U.S. patent application number 17/405685 was filed with the patent office on 2022-03-24 for acyl-acp reductase with improved properties.
The applicant listed for this patent is Genomatica, Inc.. Invention is credited to Jacob GANO, Mathew RUDE, Andreas SCHIRMER, Na TRINH.
Application Number | 20220090028 17/405685 |
Document ID | / |
Family ID | |
Filed Date | 2022-03-24 |
United States Patent
Application |
20220090028 |
Kind Code |
A1 |
RUDE; Mathew ; et
al. |
March 24, 2022 |
ACYL-ACP Reductase With Improved Properties
Abstract
The disclosure relates to acyl-ACP reductase (AAR) enzyme
variants that result in improved fatty aldehyde and fatty alcohol
production when expressed in recombinant host cells. The disclosure
further relates to methods of making and using such AAR variants
for the production of fatty alcohol compositions having particular
characteristics.
Inventors: |
RUDE; Mathew; (San Diego,
CA) ; TRINH; Na; (Burlingame, CA) ; SCHIRMER;
Andreas; (San Diego, CA) ; GANO; Jacob; (San
Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Genomatica, Inc. |
San Diego |
CA |
US |
|
|
Appl. No.: |
17/405685 |
Filed: |
August 18, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16272303 |
Feb 11, 2019 |
11130944 |
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17405685 |
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15222657 |
Jul 28, 2016 |
10208294 |
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16272303 |
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14761299 |
Jul 15, 2015 |
9683219 |
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PCT/US2014/011859 |
Jan 16, 2014 |
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15222657 |
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61753273 |
Jan 16, 2013 |
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International
Class: |
C12N 9/02 20060101
C12N009/02; C12P 7/24 20060101 C12P007/24; C12P 7/04 20060101
C12P007/04; C12P 7/64 20060101 C12P007/64 |
Claims
1.-82. (canceled)
83. A recombinant host cell expressing a variant acyl-ACP reductase
(AAR) polypeptide comprising at least 90% sequence identity to the
amino acid sequence of SEQ ID NO: 80, wherein said variant AAR
polypeptide comprises a mutation at an amino acid position selected
from the group consisting of Q40V, G52V, G273E, K303G, H340P,
L344A, L344D, L344S, L344T, A345R, V346P, V346G, and A345*, and
wherein said AAR polypeptide catalyzes the conversion of an
acyl-ACP to a fatty aldehyde.
84. The recombinant host cell of claim 83, wherein the recombinant
host cell produces a fatty aldehyde or fatty alcohol composition
with a titer that is at least 10% greater, at least 15% greater, at
least 20% greater, at least 25% greater, or at least 30% greater
than the titer of a fatty aldehyde or alcohol composition produced
by a host cell expressing a corresponding wild type AAR
polypeptide, when cultured in medium containing a carbon source
under conditions effective to express the variant AAR
polypeptide.
85. The recombinant host cell of claim 84, wherein the fatty
alcohol composition is produced at a titer of about 30 g/L to about
250 g/L.
86. The recombinant host cell of claim 84, wherein the fatty
alcohol composition is released from the cell.
87. A cell culture comprising a recombinant host cell that
expresses a variant acyl-ACP reductase (AAR) polypeptide comprising
at least 90% sequence identity to the amino acid sequence of SEQ ID
NO: 80, wherein said variant AAR polypeptide comprises a mutation
at an amino acid position selected from the group consisting of
Q40V, G52V, G273E, K303G, H340P, L344A, L344D, L344S, L344T, A345R,
V346P, V346G, and A345*, and wherein said AAR polypeptide catalyzes
the conversion of an acyl-ACP to a fatty aldehyde.
88. The cell culture of claim 87, wherein the recombinant host cell
produces a fatty alcohol composition that comprises one or more of
a C6, C8, C10, C12, C13, C14, C15, C16, C17, and a C18 fatty
alcohol.
89. The cell culture of claim 88, wherein the fatty alcohol
composition comprises one or more of a C10:1, C12:1, C14:1, C16:1,
and a C18:1 unsaturated fatty alcohol.
90. The cell culture of claim 88, wherein the fatty alcohol
composition comprises an unsaturated fatty alcohol.
91. The cell culture of claim 88, wherein the fatty alcohol
composition comprises a fatty alcohol having a double bond at
position 7 in the carbon chain between C7 and C8 from the reduced
end of the fatty alcohol.
92. The cell culture of claim 88, wherein the fatty alcohol
composition comprises a saturated fatty alcohol.
93. A method of producing a fatty alcohol composition having an
increase in titer, comprising: i. culturing with a carbon source, a
recombinant host cell that expresses a variant acyl-ACP reductase
(AAR) polypeptide comprising at least 90% sequence identity to the
amino acid sequence of SEQ ID NO: 80, wherein said variant AAR
polypeptide comprises a mutation at an amino acid position selected
from the group consisting of Q40V, G52V, G273E, K303G, H340P,
L344A, L344D, L344S, L344T, A345R, V346P, V346G, and A345*, and
wherein said AAR polypeptide catalyzes the conversion of an
acyl-ACP to a fatty aldehyde, and ii. harvesting a fatty alcohol
composition.
94. The method of claim 93, wherein the fatty alcohol composition
has a fatty alcohol titer that is at least 20% to 30% greater than
a fatty alcohol titer of a fatty alcohol composition produced by a
wild-type AAR-expressing host cell.
95. The method of claim 93, wherein the fatty alcohol composition
has an increased fraction of C14 fatty alcohols.
Description
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/753,273, filed Jan. 16, 2013, the contents of
which are hereby incorporated by reference in their entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Jan. 13, 2014, is named LS00046PCT_SL.txt and is 155,731 bytes
in size.
FIELD
[0003] The disclosure relates to acyl-ACP reductase (AAR) enzyme
variants that result in improved fatty aldehyde and/or fatty
alcohol production when expressed in recombinant host cells. The
disclosure further relates to methods of making and using such AAR
variants for the production of fatty alcohol compositions having
particular characteristics.
BACKGROUND
[0004] Fatty alcohols denote an important category of industrial
biochemical. For example, worldwide annual sales of fatty alcohols
and their derivatives are in excess of US$1 billion. These
molecules and their derivatives have numerous applications,
including use as surfactants, lubricants, plasticizers, solvents,
emulsifiers, emollients, thickeners, flavors, fragrances, and
fuels. Due to their amphiphilic nature, fatty alcohols behave as
nonionic surfactants, which are useful in personal care and
household products, for example, detergents. The shorter chain
fatty alcohols are used in the cosmetic and food industries as
emulsifiers, emollients, and thickeners.
[0005] In nature, fatty alcohols are made by enzymes that are able
to reduce various acyl-ACP or acyl-CoA molecules to the
corresponding primary alcohols (e.g., U.S. Pat. Nos. 8,323,924;
8,268,599 and 8,097,439; and U.S. Patent Publication Nos.
20120282663 and 20100105963, incorporated by reference herein).
However, current technologies involve mostly inorganic
catalyst-mediated reduction of fatty acids to the corresponding
primary alcohols. These fatty alcohols are produced via catalytic
hydrogenation of fatty acids produced from natural sources, such as
coconut oil, palm oil, palm kernel oil, tallow and lard, or by
chemical hydration of alpha-olefins produced from petrochemical
feedstocks. Fatty alcohols derived from natural sources have
varying chain lengths, which are relevant and specific to
particular applications. Dehydration of fatty alcohols to
alpha-olefins can be accomplished by chemical catalysis.
[0006] Fatty aldehydes can be used to produce industrial specialty
chemicals. For example, aldehydes are commonly used to produce
polymers, resins, dyes, flavorings, plasticizers, perfumes, and
pharmaceuticals. Aldehydes can also be used as solvents,
preservatives, and disinfectants. Certain natural and synthetic
compounds, such as vitamins and hormones, are aldehydes, and many
sugars contain aldehyde groups. Fatty aldehydes can be converted to
fatty alcohols by chemical or enzymatic reduction.
[0007] A greener and cleaner alternative to the production of fatty
aldehydes and fatty alcohols is via fermentable sugars and/or
biomass. However, in order for the production of fatty aldehydes
and fatty alcohols from fermentable sugars or biomass to be
commercially viable, industrial processes must be optimized for
efficient conversion and recovery of the final product. The present
disclosure addresses this need by providing compositions and
methods for improved production of fatty aldehydes and fatty
alcohols by using engineered host cells as biocatalysts.
SUMMARY
[0008] The present disclosure provides photosynthetic and
heterotrophic host cells that directly produce fatty aldehydes
and/or fatty alcohols of specific chain lengths such that catalytic
conversion of purified fatty acids is not necessary. This
biological route provides a higher quality product, a significant
cost reduction and a lesser impact on the environment. More
specifically, the present disclosure provides novel acyl-ACP
reductase (AAR) enzyme variants that produce fatty aldehydes and/or
fatty alcohols and compositions thereof. Also provided are specific
AAR variant nucleic acid and protein sequences as well as novel
recombinant host cells and cell cultures that encompass such
engineered AAR enzyme variants. The disclosure also provides
methods of using the recombinant AAR variant-expressing host cells
in order to make fatty aldehyde and/or fatty alcohol compositions
with particular characteristics.
[0009] One aspect of the disclosure provides variant acyl-ACP
reductase (AAR) polypeptides that catalyze the conversion of an
acyl-ACP to a fatty aldehyde, wherein the AAR polypeptide has at
least 90% sequence identity to the corresponding wild type AAR
polypeptide sequence presented as SEQ ID NO: 28, SEQ ID NO: 30, SEQ
ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO:
40, SEQ ID NO: 42 or SEQ ID NO: 44, and methods for expressing the
variant AAR polypeptides in a recombinant host cell resulting in a
higher titer of fatty aldehyde and/or fatty alcohol composition as
compared to the titer of a fatty aldehyde and/or fatty alcohol
composition produced by expression of a wild type AAR polypeptide
in a corresponding host cell. In one embodiment, the genetically
engineered variant AAR polypeptide has at least 90% sequence
identity to the corresponding wild type AAR polypeptide sequence
presented as SEQ ID NO: 28 and expression of the variant AAR
polypeptide in a recombinant host cell results in a higher titer of
fatty aldehyde and/or fatty alcohol composition or a higher titer
of C12, C14 or C16 fatty alcohols as compared to the titer produced
by expression of a wild type AAR polypeptide in a corresponding
host cell.
[0010] In one aspect, the variant AAR polypeptide has a mutation at
one or more amino acid positions of amino acids 18, 24, 31, 34, 35,
43, 50, 63, 86, 112, 113, 116, 118, 120, 135, 148, 153, 155, 157,
159, 168, 172, 187, 188, 191, 209, 210, 211, 236, 277, 283, 285,
291, 324, 328, 335, 337 and 338 of SEQ ID NO: 28. In one preferred
embodiment, the genetically engineered variant AAR polypeptide has
an S18W mutation. In another preferred embodiment, the genetically
engineered variant AAR polypeptide has an S18W mutation and further
comprises a mutation such as M21L, D24E, D24Y, L31V, W34F, W35F,
D43E, A50Q, C63A, C63G, C63Y, S86G, A112R, S113K, Q116G, R118Q,
T120S, A135S, T148C, T148E, T148V, I153P, Q155C, Q155L, T157V,
A159V, I168V, C172L, T187V, T188H, T188V, Q191A, L209R, E210Y,
A211W, T236C, Q277V, E283G, E283S, A285V, M291V, A324T, A328S,
Q335N, L337V and/or L338W.
[0011] In another aspect, the variant AAR polypeptide that has at
least 90% sequence identity to the corresponding wild type AAR
polypeptide sequence presented as SEQ ID NO: 34 and expression of
the variant AAR polypeptide in a recombinant host cell results in a
higher titer of fatty aldehyde and/or fatty alcohol or a higher
titer of C12 fatty alcohol as compared to the titer produced by
expression of a wild type AAR polypeptide in a corresponding host
cell. The variant AAR polypeptide has a mutation at an amino acid
position including amino acid 40, 52, 58, 61, 273, 303, 339, 340,
344, 345, 346 and 588 of SEQ ID NO: 34. In one preferred
embodiment, the variant AAR polypeptide has a mutation at amino
acid position Q40V, G52V, S58V, D61E, G273E, K303G, K339L, H340P,
L344A, L344D, L344S, L344T, L345R, V346P, V346G, and/or S588V.
[0012] Another aspect of the disclosure provides a recombinant host
cell that has one or more mutations as described above and wherein
when the host cell is engineered to express a variant AAR
polypeptide of SEQ ID NO: 28 or SEQ ID NO: 34. This recombinant
host cell produces a fatty aldehyde and/or fatty alcohol
composition with a titer that is at least 10% greater, at least 15%
greater, at least 20% greater, at least 25% greater, or at least
30% greater than the titer of a fatty aldehyde and/or fatty alcohol
composition produced by a host cell expressing the corresponding
wild type AAR polypeptide, when cultured in medium containing a
carbon source under conditions effective to express the variant AAR
polypeptide. In one embodiment, the fatty aldehyde and or fatty
alcohol composition is produced at a titer of 30 g/L to 250 g/L,
e.g., a titer of at least 100 mg/L. In another embodiment, the
fatty alcohol composition is produced extracellularly.
[0013] The disclosure further encompasses a cell culture that
includes the recombinant host cell as described above, wherein the
fatty alcohol composition includes one or more of a C6, C8, C10,
C12, C13, C14, C15, C16, C17, and a C18 fatty alcohol, e.g., a
C10:1, C12:1, C14:1, C16:1, or a C18:1 unsaturated fatty alcohol.
In yet another embodiment, the fatty alcohol composition comprises
a saturated fatty alcohol.
[0014] Another aspect of the disclosure provides a variant acyl-ACP
reductase (AAR) polypeptide having at least 90% sequence identity
to SEQ ID NO: 57, wherein the AAR polypeptide catalyzes the
conversion of an acyl-ACP to a fatty aldehyde. In one embodiment,
the expression of the variant AAR polypeptide in a recombinant host
cell results in a higher titer of a fatty aldehyde or fatty alcohol
composition as compared to a titer of a fatty aldehyde or fatty
alcohol composition produced by expression of a wild type AAR
polypeptide in a corresponding wild type host cell. In another
embodiment, the expression of the variant AAR polypeptide in a
recombinant host cell results in a higher titer of a fatty aldehyde
or fatty alcohol composition that is a C12, C14 and/or C16 fatty
alcohol composition as compared to a titer of a fatty aldehyde or
fatty alcohol composition produced by expression of a wild type AAR
polypeptide in a corresponding wild type host cell. In another
embodiment, the variant AAR polypeptide has a mutation at amino
acid position 18, wherein the mutation is S18W.
[0015] Another aspect of the disclosure provides a variant acyl-ACP
reductase (AAR) polypeptide having at least 90% sequence identity
to SEQ ID NO: 57, wherein the variant AAR polypeptide has another
mutation at an amino acid position at amino acid 8, 16, 21, 24, 31,
34, 35, 43, 50, 63, 86, 112, 113, 116, 118, 120, 135, 148, 153,
154, 155, 157, 159, 168, 172, 187, 188, 191, 209, 210, 211, 236,
277, 281, 283, 285, 291, 324, 328, 335, 337 and/or 338. In one
embodiment, the mutation is selected from L8A, D16L, M21L, D24E,
D24Y, D24V, D24P, L31V, L31M, W34F, W35F, D43E, A50Q, C63A, C63G,
C63Y, S86G, A112R, S113K, Q116G, R118Q, T120S, A135S, T148C, T148E,
T148V, I153P, T154A, Q155C, Q155L, T157V, A159V, I168V, C172L,
T187V, T188H, T188V, Q191A, L209R, E210Y, A211W, T236C, Q277V,
A281L, E283G, E283S, A285V, M291V, A324T, A328S, Q335N, L337V
and/or L338W. In one preferred embodiment, the variant AAR
polypeptide has a M21L mutation, a C63G mutation, a S113K mutation,
T154A, and an A281L mutation (SEQ ID NO: 58). In another preferred
embodiment, the variant AAR polypeptide has an L8A mutation, an
M21L mutation, a C63G mutation, a S113K mutation, a T154A mutation,
and an A281L mutation (SEQ ID NO: 59). In another preferred
embodiment, the variant AAR polypeptide has a D16L mutation, a M21L
mutation, a C63G mutation, a S113K mutation, T154A, and an A281L
mutation (SEQ ID NO: 60).). In another preferred embodiment, the
variant AAR polypeptide has a L8A mutation, a D24V mutation, a C63G
mutation, a S113K mutation, a Q155L mutation, and an A281L mutation
(SEQ ID NO: 61). In another preferred embodiment, the variant AAR
polypeptide has a D24P mutation, a L31M mutation, a C63G mutation,
a S113K mutation, a T154A mutation, and an A281L mutation (SEQ ID
NO: 62). In another preferred embodiment, the variant AAR
polypeptide has a L8A mutation, a D16L mutation, a D24V mutation, a
C63G mutation, a S113K mutation, a T154A mutation, and an A281L
mutation (SEQ ID NO: 63). In another preferred embodiment, the
variant AAR polypeptide has a D24E mutation, a C63G mutation, a
S113K mutation, a T154A mutation, and an A281L mutation (SEQ ID NO:
64).
