-1197 of SEQ ID NO: 98 (due to a truncation of
nucleotides 1-2 of SEQ ID NO: 98). Translation of this sequence is
expected to produce a truncated version of a protein corresponding
to SEQ ID NO. 82. The protein sequence of SEQ ID NO: 82 corresponds
to the protein sequence provided by UniProt Accession No.
A0A059VFD5, from Punica granatum (Pomegranate) t394351 This strain
comprises a codon-optimized nucleic acid that corresponds to
nucleotides 3-1167 of SEQ ID NO: 99 (due to a truncation of
nucleotides 1-2 of SEQ ID NO: 99). Translation of this sequence is
expected to produce a truncated version of a protein corresponding
to SEQ ID NO. 83. The protein sequence of SEQ ID NO: 83 corresponds
to the protein sequence provided by UniProt Accession No. Q1G6T7,
from Cardamine apennina t394414 This strain comprises a
codon-optimized nucleic acid that corresponds to nucleotides 3-1068
of SEQ ID NO: 100 (due to a truncation of nucleotides 1-2 of SEQ ID
NO: 100). Translation of this sequence is expected to produce a
truncated version of a protein corresponding to SEQ ID NO. 84. The
protein sequence of SEQ ID NO: 84 corresponds to the protein
sequence provided by UniProt Accession No. A0A2T5VUN1, from
Mycobacterium sp. YR782 t393402 This strain comprises a
codon-optimized nucleic acid that corresponds to nucleotides 3-1173
of SEQ ID NO: 101 (due to a truncation of nucleotides 1-2 of SEQ ID
NO: 101). Translation of this sequence is expected to produce a
truncated version of a protein corresponding to SEQ ID NO. 85. The
protein sequence of SEQ ID NO: 85 corresponds to the protein
sequence provided by UniProt Accession No. A0A1Q9SCX4, from Kocuria
sp. CNJ-770 t394035 This strain comprises a codon-optimized nucleic
acid that corresponds to nucleotides 3-675 of SEQ ID NO: 102 (due
to a truncation of nucleotides 1-2 of SEQ ID NO: 102). Translation
of this sequence is expected to produce a truncated version of a
protein corresponding to SEQ ID NO. 86. The protein sequence of SEQ
ID NO: 86 corresponds to the protein sequence provided by UniProt
Accession No. A0A0K8QHJ1 from Arthrobacter sp. Hiyo1 t394155 This
strain comprises a codon-optimized nucleic acid that corresponds to
nucleotides 3-1176 of SEQ ID NO: 103 (due to a truncation of
nucleotides 1-2 of SEQ ID NO: 103). Translation of this sequence is
expected to produce a truncated version of a protein corresponding
to SEQ ID NO. 87. The protein sequence of SEQ ID NO: 87 corresponds
to the protein sequence provided by UniProt Accession No. Q9XJ57
from Citrus sinensis (Sweet orange) (Citrus aurantium var.
sinensis) t394137 This strain comprises a codon-optimized nucleic
acid that corresponds to nucleotides 3-1173 of SEQ ID NO: 104 (due
to a truncation of nucleotides 1-2 of SEQ ID NO: 104). Translation
of this sequence is expected to produce a truncated version of a
protein corresponding to SEQ ID NO. 88. The protein sequence of SEQ
ID NO: 88 corresponds to the protein sequence provided by UniProt
Accession No. I6R2S0 from Narcissus tazetta var. chinensis t393976
This strain comprises a codon-optimized nucleic acid that
corresponds to nucleotides 3-1188 of SEQ ID NO: 105 (due to a
truncation of nucleotides 1-2 of SEQ ID NO: 105). Translation of
this sequence is expected to produce a truncated version of a
protein corresponding to SEQ ID NO. 89. The protein sequence of SEQ
ID NO: 89 corresponds to the protein sequence provided by UniProt
Accession No. Q2ENA5 from Abies alba (Edeltanne) (European silver
fir) t394689 This strain comprises a codon-optimized nucleic acid
that corresponds to nucleotides 3-1173 of SEQ ID NO: 106 (due to a
truncation of nucleotides 1-2 of SEQ ID NO: 106). Translation of
this sequence is expected to produce a truncated version of a
protein corresponding to SEQ ID NO. 90. The protein sequence of SEQ
ID NO: 90 corresponds to the protein sequence provided by UniProt
Accession No. A0A022RTH3 from Erythranthe guttata (Yellow monkey
flower) t393400 This strain comprises a codon-optimized nucleic
acid that corresponds to nucleotides 3-1053 of SEQ ID NO: 107 (due
to a truncation of nucleotides 1-2 of SEQ ID NO: 107). Translation
of this sequence is expected to produce a truncated version of a
protein corresponding to SEQ ID NO. 91. The protein sequence of SEQ
ID NO: 91 corresponds to the protein sequence provided by UniProt
Accession No. A0A2T7T652 from Streptomyces scopuliridis RB72
t394693 This strain comprises a codon-optimized nucleic acid that
corresponds to nucleotides 3-1188 of SEQ ID NO: 108 (due to a
truncation of nucleotides 1-2 of SEQ ID NO: 108). Translation of
this sequence is expected to produce a truncated version of a
protein corresponding to SEQ ID NO: 92. The protein sequence of SEQ
ID NO: 92 corresponds to the protein sequence provided by UniProt
Accession No. Q2EFKO, from Abies alba (Edeltanne) (European silver
fir)
TABLE-US-00021 TABLE 6 Sequence Information for Strains Described
in Example 3 and TABLE 7 and 8 OLS Nucleotide OLS Protein Sequence
Sequence Strain (SEQ ID NO) (SEQ ID NO) t527340 62 5 t527346 38 7
t599285 172 17 t598244 173 143 t598490 174 144 t598916 175 145
t598301 176 7 t598212 177 146 t598424 178 147 t598578 179 148
t598836 180 149 t597770 181 150 t597768 182 151 t599210 183 152
t597806 184 153 t598184 185 154 t598084 186 6 t598989 187 155
t598609 188 156 t598907 189 157 t598159 190 158 t598607 191 159
t598132 192 160 t598202 193 161 t598224 194 162 t598242 195 163
t598265 196 13 t598502 197 164 t598669 198 165 t598828 199 166
t598888 200 167 t598890 201 168 t598897 202 169 t598965 203 170
t598976 204 8 t599231 205 15 t599271 206 171
TABLE-US-00022 TABLE 7 Production of Olivetol and Olivetolic Acid
in Secondary Screen of Full-Length OLS Library (with sodium
hexanoate supplementation) Standard Average Standard Average
Standard Average Deviation Normalized Deviation Olivetol Deviation
Olivetolic Olivetolic Olivetol Normalized Strain Strain type [ug/L]
Olivetol [ug/L] Acid [ug/L] Acid [ug/L] (per OD) Olivetol t527338
GFP Negative 0 0 0 0 0 0 Control t527340 Cannabis OLS 19907.37
3392.375 464.2222 119.0458 1 0.17338 Positive Control t527346
Library 47674.72 7310.215 1126.656 275.7896 2.577378 0.5328 t599285
Library 40719.68 5413.028 1006.4 205.3059 1.570539 0.12684 t598244
Library 30067.48 2678.216 1328.625 391.3286 2.322177 0.24813
t598490 Library 29983.43 7816.659 895.1 368.4323 1.423788 0.286677
t598916 Library 23515.18 1702.502 680.575 66.45968 0.938026
0.042088 t598301 Library 21070.73 12453.97 512.175 274.8916
0.881438 0.570619 t598212 Library 19864.23 6981.826 582.3 129.3529
1.315837 0.25006 t598424 Library 18263.73 3738.191 661.325 148.2432
0.782173 0.157387 t598578 Library 18167.93 1534.837 614.4 62.65115
0.733176 0.065004 t598836 Library 17825.58 3298.654 614.75 139.553
0.67585 0.216607 t597770 Library 16611.23 2805.423 565.575 87.49584
1.203593 0.740925 t597768 Library 16140.08 2088.271 469.65 58.01394
0.72298 0.123509 t599210 Library 3019.65 187.1009 4913.925 344.6286
0.0939 0.012992 t597806 Library 1452.425 194.0261 872.65 58.60117
0.096846 0.040743 t598184 Library 466.15 76.41424 6016.625 5727.47
0.033016 0.009583 t598084 Library 298.6 38.96913 711.85 86.71242
0.012889 0.003159 t598989 Library 192.725 3.557504 981.225 924.6046
0.008438 0.000605 t598609 Library 97.025 65.64777 490.925 484.0728
0.003307 0.002213 t598907 Library 97 66.37625 539.75 809.4711
0.004664 0.0034 t598159 Library 73.825 50.2624 1014.55 98.44669
0.003517 0.002408 t598607 Library 57.9 67.11512 1006.4 227.8435
0.0017 0.001963
TABLE-US-00023 TABLE 8 Production of Olivetol and Olivetolic Acid
in Secondary Screen of Full-Length OLS Library (without sodium
hexanoate supplementation) Standard Standard Average Standard
Average Deviation Average Deviation Normalized Deviation Olivetol
Olivetol Olivetolic Olivetolic Olivetol Normalized Strain Strain
type [ug/L] [ug/L] Acid [ug/L] Acid [ug/L] (per OD) Olivetol
t527338 GFP Negative 0 0 0 0 0 0 Control t527340 Cannabis OLS
233.5102 24.98585 0.139276 0.5909 1 0.15846 Positive Control
t527346 Library 726.4307 68.37505 0.431449 1.830482 3.072299
0.594866 t597768 Library 313.8253 41.20191 0 0 1.887013 0.355292
t597770 Library 600.919 84.71989 0 0 2.958839 0.883218 t598084
Library 41.65336 27.90324 143.5728 17.13177 0.203694 0.149696
t598132 Library 430.7581 98.35626 0 0 2.045727 0.229508 t598202
Library 629.8948 43.73374 0 0 2.319367 0.145214 t598212 Library
439.2199 23.32112 0 0 2.133233 0.157933 t598224 Library 535.1348
45.55404 4.682865 1.486543 2.001014 0.151286 t598242 Library
444.4074 36.07498 0 0 2.509074 0.247446 t598244 Library 780.646
10.19476 21.86307 21.3525 4.250859 0.503227 t598265 Library
399.8005 24.89199 0 0 2.005431 0.247479 t598301 Library 523.4047
10.23296 0 0 2.980013 0.751203 t598424 Library 1039.723 20.19861
22.23645 1.606142 6.95869 1.19209 t598490 Library 1042.654 162.3917
12.97602 6.917579 5.733983 1.383711 t598502 Library 649.3324
105.1879 0 0 3.49989 0.626512 t598578 Library 666.2518 167.478 0 0
3.35228 0.956232 t598669 Library 555.1264 124.4665 0 0 3.070987
0.828156 t598828 Library 362.7059 53.2614 0 0 1.702596 0.290853
t598836 Library 544.9485 38.11311 0 0 2.522761 0.438238 t598888
Library 0 0 1.036494 2.072988 0 0 t598890 Library 216.4236 177.1733
0 0 1.067688 0.958823 t598897 Library 303.5841 49.89216 0 0 1.9597
0.712481 t598916 Library 528.2121 40.26965 3.166338 6.332675
2.217625 0.308839 t598965 Library 259.0065 16.53317 0 0 1.82891
0.368218 t598976 Library 262.3653 15.67957 0 0 1.252384 0.144212
t599210 Library 137.3035 10.28164 453.0067 13.07732 0.545126
0.127023 t599231 Library 575.119 59.46478 0 0 2.438902 0.437276
t599271 Library 644.8095 74.40164 0 0 2.902135 0.475949 t599285
Library 670.6119 86.9222 0 0 2.154638 0.264646
TABLE-US-00024 TABLE 9 Results of Secondary Screen of C. Sativa OLS
Protein Engineering Library Amino Acid mutations from Standard
Average Standard wild-type Average Deviation Olivetolic Deviation
Cannabis protein Olivetol Olivetol Acid Olivetolic Strain Strain
type (SEQ ID NO: 5) [ug/L] [ug/L] [ug/L] Acid [ug/L] t346317 GFP 0
0 0 0 Negative Ctrl t405417 Library T335C 29155.65 1352.507
925.5075 84.12739 t404953 Library S334P 16467.14 2021.617 473.085
68.7974 t405220 Library Y153G 15190.5 1885.253 437.9325 84.3569
t404192 Library I284Y 14380.39 1956.468 390.18 39.47375 t404323
Library K100L 14246.96 544.9192 456.55 35.50002 t404196 Library
K116R 14068.84 2527.921 380.4225 42.26844 t404209 Library I278E
13888.94 2872.84 439.1325 66.1486 t404164 Library K108D 13824.77
2873.633 292.71 197.9307 t404170 Library L348S 13625.61 1648.021
291.5625 195.0828 t404384 Library K71R 13619.49 3039.582 372.775
42.82276 t405397 Library V92G 13537.85 363.1012 414.8725 26.8083
t405164 Library T128V 13374.17 1328.433 385.2925 59.52873 t404191
Library K100M 13326.69 1006.193 280.65 188.8515 t405340 Library
Y135V 13234.48 1441.185 393.945 57.71197 t404421 Library P229A
13099.17 2790.466 280.175 190.3239 t404631 Library L241I 13096.36
2072.187 425.4825 86.29982 t405133 Library T128A 13050.31 1267.354
408.1175 30.00599 t405081 Library T128I 12839.46 770.8077 409.595
60.20914 t404898 Library S334A 12549.31 2014.497 392.02 20.58724
t405017 Library S326R 12437.99 1793.811 291.3725 198.041 t405140
Library A125S 12379.56 2038.247 579.6825 87.43007 t404276 Library
I273V 12341.81 673.6841 344.5 243.9399 t404405 Library K51R
12305.55 2024.022 401.0325 52.97083 t405079 Library H328Y 11965.56
636.541 380.795 28.50397 t404978 Library F64Y 11905.29 408.7996
208.145 241.0289 t405347 Library T17K 11875.39 1484.666 353.8775
41.08276 t404855 Library I207L 11774.75 1252.291 380.1475 30.28471
t405362 Library V324I 11489.89 2109.012 350.79 78.35325 t404523
Library L25R 11375.9 2136.219 212.6875 248.5159 t404951 Library
T296A 11266.41 958.6764 269.73 181.7056 t405308 Library D320A
11140.11 1318.646 346.7825 18.1635 t405201 Library V307I 11054.69
3152.4 194.21 241.925 t404219 Library I23C 11046.19 1061.09 380.345
25.59692 t404673 Library M267K 11004.45 2014.531 382.22 111.9515
t404274 Library L277M 10942.46 930.1003 360.4375 80.2903 t405042
Library T123C 10940.26 899.6448 314.7975 212.1934 t404528 Library
M267G 10521.73 2186.36 260.765 189.9513 t405312 Library E196K
10503.49 1491.483 321.1425 43.88772 t404725 Library R375T 10474.14
637.7002 181.37 209.4388 t405303 Library T247A 10412.99 1484.527
353.6075 15.69949 t405395 Library V95A 10397.39 1215.177 299.155
49.56504 t405326 Library D54R 10116.47 1327.968 316.9625 42.33512
t404599 Library L201C 10033.37 1349.986 368.615 82.31389
TABLE-US-00025 TABLE 10A Results of Secondary Screen of Protein
Engineering Library Using C. sativa, Cochorus, and Cymbidium
Templates (supplemented with sodium hexanoate) Standard Standard
Amino Acid Average Deviation Average Deviation Wild-type mutations
from Olivetol Olivetol Olivetolic Olivetolic Strain template used
wild-type [ug/L] [ug/L] Acid [ug/L] Acid [ug/L] t527338 22.72917
79.04841 0 0 GFP t606794 Cannabis wild-type 15598.47 5507.375
337.3906 163.6388 sativa (Hemp) (Marijuana) t527340 Cannabis
wild-type 15534.95 6243.926 359.0063 194.9093 Cannabis sativa OLS
t607067 Cannabis F367L 10907.1 3397.082 310.75 114.4806 sativa
t607367 Cannabis G366A 8394.975 2661.736 137.95 44.65143 sativa
t607391 Cannabis P298N 8381.075 1668.07 167.15 37.8978 sativa
t606801 Cannabis S334P 22691 2379.928 572.025 55.64575 sativa
t606984 Cannabis I248M 384.975 256.6688 124.475 6.990649 sativa
t606899 Cannabis S332W 55.1 110.2 19.4 38.8 sativa t606797
Corchorus wild-type 448.8643 190.2768 665.7071 222.9162 Corchorus
olitorius OLS t606807 Corchorus d1-8 Y142C 8173.95 11559.71 639.55
114.0563 olitorius t607179 Corchorus Y301W 1487.6 1289.575 528.325
51.91142 olitorius V302I V303T N305P P308K T309A t607149 Corchorus
d1-8 W339S 925.4 1638.079 108.825 35.84888 olitorius t607139
Corchorus d1-8 Y266F 539.55 467.458 383.3 31.26137 olitorius
t607112 Corchorus W339S 517.075 737.3064 81.225 37.55896 olitorius
t607332 Corchorus d1-8 408.25 41.92648 393.6 66.39794 olitorius
Y301W V302I V303T N305P P308K T309A t607153 Corchorus d1-8 A373G
334.35 19.09633 529.5 33.9143 olitorius t607158 Corchorus A373G
314.8 15.06143 423.9 57.00158 olitorius t607236 Corchorus M255I
315.325 23.9742 360.4 7.086607 olitorius t607141 Corchorus d1-8
Y266F 168.575 337.15 69.325 8.448422 olitorius W339S t607176
Corchorus L374F 130.45 150.9282 97.05 6.728298 olitorius t606930
Corchorus d1-8 M255I 265.85 306.9972 735.6 53.30647 olitorius
t607193 Corchorus d1-8 T12Y 65.675 131.35 39.3 28.53571 olitorius
F39Y Q42R L43A Q47E Q51D Q57K I77L G79E S84C E96D T100E L121K N123K
A135V A137M T139G H143Q N146K K151R H152P K156R F158M S174A V182R
D183G S184A N231T K232N I241V T253D C260G M287E M353R Q357E S395N
t607006 Corchorus Y266F 265.975 178.2236 505.325 21.29575 olitorius
t606993 Corchorus d1-8 N305P 165.875 191.5369 427.15 17.27937
olitorius t606852 Corchorus N305P 73.375 146.75 404.3 58.06772
olitorius t607119 Corchorus d1-8 L374F 0 0 111.725 18.69962
olitorius t607371 Corchorus Y266F 0 0 37.25 3.421988 olitorius
W339S t527346 Cymbidium wild-type 29779.86 10784.18 631.1694
320.6464 Cymbidium hybrid OLS cultivar t606952 Cymbidium V71Y
40374.05 3947.169 1177.275 107.3831 hybrid cultivar t607284
Cymbidium F70M 18119.03 1257.566 374.85 15.30109 hybrid cultivar
t607262 Cymbidium L385M 18869.45 2300.831 394.425 43.38251 hybrid
cultivar t606938 Cymbidium D88A 30129.05 421.2235 773.1 17.39483
hybrid cultivar t607260 Cymbidium E285A 15646.4 1341.912 319.1
21.50364 hybrid cultivar t607159 Cymbidium L76I 25322 7151.452
633.5 130.6911 hybrid cultivar t606946 Cymbidium N151P 29738.73
5548.193 778.9 116.4196 hybrid cultivar t606861 Cymbidium E203K
44399.18 12437.5 1252.525 394.7546 hybrid cultivar t606918
Cymbidium V50N 27251.28 1711.322 732.125 88.38052 hybrid cultivar
t607135 Cymbidium E28P 24306.43 6961.951 615.45 163.6961 hybrid
cultivar t607286 Cymbidium S34Q 13463.55 871.8021 287.675 21.0191
hybrid cultivar t606942 Cymbidium R100P 34937.75 2136.806 968.7
20.64752 hybrid cultivar t606959 Cymbidium A219C 32778.4 1462.567
812.475 54.31368 hybrid cultivar t607294 Cymbidium K359M 13309.98
473.943 279 10.60157 hybrid cultivar t607282 Cymbidium R100T
13723.58 1657.273 282.825 20.36768 hybrid cultivar t607230
Cymbidium E116D 21772.93 983.0294 469.875 34.1937 hybrid cultivar
t606965 Cymbidium Y142V 26443.03 1993.229 918 96.78736 hybrid
cultivar t607288 Cymbidium T289D 13669.95 750.4718 290.775 34.8278
hybrid cultivar t607228 Cymbidium M135I 21348.6 702.6819 604.6
19.56238 hybrid cultivar t606909 Cymbidium W368H 40713.38 2212.329
998.95 50.99526 hybrid cultivar t606962 Cymbidium D229E 28238.98
1771.039 765.65 43.92285 hybrid cultivar t607150 Cymbidium E285K
25386.95 2387.108 593.6 71.86895 hybrid cultivar t607361 Cymbidium
E323Q 20209.88 3057.447 378.4 72.17622 hybrid cultivar t606932
Cymbidium S18T 26673.38 4632.723 726.825 106.151 hybrid cultivar
t606940 Cymbidium A13S 29094.85 3024.508 737.1 40.72935 hybrid
cultivar t607269 Cymbidium A333R 12947.23 883.2885 268.075 35.74207
hybrid cultivar t607186 Cymbidium S180N 23191.9 4783.221 594.2
135.8793 hybrid cultivar t607476 Cymbidium L20F 17200 1998.783
350.375 25.15451 hybrid cultivar t607031 Cymbidium N80H 25388.63
6003.712 611.825 130.4566 hybrid cultivar t606916 Cymbidium R100A
25282.83 1244.898 638.4 21.58997 hybrid cultivar t607292 Cymbidium
V331I 12013.93 2802.682 241.875 58.81765 hybrid cultivar t606908
Cymbidium I22M 32699.68 21947.9 1120.825 82.20671 hybrid cultivar
t607248 Cymbidium E155C 20670.43 1761.502 477.55 41.49647 hybrid
cultivar t607023 Cymbidium V71H 27924.45 1393.419 761.2 34.79224
hybrid cultivar t606936 Cymbidium T111K 26953.6 3174.107 689.975