[0016] Another aspect of the present disclosure provides a
recombinant host cell expressing the variant AAR polypeptides as
described above (supra). In one embodiment, the recombinant host
cell produces a fatty aldehyde or fatty alcohol composition with a
titer that is at least 10% greater, at least 15% greater, at least
20% greater, at least 25% greater, or at least 30% greater than the
titer of a fatty aldehyde or fatty alcohol composition produced by
a host cell expressing a corresponding wild type AAR polypeptide,
when cultured in medium containing a carbon source under conditions
effective to express the variant AAR polypeptide. In one
embodiment, the fatty aldehyde or fatty alcohol composition
produced by the recombinant host cell is produced at a titer of
about 30 g/L to about 250 g/L. In another embodiment, the fatty
aldehyde or fatty alcohol composition is produced
extracellularly.
[0017] The disclosure further contemplates a cell culture including
the recombinant host cell expressing the variant AAR polypeptides
as described above (supra). In one embodiment, the fatty alcohol
composition includes a saturated and/or unsaturated fatty alcohol.
In one embodiment, the cell culture includes a fatty alcohol
composition that includes one or more of a C6, C8, C10, C12, C13,
C14, C15, C16, C17, and a C18 fatty alcohol. In another embodiment,
the fatty alcohol composition includes one or more of a C10:1,
C12:1, C14:1, C16:1, and a C18:1 unsaturated fatty alcohol. In yet
another embodiment, the fatty alcohol composition includes a fatty
alcohol having a double bond at position 7 in the carbon chain
between C7 and C8 from the reduced end of the fatty alcohol.
[0018] The disclosure further encompasses a method of producing a
fatty alcohol composition having an increase in titer, including
culturing the variant AAR-expressing host cell (as described above)
with a carbon source; and harvesting a fatty alcohol composition.
In one embodiment, the titer of the fatty alcohol is at least 20%
to 30% greater than the titer of a fatty alcohol composition
produced by a wild-type AAR-expressing host cell.
[0019] Another aspect of the disclosure provides a variant acyl-ACP
reductase (AAR) polypeptide having at least 90% sequence identity
to SEQ ID NO: 65, wherein the polypeptide catalyzes the conversion
of an acyl-ACP to a fatty aldehyde. In one embodiment, the
expression of the variant AAR polypeptide in a recombinant host
cell results in a higher titer of a fatty aldehyde or fatty alcohol
composition as compared to a titer of a fatty aldehyde or fatty
alcohol composition produced by expression of a wild type AAR
polypeptide in a corresponding wild type host cell. In another
embodiment, the expression of the variant AAR polypeptide in a
recombinant host cell results in a higher titer of a C12, C14
and/or C16 fatty alcohol composition as compared to a titer of a
fatty aldehyde or fatty alcohol composition produced by expression
of a wild type AAR polypeptide in a corresponding wild type host
cell. In one particular aspect, the disclosure provides a variant
acyl-ACP reductase (AAR) polypeptide having at least 90% sequence
identity to SEQ ID NO: 65, wherein the polypeptide has a mutation
at amino acid position 61. In one preferred embodiment the mutation
is D61E.
[0020] The disclosure further encompasses a variant acyl-ACP
reductase (AAR) polypeptide having at least 90% sequence identity
to SEQ ID NO: 34, wherein expression of the variant AAR polypeptide
in a recombinant host cell results in a higher titer of a fatty
aldehyde or fatty alcohol composition or a higher titer of a C12,
C14 and/or C16 fatty alcohol composition as compared to a titer of
a fatty alcohol composition produced by expression of a wild type
AAR polypeptide in a corresponding wild type host cell, and wherein
the AAR polypeptide has a mutation at amino acid position 40, 52,
273, 303, 340, 344, 345, or 346. In one embodiment, the variant AAR
polypeptide has a mutation selected Q40V, G52V, G273E, K303G,
H340P, L344A, L344D, L344S, L344T, L345R, V346P, and V346G. In one
preferred embodiment, the variant AAR polypeptide has a mutation at
V346P (SEQ ID NO: 66). In another preferred embodiment, the variant
AAR polypeptide has a mutation at Q40V (SEQ ID NO: 67). In another
preferred embodiment, the variant AAR polypeptide has a mutation
A345R (SEQ ID NO: 68). In another preferred embodiment, the variant
AAR polypeptide has a mutation at L344S (SEQ ID NO: 69). In another
preferred embodiment, the variant AAR polypeptide has a mutation at
V346G (SEQ ID NO: 70). In another preferred embodiment, the variant
AAR polypeptide has a mutation at L344D (SEQ ID NO: 71). In another
preferred embodiment, the variant AAR polypeptide has a mutation at
G52V (SEQ ID NO: 72). In another preferred embodiment, the variant
AAR polypeptide has a mutation at L344T (SEQ ID NO: 73). In another
preferred embodiment, the variant AAR polypeptide has a mutation at
K303G (SEQ ID NO: 74). In another preferred embodiment, the variant
AAR polypeptide has a mutation at L344A (SEQ ID NO: 75). In another
preferred embodiment, the variant AAR polypeptide has a mutation at
H340P (SEQ ID NO: 76). In another preferred embodiment, the variant
AAR polypeptide has a mutation at G273E (SEQ ID NO: 77).
[0021] Yet another aspect of the disclosure provides a recombinant
host cell expressing the variant AAR polypeptide as described above
(supra). In one embodiment, the recombinant host cell produces a
fatty aldehyde or fatty alcohol composition with a titer that is at
least 10% greater, at least 15% greater, at least 20% greater, at
least 25% greater, or at least 30% greater than the titer of a
fatty aldehyde or alcohol composition produced by a host cell
expressing a corresponding wild type AAR polypeptide, when cultured
in medium containing a carbon source under conditions effective to
express the variant AAR polypeptide. In another embodiment, the
fatty alcohol composition is produced at a titer of about 30 g/L to
about 250 g/L. In another embodiment, the fatty alcohol composition
is produced extracellularly.
[0022] The disclosure further contemplates a cell culture with the
recombinant host cell expressing the variant AAR polypeptide as
described above (supra). In one embodiment, the fatty alcohol
composition includes one or more of a C6, C8, C10, C12, C13, C14,
C15, C16, C17, and a C18 fatty alcohol. In another embodiment, the
fatty alcohol composition includes an unsaturated or saturated
fatty alcohol. In another embodiment, the fatty alcohol composition
includes one or more of a C10:1, C12:1, C14:1, C16:1, and a C18:1
unsaturated fatty alcohol. In another embodiment, the fatty alcohol
composition includes a fatty alcohol having a double bond at
position 7 in the carbon chain between C7 and C8 from the reduced
end of the fatty alcohol.
[0023] Another aspect of the disclosure provides a method of
producing a fatty alcohol composition having an increase in titer,
including culturing the host cell expressing the AAR (as described
above) with a carbon source; and harvesting a fatty alcohol
composition. In one embodiment, the fatty alcohol is at least 20%
to 30% greater than the titer of a fatty alcohol composition
produced by a wild-type AAR-expressing host cell.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] The present disclosure is best understood when read in
conjunction with the accompanying figures, which serve to
illustrate the preferred embodiments. It is understood, however,
that the disclosure is not limited to the specific embodiments
disclosed in the figures.
[0025] FIG. 1 is a schematic overview of an exemplary biosynthetic
pathway for use in production of acyl-CoA as a precursor to fatty
acid derivatives in a recombinant host cell. The cycle is initiated
by condensation of malonyl-ACP and acetyl-CoA.
[0026] FIG. 2 is a schematic overview of an exemplary fatty acid
biosynthetic cycle, wherein elongation cycles begin with the
condensation of malonyl-ACP and an acyl-ACP catalyzed by
.beta.-ketoacyl-ACP synthase I (fabB) and .beta.-ketoacyl-ACP
synthase II (fabF) to produce a .beta.-keto-acyl-ACP, then the
.beta.-keto-acyl-ACP is reduced by a NADPH-dependent
.beta.-ketoacyl-ACP reductase (fabG) to produce a
.beta.-hydroxy-acyl-ACP, which is dehydrated to a
trans-2-enoyl-acyl-ACP by J3-hydroxyacyl-ACP dehydratase (fabA or
fabZ). FabA can also isomerize trans-2-enoyl-acyl-ACP to
cis-3-enoyl-acyl-ACP, which can bypass fabl and can used by fabB
(typically for up to an aliphatic chain length of C16) to produce
.beta.-keto-acyl-ACP. The final step in each cycle is catalyzed by
a NADH or NADHPH-dependent enoyl-ACP reductase (fabl) that converts
trans-2-enoyl-acyl-ACP to acyl-ACP. In the methods described
herein, termination of fatty acid synthesis occurs by thioesterase
removal of the acyl group from acyl-ACP to release free fatty acids
(FFA). Thioesterases (e.g., tesA) hydrolyze thioester bonds, which
occur between acyl chains and ACP through sulfhydryl bonds.
[0027] FIG. 3 illustrates the structure and function of the
acetyl-CoA carboxylase (accABCD) enzyme complex. Biotin carboxylase
is encoded by the accC gene, whereas biotin carboxyl carrier
protein (BCCP) is encoded by the accB gene. The two subunits
involved in carboxyl transferase activity are encoded by the accA
and accD genes. The covalently bound biotin of BCCP carries the
carboxylate moiety. The birA gene biotinylates holo-accB.
[0028] FIG. 4 presents a schematic overview of an exemplary
biosynthetic pathway for production of fatty alcohol starting with
acyl-ACP, where the production of fatty aldehyde is catalyzed by
the enzymatic activity of acyl-ACP reductase (AAR) or thioesterase
and carboxylic acid reductase (Car). The fatty aldehyde is
converted to fatty alcohol by aldehyde reductase (also referred to
as alcohol dehydrogenase). This pathway does not include fatty
acyl-CoA synthetase (fadD).
[0029] FIG. 5 shows fatty alcohol production in E. coli DV2
expressing Synechococcus elongatus acyl-ACP reductase (AAR_7942)
and coexpressing various cyanobacterial acyl carrier proteins
(ACPs).
[0030] FIG. 6 shows fatty alcohol production in E. coli DV2
expressing Synechococcus elongatus acyl-ACP reductase (AAR_7942)
(pLS9-185) and coexpressing the acetyl carboxylase complex from
Corynebacterium glutamicum (pSL9-185-D+) (three individual strains
are shown, see D+1, D+2 and D+3).
[0031] FIG. 7 presents results that illustrate improved fatty
alcohol production of recombinant host cells that rely on
AAR-mediated by ifab and ifadR overexpression.
[0032] FIG. 8 presents results showing elevated fatty alcohol
levels in recombinant host cells that express AAR_7942 variants
derived from combination libraries (AAR_Com 2a-d) which are
coexpressed with ACP, AlrA (alcohol dehydrogenase (ADH)) and a
synthetic acc operon in strain Shu2. A number of alcohol
dehydrogenase polypeptides are useful in accordance with the
disclosure and include, but are not limited to AlrA of
Acinetobacter sp. M-1 (SEQ ID NO: 52) and an AlrA homolog such as
AlrAadp1 (SEQ ID NO: 53)
[0033] FIGS. 9A and 9B present results from a tank fermentation of
AAR_7942 variant S18W that illustrate FALC titer (9A) and yield on
glucose (9B) showing that expression of AAR_7942 variant S18W
results in altered activity and chain length distribution.
[0034] FIG. 10 presents results that illustrate a shift in chain
length distribution for FALC from C16 to C14 when the D61E variant
of MED4_AAR was expressed in recombinant host cells.
DETAILED DESCRIPTION
[0035] General Overview
[0036] One way of eliminating our dependency on petrochemicals is
to produce fatty acid derivatives such as fatty aldehydes and fatty
alcohols through environmentally friendly microorganisms that serve
as miniature production hosts. Such cellular hosts (i.e.,
recombinant host cells or production strains) have been engineered
to produce fatty aldehydes and/or fatty alcohols from renewable
sources such as renewable feedstock (e.g., fermentable sugars,
carbohydrates, biomass, cellulose, glycerol, CO, CO.sub.2, etc.).
These fatty aldehydes and fatty alcohols are the raw materials for
many industrial products including detergents and fuels.
[0037] The present disclosure relates to acyl-ACP reductase (AAR)
enzyme variants that result in improved titer, yield and/or
productivity of fatty aldehyde and/or fatty alcohol compositions
when expressed in recombinant host cells. Herein, enhanced fatty
aldehyde and/or fatty alcohol biosynthesis is accomplished by
transforming host cells such that they express a variant acyl-ACP
reductase (AAR) protein, which catalyzes the reaction of an
acyl-ACP to a fatty aldehyde and/or a fatty alcohol. The disclosure
further relates to the recombinant host cells or production strains
that express the AAR enzyme variants.
Definitions
[0038] As used in this specification and the appended claims, the
singular forms "a," "an" and "the" include plural referents unless
the context clearly dictates otherwise. Thus, for example,
reference to "a host cell" includes two or more such host cells,
reference to "a fatty ester" includes one or more fatty esters, or
mixtures of esters, reference to "a nucleic acid sequence" includes
one or more nucleic acid sequences, reference to "an enzyme"
includes one or more enzymes, and the like.
[0039] Accession numbers throughout this description were obtained
from databases provided by the NCBI (National Center for
Biotechnology Information) maintained by the National Institutes of
Health, U.S.A. (which are identified herein as "NCBI Accession
Numbers" or alternatively as "GenBank Accession Numbers"), and from
the UniProt Knowledgebase (UniProtKB) and Swiss-Prot databases
provided by the Swiss Institute of Bioinformatics (which are
identified herein as "UniProtKB Accession Numbers").
[0040] Enzyme Classification (EC) numbers are established by the
Nomenclature Committee of the International Union of Biochemistry
and Molecular Biology (IUBMB), description of which is available on
the IUBMB Enzyme Nomenclature website on the World Wide Web. EC
numbers classify enzymes according to the reaction they catalyze.
For example, the acyl-ACP reductase (AAR) enzymatic activity is
classified under E.C. 1.2.1.80 (also known as long-chain
acyl-[acyl-carrier-protein] reductase) or EC 1.2.1.42. The
functionality of AAR is conserved in most prokaryotes from one
species to the next. Thus, different microbial species can carry
out the same AAR enzymatic activity that is classified under E.C.
1.2.1.80 or EC 1.2.1.42.
[0041] As used herein, the term "nucleotide" refers to a monomeric
unit of a polynucleotide that consists of a heterocyclic base, a
sugar, and one or more phosphate groups. The naturally occurring
bases (guanine, (G), adenine, (A), cytosine, (C), thymine, (T), and
uracil (U)) are typically derivatives of purine or pyrimidine,
though it should be understood that naturally and non-naturally
occurring base analogs are also included. The naturally occurring
sugar is the pentose (five-carbon sugar) deoxyribose (which forms
DNA) or ribose (which forms RNA), though it should be understood
that naturally and non-naturally occurring sugar analogs are also
included. Nucleic acids are typically linked via phosphate bonds to
form nucleic acids or polynucleotides, though many other linkages
are known in the art (e.g., phosphorothioates, boranophosphates,
and the like).
[0042] The term "polynucleotide" refers to a polymer of
ribonucleotides (RNA) or deoxyribonucleotides (DNA), which can be
single-stranded or double-stranded and which can contain
non-natural or altered nucleotides. The terms "polynucleotide,"
"nucleic acid sequence," and "nucleotide sequence" are used
interchangeably herein to refer to a polymeric form of nucleotides
of any length, either RNA or DNA. These terms refer to the primary
structure of the molecule, and thus include double- and
single-stranded DNA, and double- and single-stranded RNA. The terms
include, as equivalents, analogs of either RNA or DNA made from
nucleotide analogs and modified polynucleotides such as, though not
limited to methylated and/or capped polynucleotides. The
polynucleotide can be in any form, including but not limited to,
plasmid, viral, chromosomal, EST, cDNA, mRNA, and rRNA.
[0043] As used herein, the terms "polypeptide" and "protein" are
used interchangeably to refer to a polymer of amino acid residues.
The term "recombinant polypeptide" refers to a polypeptide that is
produced by recombinant techniques, wherein generally DNA or RNA
encoding the expressed protein is inserted into a suitable
expression vector that is in turn used to transform a host cell to
produce the polypeptide. Similarly, the terms "recombinant
polynucleotide" or "recombinant nucleic acid" or "recombinant DNA"
are produced by recombinant techniques that are known to those of
skill in the art.
[0044] As used herein, the terms "homolog," and "homologous" refer
to a polynucleotide or a polypeptide comprising a sequence that is
at least about 50 percent (%) identical to the corresponding
polynucleotide or polypeptide sequence. Preferably homologous
polynucleotides or polypeptides have polynucleotide sequences or
amino acid sequences that have at least about 80%, 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98% or at least about 99% homology to the corresponding amino
acid sequence or polynucleotide sequence. As used herein the terms
sequence "homology" and sequence "identity" are used
interchangeably. One of ordinary skill in the art is well aware of
methods to determine homology between two or more sequences.