120.0151 hybrid cultivar t607433 Cymbidium L291V 18071.78 6905.44
351.525 142.8462 hybrid cultivar t607600 Cymbidium R123A 18038.33
2576.146 333.575 50.84194 hybrid cultivar t606894 Cymbidium Y142F
36097.63 2137.695 911.05 54.27403 hybrid cultivar t606963 Cymbidium
I147E 26509.6 1811.909 657.275 14.62882 hybrid cultivar t607603
Cymbidium Y142I 18913.93 1120.707 596.475 92.27106 hybrid cultivar
t607452 Cymbidium K54E 16597 3291.995 329.3 60.58185 hybrid
cultivar t607197 Cymbidium G84C 19663.7 884.8279 459.3 27.12354
hybrid cultivar t606996 Cymbidium T170A 39067.8 2354.185 1157.575
79.54543 hybrid cultivar t607043 Cymbidium N45T 28747.38 934.8057
679.875 21.52431 hybrid cultivar t607254 Cymbidium G262T 11775.15
1040.26 246.425 18.13861 hybrid cultivar t607478 Cymbidium N11R
15894.55 2710.618 324.075 39.31619 hybrid cultivar t607132
Cymbidium D208A 24881.38 1936.584 765.75 60.52451
hybrid cultivar t607109 Cymbidium D61R 25634.95 2078.231 613.375
100.6135 hybrid cultivar t607155 Cymbidium C176D 22283.45 3073.579
526.325 67.26383 hybrid cultivar t606956 Cymbidium L83M 23728.73
2145.982 656.075 79.72258 hybrid cultivar t606906 Cymbidium E28A
33411.15 939.6742 850.55 34.71894 hybrid cultivar t607195 Cymbidium
T111R 17676.68 1327.959 403.725 50.31689 hybrid cultivar t607449
Cymbidium E203P 16032.95 2469.127 317.75 28.30789 hybrid cultivar
t607256 Cymbidium G373A 12090.73 566.8141 281.125 10.12073 hybrid
cultivar t607349 Cymbidium K282D 18699.3 3173.399 354.175 88.50621
hybrid cultivar t606960 Cymbidium N78R 25400.9 667.3651 660.875
37.90764 hybrid cultivar t607601 Cymbidium K121V 18381.8 4015.223
314.125 78.4602 hybrid cultivar t607021 Cymbidium H69Y 24261.93
2128.687 571.075 61.0977 hybrid cultivar t606874 Cymbidium D208S
28092.05 2956.394 886.05 199.1175 hybrid cultivar t607320 Cymbidium
T111E 16500.03 4257.055 303.275 77.26765 hybrid cultivar t607317
Cymbidium C82E 17852.45 4825.826 325.225 76.27321 hybrid cultivar
t607224 Cymbidium Y142C 19142.53 1455.367 540.25 54.21098 hybrid
cultivar t606912 Cymbidium D14P 31141.75 1149.49 769.2 65.33912
hybrid cultivar t607602 Cymbidium R100E 17901.3 2319.379 328.5
67.41187 hybrid cultivar t606905 Cymbidium V388A 29890.83 2522.115
747.45 83.27435 hybrid cultivar t607156 Cymbidium H269S 21143.2
1008.997 527.5 30.55083 hybrid cultivar t607474 Cymbidium G262A
15068.18 1372.284 326.075 28.24894 hybrid cultivar t607482
Cymbidium R24K 14858.45 2175.705 313.6 58.93963 hybrid cultivar
t606854 Cymbidium L144I 35182.43 2487.935 921.625 54.02563 hybrid
cultivar t607032 Cymbidium K355S 24649.8 1675.56 544.4 16.12203
hybrid cultivar t606830 Cymbidium M135V 34299.15 4732.074 977.675
108.3528 hybrid cultivar t606961 Cymbidium V43M 25357.95 678.9486
646.975 24.3909 hybrid cultivar t606868 Cymbidium L248I 35144.45
1993.999 945.425 66.1703 hybrid cultivar t607083 Cymbidium G84S
21707.73 2407.367 537.85 68.42819 hybrid cultivar t606958 Cymbidium
C82N 22576.8 2610.997 564.75 61.60793 hybrid cultivar t607273
Cymbidium V341P 10381.15 857.4199 209.325 25.9962 hybrid cultivar
t607241 Cymbidium V388T 17754.23 1010.578 444.275 22.37668 hybrid
cultivar t606857 Cymbidium A195V 36686.03 2848.052 937.825 116.369
hybrid cultivar t606901 Cymbidium D208C 30154 1882.794 883.325
84.4674 hybrid cultivar t607015 Cymbidium G84T 23550.9 1835.311
559.725 25.30183 hybrid cultivar t607586 Cymbidium I326R 13845.4
1980.772 297.075 55.00051 hybrid cultivar t607122 Cymbidium F374L
20425.3 2531.711 488.175 62.87863 hybrid cultivar t606882 Cymbidium
L222I 27445.5 1536.232 692.15 54.08453 hybrid cultivar t607146
Cymbidium G262T 18830.75 1157.57 440.025 51.65213 hybrid cultivar
t607585 Cymbidium L83I 13348.68 1378.214 274.425 30.01082 hybrid
cultivar t606828 Cymbidium A107M 32613.03 1865.271 860.575 52.56104
hybrid cultivar t607081 Cymbidium I99G 20827 2377.691 521.525
35.96761 hybrid cultivar t606887 Cymbidium Q274G 27128.45 1010.786
675.45 25.36697 hybrid cultivar t606891 Cymbidium K356Q 26852.25
420.5503 655.325 41.67072 hybrid cultivar t607160 Cymbidium V341A
19361.23 1205.165 469.725 29.27984 hybrid cultivar t607194
Cymbidium F86Y 17323.08 6000.996 483.975 161.5325 hybrid cultivar
t607377 Cymbidium S339W 0 0 46 22.64553 hybrid cultivar t607265
Cymbidium E28P F40Y 5858.45 955.8912 165.625 31.40938 hybrid N51D
K54E cultivar N80H C82E L83I G84C F86Y D88A N89P N92D F97M I99R
T111Q R123K L137M I147L N151P C176D S180N K182P Q206L H233N S237F
A252E S260G G262A G281E T289K D367E t607000 Cymbidium G373A
16989.08 11567.45 700.525 192.4804 hybrid F374L cultivar t607245
Cymbidium E28P G84C 8507.025 5710.02 210.4 141.3894 hybrid N89P
cultivar T111R N151P C176D Q206L S237F A252E G262T G281E t607318
Cymbidium E28P F40Y 6864.125 789.6743 243.25 102.741 hybrid N51D
K54E cultivar N80H C82E L83I G84C F86Y D88A N89P N92D F97M I99R
T111E R123K L137M I147L N151P C176D S180N K182P Q206L H233N S237F
A252E S260G G262A G281E T289K D367E t607435 Cymbidium E28P N51D
4888.425 3390.097 163.75 117.0815 hybrid K54E N80H cultivar C82E
L83I G84C F86Y D88A N89P N92D F97M I99R T111E R123K L137M I147L
N151P C176D S180N K182P Q206L H233N S237F A252E S260G G262A G281E
T289K D367E t607337 Cymbidium E28P N51E 5250.525 764.6064 135.05
26.46186 hybrid N80H C82E cultivar L83I G84C F86Y D88A N89P N92D
F97M I99R T111R R123K L137M I147L N151P C176D S180N K182P Q206L
H233N S237F A252E S260G G262A G281E T289K D367E t607316 Cymbidium
W301Y 5549.375 462.0676 169 15.9066 hybrid P303V cultivar P305N
R308P A309T t607124 Cymbidium I255M 0 0 0 0 hybrid A266Y
cultivar W301Y P303V P305N R308P A309T S339W V341P G373A F374L
t607280 Cymbidium A266Y 0 0 0 0 hybrid P305N cultivar S339W V341P
G373A F374L t607290 Cymbidium A266Y 0 0 0 0 hybrid S339W cultivar
t607381 Cymbidium A266Y 0 0 0 0 hybrid P305N cultivar S339W
TABLE-US-00026 TABLE 10B Results of Secondary Screen of Protein
Engineering Library Using C. sativa, Cochorus, and Cymbidium
Templates (supplemented with sodium hexanoate) Standard Average
Standard Average Olivetol Deviation Olivetol Amino Acid Normalized
Deviation Normalized to Normalized to Wild-type mutations from
Olivetol Normalized t606797_Corchorus t606797_Corchorus Strain
template used wild-type (per OD) Olivetol OLS (per OD) OLS t527338
0.001903 0.006512 GFP t606794 Cannabis wild-type 1.166072 0.265739
sativa (Hemp) (Marijuana) t527340 Cannabis wild-type 1.02159
0.125216 Cannabis sativa OLS t607067 Cannabis F367L 0.761216
0.189784 sativa t607367 Cannabis G366A 0.745615 0.241737 sativa
t607391 Cannabis P298N 0.708056 0.110021 sativa t606801 Cannabis
S334P 0.674894 0.108203 sativa t606984 Cannabis I248M 0.00995
0.006837 sativa t606899 Cannabis S332W 0.001802 0.003605 sativa
t606797 Corchorus wild-type 0.027033 0.012052 1 0.520764 Corchorus
olitorius OLS t606807 Corchorus d1-8 Y142C 0.275531 0.38966 29.9306
42.32826 olitorius t607179 Corchorus Y301W V302I 0.103413 0.091496
2.842431 2.514883 olitorius V303T N305P P308K T309A t607149
Corchorus d1-8 W339S 0.075566 0.133268 2.077017 3.663029 olitorius
t607139 Corchorus d1-8 Y266F 0.038008 0.033998 1.044701 0.934465
olitorius t607112 Corchorus W339S 0.033504 0.050586 1.29632
1.957247 olitorius t607332 Corchorus d1-8 Y301W 0.027999 0.00441
0.919863 0.144887 olitorius V302I V303T N305P P308K T309A t607153
Corchorus d1-8 A373G 0.025872 0.001554 0.711133 0.042715 olitorius
t607158 Corchorus A373G 0.0221 0.001298 0.607436 0.035688 olitorius
t607236 Corchorus M255I 0.019521 0.001797 0.701647 0.064597
olitorius t607141 Corchorus d1-8 Y266F 0.012974 0.025949 0.356615
0.71323 olitorius W339S t607176 Corchorus L374F 0.010369 0.012076
0.285013 0.33193 olitorius t606930 Corchorus d1-8 M255I 0.009039
0.010752 0.948726 1.128513 olitorius t607193 Corchorus d1-8 T12Y
0.005613 0.011226 0.154285 0.30857 olitorius F39Y Q42R L43A Q47E
Q51D Q57K I77L G79E S84C E96D T100E L121K N123K A135V A137M T139G
H143Q N146K K151R H152P K156R F158M S174A V182R D183G S184A N231T
K232N I241V T253D C260G M287E M353R Q357E S395N t607006 Corchorus
Y266F 0.004498 0.003084 0.58364 0.400247 olitorius t606993
Corchorus d1-8 N305P 0.003933 0.004545 0.510385 0.58981 olitorius
t606852 Corchorus N305P 0.001714 0.003429 0.186231 0.372463
olitorius t607119 Corchorus d1-8 L374F 0 0 0 0 olitorius t607371
Corchorus Y266F W339S 0 0 0 0 olitorius t527346 Cymbidium wild-type
0.90183 0.165441 Cymbidium hybrid OLS cultivar t606952 Cymbidium
V71Y 0.967928 0.215533 hybrid cultivar t607284 Cymbidium F70M
0.949145 0.075505 hybrid cultivar t607262 Cymbidium L385M 0.890722
0.082131 hybrid cultivar t606938 Cymbidium D88A 0.871 0.251207
hybrid cultivar t607260 Cymbidium E285A 0.806964 0.103286 hybrid
cultivar t607159 Cymbidium L76I 0.760304 0.251978 hybrid cultivar
t606946 Cymbidium N151P 0.754797 0.320899 hybrid cultivar t606861
Cymbidium E203K 0.745482 0.268545 hybrid cultivar t606918 Cymbidium
V50N 0.734491 0.069332 hybrid cultivar t607135 Cymbidium E28P
0.731258 0.315887 hybrid cultivar t607286 Cymbidium S34Q 0.716576
0.071466 hybrid cultivar t606942 Cymbidium R100P 0.712487 0.038444
hybrid cultivar t606959 Cymbidium A219C 0.709144 0.063379 hybrid
cultivar t607294 Cymbidium K359M 0.707812 0.023224 hybrid cultivar
t607282 Cymbidium R100T 0.703105 0.073566 hybrid cultivar t607230
Cymbidium E116D 0.694814 0.046965 hybrid cultivar t606965 Cymbidium
Y142V 0.690425 0.129346 hybrid cultivar t607288 Cymbidium T289D
0.68465 0.060985 hybrid cultivar t607228 Cymbidium M135I 0.682179
0.118232 hybrid cultivar t606909 Cymbidium W368H 0.676488 0.062146
hybrid cultivar t606962 Cymbidium D229E 0.675087 0.074269 hybrid
cultivar t607150 Cymbidium E285K 0.664425 0.076686 hybrid cultivar
t607361 Cymbidium E323Q 0.663279 0.09616 hybrid cultivar t606932
Cymbidium S18T 0.660218 0.135435 hybrid cultivar t606940 Cymbidium
A13S 0.657782 0.010521 hybrid cultivar t607269 Cymbidium A333R
0.652497 0.051357 hybrid cultivar t607186 Cymbidium S180N 0.649029
0.165224 hybrid cultivar t607476 Cymbidium L20F 0.647873 0.057907
hybrid cultivar t607031 Cymbidium N80H 0.629308 0.25658 hybrid
cultivar t606916 Cymbidium R100A 0.628769 0.063422 hybrid cultivar
t607292 Cymbidium V331I 0.628464 0.157777 hybrid cultivar t606908
Cymbidium I22M 0.624153 0.422076 hybrid cultivar t607248 Cymbidium
E155C 0.622639 0.035079 hybrid cultivar t607023 Cymbidium V71H
0.620539 0.024017 hybrid cultivar t606936 Cymbidium T111K 0.620218
0.070587 hybrid cultivar t607433 Cymbidium L291V 0.61928 0.21641
hybrid cultivar t607600 Cymbidium R123A 0.617083 0.084619 hybrid
cultivar t606894 Cymbidium Y142F 0.617067 0.042344 hybrid cultivar
t606963 Cymbidium I147E 0.616751 0.065014 hybrid cultivar t607603
Cymbidium Y142I 0.615776 0.03639 hybrid cultivar t607452 Cymbidium
K54E 0.609431 0.073608 hybrid cultivar t607197 Cymbidium G84C
0.607758 0.04262 hybrid cultivar t606996 Cymbidium T170A 0.606281
0.062689 hybrid cultivar t607043 Cymbidium N45T 0.601817 0.034499
hybrid cultivar t607254 Cymbidium G262T 0.599911 0.07071 hybrid
cultivar t607478 Cymbidium N11R 0.593189 0.06751 hybrid cultivar
t607132 Cymbidium D208A 0.592492 0.112514 hybrid cultivar t607109
Cymbidium D61R 0.591253 0.021319 hybrid cultivar t607155 Cymbidium
C176D 0.589853 0.117858 hybrid cultivar t606956 Cymbidium L83M
0.588247 0.063594 hybrid cultivar t606906 Cymbidium E28A 0.588171
0.068304 hybrid cultivar
t607195 Cymbidium T111R 0.587434 0.080522 hybrid cultivar t607449
Cymbidium E203P 0.583681 0.065069 hybrid cultivar t607256 Cymbidium
G373A 0.582906 0.069959 hybrid cultivar t607349 Cymbidium K282D
0.581651 0.053625 hybrid cultivar t606960 Cymbidium N78R 0.571233
0.098482 hybrid cultivar t607601 Cymbidium K121V 0.569034 0.179725
hybrid cultivar t607021 Cymbidium H69Y 0.563625 0.026338 hybrid
cultivar t606874 Cymbidium D208S 0.562002 0.072513 hybrid cultivar
t607320 Cymbidium T111E 0.561861 0.128738 hybrid cultivar t607317
Cymbidium C82E 0.559312 0.140085 hybrid cultivar t607224 Cymbidium
Y142C 0.557739 0.033377 hybrid cultivar t606912 Cymbidium D14P
0.557253 0.024918 hybrid cultivar t607602 Cymbidium R100E 0.553566
0.029107 hybrid cultivar t606905 Cymbidium V388A 0.552755 0.038488
hybrid cultivar t607156 Cymbidium H269S 0.552698 0.060754 hybrid
cultivar t607474 Cymbidium G262A 0.55226 0.080327 hybrid cultivar
t607482 Cymbidium R24K 0.551229 0.059821 hybrid cultivar t606854
Cymbidium L144I 0.550953 0.054635 hybrid cultivar t607032 Cymbidium
K355S 0.543176 0.043908 hybrid cultivar t606830 Cymbidium M135V
0.542887 0.080821 hybrid cultivar t606961 Cymbidium V43M 0.535062
0.061363 hybrid cultivar t606868 Cymbidium L248I 0.532705 0.006774
hybrid cultivar t607083 Cymbidium G84S 0.529811 0.024702 hybrid
cultivar t606958 Cymbidium C82N 0.525334 0.152553 hybrid cultivar
t607273 Cymbidium V341P 0.522317 0.046732 hybrid cultivar t607241
Cymbidium V388T 0.519025 0.016822 hybrid cultivar t606857 Cymbidium
A195V 0.518645 0.087321 hybrid cultivar t606901 Cymbidium D208C
0.51686 0.063709 hybrid cultivar t607015 Cymbidium G84T 0.515084
0.042136 hybrid cultivar t607586 Cymbidium I326R 0.514156 0.080245
hybrid cultivar t607122 Cymbidium F374L 0.513354 0.048872 hybrid
cultivar t606882 Cymbidium L222I 0.510184 0.011174 hybrid cultivar
t607146 Cymbidium G262T 0.506767 0.057007 hybrid cultivar t607585
Cymbidium L83I 0.506322 0.049736 hybrid cultivar t606828 Cymbidium
A107M 0.505604 0.006906 hybrid cultivar t607081 Cymbidium I99G
0.504811 0.041631 hybrid cultivar t606887 Cymbidium Q274G 0.50406
0.082289 hybrid cultivar t606891 Cymbidium K356Q 0.501544 0.043785
hybrid cultivar t607160 Cymbidium V341A 0.501469 0.056063 hybrid
cultivar t607194 Cymbidium F86Y 0.500062 0.184291 hybrid cultivar
t607377 Cymbidium S339W 0 0 hybrid cultivar t607265 Cymbidium E28P
F40Y 0.298621 0.029421 hybrid N51D K54E cultivar N80H C82E L83I
G84C F86Y D88A N89P N92D F97M I99R T111Q R123K L137M I147L N151P
C176D S180N K182P Q206L H233N S237F A252E S260G G262A G281E T289K
D367E t607000 Cymbidium G373A F374L 0.279083 0.189883 hybrid
cultivar t607245 Cymbidium E28P G84C 0.246285 0.179183 hybrid N89P
T111R cultivar N151P C176D Q206L S237F A252E G262T G281E t607318
Cymbidium E28P F40Y 0.205093 0.035905 hybrid N51D K54E cultivar
N80H C82E L83I G84C F86Y D88A N89P N92D F97M I99R T111E R123K L137M
I147L N151P C176D S180N K182P Q206L H233N S237F A252E S260G G262A
G281E T289K D367E t607435 Cymbidium E28P N51D 0.181914 0.136726
hybrid K54E N80H cultivar C82E L83I G84C F86Y D88A N89P N92D F97M
I99R T111E R123K L137M I147L N151P C176D S180N K182P Q206L H233N
S237F A252E S260G G262A G281E T289K D367E t607337 Cymbidium E28P
N51E 0.163476 0.021163 hybrid N80H C82E cultivar L83I G84C F86Y
D88A N89P N92D F97M I99R T111R R123K L137M I147L N151P C176D S180N
K182P Q206L H233N S237F A252E S260G G262A G281E T289K D367E t607316
Cymbidium W301Y 0.161295 0.013439 hybrid P303V P305N cultivar R308P
A309T t607124 Cymbidium I255M A266Y 0 0 hybrid W301Y cultivar P303V
P305N R308P A309T S339W V341P G373A F374L t607280 Cymbidium A266Y
P305N 0 0 hybrid S339W V341P cultivar G373A F374L t607290 Cymbidium
A266Y 0 0 hybrid S339W cultivar t607381 Cymbidium A266Y P305N 0 0
hybrid S339W cultivar
TABLE-US-00027 TABLE 11A Results of Secondary Screen of Protein
Engineering Library Using C. sativa, Cochorus, and Cymbidium
Templates (not supplemented with sodium hexanoate) Amino Acid
Standard Standard Wild-type mutations Average Deviation Average
Deviation template from wild- Olivetol Olivetol Olivetolic
Olivetolic Strain used type [.mu.g/L] [.mu.g/L] Acid [.mu.g/L] Acid
[.mu.g/L] t527338 GFP 0.620833 3.04145 0 0 t527340 Cannabis
wild-type 127.8438 14.41426 0 0 Cannabis sativa OLS t606801
Cannabis S334P 220.05 20.91355 0 0 sativa (Hemp) (Marijuana)
t607067 Cannabis F367L 160.1 15.69777 0 0 sativa (Hemp) (Marijuana)
t607367 Cannabis G366A 142.075 3.482695 0 0 sativa (Hemp)
(Marijuana) t606794 Cannabis wild-type 150.8156 12.9258 0 0 sativa
(Hemp) (Marijuana) t607391 Cannabis P298N 82.95 5.945587 0 0 sativa
(Hemp) (Marijuana) t606984 Cannabis I248M 14.025 0.745542 0 0
sativa (Hemp) (Marijuana) t606899 Cannabis S332W 0 0 0 0 sativa
(Hemp) (Marijuana) t606797 Corchorus wild-type 39.93571 6.066497
128.175 20.00325 Corchorus olitorius OLS t606807 Corchorus d1-8
Y142C 170.75 207.3944 44.3 26.72864 olitorius t606930 Corchorus
d1-8M255I 43.925 4.811358 126.175 8.854519 olitorius t607179
Corchorus Y301W 40.725 8.259288 111.775 6.540833 olitorius V302I
V303T N305P P308K T309A t607332 Corchorus d1-8 Y301W 43.375
4.289814 143.675 17.99711 olitorius V302I V303T N305P P308K T309A
t607236 Corchorus M255I 38.825 0.869387 101.25 4.184495 olitorius
t607006 Corchorus Y266F 25.425 1.011187 84.475 1.575595 olitorius
t606993 Corchorus d1-8 N305P 24.35 1.053565 77.375 1.817278
olitorius t607139 Corchorus d1-8 Y266F 21.525 2.692428 62.375
2.57083 olitorius t607158 Corchorus A373G 17.725 1.705628 46.75
5.257059 olitorius t607153 Corchorus d1-8 A373G 16.875 1.327592
51.575 2.379601 olitorius t606852 Corchorus N305P 25.675 1.613227
75.975 4.164433 olitorius t607112 Corchorus W339S 0 0 0 0 olitorius
t607119 Corchorus d1-8 L374F 0 0 20.725 0.780491 olitorius t607141
Corchorus d1-8 Y266F 0 0 0 0 olitorius W339S t607149 Corchorus d1-8
W339S 0 0 0 0 olitorius t607176 Corchorus L374F 0 0 17.975 0.684957
olitorius t607193 Corchorus d1-8 T12Y 0 0 0 0 olitorius F39Y Q42R
L43A Q47E Q51D Q57K I77L G79E S84C E96D T100E L121K N123K A135V
A137M T139G H143Q N146K K151R H152P K156R F158M S174A V182R D183G
S184A N231T K232N I241V T253D C260G M287E M353R Q357E S395N t607371
Corchorus Y266F 0 0 0 0 olitorius W339S t527346 Cymbidium wild-type
406.4417 80.3967 3.658333 1.293141 Cymbidium hybrid OLS cultivar
t607221 Cymbidium N92D 509.9 0 6.5 0.424264 hybrid cultivar t607228
Cymbidium M135I 524.425 26.05281 6.825 0.861684 hybrid cultivar
t606878 Cymbidium P303A 632.95 28.30365 8.125 5.596055 hybrid