Briefly, calculations of "homology" between two sequences can be
performed as follows. The sequences are aligned for optimal
comparison purposes (e.g., gaps can be introduced in one or both of
a first and a second amino acid or nucleic acid sequence for
optimal alignment and non-homologous sequences can be disregarded
for comparison purposes). In one preferred embodiment, the length
of a first sequence that is aligned for comparison purposes is at
least about 30%, preferably at least about 40%, more preferably at
least about 50%, even more preferably at least about 60%, and even
more preferably at least about 70%, at least about 80%, at least
about 85%, at least about 90%, at least about 95%, at least about
98%, or about 100% of the length of a second sequence. The amino
acid residues or nucleotides at corresponding amino acid positions
or nucleotide positions of the first and second sequences are then
compared. When a position in the first sequence is occupied by the
same amino acid residue or nucleotide as the corresponding position
in the second sequence, then the molecules are identical at that
position. The percent homology between the two sequences is a
function of the number of identical positions shared by the
sequences, taking into account the number of gaps and the length of
each gap, that need to be introduced for optimal alignment of the
two sequences. The comparison of sequences and determination of
percent homology between two sequences can be accomplished using a
mathematical algorithm, such as BLAST (Altschul et al. (1990) J.
Mol. Biol. 215(3):403-410). The percent homology between two amino
acid sequences also can be determined using the Needleman and
Wunsch algorithm that has been incorporated into the GAP program in
the GCG software package, using either a Blossum 62 matrix or a
PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a
length weight of 1, 2, 3, 4, 5, or 6 (Needleman and Wunsch (1970)
J. Mol. Biol. 48:444-453). The percent homology between two
nucleotide sequences also can be determined using the GAP program
in the GCG software package, using a NWSgapdna.CMP matrix and a gap
weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4,
5, or 6. One of ordinary skill in the art can perform initial
homology calculations and adjust the algorithm parameters
accordingly. A preferred set of parameters (and the one that should
be used if a practitioner is uncertain about which parameters
should be applied to determine if a molecule is within a homology
limitation of the claims) are a Blossum 62 scoring matrix with a
gap penalty of 12, a gap extend penalty of 4, and a frameshift gap
penalty of 5. Additional methods of sequence alignment are known in
the biotechnology arts (see, e.g., Rosenberg (2005) BMC
Bioinforinatics 6:278; Altschul et al. (2005) FEBS J.
272(20):5101-5109).
[0045] The term "hybridizes under low stringency, medium
stringency, high stringency, or very high stringency conditions"
describes conditions for hybridization and washing. Guidance for
performing hybridization reactions can be found in Current
Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989),
6.3.1-6.3.6. Aqueous and non-aqueous methods are described in that
reference and either method can be used. Specific hybridization
conditions referred to herein are as follows: (1) low stringency
hybridization conditions--6.times. sodium chloride/sodium citrate
(SSC) at about 45.degree. C., followed by two washes in
0.2.times.SSC, 0.1% SDS at least at 50.degree. C. (the temperature
of the washes can be increased to 55.degree. C. for low stringency
conditions); (2) medium stringency hybridization
conditions--6.times.SSC at about 45.degree. C., followed by one or
more washes in 0.2.times.SSC, 0.1% SDS at 60.degree. C.; (3) high
stringency hybridization conditions--6.times.SSC at about
45.degree. C., followed by one or more washes in 0.2..times.SSC,
0.1% SDS at 65.degree. C.; and (4) very high stringency
hybridization conditions--0.5M sodium phosphate, 7% SDS at
65.degree. C., followed by one or more washes at 0.2.times.SSC, 1%
SDS at 65.degree. C. Very high stringency conditions (4) are the
preferred conditions unless otherwise specified.
[0046] An "endogenous" polypeptide refers to a polypeptide encoded
by the genome of the parental cell (or host cell). An "exogenous"
polypeptide refers to a polypeptide which is not encoded by the
genome of the parental cell. A variant or mutant polypeptide is an
example of an exogenous polypeptide. Thus, a
non-naturally-occurring nucleic acid molecule is considered to be
exogenous to a cell once introduced into the cell. A nucleic acid
molecule that is naturally-occurring can also be exogenous to a
particular cell. For example, an entire coding sequence isolated
from cell X is an exogenous nucleic acid with respect to cell Y
once that coding sequence is introduced into cell Y, even if X and
Y are the same cell type.
[0047] The term "overexpressed" means that a gene is caused to be
transcribed at an elevated rate compared to the endogenous
transcription rate for that gene. In some examples, overexpression
additionally includes an elevated rate of translation of the gene
compared to the endogenous translation rate for that gene. Methods
of testing for overexpression are well known in the art, for
example transcribed RNA levels can be assessed using rtPCR and
protein levels can be assessed using SDS page gel analysis.
[0048] The term "heterologous" means derived from a different
organism, different cell type, or different species. As used herein
it refers to a nucleotide-, polynucleotide-, polypeptide- or
protein sequence, not naturally present in a given organism. For
example, a polynucleotide sequence that is native to cyanobacteria
can be introduced into a host cell of E. coli by recombinant
methods, and the polynucleotide from cyanobacteria is then
heterologous to the E. coli cell (e.g., recombinant cell). The term
"heterologous" may also be used with reference to a nucleotide-,
polynucleotide-, polypeptide-, or protein sequence which is present
in a recombinant host cell in a non-native state. For example, a
"heterologous" nucleotide, polynucleotide, polypeptide or protein
sequence may be modified relative to the wild type sequence
naturally present in the corresponding wild type host cell, e.g., a
modification in the level of expression or in the sequence of a
nucleotide, polynucleotide, polypeptide or protein.
[0049] As used herein, the term "fragment" of a polypeptide refers
to a shorter portion of a full-length polypeptide or protein
ranging in size from two amino acid residues to the entire amino
acid sequence minus one amino acid residue. In certain embodiments
of the disclosure, a fragment refers to the entire amino acid
sequence of a domain of a polypeptide or protein (e.g., a substrate
binding domain or a catalytic domain).
[0050] The term "mutagenesis" refers to a process by which the
genetic information of an organism is changed in a stable manner.
Mutagenesis of a protein coding nucleic acid sequence produces a
mutant protein. Mutagenesis also refers to changes in non-coding
nucleic acid sequences that result in modified protein
activity.
[0051] A "mutation", as used herein, refers to a permanent change
in a nucleic acid position of a gene or in an amino acid position
of a polypeptide or protein. Mutations include substitutions,
additions, insertions, and deletions. For example, a mutation in an
amino acid position can be a substitution of one type of amino acid
with another type of amino acid (e.g., a serine (S) may be
substituted with an alanine (A); a lysine (L) may be substituted
with an T (Threonine); etc.). As such, a polypeptide or a protein
can have one or more mutations wherein one amino acid is
substituted with another amino acid.
[0052] The terms "acyl-ACP reductase (AAR) variant" and "variant
acyl-ACP reductase (AAR)" are used interchangeably herein and mean
an AAR related polypeptide or protein that has one or more
mutations in its amino acid sequence. The AAR refers to an enzyme
that catalyzes the reduction of an acyl-ACP to a fatty aldehyde
and/or a fatty alcohol. The AAR variant may encompass a mutation in
one or more amino acid of its polypeptide sequence. When a cell has
been transformed with an AAR variant it is a cell that expresses
the AAR variant (e.g., a recombinant cell). In one embodiment, the
titer and/or yield of a fatty alcohol produced by a cell that
expresses the AAR variant is at least twice that of a corresponding
wild type cell (i.e., a corresponding cell that does not express
the AAR variant). In a heterologous host such as Escherichia coli,
fatty aldehydes may be converted to fatty alcohols by endogenous
alcohol dehydrogenases. In another embodiment, the titer and/or
yield of a fatty alcohol produced by a cell that expresses the AAR
variant is at least about 1 times, at least about 2 times, at least
about 3 times, at least about 4 times, at least about 5 times, at
least about 6 times, at least about 7 times, at least about 8
times, at least about 9 times, or at least about 10 times greater
than that of a corresponding wild type cell. In one embodiment, the
titer and/or yield of a fatty alcohol produced by a cell expressing
the AAR variant is at least about 1 percent, at least about 2
percent, at least about 3 percent, at least about 4 percent, at
least about 5 percent, at least about 6 percent, at least about 7
percent, at least about 8 percent, at least about 9 percent, or
about 10 percent greater than that of a corresponding wild type
cell. In another embodiment, the titer and/or yield of fatty
alcohols produced in a recombinant cell due to the expression of an
AAR variant is at least about 20 percent to at least about 100
percent greater than that of a wild type cell. In a particular
embodiments, the titer and/or yield of a fatty alcohol produced by
a cell is at least about 20 percent, at least about 25 percent, at
least about 30 percent, at least about 35 percent, at least about
40 percent, at least about 45 percent at least about 50 percent, at
least about 55 percent, at least about 60 percent, at least about
65 percent, at least about 70 percent, at least about 75 percent,
at least about 80 percent, at least about 85 percent, at least
about 90 percent, at least about 95 percent, at least about 97
percent, at least about 98 percent, or at least about 100 percent
greater than that of the corresponding wild type cell.
[0053] As used herein, the term "gene" refers to nucleic acid
sequences encoding either an RNA product or a protein product, as
well as operably-linked nucleic acid sequences affecting the
expression of the RNA or protein (e.g., such sequences include but
are not limited to promoter or enhancer sequences) or
operably-linked nucleic acid sequences encoding sequences that
affect the expression of the RNA or protein (e.g., such sequences
include but are not limited to ribosome binding sites or
translational control sequences).
[0054] Expression control sequences are known in the art and
include, for example, promoters, enhancers, polyadenylation
signals, transcription terminators, internal ribosome entry sites
(IRES), and the like, that provide for the expression of the
polynucleotide sequence in a host cell. Expression control
sequences interact specifically with cellular proteins involved in
transcription (Maniatis et al. (1987) Science 236:1237-1245).
Exemplary expression control sequences are described in, for
example, Goeddel, Gene Expression Technology: Methods in
Enzymology, Vol. 185, Academic Press, San Diego, Calif. (1990). In
the methods of the disclosure, an expression control sequence is
operably linked to a polynucleotide sequence. By "operably linked"
is meant that a polynucleotide sequence and an expression control
sequence are connected in such a way as to permit gene expression
when the appropriate molecules (e.g., transcriptional activator
proteins) are bound to the expression control sequence. Operably
linked promoters are located upstream of the selected
polynucleotide sequence in terms of the direction of transcription
and translation. Operably linked enhancers can be located upstream,
within, or downstream of the selected polynucleotide.
[0055] As used herein, the term "vector" refers to a nucleic acid
molecule capable of transporting another nucleic acid, i.e., a
polynucleotide sequence, to which it has been linked. One type of
useful vector is an episome (i.e., a nucleic acid capable of
extra-chromosomal replication). Useful vectors are those capable of
autonomous replication and/or expression of nucleic acids to which
they are linked. Vectors capable of directing the expression of
genes to which they are operatively linked are referred to herein
as "expression vectors." In general, expression vectors of utility
in recombinant DNA techniques are often in the form of "plasmids,"
which refer generally to circular double stranded DNA loops that,
in their vector form, are not bound to the chromosome. Other useful
expression vectors are provided in linear form. Also included are
such other forms of expression vectors that serve equivalent
functions and that have become known in the art subsequently
hereto. In some embodiments, a recombinant vector further includes
a promoter operably linked to the polynucleotide sequence. In some
embodiments, the promoter is a developmentally-regulated promoter,
an organelle-specific promoter, a tissue-specific promoter, an
inducible promoter, a constitutive promoter, or a cell-specific
promoter. The recombinant vector typically comprises at least one
sequence selected from an expression control sequence operatively
coupled to the polynucleotide sequence; a selection marker
operatively coupled to the polynucleotide sequence; a marker
sequence operatively coupled to the polynucleotide sequence; a
purification moiety operatively coupled to the polynucleotide
sequence; a secretion sequence operatively coupled to the
polynucleotide sequence; and a targeting sequence operatively
coupled to the polynucleotide sequence. In certain embodiments, the
nucleotide sequence is stably incorporated into the genomic DNA of
the host cell, and the expression of the nucleotide sequence is
under the control of a regulated promoter region. The expression
vectors as used herein include a particular polynucleotide sequence
as described herein in a form suitable for expression of the
polynucleotide sequence in a host cell. It will be appreciated by
those skilled in the art that the design of the expression vector
can depend on such factors as the choice of the host cell to be
transformed, the level of expression of polypeptide desired, etc.
The expression vectors described herein can be introduced into host
cells to produce polypeptides, including fusion polypeptides,
encoded by the polynucleotide sequences as described herein.
Expression of genes encoding polypeptides in prokaryotes, for
example, E. coli, is most often carried out with vectors containing
constitutive or inducible promoters directing the expression of
either fusion or non-fusion polypeptides. Fusion vectors add a
number of amino acids to a polypeptide encoded therein, usually to
the amino- or carboxy-terminus of the recombinant polypeptide. Such
fusion vectors typically serve one or more of the following three
purposes, including to increase expression of the recombinant
polypeptide; to increase the solubility of the recombinant
polypeptide; and to aid in the purification of the recombinant
polypeptide by acting as a ligand in affinity purification. Often,
in fusion expression vectors, a proteolytic cleavage site is
introduced at the junction of the fusion moiety and the recombinant
polypeptide. This enables separation of the recombinant polypeptide
from the fusion moiety after purification of the fusion
polypeptide. In certain embodiments, a polynucleotide sequence of
the disclosure is operably linked to a promoter derived from
bacteriophage T5.
[0056] In certain embodiments, the host cell is a yeast cell, and
the expression vector is a yeast expression vector. Examples of
vectors for expression in yeast S. cerevisiae include pYepSec1
(Baldari et al. (1987) EMBO J 6:229-234); pMFa (Kurjan et al.
(1982) Cell 30:933-943); pJRY88 (Schultz et al. (1987) Gene 54:
113-123); pYES2 (Invitrogen Corp., San Diego, Calif.), and picZ
(Invitrogen Corp., San Diego, Calif.). In other embodiments, the
host cell is an insect cell, and the expression vector is a
baculovirus expression vector. Baculovirus vectors available for
expression of proteins in cultured insect cells (e.g., Sf9 cells)
include, for example, the pAc series (Smith et al. (1983) Mol. Cell
Biol. 3:2156-2165) and the pVL series (Lucklow et al. (1989)
Virology 170:31-39). In yet another embodiment, the polynucleotide
sequences described herein can be expressed in mammalian cells
using a mammalian expression vector. Other suitable expression
systems for both prokaryotic and eukaryotic cells are well known in
the art; see, e.g., Sambrook et al., "Molecular Cloning: A
Laboratory Manual," second edition, Cold Spring Harbor Laboratory,
(1989).
[0057] As used herein, the term "CoA" or "acyl-CoA" refers to an
acyl thioester formed between the carbonyl carbon of alkyl chain
and the sulfhydryl group of the 4'-phosphopantethionyl moiety of
coenzyme A (CoA), which has the formula R--C(O)S--CoA, where R is
any alkyl group having at least 4 carbon atoms.
[0058] The term "ACP" means acyl carrier protein. ACP is a highly
conserved carrier of acyl intermediates during fatty acid
biosynthesis, wherein the growing chain is bound during synthesis
as a thiol ester at the distal thiol of a 4'-phosphopantetheine
moiety. The protein exists in two forms, i.e., apo-ACP (inactive in
fatty acid biosynthesis) and ACP or holo-ACP (active in fatty acid
biosynthesis). The terms "ACP" and "holo-ACP" are used
interchangeably herein and refer to the active form of the protein.
An enzyme called a phosphopantetheinyltransferase is involved in
the conversion of the inactive apo-ACP to the active holo-ACP. More
specifically, ACP is expressed in the inactive apo-ACP form and a
4'-phosphopantetheine moiety must be post-translationally attached
to a conserved serine residue on the ACP by the action of holo-acyl
carrier protein synthase (ACPS), a phosphopantetheinyltransferase,
in order to produce holo-ACP.
[0059] As used herein, the term "acyl-ACP" refers to an acyl
thioester formed between the carbonyl carbon of an alkyl chain and
the sulfhydryl group of the phosphopantetheinyl moiety of an acyl
carrier protein (ACP). In some embodiments an ACP is an
intermediate in the synthesis of fully saturated acyl-ACPs. In
other embodiments an ACP is an intermediate in the synthesis of
unsaturated acyl-ACPs. In some embodiments, the carbon chain will
have about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, or 26 carbons.
[0060] As used herein, the term "fatty acid derivative" means a
"fatty acid" or a "fatty acid derivative", which may be referred to
as a "fatty acid or derivative thereof". The term "fatty acid"
means a carboxylic acid having the formula RCOOH. R represents an
aliphatic group, preferably an alkyl group. R can include between
about 4 and about 22 carbon atoms. Fatty acids can be saturated,
monounsaturated, or polyunsaturated. A "fatty acid derivative" is a
product made in part from the fatty acid biosynthetic pathway of
the production host organism. "Fatty acid derivatives" includes
products made in part from ACP, acyl-ACP or acyl-ACP derivatives.