cultivar t606986 Cymbidium E323G 551.95 42.85468 8.6 1.411855
hybrid cultivar t606999 Cymbidium N151P 508.3 23.17873 8.4 0.365148
hybrid cultivar t607224 Cymbidium Y142C 493.525 14.39546 7.725
0.556028 hybrid cultivar t606976 Cymbidium Q274K 541.475 26.72569
6.525 4.353064 hybrid cultivar t607241 Cymbidium V388T 455.325
14.62244 5.75 0.420317 hybrid cultivar t607603 Cymbidium Y142I
540.975 14.56237 11.475 0.853913 hybrid cultivar t607222 Cymbidium
N11K 479.35 10.11163 6.3 0.424264 hybrid cultivar t607014 Cymbidium
A287M 511.8 26.34755 7.975 1.388944 hybrid cultivar t606994
Cymbidium T289A 496.475 22.44614 8.225 0.57373 hybrid cultivar
t606982 Cymbidium V314I 546.875 39.64899 8.9 1.177568 hybrid
cultivar t606995 Cymbidium I147L 480.9 23.94006 7.225 0.7932 hybrid
cultivar t607007 Cymbidium G281E 489.6 12.27436 8.15 0.544671
hybrid cultivar t607008 Cymbidium I390Q 491.625 18.42107 6.575
0.125831 hybrid cultivar t606965 Cymbidium Y142V 570.85 25.65599
10.1 0.547723 hybrid cultivar t607107 Cymbidium A79E 431.575
22.92108 4.575 0.877021 hybrid cultivar t607194 Cymbidium F86Y
437.325 4.76891 4.875 0.727438 hybrid cultivar t606981 Cymbidium
E96R 512.85 23.07993 3.825 4.439501 hybrid cultivar t606979
Cymbidium E32R 492 40.898 6.2 1.249 hybrid cultivar t606975
Cymbidium K182P 532.825 6.97824 7.75 0.3 hybrid cultivar t607230
Cymbidium E116D 461.35 10.56362 5.175 0.499166 hybrid cultivar
t607004 Cymbidium L291Y 487.475 6.717825 8.25 0.619139 hybrid
cultivar t606996 Cymbidium T170A 485.075 15.50965 8 0.559762 hybrid
cultivar t607046 Cymbidium K359R 527.9 18.45842 7.775 0.556028
hybrid cultivar t607043 Cymbidium N45T 524.225 15.59837 6.175
1.040433 hybrid cultivar t607021 Cymbidium H69Y 530.025 26.2045
6.95 0.635085 hybrid cultivar t607109 Cymbidium D61R 446.85
18.05076 5.275 0.873212 hybrid cultivar t607036 Cymbidium K321E
521.625 5.105797 8.05 0.331662 hybrid cultivar t606912 Cymbidium
D14P 538.775 35.94546 8.975 0.910586 hybrid cultivar t607602
Cymbidium R100E 465.225 21.39554 5.75 0.83865 hybrid cultivar
t607361 Cymbidium E323Q 453.35 6.413787 4.775 0.411299 hybrid
cultivar t606882 Cymbidium L222I 501.55 28.07757 7.6 0.559762
hybrid cultivar t606962 Cymbidium D229E 523.325 17.80587 7.225
0.330404 hybrid cultivar t607150 Cymbidium E285K 418.225 12.1393
4.225 0.330404 hybrid cultivar t607252 Cymbidium F40Y 435.9
14.81486 5.275 0.713559 hybrid cultivar t607225 Cymbidium S260G
418.925 13.55221 4.175 0.170783 hybrid cultivar t607032 Cymbidium
K355S 500.8 16.68772 6.75 1.767767 hybrid cultivar t607248
Cymbidium E155C 405.15 11.08708 3.3 1.036018 hybrid cultivar
t607155 Cymbidium C176D 423 7.037045 4.475 0.095743 hybrid cultivar
t606958 Cymbidium C82N 536.2 25.07761 7.575 0.634429 hybrid
cultivar t607023 Cymbidium V71H 511.725 10.15755 7 0.216025
hybrid
cultivar t607027 Cymbidium T289K 520.675 13.61503 8.1 0.535413
hybrid cultivar t606892 Cymbidium Y142T 572.6 39.21003 10.9
0.702377 hybrid cultivar t607035 Cymbidium Y160G 513.55 23.95308
11.825 0.93586 hybrid cultivar t607237 Cymbidium T289S 424.05
16.8777 4.45 0.556776 hybrid cultivar t607189 Cymbidium N51E 420
11.2116 4.55 0.619139 hybrid cultivar t607118 Cymbidium T289Q 404.3
9.899495 4.25 0.070711 hybrid cultivar t607018 Cymbidium I390N
493.25 17.93813 7.025 1.297112 hybrid cultivar t607045 Cymbidium
F30C 522.175 29.45996 8 0.930949 hybrid cultivar t606888 Cymbidium
A107L 521.125 20.05731 6.85 0.404145 hybrid cultivar t607220
Cymbidium A13T 432.725 9.603602 4.825 0.206155 hybrid cultivar
t606830 Cymbidium M135V 568.425 18.5615 7.6 0.432049 hybrid
cultivar t606832 Cymbidium E155Q 552.975 20.81688 7.225 0.741058
hybrid cultivar t607601 Cymbidium K121V 429.25 19.20772 5.025 0.15
hybrid cultivar t606857 Cymbidium A195V 607.575 22.90988 8.125
0.567891 hybrid cultivar t607452 Cymbidium K54E 436.75 12.72962
5.325 0.623832 hybrid cultivar t607218 Cymbidium F97M 451.85
12.7291 5 0.787401 hybrid cultivar t607186 Cymbidium S180N 435.525
20.91274 4.8 0.535413 hybrid cultivar t607123 Cymbidium S237F
421.475 3.484609 4.875 0.386221 hybrid cultivar t607286 Cymbidium
S34Q 452.15 17.4135 5.35 0.412311 hybrid cultivar t606918 Cymbidium
V50N 506.275 26.08453 6.5 0.752773 hybrid cultivar t606916
Cymbidium R100A 509.775 27.51986 7.05 0.988264 hybrid cultivar
t606990 Cymbidium L291W 457.875 21.00831 6.925 1.001249 hybrid
cultivar t606908 Cymbidium I22M 523.45 43.08074 4.35 5.05536 hybrid
cultivar t606963 Cymbidium I147E 502.075 20.19032 6.4 0.752773
hybrid cultivar t607226 Cymbidium Q115D 435.7 13.37934 5.125
0.359398 hybrid cultivar t606961 Cymbidium V43M 537.45 13.96675 6.8
0.355903 hybrid cultivar t607260 Cymbidium E285A 454.15 7.783101
5.2 0.244949 hybrid cultivar t607160 Cymbidium V341A 405.65
13.91893 4.4 0.541603 hybrid cultivar t607156 Cymbidium H269S
415.35 17.57546 3.925 0.464579 hybrid cultivar t607478 Cymbidium
N11R 448.1 9.988994 5.05 0.772442 hybrid cultivar t606887 Cymbidium
Q274G 481.45 22.29716 5.7 3.81663 hybrid cultivar t606861 Cymbidium
E203K 594.35 17.56407 8.25 0.82664 hybrid cultivar t607217
Cymbidium D367E 421.1 11.77427 4.375 0.805709 hybrid cultivar
t606894 Cymbidium Y142F 512.875 39.92705 7.9 0.752773 hybrid
cultivar t607288 Cymbidium T289D 447.875 12.13875 4.8 0.547723
hybrid cultivar t606952 Cymbidium V71Y 514.9 31.88239 5.225
3.545302 hybrid cultivar t607197 Cymbidium G84C 444.2 14.33806
4.825 0.262996 hybrid cultivar t607146 Cymbidium G262T 399.375
11.43864 3.925 0.394757 hybrid cultivar t607017 Cymbidium N78E
517.475 8.940311 8.9 1.75119 hybrid cultivar t607456 Cymbidium
T111Q 466.95 34.97175 5.375 0.512348 hybrid cultivar t606854
Cymbidium L144I 581.275 44.29946 5.925 4.19871 hybrid cultivar
t606838 Cymbidium R123N 529.65 7.364102 6.85 0.896289 hybrid
cultivar t607213 Cymbidium T289G 416.8 8.30542 4.375 0.531507
hybrid cultivar t606932 Cymbidium S18T 495.975 39.19034 6.075
1.189888 hybrid cultivar t607349 Cymbidium K282D 419.1 9.083685
4.075 0.419325 hybrid cultivar t607585 Cymbidium L83I 450.375
20.91226 5.175 0.287228 hybrid cultivar t606956 Cymbidium L83M
475.55 9.733276 6.3 0.535413 hybrid cultivar t607586 Cymbidium
I326R 443.525 14.63361 5.025 0.330404 hybrid cultivar t607025
Cymbidium I99T 473.65 23.0925 6.4 0.408248 hybrid cultivar t607322
Cymbidium R123K 425.975 8.165935 4.6 0.616441 hybrid cultivar
t606905 Cymbidium V388A 489.825 32.71823 6.075 4.224827 hybrid
cultivar t606835 Cymbidium Q161F 562.35 16.93999 4.05 2.763452
hybrid cultivar t607104 Cymbidium E323H 406.55 15.15443 4.85
0.74162 hybrid cultivar t607135 Cymbidium E28P 392.8 21.08159 4.05
0.789515 hybrid cultivar t606891 Cymbidium K356Q 484.225 22.33762
7.45 0.660808 hybrid cultivar t607031 Cymbidium N80H 508.1 14.36825
7.05 0.613732 hybrid cultivar t607317 Cymbidium C82E 472.45
13.27466 6.4 0.909212 hybrid cultivar t607088 Cymbidium P303V
402.225 18.42487 3.225 0.826136 hybrid cultivar t607262 Cymbidium
L385M 467.625 36.03085 4.5 1.048809 hybrid cultivar t606896
Cymbidium Q115S 500.675 15.10968 6.15 4.194043 hybrid cultivar
t607269 Cymbidium A333R 455.775 3.975236 5.15 0.597216 hybrid
cultivar t607294 Cymbidium K359M 441.65 4.773887 5.125 0.25 hybrid
cultivar t607344 Cymbidium A252E 421.125 19.75135 4.675 0.670199
hybrid cultivar t607159 Cymbidium L76I 411.4 24.21955 4.05 0.660808
hybrid cultivar t606890 Cymbidium K112R 519.575 20.66985 8.55
0.675771 hybrid cultivar t607284 Cymbidium F70M 422.125 9.036731
3.925 0.419325 hybrid cultivar t607292 Cymbidium V331I 439.375
22.82475 4.825 0.618466 hybrid cultivar t607476 Cymbidium L20F
438.925 17.62978 4.075 0.464579 hybrid cultivar t606946 Cymbidium
N151P 484.525 10.50599 5.875 0.699405 hybrid cultivar t607320
Cymbidium T111E 427.525 18.14412 5.55 1.021437 hybrid cultivar
t607083 Cymbidium G84S 379.5 14.5952 4.175 1.284199 hybrid cultivar
t607480 Cymbidium A13V 437.5 12.94656 4.85 0.759386 hybrid cultivar
t606909 Cymbidium W368H 496.825 40.68967 4.975 3.350995 hybrid
cultivar t607449 Cymbidium E203P 433.675 17.36613 4.35 0.331662
hybrid cultivar t606851 Cymbidium I293V 514.65 43.13626 6.35
1.369915 hybrid cultivar t607079 Cymbidium S34E 466.325 54.18348
7.175 1.25 hybrid cultivar t607282 Cymbidium R100T 433.175 10.15099
4.525 0.655108 hybrid cultivar t606967 Cymbidium M135A 446.85
27.16143 2.8 3.237283 hybrid cultivar t606938 Cymbidium D88A 483.9
1.131371 6.05 0.070711 hybrid cultivar t607433 Cymbidium L291V
424.875 12.5492 4.475 0.394757 hybrid cultivar t607357 Cymbidium
T243A 419.45 12.8108 4.5 0.294392 hybrid cultivar t607122 Cymbidium
F374L 381.9 18.88686 2.9 0.678233 hybrid cultivar t607110 Cymbidium
R317T 374.025 4.807199 3.575 0.221736 hybrid cultivar t607015
Cymbidium G84T 514.575 14.52111 7.575 1.367175
hybrid cultivar t607087 Cymbidium E96A 393.975 20.30195 4.375
1.004573 hybrid cultivar t607019 Cymbidium I99A 462.175 18.38104
6.175 0.543906 hybrid cultivar t606839 Cymbidium D207S 557.425
29.43245 7.425 0.818026 hybrid cultivar t606942 Cymbidium R100P
461.85 7.707464 4.05 0.212132 hybrid cultivar t607164 Cymbidium
V327A 393.05 14.87672 3.225 0.35 hybrid cultivar t606906 Cymbidium
E28A 482 14.14214 6.85 0.494975 hybrid cultivar t606868 Cymbidium
L248I 555.5 18.09586 9 0.955685 hybrid cultivar t607089 Cymbidium
L77I 380.525 6.542871 4.125 1.170114 hybrid cultivar t606910
Cymbidium N89P 471.825 23.09998 7.025 1.611159 hybrid cultivar
t606936 Cymbidium T111K 468.95 8.328465 5.125 0.287228 hybrid
cultivar t606856 Cymbidium V157T 551.4 18.24573 8.525 0.78475
hybrid cultivar t607450 Cymbidium I99R 406.45 13.84786 3.5 0.637704
hybrid cultivar t606960 Cymbidium N78R 456.45 26.30089 4.225
3.001527 hybrid cultivar t607600 Cymbidium R123A 413.025 13.12945
4.675 0.457347 hybrid cultivar t606940 Cymbidium A13S 481.65
28.77925 6.3 0.424264 hybrid cultivar t607085 Cymbidium K55R 349.1
16.14745 3.125 0.543906 hybrid cultivar t607474 Cymbidium G262A
397.225 2.348581 3.4 0.294392 hybrid cultivar t606959 Cymbidium
A219C 411.275 14.18506 2.25 0.544671 hybrid cultivar t606859
Cymbidium I99E 511.05 22.442 7.325 0.543906 hybrid cultivar t606904
Cymbidium S10R 467.15 52.82088 7.25 1.626346 hybrid cultivar
t607195 Cymbidium T111R 404.8 17.43885 4.25 0.493288 hybrid
cultivar t607445 Cymbidium P305N 341.875 14.41281 2.725 0.55 hybrid
cultivar t607273 Cymbidium V341P 395.325 9.541619 3.625 0.655108
hybrid cultivar t606834 Cymbidium V157I 516.05 31.53966 6.625
1.192686 hybrid cultivar t607254 Cymbidium G262T 418.575 14.71991
4.35 0.574456 hybrid cultivar t606828 Cymbidium A107M 503.3
16.92986 5.5 1.191638 hybrid cultivar t606836 Cymbidium K104Q 543.5
39.43129 7.1 1.174734 hybrid cultivar t607081 Cymbidium I99G
347.025 12.30864 2.725 0.567891 hybrid cultivar t607482 Cymbidium
R24K 405.575 10.66908 4.175 0.434933 hybrid cultivar t607132
Cymbidium D208A 328.9 30.48748 2.675 0.634429 hybrid cultivar
t606874 Cymbidium D208S 395.55 19.92327 4.2 0.6733 hybrid cultivar
t607190 Cymbidium L137M 292.8 8.173942 1.5 0.216025 hybrid cultivar
t607028 Cymbidium D208N 356.3 20.88349 3.125 0.377492 hybrid
cultivar t607370 Cymbidium L264F 297.2 7.708869 4.575 0.411299
hybrid cultivar t606898 Cymbidium I102A 371.3 14.25669 2.75
0.834666 hybrid cultivar t607216 Cymbidium Q206L 280.95 22.84637
2.325 0.359398 hybrid cultivar t606901 Cymbidium D208C 351.525
23.67324 1.8 1.275408 hybrid cultivar t607256 Cymbidium G373A
236.425 5.235376 0 0 hybrid cultivar t607131 Cymbidium N51D 191.275
227.01 1.9 2.941655 hybrid cultivar t607604 Cymbidium I99K 203.675
235.3296 2.225 hybrid cultivar t606914 Cymbidium A13N 246.7
285.4356 3.825 4.470925 hybrid cultivar t606934 Cymbidium S10N
230.525 266.3877 2.975 3.435477 hybrid cultivar t607312 Cymbidium
I255M 60.1 3.31763 0 0 hybrid cultivar t607377 Cymbidium S339W 0 0
0 0 hybrid cultivar t607318 Cymbidium E28P F40Y hybrid N51D K54E
cultivar N80H C82E L83I G84C F86Y D88A N89P N92D F97M I99R T111E
R123K L137M I147L N151P C176D 260.2 9.255269 3.3 0.702377 S180N
K182P Q206L H233N S237F A252E S260G G262A G281E T289K D367E t607245
Cymbidium E28P G84C 265.1 7.813237 0.225 0.45 hybrid N89P T111R
cultivar N151P C176D Q206L S237F A252E G262T G281E t607000
Cymbidium G373A 253.05 22.66282 0 0 hybrid F374L cultivar t607337
Cymbidium E28P N51E 214.225 6.286162 1.6 0.141421 hybrid N80H C82E
cultivar L83I G84C F86Y D88A N89P N92D F97M I99R T111R R123K L137M
I147L N151P C176D S180N K182P Q206L H233N S237F A252E S260G G262A
G281E T289K D367E t607265 Cymbidium E28P F40Y 225.35 4.919011 1.8
0.374166 hybrid N51D K54E cultivar N80H C82E L83I G84C F86Y D88A
N89P N92D F97M I99R T111Q R123K L137M I147L N151P C176D S180N K182P
Q206L H233N S237F A252E S260G G262A G281E T289K D367E t607316
Cymbidium W301Y 177.7 14.90526 2.075 0.801561 hybrid P303V cultivar
P305N R308P A309T t607435 Cymbidium E28P N51D 156.525 104.7564
1.075 0.813941 hybrid K54E N80H cultivar C82E L83I G84C F86Y D88A
N89P N92D F97M I99R T111E R123K L137M I147L N151P C176D S180N K182P
Q206L H233N S237F A252E S260G G262A G281E T289K D367E t607381
Cymbidium A266Y 4.825 9.65 0 0 hybrid P305N cultivar S339W t607124
Cymbidium I255M 3.625 7.25 0 0 hybrid A266Y cultivar W301Y P303V
P305N R308P A309T
S339W V341P G373A F374L t607280 Cymbidium A266Y 0 0 0 0 hybrid
P305N cultivar S339W V341P G373A F374L t607290 Cymbidium A266Y 0 0
0 0 hybrid S339W cultivar
TABLE-US-00028 TABLE 11B Results of Secondary Screen of Protein
Engineering Library Using C. sativa, Cochorus, and Cymbidium
Templates (not supplemented with sodium hexanoate) Average Standard
Olivetol Deviation Normalized Olivetol Amino Acid Average Standard
to t606797_ Normalized Wild-type mutations Normalized Deviation
Corchorus to t606797_ template from wild- Olivetol Normalized OLS
Corchorus Strain used type (per OD) Olivetol (per OD) OLS t527338
0.0048 0.0235 GFP t527340 Cannabis wild-type 1.05197 0.13292
Cannabis sativa OLS t606801 Cannabis S334P 1.9025 0.12934 sativa
t607067 Cannabis F367L 1.49374 0.07849 sativa t607367 Cannabis
G366A 1.32919 0.09047 sativa) t606794 Cannabis wild-type 1.30342
0.19421 sativa (Hemp) (Marijuana) t607391 Cannabis P298N 0.8132
0.03261 sativa t606984 Cannabis I248M 0.15063 0.02208 sativa
t606899 Cannabis S332W 0 0 sativa t606797 Corchorus wild-type
0.31178 0.05119 1 0.14396 Corchorus olitorius OLS t606807 Corchorus
d1-8 Y142C 1.36312 1.64905 5.49321 6.64551 olitorius t606930
Corchorus d1-8 M255I 0.44815 0.04224 1.40843 0.13275 olitorius
t607179 Corchorus Y301W 0.42428 0.08973 1.45657 0.30806 olitorius
V302I V303T N305P P308K T309A t607332 Corchorus d1-8 Y301W 0.39884
0.06665 1.15654 0.19327 olitorius V302I V303T N305P P308K T309A
t607236 Corchorus M255I 0.37718 0.03147 1.27535 0.1064 olitorius
t607006 Corchorus Y266F 0.2237 0.00789 0.72435 0.02554 olitorius
t606993 Corchorus d1-8 N305P 0.20738 0.02797 0.67149 0.09057
olitorius t607139 Corchorus d1-8 Y266F 0.19496 0.02599 0.66932
0.08921 olitorius t607158 Corchorus A373G 0.18139 0.0179 0.62271
0.06145 olitorius t607153 Corchorus d1-8 A373G 0.16382 0.01353
0.56242 0.04646 olitorius t606852 Corchorus N305P 0.16244 0.00836
0.65462 0.03369 olitorius t607112 Corchorus W339S 0 0 0 0 olitorius
t607119 Corchorus d1-8 L374F 0 0 0 0 olitorius t607141 Corchorus
d1-8 Y266F 0 0 0 0 olitorius W339S t607149 Corchorus d1-8 W339S 0 0
0 0 olitorius t607176 Corchorus L374F 0 0 0 0 olitorius t607193
Corchorus d1-8 T12Y 0 0 0 0 olitorius F39Y Q42R L43A Q47E Q51D Q57K
I77L G79E S84C E96D T100E L121K N123K A135V A137M T139G H143Q N146K
K151R H152P K156R F158M S174A V182R D183G S184A N231T K232N I241V
T253D C260G M287E M353R Q357E S395N t607371 Corchorus Y266F 0 0 0 0
olitorius W339S t527346 Cymbidium wild-type 1.02322 0.20846
Cymbidium hybrid OLS cultivar t607221 Cymbidium N92D 1.67744
0.07244 hybrid cultivar t607228 Cymbidium M135I 1.64252 0.01538
hybrid cultivar t606878 Cymbidium P303A 1.56942 0.06675 hybrid
cultivar t606986 Cymbidium E323G 1.56671 0.16314 hybrid cultivar
t606999 Cymbidium N151P 1.5521 0.09794 hybrid cultivar t607224
Cymbidium Y142C 1.54592 0.10755 hybrid cultivar t606976 Cymbidium
Q274K 1.54274 0.07232 hybrid cultivar t607241 Cymbidium V388T
1.53854 0.07404 hybrid cultivar t607603 Cymbidium Y142I 1.53604
0.11347 hybrid cultivar t607222 Cymbidium N11K 1.52325 0.04008
hybrid cultivar t607014 Cymbidium A287M 1.49053 0.07415 hybrid
cultivar t606994 Cymbidium T289A 1.46316 0.12925 hybrid cultivar
t606982 Cymbidium V314I 1.46045 0.08295 hybrid cultivar t606995
Cymbidium I147L 1.4549 0.07909 hybrid cultivar t607007 Cymbidium
G281E 1.45405 0.06194 hybrid cultivar t607008 Cymbidium I390Q
1.45278 0.0047 hybrid cultivar t606965 Cymbidium Y142V 1.42708
0.18261 hybrid cultivar t607107 Cymbidium A79E 1.4258 0.07329
hybrid cultivar t607194 Cymbidium F86Y 1.42238 0.02094 hybrid
cultivar t606981 Cymbidium E96R 1.42122 0.04842 hybrid cultivar
t606979 Cymbidium E32R 1.416 0.12096 hybrid cultivar t606975
Cymbidium K182P 1.4052 0.03345 hybrid cultivar t607230 Cymbidium
E116D 1.40132 0.0774 hybrid cultivar t607004 Cymbidium L291Y
1.40103 0.06311 hybrid cultivar t606996 Cymbidium T170A 1.38574
0.05793 hybrid cultivar t607046 Cymbidium K359R 1.38299 0.01508
hybrid cultivar t607043 Cymbidium N45T 1.38195 0.03083 hybrid
cultivar t607021 Cymbidium H69Y 1.37598 0.10301 hybrid cultivar
t607109 Cymbidium D61R 1.37011 0.10414 hybrid cultivar t607036
Cymbidium K321E 1.36745 0.04988 hybrid cultivar t606912 Cymbidium
D14P 1.36318 0.09371 hybrid cultivar t607602 Cymbidium R100E
1.35489 0.07769 hybrid cultivar t607361 Cymbidium E323Q 1.35416
0.07809 hybrid cultivar t606882 Cymbidium L222I 1.35377 0.07907
hybrid cultivar t606962 Cymbidium D229E 1.34991 0.03688 hybrid
cultivar t607150 Cymbidium E285K 1.3445 0.07571 hybrid cultivar
t607252 Cymbidium F40Y 1.34329 0.11563 hybrid cultivar t607225
Cymbidium S260G 1.34171 0.08715 hybrid cultivar t607032 Cymbidium