Exemplary fatty acid derivatives include, for example, acyl-CoA,
fatty acids, fatty aldehydes, short and long chain alcohols, fatty
alcohols, hydrocarbons, esters (e.g., waxes, fatty acid esters, or
fatty esters), terminal olefins, internal olefins, and ketones.
[0061] As used herein, the term "fatty acid biosynthetic pathway"
means a biosynthetic pathway that produces fatty acids and
derivatives thereof. The fatty acid biosynthetic pathway may
include additional enzymes to produce fatty acids derivatives
having desired characteristics.
[0062] As used herein, "fatty aldehyde" means an aldehyde having
the formula RCHO characterized by a carbonyl group (C.dbd.O). In
some embodiments, the fatty aldehyde is any aldehyde made from a
fatty alcohol. In certain embodiments, the R group is 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, or at least 19, carbons in
length. Alternatively, or in addition, the R group is 20 or less,
19 or less, 18 or less, 17 or less, 16 or less, 15 or less, 14 or
less, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8
or less, 7 or less, or 6 or less carbons in length. Thus, the R
group can have an R group bounded by any two of the above
endpoints. For example, the R group can be 6-16 carbons in length,
10-14 carbons in length, or 12-18 carbons in length. In some
embodiments, the fatty aldehyde is a C.sub.6, C.sub.7, C.sub.8,
C.sub.9, C.sub.10, C.sub.11, C.sub.12, C.sub.13, C.sub.14,
C.sub.15, C.sub.16, C.sub.17, C.sub.18, C.sub.19, C.sub.20,
C.sub.21, C.sub.22, C.sub.23, C.sub.24, C.sub.25, or a C.sub.26
fatty aldehyde. In certain embodiments, the fatty aldehyde is a
C.sub.6, C.sub.8, C.sub.10, C.sub.12, C.sub.13, C.sub.14, C.sub.15,
C.sub.16, C.sub.17, or C.sub.18 fatty aldehyde.
[0063] As used herein, "fatty alcohol" means an alcohol having the
formula ROH. In some embodiments, the R group is 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, or at least 19, carbons in length.
Alternatively, or in addition, the R group is 20 or less, 19 or
less, 18 or less, 17 or less, 16 or less, 15 or less, 14 or less,
13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 or
less, 7 or less, or 6 or less carbons in length. Thus, the R group
can have an R group bounded by any two of the above endpoints. For
example, the R group can be 6-16 carbons in length, 10-14 carbons
in length, or 12-18 carbons in length. In some embodiments, the
fatty alcohol is a C.sub.6, C.sub.7, C.sub.8, C.sub.9, C.sub.10,
C.sub.11, C.sub.12, C.sub.13, C.sub.14, C.sub.15, C.sub.16,
C.sub.17, C.sub.18, C.sub.19, C.sub.20, C.sub.21, C.sub.22,
C.sub.23, C.sub.24, C.sub.25, or a C.sub.26 fatty alcohol. In
certain embodiments, the fatty alcohol is a C.sub.6, C.sub.8,
C.sub.10, C.sub.12, C.sub.13, C.sub.14, C.sub.15, C.sub.16,
C.sub.17, or C.sub.18 fatty alcohol.
[0064] A "fatty alcohol composition" as referred to herein is
produced by a recombinant host cell and typically comprises a
mixture of fatty alcohols. In some cases, the mixture includes more
than one type of product (e.g., fatty alcohols and fatty acids). In
other cases, the fatty acid derivative compositions may comprise,
for example, a mixture of fatty alcohols with various chain lengths
and saturation or branching characteristics. In still other cases,
the fatty alcohol composition comprises a mixture of both more than
one type of product and products with various chain lengths and
saturation or branching characteristics.
[0065] A host cell engineered to produce a fatty aldehyde will
typically convert some of the fatty aldehyde to a fatty alcohol. In
one exemplary embodiment, acyl-ACP is converted to a fatty aldehyde
via the action of AAR. Conversion of fatty aldehydes to fatty
alcohols can be further facilitated, for example, via a fatty
alcohol biosynthetic polypeptide. In some embodiments, a gene
encoding a fatty alcohol biosynthetic polypeptide is expressed or
overexpressed in the host cell. In certain embodiments, the fatty
alcohol biosynthetic polypeptide has aldehyde reductase or alcohol
dehydrogenase activity.
[0066] Examples of alcohol dehydrogenase polypeptides useful in
accordance with the disclosure include, but are not limited to AlrA
of Acinetobacter sp. M-1 (SEQ ID NO: 52) or AlrA homologs, such as
AlrAadp1 (SEQ ID NO:53) and endogenous E. coli alcohol
dehydrogenases such as YjgB, (AAC77226), DkgA (NP_417485), DkgB
(NP_414743), YdjL (AAC74846), YdjJ (NP_416288), AdhP (NP_415995),
YhdH (NP_417719), YahK (NP_414859), YphC (AAC75598), YqhD (446856)
and YbbO [AAC73595.1]. Additional examples are described in
International Patent Application Publication Nos. WO 2007/136762,
WO2008/119082 and WO 2010/062480. In certain embodiments, the fatty
alcohol biosynthetic polypeptide has aldehyde reductase or alcohol
dehydrogenase activity (EC 1.1.1.1).
[0067] The R group of a fatty acid, fatty aldehyde, or fatty
alcohol can be a straight chain or a branched chain. Branched
chains may have more than one point of branching and may include
cyclic branches. In some embodiments, the branched fatty acid,
branched fatty aldehyde, or branched fatty alcohol is a C.sub.6,
C.sub.7, C.sub.8, C.sub.9, C.sub.10, C.sub.11, C.sub.12, C.sub.13,
C.sub.14, C.sub.15, C.sub.16, C.sub.17, C.sub.18, C.sub.19,
C.sub.20, C.sub.21, C.sub.22, C.sub.23, C.sub.24, C.sub.25, or a
C.sub.26 branched fatty acid, branched fatty aldehyde, or branched
fatty alcohol. In particular embodiments, the branched fatty acid,
branched fatty aldehyde, or branched fatty alcohol is a C.sub.6,
C.sub.8, C.sub.10, C.sub.12, C.sub.13, C.sub.14, C.sub.15,
C.sub.16, C.sub.17, or C.sub.18 branched fatty acid, branched fatty
aldehyde, or branched fatty alcohol. In certain embodiments, the
hydroxyl group of the branched fatty acid, branched fatty aldehyde,
or branched fatty alcohol is in the primary (C.sub.1) position.
[0068] In certain embodiments, the branched fatty acid, branched
fatty aldehyde, or branched fatty alcohol is an iso-fatty acid,
iso-fatty aldehyde, or iso-fatty alcohol, or an anteiso-fatty acid,
an anteiso-fatty aldehyde, or anteiso-fatty alcohol. In exemplary
embodiments, the branched fatty acid, branched fatty aldehyde, or
branched fatty alcohol is selected from iso-C.sub.7:0,
iso-C.sub.8:0, iso-C.sub.9:0, iso-C.sub.10:0, iso-C.sub.11:0,
iso-C.sub.12:0, iso-C.sub.13:0, iso-C.sub.14:0, iso-C.sub.15:0,
iso-C.sub.16:0, iso-C.sub.17:0, iso-C.sub.18:0, iso-C.sub.19:0,
anteiso-C.sub.7:0, anteiso-C.sub.8:0, anteiso-C.sub.9:0,
anteiso-C.sub.10:0, anteiso-C.sub.11:0, anteiso-C.sub.12:0,
anteiso-C.sub.13:0, anteiso-C.sub.14:0, anteiso-C.sub.15:0,
anteiso-C.sub.16:0, anteiso-C.sub.17:0, anteiso-C.sub.18:0, and
anteiso-C.sub.19:0 branched fatty acid, branched fatty aldehyde or
branched fatty alcohol.
[0069] The R group of a branched or unbranched fatty acid, branched
or unbranched fatty aldehyde, or branched or unbranched fatty
alcohol can be saturated or unsaturated. If unsaturated, the R
group can have one or more than one point of unsaturation. In some
embodiments, the unsaturated fatty acid, unsaturated fatty
aldehyde, or unsaturated fatty alcohol is a monounsaturated fatty
acid, monounsaturated fatty aldehyde, or monounsaturated fatty
alcohol. In certain embodiments, the unsaturated fatty acid,
unsaturated fatty aldehyde, or unsaturated fatty alcohol is a C6:1,
C7:1, C8:1, C9:1, C10:1, C11:1, C12:1, C13:1, C14:1, C15:1, C16:1,
C17:1, C18:1, C19:1, C20:1, C21:1, C22:1, C23:1, C24:1, C25:1, or a
C26:1 unsaturated fatty acid, unsaturated fatty aldehyde, or
unsaturated fatty alcohol. In certain preferred embodiments, the
unsaturated fatty acid, unsaturated fatty aldehyde, or unsaturated
fatty alcohol is C10:1, C12:1, C14:1, C16:1, or C18:1. In yet other
embodiments, the unsaturated fatty acid, unsaturated fatty
aldehyde, or unsaturated fatty alcohol is unsaturated at the
omega-7 position. In certain embodiments, the unsaturated fatty
acid, unsaturated fatty aldehyde, or unsaturated fatty alcohol
comprises a cis double bond.
[0070] As used herein, a "recombinant host cell" or "engineered
host cell" is a host cell, e.g., a microorganism that has been
modified such that it produces fatty alcohols. In some embodiments,
the recombinant host cell comprises one or more polynucleotides,
each polynucleotide encoding a polypeptide having fatty aldehyde
and/or fatty alcohol biosynthetic enzyme activity, wherein the
recombinant host cell produces a fatty alcohol composition when
cultured in the presence of a carbon source under conditions
effective to express the polynucleotides.
[0071] As used herein, the term "clone" typically refers to a cell
or group of cells descended from and essentially genetically
identical to a single common ancestor, for example, the bacteria of
a cloned bacterial colony arose from a single bacterial cell.
[0072] As used herein, the term "culture" typically refers to a
liquid media comprising viable cells. In one embodiment, a culture
comprises cells reproducing in a predetermined culture media under
controlled conditions, for example, a culture of recombinant host
cells grown in liquid media comprising a selected carbon source
and/or nitrogen.
[0073] The terms "culturing" or "cultivation" refers to growing a
population of cells (e.g., microbial cells) under suitable
conditions in a liquid or solid medium. In particular embodiments,
culturing refers to the fermentative bioconversion of a substrate
to an end-product. Culturing media are well known and individual
components of such culture media are available from commercial
sources, e.g., under the DIFCO media and BBL media. In one
non-limiting example, the aqueous nutrient medium is a "rich
medium" comprising complex sources of nitrogen, salts, and carbon,
such as YP medium, comprising 10 g/L of peptone and 10 g/L yeast
extract of such a medium.
[0074] The host cell can be additionally engineered to assimilate
carbon efficiently and use cellulosic materials as carbon sources
according to methods described, for example, in U.S. Pat. Nos.
5,000,000; 5,028,539; 5,424,202; 5,482,846; 5,602,030 and
WO2010127318. In addition, the host cell can be engineered to
express an invertase so that sucrose can be used as a carbon
source.
[0075] As used herein, the term "under conditions effective to
express said heterologous nucleotide sequences" means any
conditions that allow a host cell to produce a desired fatty
aldehyde or fatty alcohol. Suitable conditions include, for
example, fermentation conditions.
[0076] As used herein, "modified" or an "altered level of" activity
of a protein, for example an enzyme, in a recombinant host cell
refers to a difference in one or more characteristics in the
activity determined relative to the parent or native host cell.
Typically differences in activity are determined between a
recombinant host cell, having modified activity, and the
corresponding wild-type host cell (e.g., comparison of a culture of
a recombinant host cell relative to wild-type host cell). Modified
activities can be the result of, for example, modified amounts of
protein expressed by a recombinant host cell (e.g., as the result
of increased or decreased number of copies of DNA sequences
encoding the protein, increased or decreased number of mRNA
transcripts encoding the protein, and/or increased or decreased
amounts of protein translation of the protein from mRNA); changes
in the structure of the protein (e.g., changes to the primary
structure, such as, changes to the protein's coding sequence that
result in changes in substrate specificity, changes in observed
kinetic parameters); and changes in protein stability (e.g.,
increased or decreased degradation of the protein). In some
embodiments, the polypeptide is a mutant or a variant of any of the
polypeptides described herein. In certain instances, the coding
sequence for the polypeptides as described herein are codon
optimized for expression in a particular host cell. For example,
for expression in E. coli, one or more codons can be optimized
(Grosjean et al. (1982) Gene 18:199-209).
[0077] The term "regulatory sequences" as used herein typically
refers to a sequence of bases in DNA, operably-linked to DNA
sequences encoding a protein that ultimately controls the
expression of the protein. Examples of regulatory sequences
include, but are not limited to, RNA promoter sequences,
transcription factor binding sequences, transcription termination
sequences, modulators of transcription (such as enhancer elements),
nucleotide sequences that affect RNA stability, and translational
regulatory sequences (such as, ribosome binding sites (e.g.,
Shine-Dalgarno sequences in prokaryotes or Kozak sequences in
eukaryotes), initiation codons, termination codons).
[0078] As used herein, the phrase "the expression of said
nucleotide sequence is modified relative to the wild type
nucleotide sequence," means an increase or decrease in the level of
expression and/or activity of an endogenous nucleotide sequence or
the expression and/or activity of a heterologous or non-native
polypeptide-encoding nucleotide sequence.
[0079] As used herein, the term "express" with respect to a
polynucleotide is to cause it to function. A polynucleotide which
encodes a polypeptide (or protein) will, when expressed, be
transcribed and translated to produce that polypeptide (or
protein). As used herein, the term "overexpress" means to express
(or cause to express) a polynucleotide or polypeptide in a cell at
a greater concentration than is normally expressed in a
corresponding wild-type cell under the same conditions.
[0080] The terms "altered level of expression" and "modified level
of expression" are used interchangeably and mean that a
polynucleotide, polypeptide, or hydrocarbon is present in a
different concentration in an engineered host cell as compared to
its concentration in a corresponding wild-type cell under the same
conditions.
[0081] As used herein, the term "titer" refers to the quantity of
fatty aldehyde or fatty alcohol produced per unit volume of host
cell culture. In any aspect of the compositions and methods
described herein, a fatty alcohol is produced at a titer of about
25 mg/L, about 50 mg/L, about 75 mg/L, about 100 mg/L, about 125
mg/L, about 150 mg/L, about 175 mg/L, about 200 mg/L, about 225
mg/L, about 250 mg/L, about 275 mg/L, about 300 mg/L, about 325
mg/L, about 350 mg/L, about 375 mg/L, about 400 mg/L, about 425
mg/L, about 450 mg/L, about 475 mg/L, about 500 mg/L, about 525
mg/L, about 550 mg/L, about 575 mg/L, about 600 mg/L, about 625
mg/L, about 650 mg/L, about 675 mg/L, about 700 mg/L, about 725
mg/L, about 750 mg/L, about 775 mg/L, about 800 mg/L, about 825
mg/L, about 850 mg/L, about 875 mg/L, about 900 mg/L, about 925
mg/L, about 950 mg/L, about 975 mg/L, about 1000 mg/L, about 1050
mg/L, about 1075 mg/L, about 1100 mg/L, about 1125 mg/L, about 1150
mg/L, about 1175 mg/L, about 1200 mg/L, about 1225 mg/L, about 1250
mg/L, about 1275 mg/L, about 1300 mg/L, about 1325 mg/L, about 1350
mg/L, about 1375 mg/L, about 1400 mg/L, about 1425 mg/L, about 1450
mg/L, about 1475 mg/L, about 1500 mg/L, about 1525 mg/L, about 1550
mg/L, about 1575 mg/L, about 1600 mg/L, about 1625 mg/L, about 1650
mg/L, about 1675 mg/L, about 1700 mg/L, about 1725 mg/L, about 1750
mg/L, about 1775 mg/L, about 1800 mg/L, about 1825 mg/L, about 1850
mg/L, about 1875 mg/L, about 1900 mg/L, about 1925 mg/L, about 1950
mg/L, about 1975 mg/L, about 2000 mg/L (2 g/L), 3 g/L, 5 g/L, 10
g/L, 20 g/L, 30 g/L, 40 g/L, 50 g/L, 60 g/L, 70 g/L, 80 g/L, 90
g/L, 100 g/L or a range bounded by any two of the foregoing values.
In other embodiments, a fatty aldehyde or fatty alcohol is produced
at a titer of more than 100 g/L, more than 200 g/L, more than 300
g/L, or higher, such as 500 g/L, 700 g/L, 1000 g/L, 1200 g/L, 1500
g/L, or 2000 g/L. The preferred titer of fatty aldehyde or fatty
alcohol produced by a recombinant host cell according to the
methods of the disclosure is from 5 g/L to 200 g/L, 10 g/L to 150
g/L, 20 g/L to 120 g/L and 30 g/L to 100 g/L.