K355S 1.34146 0.08572 hybrid cultivar t607248 Cymbidium E155C
1.3346 0.03904 hybrid cultivar t607155 Cymbidium C176D 1.33173
0.01853 hybrid cultivar t606958 Cymbidium C82N 1.32771 0.01591
hybrid cultivar t607023 Cymbidium V71H 1.32703 0.0343 hybrid
cultivar t607027 Cymbidium T289K 1.32433 0.08328 hybrid cultivar
t606892 Cymbidium Y142T 1.31787 0.21329 hybrid cultivar t607035
Cymbidium Y160G 1.31694 0.07538
hybrid cultivar t607237 Cymbidium T289S 1.31692 0.10049 hybrid
cultivar t607189 Cymbidium N51E 1.31519 0.10593 hybrid cultivar
t607118 Cymbidium T289Q 1.31279 0.05154 hybrid cultivar t607018
Cymbidium I390N 1.31146 0.00884 hybrid cultivar t607045 Cymbidium
F30C 1.31145 0.13007 hybrid cultivar t606888 Cymbidium A107L
1.31126 0.05185 hybrid cultivar t607220 Cymbidium A13T 1.3073
0.0685 hybrid cultivar t606830 Cymbidium M135V 1.30213 0.13463
hybrid cultivar t606832 Cymbidium E155Q 1.29883 0.18596 hybrid
cultivar t607601 Cymbidium K121V 1.29806 0.0748 hybrid cultivar
t606857 Cymbidium A195V 1.29426 0.3015 hybrid cultivar t607452
Cymbidium K54E 1.29134 0.03462 hybrid cultivar t607218 Cymbidium
F97M 1.29082 0.07837 hybrid cultivar t607186 Cymbidium S180N
1.28937 0.10055 hybrid cultivar t607123 Cymbidium S237F 1.28824
0.07181 hybrid cultivar t607286 Cymbidium S34Q 1.28672 0.12162
hybrid cultivar t606918 Cymbidium V50N 1.28475 0.02954 hybrid
cultivar t606916 Cymbidium R100A 1.28388 0.03268 hybrid cultivar
t606990 Cymbidium L291W 1.27925 0.06685 hybrid cultivar t606908
Cymbidium I22M 1.27921 0.08525 hybrid cultivar t606963 Cymbidium
I147E 1.27917 0.09902 hybrid cultivar t607226 Cymbidium Q115D
1.27909 0.05228 hybrid cultivar t606961 Cymbidium V43M 1.27574
0.04471 hybrid cultivar t607260 Cymbidium E285A 1.27099 0.05173
hybrid cultivar t607160 Cymbidium V341A 1.26952 0.05458 hybrid
cultivar t607156 Cymbidium H269S 1.26921 0.0896 hybrid cultivar
t607478 Cymbidium N11R 1.26856 0.08255 hybrid cultivar t606887
Cymbidium Q274G 1.26839 0.09424 hybrid cultivar t606861 Cymbidium
E203K 1.26653 0.2508 hybrid cultivar t607217 Cymbidium D367E
1.26643 0.04114 hybrid cultivar t606894 Cymbidium Y142F 1.26586
0.09867 hybrid cultivar t607288 Cymbidium T289D 1.26389 0.05999
hybrid cultivar t606952 Cymbidium V71Y 1.26229 0.12989 hybrid
cultivar t607197 Cymbidium G84C 1.26224 0.07225 hybrid cultivar
t607146 Cymbidium G262T 1.26188 0.03879 hybrid cultivar t607017
Cymbidium N78E 1.26073 0.09115 hybrid cultivar t607456 Cymbidium
T111Q 1.25899 0.07745 hybrid cultivar t606854 Cymbidium L144I
1.25807 0.22743 hybrid cultivar t606838 Cymbidium R123N 1.25683
0.23657 hybrid cultivar t607213 Cymbidium T289G 1.25405 0.08316
hybrid cultivar t606932 Cymbidium S18T 1.25389 0.12779 hybrid
cultivar t607349 Cymbidium K282D 1.25244 0.08104 hybrid cultivar
t607585 Cymbidium L83I 1.2509 0.04892 hybrid cultivar t606956
Cymbidium L83M 1.24841 0.05473 hybrid cultivar t607586 Cymbidium
I326R 1.24784 0.04812 hybrid cultivar t607025 Cymbidium I99T
1.24421 0.09819 hybrid cultivar t607322 Cymbidium R123K 1.24342
0.08106 hybrid cultivar t606905 Cymbidium V388A 1.24241 0.09318
hybrid cultivar t606835 Cymbidium Q161F 1.24193 0.10262 hybrid
cultivar t607104 Cymbidium E323H 1.24084 0.05988 hybrid cultivar
t607135 Cymbidium E28P 1.24063 0.06484 hybrid cultivar t606891
Cymbidium K356Q 1.24008 0.09459 hybrid cultivar t607031 Cymbidium
N80H 1.23893 0.09737 hybrid cultivar t607317 Cymbidium C82E 1.23849
0.03065 hybrid cultivar t607088 Cymbidium P303V 1.23838 0.07685
hybrid cultivar t607262 Cymbidium L385M 1.23811 0.14991 hybrid
cultivar t606896 Cymbidium Q115S 1.2378 0.10758 hybrid cultivar
t607269 Cymbidium A333R 1.23132 0.05599 hybrid cultivar t607294
Cymbidium K359M 1.22923 0.04507 hybrid cultivar t607344 Cymbidium
A252E 1.22825 0.1572 hybrid cultivar t607159 Cymbidium L76I 1.22743
0.10259 hybrid cultivar t606890 Cymbidium K112R 1.22535 0.04006
hybrid cultivar t607284 Cymbidium F70M 1.22458 0.03416 hybrid
cultivar t607292 Cymbidium V331I 1.22387 0.05296 hybrid cultivar
t607476 Cymbidium L20F 1.22172 0.06972 hybrid cultivar t606946
Cymbidium N151P 1.22164 0.0856 hybrid cultivar t607320 Cymbidium
T111E 1.22064 0.07826 hybrid cultivar t607083 Cymbidium G84S
1.21542 0.11127 hybrid cultivar t607480 Cymbidium A13V 1.21428
0.00581 hybrid cultivar t606909 Cymbidium W368H 1.21403 0.1423
hybrid cultivar t607449 Cymbidium E203P 1.21329 0.09644 hybrid
cultivar t606851 Cymbidium I293V 1.21178 0.19419 hybrid cultivar
t607079 Cymbidium S34E 1.21014 0.05037 hybrid cultivar t607282
Cymbidium R100T 1.20952 0.03641 hybrid cultivar t606967 Cymbidium
M135A 1.20347 0.12224 hybrid cultivar t606938 Cymbidium D88A
1.19819 0.02307 hybrid cultivar t607433 Cymbidium L291V 1.19755
0.06191 hybrid cultivar t607357 Cymbidium T243A 1.19607 0.10095
hybrid cultivar t607122 Cymbidium F374L 1.19551 0.12138 hybrid
cultivar t607110 Cymbidium R317T 1.19291 0.05771 hybrid cultivar
t607015 Cymbidium G84T 1.19021 0.12242 hybrid cultivar t607087
Cymbidium E96A 1.18792 0.06196 hybrid cultivar t607019 Cymbidium
I99A 1.18519 0.12123 hybrid cultivar
t606839 Cymbidium D207S 1.17855 0.17153 hybrid cultivar t606942
Cymbidium R100P 1.17753 0.09144 hybrid cultivar t607164 Cymbidium
V327A 1.17683 0.0485 hybrid cultivar t606906 Cymbidium E28A 1.1681
0.05241 hybrid cultivar t606868 Cymbidium L248I 1.16589 0.03553
hybrid cultivar t607089 Cymbidium L77I 1.16044 0.02039 hybrid
cultivar t606910 Cymbidium N89P 1.15844 0.18953 hybrid cultivar
t606936 Cymbidium T111K 1.15338 0.07375 hybrid cultivar t606856
Cymbidium V157T 1.14804 0.06609 hybrid cultivar t607450 Cymbidium
I99R 1.13733 0.02723 hybrid cultivar t606960 Cymbidium N78R 1.13483
0.06431 hybrid cultivar t607600 Cymbidium R123A 1.13448 0.08691
hybrid cultivar t606940 Cymbidium A13S 1.13438 0.04862 hybrid
cultivar t607085 Cymbidium K55R 1.12814 0.06807 hybrid cultivar
t607474 Cymbidium G262A 1.12724 0.09746 hybrid cultivar t606959
Cymbidium A219C 1.1175 0.07755 hybrid cultivar t606859 Cymbidium
I99E 1.10483 0.19373 hybrid cultivar t606904 Cymbidium S10R 1.10411
0.13592 hybrid cultivar t607195 Cymbidium T111R 1.10148 0.06594
hybrid cultivar t607445 Cymbidium P305N 1.0924 0.04527 hybrid
cultivar t607273 Cymbidium V341P 1.09021 0.01522 hybrid cultivar
t606834 Cymbidium V157I 1.07903 0.07146 hybrid cultivar t607254
Cymbidium G262T 1.07703 0.05546 hybrid cultivar t606828 Cymbidium
A107M 1.07407 0.12564 hybrid cultivar t606836 Cymbidium K104Q
1.07292 0.1499 hybrid cultivar t607081 Cymbidium I99G 1.0581 0.0688
hybrid cultivar t607482 Cymbidium R24K 1.05409 0.07847 hybrid
cultivar t607132 Cymbidium D208A 1.01083 0.07712 hybrid cultivar
t606874 Cymbidium D208S 0.95301 0.04077 hybrid cultivar t607190
Cymbidium L137M 0.90492 0.02932 hybrid cultivar t607028 Cymbidium
D208N 0.88114 0.04406 hybrid cultivar t607370 Cymbidium L264F
0.87471 0.07388 hybrid cultivar t606898 Cymbidium I102A 0.85753
0.12759 hybrid cultivar t607216 Cymbidium Q206L 0.83138 0.05303
hybrid cultivar t606901 Cymbidium D208C 0.82507 0.10608 hybrid
cultivar t607256 Cymbidium G373A 0.65595 0.02379 hybrid cultivar
t607131 Cymbidium N51D 0.5808 0.70692 hybrid cultivar t607604
Cymbidium I99K 0.57364 0.66547 hybrid cultivar t606914 Cymbidium
A13N 0.56823 0.65663 hybrid cultivar t606934 Cymbidium S10N 0.54561
0.63028 hybrid cultivar t607312 Cymbidium I255M 0.16984 0.01174
hybrid cultivar t607377 Cymbidium S339W 0 0 hybrid cultivar t607318
Cymbidium E28P F40Y 0.78529 0.05769 hybrid N51D K54E cultivar N80H
C82E L83I G84C F86Y D88A N89P N92D F97M I99R T111E R123K L137M
I147L N151P C176D S180N K182P Q206L H233N S237F A252E S260G G262A
G281E T289K D367E t607245 Cymbidium E28P G84C 0.77803 0.02842
hybrid N89P T111R cultivar N151P C176D Q206L S237F A252E G262T
G281E t607000 Cymbidium G373A 0.71402 0.06528 hybrid F374L cultivar
t607337 Cymbidium E28P N51E 0.66352 0.03712 hybrid N80H C82E
cultivar L83I G84C F86Y D88A N89P N92D F97M I99R T111R R123K L137M
I147L N151P C176D S180N K182P Q206L H233N S237F A252E S260G G262A
G281E T289K D367E t607265 Cymbidium E28P F40Y 0.64379 0.0247 hybrid
N51D K54E cultivar N80H C82E L83I G84C F86Y D88A N89P N92D F97M
I99R T111Q R123K L137M I147L N151P C176D S180N K182P Q206L H233N
S237F A252E S260G G262A G281E T289K D367E t607316 Cymbidium W301Y
0.49342 0.01579 hybrid P303V cultivar P305N R308P A309T t607435
Cymbidium E28P N51D 0.46307 0.30921 hybrid K54E N80H cultivar C82E
L83I G84C F86Y D88A N89P N92D F97M I99R T111E R123K L137M I147L
N151P C176D S180N K182P Q206L H233N S237F A252E S260G G262A G281E
T289K D367E t607381 Cymbidium A266Y 0.01389 0.02777 hybrid P305N
cultivar S339W t607124 Cymbidium I255M 0.01165 0.02329 hybrid A266Y
cultivar W301Y P303V P305N R308P A309T S339W V341P G373A F374L
t607280 Cymbidium A266Y 0 0 hybrid P305N cultivar S339W V341P G373A
F374L t607290 Cymbidium A266Y 0 0 hybrid S339W cultivar
TABLE-US-00029 TABLE 12 Screening Results in Prototrophic S.