[0082] As used herein, the term "yield of the fatty aldehyde or
fatty alcohol produced by a host cell" refers to the efficiency by
which an input carbon source is converted to product (i.e., fatty
alcohol or fatty aldehyde) in a host cell. Host cells engineered to
produce fatty alcohols and/or fatty aldehydes according to the
methods of the disclosure have a yield of 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%, or at least 30% or a range bounded by any two of the
foregoing values. In other embodiments, a fatty aldehyde or fatty
alcohol is produced at a yield of more than 30%, 40%, 50%, 60%,
70%, 80%, 90% or more. Alternatively, or in addition, the yield is
about 30% or less, about 27% or less, about 25% or less, or about
22% or less. Thus, the yield can be bounded by any two of the above
endpoints. For example, the yield of the fatty alcohol or fatty
aldehyde produced by the recombinant host cell according to the
methods of the disclosure can be 5% to 15%, 10% to 25%, 10% to 22%,
15% to 27%, 18% to 22%, 20% to 28%, or 20% to 30%. The preferred
yield of fatty alcohol produced by the recombinant host cell
according to the methods of the disclosure is from 10% to 30%.
[0083] As used herein, the term "productivity" refers to the
quantity of fatty aldehyde or fatty alcohol produced per unit
volume of host cell culture per unit time. In any aspect of the
compositions and methods described herein, the productivity of
fatty aldehyde or fatty alcohol produced by a recombinant host cell
is at least 100 mg/L/hour, at least 200 mg/L/hour.sub.0, at least
300 mg/L/hour, at least 400 mg/L/hour, at least 500 mg/L/hour, at
least 600 mg/L/hour, at least 700 mg/L/hour, at least 800
mg/L/hour, at least 900 mg/L/hour, at least 1000 mg/L/hour, at
least 1100 mg/L/hour, at least 1200 mg/L/hour, at least 1300
mg/L/hour, at least 1400 mg/L/hour, at least 1500 mg/L/hour, at
least 1600 mg/L/hour, at least 1700 mg/L/hour, at least 1800
mg/L/hour, at least 1900 mg/L/hour, at least 2000 mg/L/hour, at
least 2100 mg/L/hour, at least 2200 mg/L/hour, at least 2300
mg/L/hour, at least 2400 mg/L/hour, or at least 2500 mg/L/hour.
Alternatively, or in addition, the productivity is 2500 mg/L/hour
or less, 2000 mg/L/OD.sub.600 or less, 1500 mg/L/OD.sub.600 or
less, 120 mg/L/hour, or less, 1000 mg/L/hour or less, 800
mg/L/hour, or less, or 600 mg/L/hour or less. Thus, the
productivity can be bounded by any two of the above endpoints. For
example, the productivity can be 3 to 30 mg/L/hour, 6 to 20
mg/L/hour, or 15 to 30 mg/L/hour. The preferred productivity of a
fatty aldehyde or fatty alcohol produced by a recombinant host cell
according to the methods of the disclosure is selected from 500
mg/L/hour to 2500 mg/L/hour, or from 700 mg/L/hour to 2000
mg/L/hour.
[0084] The terms "total fatty species" and "total fatty acid
product" may be used interchangeably herein with reference to the
total amount of fatty alcohols, fatty aldehydes, fee fatty acids,
and fatty esters present in a sample as evaluated by GC-FID as
described in International Patent Application Publication WO
2008/119082. Samples may contain one, two, three, or four of these
compounds depending on the context.
[0085] As used herein, the term "glucose utilization rate" means
the amount of glucose used by the culture per unit time, reported
as grams/liter/hour (g/L/hr).
[0086] As used herein, the term "carbon source" refers to a
substrate or compound suitable to be used as a source of carbon for
prokaryotic or simple eukaryotic cell growth. Carbon sources can be
in various forms, including, but not limited to polymers,
carbohydrates, acids, alcohols, aldehydes, ketones, amino acids,
peptides, and gases (e.g., CO and CO.sub.2). Exemplary carbon
sources include, but are not limited to, monosaccharides, such as
glucose, fructose, mannose, galactose, xylose, and arabinose;
oligosaccharides, such as fructo-oligosaccharide and
galacto-oligosaccharide; polysaccharides such as starch, cellulose,
pectin, and xylan; disaccharides, such as sucrose, maltose,
cellobiose, and turanose; cellulosic material and variants such as
hemicelluloses, methyl cellulose and sodium carboxymethyl
cellulose; saturated or unsaturated fatty acids, succinate,
lactate, and acetate; alcohols, such as ethanol, methanol, and
glycerol, or mixtures thereof. The carbon source can also be a
product of photosynthesis, such as glucose. In certain embodiments,
the carbon source is gas mixture containing CO coming from flu gas.
In another embodiment, the carbon source is a gas mixture
containing CO coming from the reformation of a carbon containing
material, such as biomass, coal, or natural gas. In other
embodiments the carbon source is syngas, methane, or natural gas.
In certain preferred embodiments, the carbon source is biomass. In
other preferred embodiments, the carbon source is glucose. In other
preferred embodiments the carbon source is sucrose. In other
embodiments the carbon source is glycerol. In other preferred
embodiments the carbon source is sugar cane juice, sugar cane
syrup, or corn syrup. In other preferred embodiments, the carbon
source is derived from renewable feedstocks, such as CO.sub.2, CO,
glucose, sucrose, xylose, arabinose, glycerol, mannose, or mixtures
thereof. In other embodiments, the carbon source is derived from
renewable feedstocks including starches, cellulosic biomass,
molasses, and other sources of carbohydrates including carbohydrate
mixtures derived from hydrolysis of cellulosic biomass, or the
waste materials derived from plant- or natural oil processing.
[0087] As used herein, the term "biomass" refers to any biological
material from which a carbon source is derived. In some
embodiments, a biomass is processed into a carbon source, which is
suitable for bioconversion. In other embodiments, the biomass does
not require further processing into a carbon source. An exemplary
source of biomass is plant matter or vegetation, such as corn,
sugar cane, or switchgrass. Another exemplary source of biomass is
metabolic waste products, such as animal matter (e.g., cow manure).
Further exemplary sources of biomass include algae and other marine
plants. Biomass also includes waste products from industry,
agriculture, forestry, and households, including, but not limited
to, glycerol, fermentation waste, ensilage, straw, lumber, sewage,
garbage, cellulosic urban waste, and food leftovers (e.g., soaps,
oils and fatty acids). The term "biomass" also can refer to sources
of carbon, such as carbohydrates (e.g., monosaccharides,
disaccharides, or polysaccharides).
[0088] As used herein, the term "isolated," with respect to
products (such as fatty acids and derivatives thereof) refers to
products that are separated from cellular components, cell culture
media, or chemical or synthetic precursors. The fatty acids and
derivatives thereof produced by the methods described herein can be
relatively immiscible in the fermentation broth, as well as in the
cytoplasm. Therefore, the fatty acids and derivatives thereof can
collect in an organic phase either intracellularly or
extracellularly.
[0089] As used herein, the terms "purify," "purified," or
"purification" mean the removal or isolation of a molecule from its
environment by, for example, isolation or separation.
"Substantially purified" molecules are at least about 60% free
(e.g., at least about 70% free, at least about 75% free, at least
about 85% free, at least about 90% free, at least about 95% free,
at least about 97% free, at least about 99% free) from other
components with which they are associated. As used herein, these
terms also refer to the removal of contaminants from a sample. For
example, the removal of contaminants can result in an increase in
the percentage of fatty acid derivatives such as fatty alcohols in
a sample. For example, when a fatty acid derivative is produced in
a recombinant host cell, the fatty acid derivative can be purified
by the removal of host cell proteins or other host cell materials.
After purification, the percentage of fatty acid derivative in the
sample is increased. The terms "purify", "purified," and
"purification" are relative terms which do not require absolute
purity. Thus, for example, when a fatty acid derivative is produced
in recombinant host cells, a purified fatty acid derivative is a
fatty acid derivative that is substantially separated from other
cellular components (e.g., nucleic acids, polypeptides, lipids,
carbohydrates, or other hydrocarbons).
[0090] Increased Fatty Alcohol Production
[0091] The disclosure provides for the production of a fatty
alcohol composition that is enhanced as a result of the modified
expression of an acyl-ACP reductase (AAR) gene in a host cell. AAR
is involved in a biosynthetic pathway for the production of fatty
aldehydes and fatty alcohols. Variant AAR is used alone or in
combination with the modified expression of another gene involved
in the biosynthetic pathway that converts a fatty aldehyde to a
fatty alcohol. Herein, the disclosure provides recombinant host
cells, which have been engineered to express a variant AAR to
provide enhanced fatty alcohol biosynthesis relative to
non-engineered or native or wild type host cells that express wild
type AAR or other fatty alcohol biosynthetic polypeptides with the
same function as AAR. The disclosure identifies AAR related
polynucleotides and polypeptides useful in the recombinant host
cells. However, it will be recognized that absolute sequence
identity to AAR related polynucleotides is not necessary. For
example, changes in a particular polynucleotide sequence can be
made and the encoded polypeptide screened for activity. Such
changes typically include conservative mutations and silent
mutations such as, for example, through codon optimization.
Modified or mutated (i.e., mutant) polynucleotides and encoded
variant polypeptides can be screened for a desired function, such
as, an improved function compared to the parent polypeptide,
including but not limited to increased catalytic activity,
increased stability, or decreased inhibition (e.g., decreased
feedback inhibition), using methods known in the art. The
disclosure identifies enzymatic activities involved in various
steps (i.e., reactions) of the fatty acid biosynthetic pathways
described herein according to Enzyme Classification (EC) number,
and provides exemplary polypeptides (enzymes) categorized by such
EC numbers, and exemplary polynucleotides encoding such
polypeptides. Such exemplary polypeptides and polynucleotides,
which are identified herein by Accession Numbers and/or Sequence
Identifier Numbers (SEQ ID NOs), are useful for engineering fatty
acid pathways in parental host cells to obtain the recombinant host
cells described herein. It is to be understood, however, that
polypeptides and polynucleotides described herein are exemplary
and, thus, non-limiting. The sequences of homologues of exemplary
polypeptides described herein are available to those of skill in
the art using databases such as, for example, the Entrez databases
provided by the National Center for Biotechnology Information
(NCBI), the ExPasy databases provided by the Swiss Institute of
Bioinformatics, the BRENDA database provided by the Technical
University of Braunschweig, and the KEGG database provided by the
Bioinformatics Center of Kyoto University and University of Tokyo,
all which are available on the World Wide Web. A variety of
different host cells can be modified to express variant AAR fatty
alcohol biosynthetic enzymes such as those described herein,
resulting in recombinant host cells suitable for the enhanced
production of fatty alcohol compositions. It is understood that a
variety of cells can provide sources of genetic material, including
polynucleotide sequences that encode polypeptides suitable for use
in a recombinant host cell as described herein.
[0092] Acyl-ACP Reductase (AAR) Polypeptides and Variants
Thereof
[0093] In one aspect the disclosure relates to improved production
of fatty acid derivatives such as fatty aldehydes and/or fatty
alcohols by engineering a host cell to express a native or
non-native acyl-ACP reductase (AAR) protein. The AAR protein
catalyzes the reduction of an acyl-ACP to a fatty aldehyde and may
also catalyze the conversion of a fatty aldehyde to a fatty alcohol
(see U.S. Patent Publication No. 20120282663, incorporated herein
by reference). The AAR polypeptide or the polynucleotide sequence
that encodes the AAR polypeptide may be native (e.g., endogenous)
or non-native (e.g., exogenous, heterologous, etc.), i.e., it may
differ from the wild type sequence and expression thereof naturally
present in the corresponding wild type host cell. Examples include
a modification in the sequence of the AAR polynucleotide,
polypeptide or protein resulting in a variant AAR (e.g., mutant)
and/or in the level of expression thereof. The disclosure includes
AAR polypeptides, homologs, and variants.
[0094] In one embodiment, an AAR polypeptide for use in practicing
the disclosure has at least 90% sequence identity to the wild-type
AAR polypeptide sequence of SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID
NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40,
SEQ ID NO: 42 or SEQ ID NO: 44. In some embodiments the AAR is
derived from a Synechococcus species or a Prochlorococcus species.
In other embodiments, an AAR polypeptide for use in practicing the
disclosure has at least 75% (e.g., at least 76%, 77%, 78%, 79%,
80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or at least 99%) sequence identity to
the wild-type AAR polypeptide sequence of SEQ ID NO: 28, SEQ ID NO:
30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ
ID NO: 40, SEQ ID NO: 42 or SEQ ID NO: 44, and may also include one
or more substitutions which results in useful characteristics
and/or properties as described herein. In one embodiment, the AAR
polypeptide for use in practicing the present disclosure has at
least 75% (e.g., at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98% or at least 99%) sequence identity to the wild-type AAR
polypeptide sequence of SEQ ID NO: 28 or SEQ ID NO: 34. In other
embodiments, an AAR polypeptide for use in practicing the
disclosure has 100% sequence identity to SEQ ID NO: 28, SEQ ID NO:
30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ
ID NO: 40, SEQ ID NO: 42 or SEQ ID NO: 44. In still other
embodiments, the improved or variant AAR polypeptide sequence is
derived from a species other than a Synechococcus species or a
Prochlorococcus species.
[0095] In a related embodiment, the disclosure includes AAR
polypeptides that have an amino acid sequence encoded by a nucleic
acid sequence that has at least 75% (e.g., at least 76%, 77%, 78%,
79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98% or and at least 99%) sequence
identity to SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO:
33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41 or
SEQ ID NO: 43. In some embodiments the nucleic acid sequence
encodes an AAR variant with one or more substitutions which results
in improved characteristics and/or properties as described herein.
In yet another related embodiment, an AAR polypeptide for use in
practicing the disclosure is encoded by a nucleotide sequence
having 100% sequence identity to SEQ ID NO: 27, SEQ ID NO: 29, SEQ
ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO:
39, SEQ ID NO: 41 or SEQ ID NO: 43. In another aspect, the
disclosure relates to AAR polypeptides that comprise an amino acid
sequence encoded by a nucleic acid that hybridizes under stringent
conditions over substantially the entire length of a nucleic acid
corresponding to SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ
ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO:
41 or SEQ ID NO: 43.
[0096] In one preferred embodiment, the disclosure provides a AAR
polypeptide for use in practicing the disclosure that has at least
90% sequence identity to the variant AAR polypeptide sequence of
any one of SEQ ID NO: 57 through SEQ ID NO: 78. In some embodiments
the variant AAR is derived from a Synechococcus species or a
Prochlorococcus species. In other embodiments, an AAR polypeptide
for use in practicing the disclosure has at least 75% (e.g., at
least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least
99%) sequence identity to the variant AAR polypeptide sequence of
any one of SEQ ID NO: 57 through SEQ ID NO: 78. The variant AAR
polypeptide may include one or more substitution(s) which result in
useful characteristics and/or properties as described herein. In
another preferred embodiment, the AAR polypeptide for use in
practicing the disclosure has at least 75% (e.g., at least 76%,
77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%)
sequence identity to the variant AAR polypeptide sequence of SEQ ID
NO: 57, SEQ ID NO: 58, SEQ ID NO: 59 or SEQ ID NO: 65. In other
embodiments, an AAR polypeptide for use in practicing the
disclosure has 100% sequence identity to any one of SEQ ID NO: 57
through SEQ ID NO: 78. In still other embodiments, the improved or
variant AAR polypeptide sequence is derived from a species other
than a Synechococcus species or a Prochlorococcus species.
[0097] The inventors built an error prone library of the acyl-ACP
reductase from Synechococcus elongatus PCC7942 (AAR_7942) in order
to screen for variants that have improvements over the wild type
AAR_7942 (see Example 3, infra). The improvements are classified as
either improving fatty alcohol titer overall or increasing the
fraction of C10, C12, C14 or C16 fatty alcohols without
significantly affecting titer. The error prone library identified
various amino acid positions including amino acid position 18.
Saturation and combination libraries were prepared to further test
these positions. SEQ ID NO: 57 codes for the amino acid sequence
for an AAR variant (mutant) that has a mutation in amino acid 18.
The mutation S18W where serine is replaced with tryptophan results
in a significant increase of fatty alcohol production when
expressed in cells (see Examples 3 and 4, infra). More
specifically, the S18W mutation lead to a 227 percent increase in
total fatty alcohol (FALC) production and a 324 percent increase in
C14 fatty alcohols compared to wild type AAR used as control (see
Tables 4A and 4B, infra).
[0098] The inventors built saturation libraries based on the S18W
mutation in order to identify variants (mutants) that further
increased overall FALC titer or the fraction of C12 fatty alcohols
(see Example 4 and Table 5, infra). Combination libraries that used
the S18W mutation (SEQ ID NO: 57) as a template produced 7
combination mutants that showed further significant increases in
the total FALC titer and/or C12 fatty alcohol production (see Table
6B, infra). The 7 combination mutants include AAR with mutation
S18W (SEQ ID NO: 57); AAR with mutations M21L, C63G, S113K, T154A,
A281L (SEQ ID NO: 58); AAR with mutations L8A, M21L, C63G, A77A
(GCC to GCA silent codon mutation), S113K, T154A, A281L (SEQ ID NO:
59); AAR with mutations D16L, M21L, C63G, S113K, T154A, A281L (SEQ
ID NO: 60); AAR with mutations L8A, D24V, C63G, S113K, Q155L, A281L
(SEQ ID NO: 61); AAR with mutations D24P, L31M, C63G, S113K, T154A,
A281L (SEQ ID NO: 62); AAR with mutations L8A, D16L, D24V, C63G,
S113K, T154A, A281L (SEQ ID NO: 63); and AAR with mutations D24E,
C63G, S113K, T154A, A281L (SEQ ID NO: 64). Notably, SEQ ID NO: 58
showed the highest fraction of C12 fatty alcohols while SEQ ID NO:
59 showed the highest titer of the combination mutants (see Table
6B, infra).
[0099] The inventors also built a full saturation library of the
acyl-ACP reductase from Prochlorococcus marinas MED4_AAR in order
to screen for variants that show improvements over the wild type
MED4_AAR (see Example 7, infra). The AAR variants were selected
based upon production of more fatty alcohols than the wild type AAR
enzyme or the ability to produce fatty alcohols with an altered
chain length profile, e.g., an increased fraction of C12, C14, or
C16 fatty alcohols. Table 8 (see Example 7, infra) shows
representative data from 16 AAR variants that produced the highest
FALC titers ranging from 1.4-fold to 2.2 fold over wild type
MED4_AAR. The inventors also screened for AAR variants that have an
altered chain length profile, wherein an increased proportion of
FALC species with a chain length shorter than C16 is of interest.
Two variant clones that lead to a 2-3-fold increase in the quantity
of FALC are shown in FIG. 10. The expression of one of these AAR
variants, i.e., the D61E mutant (SEQ ID NO: 65) in a recombinant
host cell, skewed the chain length distribution of fatty alcohol
species toward shorter carbon chains. All variants led to a higher
quantity of FALC as they have increased titer, but only SEQ ID NO:
65 had an increased fraction of C14 (and higher titer). Table 9
(see Example 7, infra) illustrates the FALC chain length
distribution produced by recombinant host cells expressing the D61E
variant of MED4_AAR compared to wild type (WT) MED4_AAR and the
V346P variant of MED4_AAR which did not produce products with
altered chain lengths.
[0100] Acyl Carrier Protein (ACP)
[0101] There are conflicting reports in the literature as to
factors that can limit fatty acid biosynthesis in host cells, such
as Escherichia coli (E. coli). Although acyl carrier proteins (ACP)
are conserved to some extent in all organisms, their primary
sequence can differ. It has been suggested that when terminal
pathway enzymes from sources other than E. coli are expressed in E.
coli in order to convert fatty acyl-ACPs to products, limitations
may exist such as in the recognition, affinity and/or turnover of
the recombinant pathway enzyme towards the fatty acyl-ACPs (see Suh
et al. (1999) The Plant Journal 17(6):679-688; Salas et al. (2002)
Archives of Biochemistry and Biophysics 403:25-34). One suggestion
is that a limitation of the main precursors for fatty acid
biosynthesis, for example, acetyl-CoA and malonyl-CoA can result in
decreased synthesis of fatty acid derivatives. One approach to
increasing the flux through fatty acid biosynthesis is to
manipulate various enzymes in the pathway (see FIGS. 1-3). The
supply of acyl-ACPs from acetyl-CoA via the acetyl-CoA carboxylase
(acc) complex and the fatty acid biosynthetic (fab) pathway may
impact the rate of fatty acid derivative production (see FIG. 2).
As detailed in the Examples (infra), the effect of overexpression
of ACP on production of fatty alcohols was evaluated for purpose of
illustration.
[0102] A host cell that is engineered to express an ACP can exhibit
an increase in the titer of a fatty aldehyde and/or fatty alcohol
composition or a specific fatty aldehyde and/or fatty alcohol
composition wherein the increase is 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%, or at least 30% greater than the titer of the fatty
aldehyde and/or fatty alcohol composition produced by a
corresponding host cell that does not express ACP when cultured
under the same conditions. In one aspect the disclosure relates to
improved production of fatty aldehyde and/or fatty alcohol
composition by engineering a host cell to express a native (e.g.,
endogenous) or non-native (e.g., exogenous, heterologous) ACP
protein. The ACP polypeptide or the polynucleotide sequence that
encodes the ACP polypeptide may be non-native, i.e., it may differ
from the wild type sequence naturally present in the corresponding
wild type host cell. Examples include, a modification in the level
of expression or in the sequence of a nucleotide, polypeptide or
protein. The disclosure includes ACP polypeptides and homologs
thereof.
[0103] In one embodiment, an ACP polypeptide for use in practicing
the disclosure has at least 70% sequence identity to SEQ ID NO: 2,
SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10. In some
embodiments the ACP is derived from Marinobacter
hydrocarbonoclasticus or E. coli. In other embodiments, an ACP
polypeptide for use in practicing the disclosure has at least 75%
(e.g., at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
at least 99%) sequence identity to the wild-type ACP polypeptide
sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8
or SEQ ID NO: 10, and may also include one or more substitutions
which results in useful characteristics and/or properties as
described herein. In one aspect, an ACP polypeptide for use in
practicing the disclosure has 100% sequence identity to SEQ ID NO:
2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10. In
other embodiments, the improved or variant ACP polypeptide sequence
is derived from a species other than M. hydrocarbonoclasticus or E.
coli. In a related aspect, an ACP polypeptide for use in practicing
the disclosure is encoded by a nucleotide sequence having 100%
sequence identity to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ
ID NO: 7, or SEQ ID NO: 9. In another related aspect, the
disclosure relates to ACP polypeptides that comprise an amino acid
sequence encoded by a nucleic acid sequence that has at least 75%
(e.g., at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
and at least 99%) sequence identity to SEQ ID NO: 1, SEQ ID NO: 3,
SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9. In some embodiments
the nucleic acid sequence encodes an ACP variant with one or more
substitutions which result in improved characteristics and/or
properties as described herein. In other embodiments, the improved
or variant ACP nucleic acid sequence is derived from a species
other than a Marinobacter species or E. coli. In another aspect,
the disclosure relates to ACP polypeptides that have an amino acid
sequence encoded by a nucleic acid that hybridizes under stringent
conditions over substantially the entire length of a nucleic acid
corresponding to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID
NO: 7, or SEQ ID NO: 9.
[0104] Variations and Mutations
[0105] In some embodiments, the AAR or ACP polypeptide is a mutant
or a variant of any of the polypeptides described herein. A variant
or mutant polypeptide as used herein refers to a polypeptide having
an amino acid sequence that differs from a wild-type polypeptide by
at least one amino acid. For example, the mutant can have one or
more of the following conservative amino acid substitutions,
including but not limited to, replacement of an aliphatic amino
acid, such as alanine, valine, leucine, and isoleucine, with
another aliphatic amino acid; replacement of a serine with a
threonine; replacement of a threonine with a serine; replacement of
an acidic residue, such as aspartic acid and glutamic acid, with
another acidic residue; replacement of a residue bearing an amide
group, such as asparagine and glutamine, with another residue
bearing an amide group; exchange of a basic residue, such as lysine
and arginine, with another basic residue; and replacement of an
aromatic residue, such as phenylalanine and tyrosine, with another
aromatic residue. In some embodiments, the variant or mutant
polypeptide has about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30,
40, 50, 60, 70, 80, 90, 100, or more amino acid substitutions,
additions, insertions, or deletions. Some preferred fragments of a
polypeptide that function as a variant or mutant retain some or all
of the biological function (e.g., enzymatic activity) of the
corresponding wild-type polypeptide. In some embodiments, the
fragment retains at least 75%, at least 80%, at least 90%, at least
95%, or at least 98% or more of the biological function of the
corresponding wild-type polypeptide. In other embodiments, the
fragment or mutant retains about 100% of the biological function of
the corresponding wild-type polypeptide. Guidance in determining
which amino acid residues may be substituted, inserted, or deleted
without affecting biological activity may be found using computer
programs well known in the art, for example, LASERGENE software
(DNASTAR, Inc., Madison, Wis.). In yet other embodiments, a
fragment exhibits increased biological function as compared to a
corresponding wild-type polypeptide. For example, a fragment may
display at least a 10%, at least a 25%, at least a 50%, at least a
75%, or at least a 90% improvement in enzymatic activity as
compared to the corresponding wild-type polypeptide. In other
embodiments, the fragment displays at least 100%, at least 200%, or
at least 500% improvement in enzymatic activity as compared to the
corresponding wild-type polypeptide.
[0106] It is understood that the polypeptides described herein may
have additional conservative or non-essential amino acid
substitutions which do not have a substantial effect on the
polypeptide function. Whether or not a particular substitution will
be tolerated (i.e., will not adversely affect the desired
biological function, such as acyl-ACP reductase activity), can be
determined as known in the art (see Bowie et al. (1990) Science,
247:1306-1310). A conservative amino acid substitution is one in
which the amino acid residue is replaced with an amino acid residue
having a similar side chain. Families of amino acid residues having
similar side chains have been defined in the art. These families
include amino acids with basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine), nonpolar side
chains (e.g., alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan), beta-branched side chains
(e.g., threonine, valine, isoleucine), and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine).
[0107] Variants can be naturally occurring or created in vitro. In
particular, such variants can be created using genetic engineering
techniques, such as site directed mutagenesis, random chemical
mutagenesis, Exonuclease III deletion procedures, or standard
cloning techniques. Alternatively, such variants, mutants,
fragments, analogs, or derivatives can be created using chemical
synthesis or modification procedures. Methods of making variants
are well known in the art. For example, variants can be prepared by
using random and site-directed mutagenesis. Random and
site-directed mutagenesis are generally known in the art (see, for
example, Arnold (1993) Curr. Opin. Biotech. 4:450-455). Random
mutagenesis can be achieved using error prone PCR (see, for
example, Leung et al. (1989) Technique 1:11-15; and Caldwell et al.
(1992) PCR Methods Applic. 2: 28-33). In error prone PCR, the
actual PCR is performed under conditions where the copying fidelity
of the DNA polymerase is low, such that a high rate of point
mutations is obtained along the entire length of the PCR product.
Briefly, in such procedures, nucleic acids to be mutagenized (e.g.,
a polynucleotide sequence encoding an AAR enzyme) are mixed with
PCR primers, reaction buffer, MgCl.sub.2, MnCl.sub.2, Taq
polymerase, and an appropriate concentration of dNTPs for achieving
a high rate of point mutation along the entire length of the PCR
product. For example, the reaction can be performed using 20 fmoles
of nucleic acid to be mutagenized, 30 pmole of each PCR primer, a
reaction buffer comprising 50 mMKCl, 10 mM Tris HCl (pH 8.3), 0.01%
gelatin, 7 mM MgCl.sub.2, 0.5 mM MnCl.sub.2, 5 units of Taq
polymerase, 0.2 mM dGTP, 0.2 mM dATP, 1 mM dCTP, and 1 mM dTTP. PCR
can be performed for 30 cycles of 94.degree. C. for 1 min,
45.degree. C. for 1 min, and 72.degree. C. for 1 min. However, it
will be appreciated by those in the art that these parameters can
be varied as appropriate. The mutagenized nucleic acids are then
cloned into an appropriate vector, and the activities of the
polypeptides encoded by the mutagenized nucleic acids are
evaluated. Site-directed mutagenesis can be achieved using
oligonucleotide-directed mutagenesis to generate site-specific
mutations in any cloned DNA of interest. Oligonucleotide
mutagenesis is described in the art (see, for example,
Reidhaar-Olson et al. (1988) Science 241:53-57). Briefly, in such
procedures a plurality of double stranded oligonucleotides bearing
one or more mutations to be introduced into the cloned DNA are
synthesized and inserted into the cloned DNA to be mutagenized
(e.g., a polynucleotide sequence encoding an AAR polypeptide).
Clones containing the mutagenized DNA are recovered, and the
activities of the polypeptides they encode are assessed.
[0108] Another method for generating variants is assembly PCR.
Assembly PCR involves the assembly of a PCR product from a mixture
of small DNA fragments. A large number of different PCR reactions
occur in parallel in the same vial, with the products of one
reaction priming the products of another reaction (see U.S. Pat.
No. 5,965,408). Still another method of generating variants is
sexual PCR mutagenesis. In sexual PCR mutagenesis, forced
homologous recombination occurs between DNA molecules of different,
but highly related, DNA sequences in vitro as a result of random
fragmentation of the DNA molecule based on sequence homology. This
is followed by fixation of the crossover by primer extension in a
PCR reaction. Sexual PCR mutagenesis is described publications
known in the art (see, for example, Stemmer (1994) Proc. Natl.
Acad. Sci. U.S.A. 91:10747-10751). Variants can also be created by
in vivo mutagenesis. In some embodiments, random mutations in a
nucleic acid sequence are generated by propagating the sequence in
a bacterial strain, such as an E. coli strain, which carries
mutations in one or more of the DNA repair pathways. Such mutator
strains have a higher random mutation rate than that of a wild-type
strain. Propagating a DNA sequence (e.g., a polynucleotide sequence
encoding an AAR polypeptide) in one of these strains will
eventually generate random mutations within the DNA. Mutator
strains suitable for use for in vivo mutagenesis are described in
publication in the art (see, for example, International Patent
Application Publication No. WO1991/016427). Variants can also be
generated using cassette mutagenesis. In cassette mutagenesis, a
small region of a double-stranded DNA molecule is replaced with a
synthetic oligonucleotide cassette that differs from the native
sequence. The oligonucleotide often contains a completely and/or
partially randomized native sequence. Recursive ensemble
mutagenesis can also be used to generate variants. Recursive
ensemble mutagenesis is an algorithm for protein engineering (i.e.,
protein mutagenesis) developed to produce diverse populations of
phenotypically related mutants whose members differ in amino acid
sequence. This method uses a feedback mechanism to control
successive rounds of combinatorial cassette mutagenesis (see, for
example, Arkin et al. (1992) Proc. Natl. Acad. Sci., U.S.A.
89:7811-7815). In some embodiments, variants are created using
exponential ensemble mutagenesis. Exponential ensemble mutagenesis
is a process for generating combinatorial libraries with a high
percentage of unique and functional mutants, wherein small groups
of residues are randomized in parallel to identify, at each altered
position, amino acids which lead to functional proteins (see, for
example, Delegrave et al. (1993) Biotech. Res. 11:1548-1552). In
some embodiments, variants are created using shuffling procedures
wherein portions of a plurality of nucleic acids that encode
distinct polypeptides are fused together to create chimeric nucleic
acid sequences that encode chimeric polypeptides (as described in,
for example, U.S. Pat. Nos. 5,965,408 and 5,939,250).
[0109] Production of Fatty Aldehydes and Fatty Alcohols
[0110] A native or recombinant host cell may comprise a
polynucleotide encoding an enzyme having fatty aldehyde
biosynthesis activity (also referred to herein as a fatty aldehyde
biosynthetic polypeptide or a fatty aldehyde biosynthetic
polypeptide or enzyme). A fatty aldehyde is produced when the fatty
aldehyde biosynthetic enzyme is expressed or overexpressed in the
host cell. Expression or overexpression of an acyl-ACP reductase
(AAR) polypeptide in a recombinant host cell may result in
production of a fatty aldehyde by the recombinant host cell. In one
embodiment, the recombinant host cell produces a fatty aldehyde. In
some embodiments, the fatty aldehyde produced by the recombinant
host cell is converted into a fatty alcohol. In some embodiments,
native (endogenous) fatty aldehyde biosynthetic polypeptides, such
as aldehyde reductases, are present in the host cell (e.g., E.
coli) and are effective to convert fatty aldehydes to fatty
alcohols. In other embodiments, a native (endogenous) fatty
aldehyde biosynthetic polypeptide is overexpressed. In still other
embodiments, an exogenous fatty aldehyde biosynthetic polypeptide
is introduced into a recombinant host cell and expressed or
overexpressed. A fatty aldehyde may be produced by expressing or
overexpressing in the recombinant host cell a polynucleotide
encoding a fatty aldehyde biosynthetic polypeptide, such as a
polypeptide having acyl-ACP reductase (AAR) activity. Expression of
AAR in a recombinant host cell results in the production of fatty
aldehydes and fatty alcohols (FIG. 4). Exemplary AAR polypeptides
are described herein and in PCT Publication Nos. WO2009/140695 and
WO/2009/140696, both of which are expressly incorporated by
reference herein.