cerevisiae strain Average Standard Deviation Strain Strain type
Olivetol [.mu.g/L] Olivetol [.mu.g/L] t473139 Negative Control 0 0
t496101 Cannabis OLS variant 20254.13 2236.483 (positive control)
t496102 Library 20566.05 2055.026 t485668 Library 24062.45 4250.129
t496079 Library 29485.08 2786.913 t485662 Library 50257.28 3891.439
t496084 t496084 Cannabis OLS 53595.65 7035.556 T335C point mutant
t485672 Library 53606.37 6230.06 t496073 Library 56729.84
4435.122
TABLE-US-00030 TABLE 13 Sequence Information for Strains described
in TABLE 9 Nucleotide Protein Sequence Sequence (SEQ ID (SEQ ID
Strain NO) NO) t405417 250 207 t404953 251 208 t405220 252 209
t404192 253 210 t404323 254 211 t404196 255 212 t404209 256 213
t404164 257 214 t404170 258 215 t404384 259 216 t405397 260 217
t405164 261 218 t404191 262 219 t405340 263 220 t404421 264 221
t404631 265 222 t405133 266 223 t405081 267 224 t404898 268 225
t405017 269 226 t405140 270 227 t404276 271 228 t404405 272 229
t405079 273 230 t404978 274 231 t405347 275 232 t404855 276 233
t405362 277 234 t404523 278 235 t404951 279 236 t405308 280 237
t405201 281 238 t404219 282 239 t404673 283 240 t404274 284 241
t405042 285 242 t404528 286 243 t405312 287 244 t404725 288 245
t405303 289 246 t405395 290 247 t405326 291 248 t404599 292 249
TABLE-US-00031 TABLE 14 Sequence Information for Strains Described
in TABLE 10A-10B Nucleotide Protein Sequence Sequence Strain (SEQ
ID NO) (SEQ ID NO) t606794 96 80 t527340 62 5 t607067 421 293
t607367 422 294 t607391 423 295 t606801 424 296 t606984 425 297
t606899 426 298 t606797 37 6 t606807 427 299 t607179 428 300
t607149 429 301 t607139 430 302 t607112 431 303 t607332 432 304
t607153 433 305 t607158 434 306 t607236 435 307 t607141 436 308
t607176 437 309 t606930 438 310 t607193 439 311 t607006 440 312
t606993 441 313 t606852 442 314 t607119 443 315 t607371 444 316
t527346 38 7 t606952 445 317 t607284 446 318 t607262 447 319
t606938 448 320 t607260 449 321 t607159 450 322 t606946 451 323
t606861 452 324 t606918 453 325 t607135 454 326 t607286 455 327
t606942 456 328 t606959 457 329 t607294 458 330 t607282 459 331
t607230 460 332 t606965 461 333 t607288 462 334 t607228 463 335
t606909 464 336 t606962 465 337 t607150 466 338 t607361 467 339
t606932 468 340 t606940 469 341 t607269 470 342 t607186 471 343
t607476 472 344 t607031 473 345 t606916 474 346 t607292 475 347
t606908 476 348 t607248 477 349 t607023 478 350 t606936 479 351
t607433 480 352 t607600 481 353 t606894 482 354 t606963 483 355
t607603 484 356 t607452 485 357 t607197 486 358 t606996 487 359
t607043 488 360 t607254 489 361 t607478 490 362 t607132 491 363
t607109 492 364 t607155 493 365 t606956 494 366 t606906 495 367
t607195 496 368 t607449 497 369 t607256 498 370 t607349 499 371
t606960 500 372 t607601 501 373 t607021 502 374 t606874 503 375
t607320 504 376 t607317 505 377 t607224 506 378 t606912 507 379
t607602 508 380 t606905 509 381 t607156 510 382 t607474 511 383
t607482 512 384 t606854 513 385 t607032 514 386 t606830 515 387
t606961 516 388 t606868 517 389 t607083 518 390 t606958 519 391
t607273 520 392 t607241 521 393 t606857 522 394 t606901 523 395
t607015 524 396 t607586 525 397 t607122 526 398 t606882 527 399
t607146 528 400 t607585 529 401 t606828 530 402 t607081 531 403
t606887 532 404 t606891 533 405 t607160 534 406 t607194 535 407
t607377 537 409 t607265 538 410 t607000 539 411 t607245 540 412
t607318 541 413 t607435 542 414 t607337 543 415 t607316 544 416
t607124 545 417 t607280 546 418 t607290 547 419 t607381 548 420
TABLE-US-00032 TABLE 15 Sequence Information for Strains Described
in TABLES 11A-11B Nucleotide Protein Sequence Sequence (SEQ ID (SEQ
ID Strain NO) NO) t527340 62 5 t606801 424 296 t607067 421 293
t607367 422 294 t606794 96 80 t607391 423 295 t606984 425 297
t606899 426 298 t606797 37 6 t606807 427 299 t606930 438 310
t607179 428 300 t607332 432 304 t607236 435 307 t607006 440 312
t606993 441 313 t607139 430 302 t607158 434 306 t607153 433 305
t606852 442 314 t607112 431 303 t607119 443 315 t607141 436 308
t607149 429 301 t607176 437 309 t607193 439 311 t607371 444 316
t527346 38 7 t607221 628 549 t607228 463 335 t606878 629 550
t606986 630 551 t606999 631 552 t607224 506 378 t606976 632 553
t607241 521 393 t607603 484 356 t607222 633 554 t607014 634 555
t606994 635 556 t606982 636 557 t606995 637 558 t607007 638 559
t607008 639 560 t606965 461 333 t607107 640 561 t607194 535 407
t606981 641 562 t606979 642 563 t606975 643 564 t607230 460 332
t607004 644 565 t606996 487 359 t607046 645 566 t607043 488 360
t607021 502 374 t607109 492 364 t607036 646 567 t606912 507 379
t607602 508 380 t607361 467 339 t606882 527 399 t606962 465 337
t607150 466 338 t607252 647 568 t607225 648 569 t607032 514 386
t607248 477 349 t607155 493 365 t606958 519 391 t607023 478 350
t607027 649 570 t606892 650 571 t607035 651 572 t607237 652 573
t607189 653 574 t607118 654 575 t607018 655 576 t607045 656 577
t606888 657 578 t607220 658 579 t606830 515 387 t606832 659 580
t607601 501 373 t606857 522 394 t607452 485 357 t607218 660 581
t607186 471 343 t607123 661 582 t607286 455 327 t606918 453 325
t606916 474 346 t606990 662 583 t606908 476 348 t606963 483 355
t607226 663 584 t606961 516 388 t607260 449 321 t607160 534 406
t607156 510 382 t607478 490 362 t606887 532 404 t606861 452 324
t607217 664 585 t606894 482 354 t607288 462 334 t606952 445 317
t607197 486 358 t607146 528 400 t607017 665 586 t607456 666 587
t606854 513 385 t606838 667 588 t607213 668 589 t606932 468 340
t607349 499 371 t607585 529 401 t606956 494 366 t607586 525 397
t607025 669 590 t607322 670 591 t606905 509 381 t606835 671 592
t607104 672 593 t607135 454 326 t606891 533 405 t607031 473 345
t607317 505 377 t607088 673 594 t607262 447 319 t606896 674 595
t607269 470 342 t607294 458 330 t607344 675 596 t607159 450 322
t606890 676 597 t607284 446 318 t607292 475 347 t607476 472 344
t606946 451 323 t607320 504 376 t607083 518 390 t607480 677 598
t606909 464 336 t607449 497 369 t606851 678 599 t607079 679 600
t607282 459 331 t606967 680 601 t606938 448 320 t607433 480 352
t607357 681 602 t607122 526 398 t607110 682 603 t607015 524 396
t607087 683 604 t607019 684 605 t606839 685 606 t606942 456 328
t607164 686 607 t606906 495 367 t606868 517 389 t607089 687 608
t606910 688 609 t606936 479 351 t606856 689 610 t607450 690 611
t606960 500 372 t607600 481 353 t606940 469 341 t607085 691 612
t607474 511 383 t606959 457 329 t606859 692 613 t606904 693 614
t607195 496 368 t607445 694 615 t607273 520 392 t606834 695 616
t607254 489 361 t606828 530 402 t606836 696 617 t607081 531 403
t607482 512 384 t607132 491 363 t606874 503 375 t607190 697 618
t607028 698 619 t607370 699 620 t606898 700 621 t607216 701 622
t606901 523 395 t607256 498 370 t607131 702 623 t607604 703 624
t606914 704 625 t606934 705 626 t607312 536 408 t607377 537 409
t607318 541 413 t607245 540 412 t607000 539 411 t607337 543 415
t607265 538 410 t607316 544 416 t607435 542 414 t607381 548 420
t607124 545 417 t607280 546 418 t607290 547 419
TABLE-US-00033 TABLE 16 Sequence Information for Strains Described
in TABLE 12 Amino Nucleotide Acid Sequence Sequence (SEQ ID (SEQ ID
Strain NO) NO) t496101 62 5 t496102 39 8 t485668 44 13 t496079 47
16 t485662 46 15 t496084 706 627 t485672 48 17 t496073 38 7
EQUIVALENTS
[0469] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
All references, including patent documents, disclosed herein are
incorporated by reference in their entirety.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20220195472A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20220195472A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References