[0111] A composition comprising fatty aldehydes (a fatty aldehyde
composition) is produced by culturing a host cell in the presence
of a carbon source under conditions effective to express the fatty
aldehyde biosynthetic enzyme, e.g., AAR. A recombinant host cell
engineered to produce a fatty aldehyde will typically convert some
of the fatty aldehyde to a fatty alcohol. In some embodiments, the
fatty aldehyde composition comprises fatty aldehydes and fatty
alcohols. Typically, the fatty aldehyde composition is recovered
from the extracellular environment of the recombinant host cell,
i.e., the cell culture medium. In some embodiments, the recombinant
host cell includes a polynucleotide encoding a polypeptide (an
enzyme) having fatty alcohol biosynthetic activity (also referred
to herein as a fatty alcohol biosynthetic polypeptide or a fatty
alcohol biosynthetic enzyme), and a fatty alcohol is produced by
the recombinant host cell. A composition including the fatty
alcohols (i.e., a fatty alcohol composition) may be produced by
culturing the recombinant host cell in the presence of a carbon
source under conditions effective to express a fatty alcohol
biosynthetic enzyme. Native (e.g., endogenous) aldehyde reductases
present in a recombinant host cell (e.g., E. coli), will convert
fatty aldehydes into fatty alcohols. In some embodiments, native
(e.g., endogenous) fatty aldehyde biosynthetic polypeptides, such
as aldehyde reductases present in the host cell are sufficient to
convert fatty aldehydes to fatty alcohols. However, in other
embodiments, a fatty alcohol is produced by expressing or
overexpressing in the recombinant host cell a polynucleotide
encoding a polypeptide having fatty alcohol biosynthetic activity
which converts a fatty aldehyde to a fatty alcohol. For example, an
alcohol dehydrogenase (also referred to herein as an aldehyde
reductase, e.g., EC 1.1.1.1), may be useful in practicing the
disclosure. As used herein, the term alcohol dehydrogenase refers
to a polypeptide capable of catalyzing the conversion of a fatty
aldehyde to a fatty alcohol. One of ordinary skill in the art will
appreciate that certain alcohol dehydrogenases are capable of
catalyzing other reactions as well, and these non-specific alcohol
dehydrogenases also are encompassed by the term alcohol
dehydrogenase. Examples of alcohol dehydrogenase polypeptides
useful in accordance with the disclosure include, but are not
limited to, AlrA of Acinetobacter sp. M-1 (SEQ ID NO: 52) or AlrA
homologs such as AlrAadp1 (SEQ ID NO: 53) and endogenous E. coli
alcohol dehydrogenases such as YjgB, (AAC77226) (SEQ ID NO: 5),
DkgA (NP_417485), DkgB (NP_414743), YdjL (AAC74846), YdjJ
(NP_416288), AdhP (NP_415995), YhdH (NP_417719), YahK (NP_414859),
YphC (AAC75598), YqhD (446856) and YbbO [AAC73595.1]. Additional
examples are described in International Patent Application
Publication Nos. WO2007/136762, WO2008/119082 and WO2010/062480,
each of which is expressly incorporated by reference herein. In
certain embodiments, the fatty alcohol biosynthetic polypeptide has
aldehyde reductase or alcohol dehydrogenase activity (EC 1.1.1.1).
In some embodiments, a native (e.g., endogenous) fatty alcohol
biosynthetic polypeptide is overexpressed and in other embodiments,
an exogenous fatty alcohol biosynthetic polypeptide is introduced
into a recombinant host cell and expressed or overexpressed.
[0112] Fatty alcohols may be produced via an acyl-CoA dependent
pathway utilizing fatty acyl-ACP and fatty acyl-CoA intermediates
and an acyl-CoA independent pathway utilizing fatty acyl-ACP
intermediates but not a fatty acyl-CoA intermediate. In particular
embodiments, the enzyme encoded by the over expressed gene is
selected from a fatty acid synthase, an acyl-ACP thioesterase, a
fatty acyl-CoA synthase and an acetyl-CoA carboxylase. Fatty
alcohols are also made in nature by enzymes that are able to reduce
various acyl-ACP or acyl-CoA molecules to the corresponding primary
alcohols (see U.S. Patent Publication Nos. 20100105963 and
20110206630, and U.S. Pat. No. 8,097,439, expressly incorporated by
reference herein). A fatty alcohol composition often includes fatty
alcohols together with other fatty acid derivatives, e.g., fatty
aldehydes and/or fatty acids. Typically, the fatty alcohol
composition is recovered from the extracellular environment of the
recombinant host cell, i.e., the cell culture medium. In certain
embodiments, the expression of a polypeptide, for example, an
enzyme directly or indirectly involved in fatty acid biosynthesis
is modulated (e.g., expressed, overexpressed or attenuated),
wherein such modulation results in a higher yield, higher titer or
higher productivity of a fatty acid derivative of interest, such as
a fatty alcohol. The enzyme may be encoded by a fatty acid
biosynthetic polynucleotide that is exogenous or heterologous
(e.g., a polypeptide originating from an organism other than the
parental host cell, or, a variant of a polypeptide native to the
parental microbial cell) or an endogenous polypeptide (e.g., a
polypeptide native to the parental host cell) wherein the
endogenous polypeptide is overexpressed in the recombinant host
cell. Table 1 provides a listing of exemplary proteins which can be
expressed in recombinant host cells to facilitate production of
particular fatty alcohol compositions.
TABLE-US-00001 TABLE 1 Gene Designations Gene Source Accession EC
Designation Organism Enzyme Name No. Number Exemplary Use Fatty
Acid Production Increase/Product Production Increase accA E. coli,
acetyl-CoA AAC73296, 6.4.1.2 increase Lactococci carboxylase,
NP_414727 Malonyl-CoA subunit A production (carboxyltransferase
alpha) accB E. coli, acetyl-CoA NP_417721 6.4.1.2 increase
Lactococci carboxylase, Malonyl-CoA subunit B production (BCCP:
biotin carboxyl carrier protein) accC E. coli, acetyl-CoA NP_417722
6.4.1.2, increase Lactococci carboxylase, 6.3.4.14 Malonyl-CoA
subunit C production (biotin carboxylase) accD E. coli, acetyl-CoA
NP_416819 6.4.1.2 increase Lactococci carboxylase, Malonyl-CoA
subunit D production (carboxyltransferase beta) fadD E. coli
acyl-CoA AP_002424 2.3.1.86, increase W3110 synthase 6.2.1.3 Fatty
acid production fabA E. coli K12 .beta.-hydroxydecanoylthio-
NP_415474 4.2.1.60 increase fatty esterdehydratase/ acyl-ACP/CoA
isomerase production fabB E. coli 3-oxoacyl-[acyl-carrier-protein]
BAA16180 2.3.1.41 increase fatty synthase I acyl-ACP/CoA production
fabD E. coli K12 [acyl-carrier-protein] AAC74176 2.3.1.39 increase
fatty S-malonyltransferase acyl-ACP/CoA production fabF E. coli K12
3-oxoacyl-[acyl-carrier-protein] AAC74179 2.3.1.179 increase fatty
synthase II acyl-ACP/CoA production fabG E. coli K12
3-oxoacyl-[acyl-carrier AAC74177 1.1.1.100 increase fatty protein]
reductase acyl-ACP/CoA production fabH E. coli K12
3-oxoacyl-[acyl-carrier-protein] AAC74175 2.3.1.180 increase fatty
synthase III acyl-ACP/CoA production fabI E. coli K12
enoyl-[acyl-carrier- NP_415804 1.3.1.9 increase fatty protein]
reductase acyl-ACP/CoA production fabR E. coli K12 transcriptional
NP_418398 none modulate Repressor unsaturated fatty acid production
fabV Vibrio enoyl-[acyl-carrier-protein] YP_001217283 1.3.1.9
increase fatty cholerae reductase acyl-ACP/CoA production fabZ E.
coli K12 (3R)-hydroxymyristol NP_414722 4.2.1.- increase fatty acyl
carrier protein acyl-ACP/CoA dehydratase production fadE E. coli
K13 acyl-CoA AAC73325 1.3.99.3, reduce fatty acid dehydrogenase
1.3.99.- degradation fadR E. coli transcriptional NP_415705 none
Block or reverse regulatory fatty acid protein degradation Chain
Length Control tesA (with or E. coli thioesterase-leader P0ADA1
3.1.2.-, C18 without sequence is amino 3.1.1.5 Chain Length leader
acids 1-26 sequence) tesA E. coli thioesterase AAC73596, 3.1.2.-,
C18:1 (without NP_415027 3.1.1.5 Chain Length leader sequence) tesA
(mutant E. coli thioesterase L109P 3.1.2.-, <C18 of E. coli
3.1.1.5 Chain Length thioesterase I complexed with octanoic acid)
fatB1 Umbellularia thioesterase Q41635 3.1.2.14 C12:0 californica
Chain Length fatB2 Cuphea thioesterase AAC49269 3.1.2.14 C8:0-C10:0
hookeriana Chain Length fatB3 Cuphea thioesterase AAC72881 3.1.2.14
C14:0-C16:0 hookeriana Chain Length fatB Cinnamomum thioesterase
Q39473 3.1.2.14 C14:0 camphora Chain Length fatB Arabidopsis
thioesterase CAA85388 3.1.2.14 C16:1 thaliana Chain Length fatA1
Helianthus thioesterase AAL79361 3.1.2.14 C18:1 annuus Chain Length
atfata Arabidopsis thioesterase NP_189147, 3.1.2.14 C18:1 thaliana
NP_193041 Chain Length fatA Brassica thioesterase CAC39106 3.1.2.14
C18:1 juncea Chain Length fatA Cuphea thioesterase AAC72883
3.1.2.14 C18:1 hookeriana Chain Length tesA Photbacterium
thioesterase YP_130990 3.1.2.14 Chain Length profundum tesB E. coli
thioesterase NP_414986 3.1.2.14 Chain Length fadM E. coli
thioesterase NP_414977 3.1.2.14 Chain Length yciA E. coli
thioesterase NP_415769 3.1.2.14 Chain Length ybgC E. coli
thioesterase NP_415264 3.1.2.14 Chain Length Saturation Level
Control* Sfa E. coli suppressor AAN79592, none increase of fabA
AAC44390 monoun- saturated fatty acids fabA E. coli K12
.beta.-hydroxydecanoylthio- NP_415474 4.2.1.60 produce
esterdehydratase/ unsaturated isomerase fatty acids GnsA E. coli
suppressors ABD18647.1 none increase of the secG unsaturated null
mutation fatty acid esters GnsB E. coli suppressors AAC74076.1 none
increase of the secG unsaturated null mutation fatty acid esters
fabB E. coli 3-oxoacyl-[acyl-carrier-protein] BAA16180 2.3.1.41
modulate synthase I unsaturated fatty acid production des Bacillus
D5 fatty acyl O34653 1.14.19 modulate subtilis desaturase
unsaturated fatty acid production Product Output: Ester Production
AT3G51970 Arabidopsis long-chain-alcohol NP_190765 2.3.1.26 wax
thaliana O-fatty-acyltransferase production ELO1 Pichia fatty acid
BAD98251 2.3.1.- produce angusta elongase very long chain length
fatty acids plsC Saccharomyces acyltransferase AAA16514 2.3.1.51
wax cerevisiae production DAGAT/ Arabidopsis diacylglycerolacyl-
AAF19262 2.3.1.20 wax DGAT thaliana transferase production hWS Homo
acyl-CoA AAX48018 2.3.1.20 wax sapiens wax alcohol production
acyltransferase aft1 Acinetobacter bifunctional AAO17391 2.3.1.20
wax sp. ADP1 wax ester production synthase/acyl-CoA:diacyl-
glycerolacyltransferase ES9 Marinobacter wax ester synthase
ABO21021 2.3.1.20 wax hydrocarbonoclasticus production mWS
Simmondsia wax ester synthase AAD38041 2.3.1.- wax chinensis
production acr1 Acinetobacter acyl-CoA reductase YP_047869 1.2.1.42
modify output sp. ADP1 yqhD E. Coli K12 alcohol dehydrogenase
AP_003562 1.1.-.- modify output AAT Fragaria x alcohol AAG13130
2.3.1.84 modify output ananassa O-acetyltransferase Product Output:
Fatty Alcohol Output thioesterases increase (see above) fatty acid/
fatty alcohol production BmFAR Bombyx mori FAR (fatty alcohol
BAC79425 1.1.1.- convert forming acyl-CoA acyl-CoA to reductase)
fatty alcohol acr1 Acinetobacter acyl-CoA reductase YP_047869
1.2.1.42 reduce fatty sp. ADP1 acyl-CoA to fatty aldehydes yqhD E.
coli W3110 alcohol dehydrogenase AP_003562 1.1.-.- reduce fatty
aldehydes to fatty alcohols; increase fatty alcohol production alrA
Acinetobacter alcohol dehydrogenase CAG70252 1.1.-.- reduce fatty
sp. ADP1 aldehydes to fatty alcohols BmFAR Bombyx mori FAR (fatty
alcohol BAC79425 1.1.1.- reduce fatty forming acyl-CoA acyl-CoA to
reductase) fatty alcohol GTNG_1865 Geobacillus long-chain aldehyde
YP_001125970 1.2.1.3 reduce fatty thermodenitrificans dehydrogenase
aldehydes to NG80-2 fatty alcohols AAR Synechococcus acyl-ACP
reductase YP_400611 1.2.1.80 reduce fatty elongatus 1.2.1.42
acyl-ACP/CoA to fatty aldehydes carB Mycobacterium carboxylic acid
YP_889972 6.2.1.3, reduce fatty smegmatis reductase (CAR) 1.2.1.42
acids to protein fatty aldehyde FadD E. coli K12 acyl-CoA
synthetase NP_416319 6.2.1.3 activates fatty acids to fatty
acyl-CoAs atoB Erwinia acetyl-CoA YP_049388 2.3.1.9 production of
carotovora acetyltransferase butanol hbd Butyrivibrio
beta-hydroxybutyryl-CoA BAD51424 1.1.1.157 production of
fibrisolvens dehydrogenase butanol CPE0095 Clostridium
crotonasebutyryl-CoA BAB79801 4.2.1.55 production of perfringens
dehydryogenase butanol bcd Clostridium butyryl-CoA AAM14583
1.3.99.2 production of beijerinckii dehydryogenase butanol ALDH
Clostridium coenzyme A-acylating AAT66436 1.2.1.3 production of
beijerinckii aldehyde butanol dehydrogenase AdhE E. coli
aldehyde-alcohol AAN80172 1.1.1.1 production of CFT073
dehydrogenase 1.2.1.10 butanol Product Export AtMRP5 Arabidopsis
Arabidopsis thaliana NP_171908 none modify product thaliana
multidrug resistance- export amount associated AmiS2 Rhodococcus
ABC transporter JC5491 none modify product sp. AmiS2 export amount
AtPGP1 Arabidopsis Arabidopsis NP_181228 none modify product
thaliana thaliana p export amount glycoprotein 1 AcrA Candidatus
putative multidrug- CAF23274 none modify product Protochlamydia
efflux transport protein export amount amoebophila acrA UWE25 AcrB
Candidatus probable CAF23275 none modify product Protochlamydia
multidrug- export amount amoebophila efflux transport UWE25
protein, acrB TolC Francisella outer membrane ABD59001 none modify
product tularensis protein export amount subsp. [Cell envelope
novicida biogenesis, AcrE Shigella transmembrane protein YP_312213
none modify product sonnei affects septum export amount Ss046
formation and cell membrane permeability AcrF E. coli acriflavine
resistance P24181 none modify product protein F export amount
tll1619 Thermosynechococcus multidrug efflux NP_682409.1 none
modify product elongatus transporter export amount [BP-1] tll0139
Thermosynechococcus multidrug efflux NP_680930.1 none modify
product elongatus transporter export amount [BP-1] Fermentation
replication increase output checkpoint efficiency genes
umuD Shigella sonnei DNA polymerase V, YP_310132 3.4.21.- increase
output Ss046 subunit efficiency umuC E. coli DNA polymerase V,
ABC42261 2.7.7.7 increase output subunit efficiency pntA, pntB
Shigella NADH:NADPH P07001, 1.6.1.2 increase output flexneri
transhydrogenase P0AB70 efficiency (alpha and beta subunits) Other
fabK Streptococcus trans-2-enoyl-ACP AAF98273 1.3.1.9 Contributes
pneumoniae reductase II to fatty acid biosynthesis fabL Bacillus
enoyl-(acyl carrier AAU39821 1.3.1.9 Contributes licheniformis
protein) reductase to fatty acid DSM 13 biosynthesis fabM
Streptococcus trans-2, DAA05501 4.2.1.17 Contributes mutans
cis-3-decenoyl-ACP to fatty acid isomerase biosynthesis
[0113] Recombinant Host Cells and Cell Cultures
[0114] Strategies to increase production of fatty aldehyde or fatty
alcohol compositions by recombinant host cells include increased
flux through the fatty acid biosynthetic pathway by overexpression
of native fatty aldehyde or fatty alcohol biosynthetic genes and
expression of exogenous fatty aldehyde and fatty alcohol
biosynthetic genes from different organisms in the production host.
As used herein, the term recombinant host cell or engineered host
cell refers to a host cell whose genetic makeup has been altered
relative to the corresponding wild-type host cell, for example, by
deliberate introduction of new genetic elements and/or deliberate
modification of genetic elements naturally present in the host
cell. The offspring of such recombinant host cells also contain
these new and/or modified genetic elements. In any of the aspects
of the disclosure described herein, the host cell can be selected
from a plant cell, insect cell, fungus cell (e.g., a filamentous
fungus, such as Candida sp., or a budding yeast, such as
Saccharomyces sp.), an algal cell and a bacterial cell. In one
preferred embodiment, recombinant host cells are recombinant
microorganisms. Examples of host cells that are microorganisms
include, but are not limited to, cells from the genus Escherichia,
Bacillus, Lactobacillus, Zymomonas, Rhodococcus, Pseudomonas,
Aspergillus, Trichoderma, Neurospora, Fusarium, Humicola,
Rhizomucor, Kluyveromyces, Pichia, Mucor, Myceliophtora,
Penicillium, Phanerochaete, Pleurotus, Trametes, Chrysosporium,
Saccharomyces, Stenotrophamonas, Schizosaccharomyces, Yarrowia, or
Streptomyces. In some embodiments, the host cell is a Gram-positive
bacterial cell. In other embodiments, the host cell is a
Gram-negative bacterial cell. In some embodiments, the host cell is
an E. coli cell. In other embodiments, the host cell is a Bacillus
lentus cell, a Bacillus brevis cell, a Bacillus stearothermophilus
cell, a Bacillus licheniformis cell, a Bacillus alkalophilus cell,
a Bacillus coagulans cell, a Bacillus circulans cell, a Bacillus
pumilis cell, a Bacillus thuringiensis cell, a Bacillus clausii
cell, a Bacillus megaterium cell, a Bacillus subtilis cell, or a
Bacillus amyloliquefaciens cell. In other embodiments, the host
cell is a Trichoderma koningii cell, a Trichoderma viride cell, a
Trichoderma reesei cell, a Trichoderma longibrachiatum cell, an
Aspergillus awamori cell, an Aspergillus fumigates cell, an
Aspergillus foetidus cell, an Aspergillus nidulans cell, an
Aspergillus niger cell, an Aspergillus oryzae cell, a Humicola
insolens cell, a Humicola lanuginose cell, a Rhodococcusopacus
cell, a Rhizomucormiehei cell, or a Mucormichei cell. In yet other
embodiments, the host cell is a Streptomyces lividans cell or a
Streptomyces murinus cell. In yet other embodiments, the host cell
is an Actinomycetes cell. In some embodiments, the host cell is a
Saccharomyces cerevisiae cell.
[0115] In other embodiments, the host cell is a eukaryotic plant
cell, an alga cell, a cyanobacterium cell, a green-sulfur bacterium
cell, a green non-sulfur bacterium cell, a purple sulfur bacterium
cell, a purple non-sulfur bacterium cell, an extremophile cell, a
yeast cell, a fungus cell, an engineered cell of any of species
described herein, or a synthetic organism. In some embodiments, the
host cell is light-dependent or fixes carbon. In some embodiments,
the host cell has autotrophic activity. In some embodiments, the
host cell has photoautotrophic activity, such as in the presence of
light. In some embodiments, the host cell is heterotrophic or
mixotrophic in the absence of light. In certain embodiments, the
host cell is a cell from Arabidopsis thaliana, Panicum virgatum,
Miscanthus giganteus, Zea mays, Botryococcuse braunii,
Chlarnydomonas reinhardtii, Dunaliela salina, Synechococcus Sp. PCC
7002, Synechococcus Sp. PCC 7942, Synechocystis Sp. PCC 6803,
Thermosynechococcus elongates BP-1, Chlorobium tepidum,
Chlorojlexus auranticus, Chromatiumm vinosum, Rhodospirillum
rubrum, Rhodobacter capsulatus, Rhodopseudomonas palusris,
Clostridium ljungdahlii, Clostridium thermocellum, Penicillium
chrysogenum, Pichia pastoris, Saccharomyces cerevisiae,
Schizosaccharomyces pombe, Pseudomonas fluorescens, or Zymomonas
mobilis.
[0116] Engineering Host Cells
[0117] In some embodiments, a polynucleotide (or gene) sequence is
provided to the host cell by way of a recombinant vector, which
includes a promoter operably linked to the polynucleotide sequence.
In certain embodiments, the promoter is a
developmentally-regulated, an organelle-specific, a
tissue-specific, an inducible, a constitutive, or a cell-specific
promoter. In some embodiments, the recombinant vector includes at
least one sequence selected from an expression control sequence
operatively coupled to the polynucleotide sequence; a selection
marker operatively coupled to the polynucleotide sequence; a marker
sequence operatively coupled to the polynucleotide sequence; a
purification moiety operatively coupled to the polynucleotide
sequence; a secretion sequence operatively coupled to the
polynucleotide sequence; and a targeting sequence operatively
coupled to the polynucleotide sequence. The expression vectors
described herein include a polynucleotide sequence in a form
suitable for expression of the polynucleotide sequence in a host
cell. It will be appreciated by those skilled in the art that the
design of the expression vector can depend on such factors as the
choice of the host cell to be transformed, the level of expression
of polypeptide desired, and the like. The expression vectors
described herein can be introduced into host cells to produce
polypeptides, including fusion polypeptides, encoded by the
polynucleotide sequences as described above (supra). Expression of
genes encoding polypeptides in prokaryotes, for example, E. coli,
is most often carried out with vectors containing constitutive or
inducible promoters directing the expression of either fusion or
non-fusion polypeptides. Fusion vectors add a number of amino acids
to a polypeptide encoded therein, usually to the amino- or
carboxy-terminus of the recombinant polypeptide. Such fusion
vectors typically serve one or more of the following three purposes
including to increase expression of the recombinant polypeptide; to
increase the solubility of the recombinant polypeptide; and to aid
in the purification of the recombinant polypeptide by acting as a
ligand in affinity purification. Often, in fusion expression
vectors, a proteolytic cleavage site is introduced at the junction
of the fusion moiety and the recombinant polypeptide. This allows
separation of the recombinant polypeptide from the fusion moiety
after purification of the fusion polypeptide. Examples of such
enzymes, and their cognate recognition sequences, include Factor
Xa, thrombin, and enterokinase. Exemplary fusion expression vectors
include pGEX vector (Pharmacia Biotech, Inc., Piscataway, N.J.;
Smith et al. (1988) Gene 67:31-40), pMAL vector (New England
Biolabs, Beverly, Mass.), and pRITS vector (Pharmacia Biotech,
Inc., Piscataway, N.J.), which fuse glutathione S-transferase
(GST), maltose E binding protein, or protein A, respectively, to
the target recombinant polypeptide.
[0118] Examples of inducible, non-fusion E. coli expression vectors
include pTrc vector (Amann et al. (1988) Gene 69:301-315) and pET
11d vector (Studier et al., Gene Expression Technology: Methods in
Enzymology 185, Academic Press, San Diego, Calif. (1990) 60-89).
Target gene expression from the pTrc vector relies on host RNA
polymerase transcription from a hybrid trp-lac fusion promoter.
Target gene expression from the pET 11d vector relies on
transcription from a T7 gn10-lac fusion promoter mediated by a
coexpressed viral RNA polymerase (T7 gn1). This viral polymerase is
supplied by host strains such as BL21(DE3) or HMS174(DE3) from a
resident .lamda. prophage harboring a T7 gn1 gene under the
transcriptional control of the lacUV 5 promoter. Suitable
expression systems for both prokaryotic and eukaryotic cells are
well known in the art (see, e.g., Sambrook et al. (1989) Molecular
Cloning: A Laboratory Manual, second edition, Cold Spring Harbor
Laboratory). Examples of inducible, non-fusion E. coli expression
vectors include pTrc vector (Amann et al. (1988) Gene 69:301-315)
and PET 11d vector (Studier et al. (1990) Gene Expression
Technology: Methods in Enzymology 185, Academic Press, San Diego,
Calif., pp. 60-89). In certain embodiments, a polynucleotide
sequence of the disclosure is operably linked to a promoter derived
from bacteriophage T5. In one embodiment, the host cell is a yeast
cell. In this embodiment, the expression vector is a yeast
expression vector. Vectors can be introduced into prokaryotic or
eukaryotic cells via a variety of art-recognized techniques for
introducing foreign nucleic acid (e.g., DNA) into a host cell.
Suitable methods for transforming or transfecting host cells can be
found in, for example, Sambrook et al. (supra). For stable
transformation of bacterial cells, it is known that (depending upon
the expression vector and transformation technique used) a certain
fraction of cells will take-up and replicate the expression vector.
In order to identify and select these transformants, a gene that
encodes a selectable marker (e.g., resistance to an antibiotic) can
be introduced into the host cells along with the gene of interest.
Selectable markers include those that confer resistance to drugs
such as, but not limited to, ampicillin, kanamycin,
chloramphenicol, or tetracycline. Nucleic acids encoding a
selectable marker can be introduced into a host cell on the same
vector as that encoding a polypeptide described herein or can be
introduced on a separate vector. Cells stably transformed with the
introduced nucleic acid can be identified by growth in the presence
of an appropriate selection drug. The engineered or recombinant
host cell as described herein (supra) is a cell used to produce a
fatty acid derivative composition such as a fatty aldehyde or a
fatty alcohol. In any of the aspects of the disclosure described
herein, the host cell can be selected from a eukaryotic plant,
bacteria, algae, cyanobacterium, green-sulfur bacterium, green
non-sulfur bacterium, purple sulfur bacterium, purple non-sulfur
bacterium, extremophile, yeast, fungus, engineered organisms
thereof, or a synthetic organism. In some embodiments, the host
cell is light dependent or fixes carbon. In some embodiments, the
host cell has autotrophic activity. Various host cells can be used
to produce fatty acid derivatives, as described herein.
[0119] The host cells or microorganisms of the disclosure include
host strains or host cells that are genetically engineered or
modified to contain alterations in order to test the efficiency of
specific mutations on enzymatic activities (i.e., recombinant cells
or microorganisms). Various optional genetic manipulations and
alterations can be used interchangeably from one host cell to
another, depending on what native enzymatic pathways are present in
the original host cell. In one embodiment, a host strain can be
used for testing the expression of an AAR polypeptide in
combination with other biosynthetic polypeptides (e.g., enzymes). A
host strain may encompasses a number of genetic alterations in
order to test specific variables, including but not limited to,
culture conditions including fermentation components, carbon source
(e.g., feedstock), temperature, pressure, reduced culture
contamination conditions, and oxygen levels.
[0120] In one embodiment, a host strain encompasses an optional
fadE and fhuA deletion. Acyl-CoA dehydrogenase (FadE) is an enzyme
that is important for metabolizing fatty acids. It catalyzes the
second step in fatty acid utilization (beta-oxidation), which is
the process of breaking long chains of fatty acids (acyl-CoAs) into
acetyl-CoA molecules. More specifically, the second step of the
.beta.-oxidation cycle of fatty acid degradation in bacteria is the
oxidation of acyl-CoA to 2-enoyl-CoA, which is catalyzed by FadE.
When E. coli lacks FadE, it cannot grow on fatty acids as a carbon
source but it can grow on acetate. The inability to utilize fatty
acids of any chain length is consistent with the reported phenotype
of fadE strains, i.e., fadE mutant strains where FadE function is
disrupted. The fadE gene can be optionally knocked out or
attenuated to assure that acyl-CoAs, which may be intermediates in
a fatty acid derivative pathway, can accumulate in the cell such
that all acyl-CoAs can be efficiently converted to fatty acid
derivatives. However, fadE attenuation is optional when sugar is
used as a carbon source since under such condition expression of
FadE is likely repressed and FadE therefore may only be present in
small amounts and not able to efficiently compete with ester
synthase or other enzymes for acyl-CoA substrates. FadE is
repressed due to catabolite repression. E. coli and many other
microbes prefer to consume sugar over fatty acids, so when both
sources are available sugar is consumed first by repressing the fad
regulon (see D. Clark, J Bacteriol. (1981) 148(2):521-6)).
Moreover, the absence of sugars and the presence of fatty acids
induces FadE expression. Acyl-CoA intermediates could be lost to
the beta oxidation pathway since the proteins expressed by the fad
regulon (including FadE) are up-regulated and will efficiently
compete for acyl-CoAs. Thus, it can be beneficial to have the fadE
gene knocked out or attenuated. Since most carbon sources are
mainly sugar based, it is optional to attenuate FadE. The gene fhuA
codes for the TonA protein, which is an energy-coupled transporter
and receptor in the outer membrane of E. coli (V. Braun (2009) J
Bacteria 191(11):3431-3436). Its deletion is optional. The fhuA
deletion allows the cell to become more resistant to phage attack
which can be beneficial in certain fermentation conditions. Thus,
it may be desirable to delete fhuA in a host cell that is likely
subject to potential contamination during fermentation runs.
[0121] In another embodiment, the host strain (supra) also
encompasses optional overexpression of one or more of the following
genes including fadR, fabA, fabD, fabG, fabH, fabV, and/or fabF.
Examples of such genes are fadR from Escherichia coli, fabA from
Salmonella typhimurium (NP_460041), fabD from Salmonella
typhimurium (NP_460164), fabG from Salmonella typhimurium
(NP_460165), fabH from Salmonella typhimurium (NP_460163), fabV
from Vibrio cholera (YP_001217283), and fabF from Clostridium
acetobutylicum (NP_350156). The overexpression of one or more of
these genes, which code for enzymes and regulators in fatty acid
biosynthesis, can serve to increase the titer of fatty-acid
derivative compounds including fatty aldehydes and fatty alcohols
under various culture conditions.
[0122] In another embodiment, E. coli strains are used as host
cells for the production of fatty acid derivatives such a fatty
aldehydes and/or fatty alcohols. Similarly, these host cells
provide optional overexpression of one or more biosynthesis genes
(i.e., genes coding for enzymes and regulators of fatty acid
biosynthesis) that can further increase or enhance the titer of
fatty-acid derivative compounds such as fatty acid derivatives
(e.g., fatty alcohols, fatty aldehydes, etc.) under various culture
conditions including, but not limited to, fadR, fabA, fabD, fabG,
fabH, fabV and/or fabF. Examples of genetic alterations include
fadR from Escherichia coli, fabA from Salmonella typhimurium
(NP_460041), fabD from Salmonella typhimurium (NP_460164), fabG
from Salmonella typhimurium (NP_460165), fabH from Salmonella
typhimurium (NP_460163), fabV from Vibrio cholera (YP_001217283),
and fabF from Clostridium acetobutylicum (NP_350156). In some
embodiments, synthetic operons that carry these biosynthetic genes
can be engineered and expressed in cells in order to test fatty
aldehyde and/or fatty alcohol overexpression under various culture
conditions and/or further enhance fatty aldehyde and/or fatty
alcohol production. Such synthetic operons contain one or more
biosynthetic gene. The ifab138 operon, for example, is an
engineered operon that contains optional fatty acid biosynthetic
genes, including fabV from Vibrio cholera, fabH from Salmonella
typhimurium, fabD from S. typhimurium, fabG from S. typhimurium,
fabA from S. typhimurium and/or fabF from Clostridium
acetobutylicum that can be used to facilitate overexpression of
fatty acid derivatives in order to test specific culture
conditions. One advantage of such synthetic operons is that the
rate of fatty acid derivative production can be further increased
or enhanced.
[0123] In some embodiments, the host cells or microorganisms that
are used to express ACP and biosynthetic enzymes (e.g., TE, ES,
CAR, AAR, ADC, etc.) will further express genes that encompass
certain enzymatic activities that can increase the production to
one or more particular fatty acid derivative(s) such as fatty
alcohols, fatty aldehydes, fatty esters, fatty amines, bifunctional
fatty acid derivatives, diacids and the like. In one embodiment,
the host cell has thioesterase activity (E.C. 3.1.2.* or E.C. 3.1.
2.14 or E.C. 3.1.1.5) for the production of fatty acids which can
be increased by overexpressing the gene. In another embodiment, the
host cell has ester synthase activity (E.C. 2.3.1.75) for the
production of fatty esters. In another embodiment, the host cell
has acyl-ACP reductase (AAR) (E.C. 1.2.1.80) activity and/or
alcohol dehydrogenase activity (E.C. 1.1.1.1.) and/or fatty alcohol
acyl-CoA reductase (FAR) (E.C. 1.1.1.*) activity and/or carboxylic
acid reductase (CAR) (EC 1.2.99.6) activity for the production of
fatty alcohols. In another embodiment, the host cell has acyl-ACP
reductase (AAR) (E.C. 1.2.1.80) activity for the production of
fatty aldehydes. In another embodiment, the host cell has acyl-ACP
reductase (AAR) (E.C. 1.2.1.80) activity and decarbonylase (ADC)
activity for the production of alkanes and alkenes. In another
embodiment, the host cell has acyl-CoA reductase (E.C. 1.2.1.50)
activity, acyl-CoA synthase (FadD) (E.C. 2.3.1.86) activity, and
thioesterase (E.C. 3.1.2.* or E.C. 3.1. 2.14 or E.C. 3.1.1.5)
activity for the production of fatty alcohols. In another
embodiment, the host cell has ester synthase activity (E.C.
2.3.1.75), acyl-CoA synthase (FadD) (E.C. 2.3.1.86) activity, and
thioesterase (E.C. 3.1.2.* or E.C. 3.1. 2.14 or E.C. 3.1.1.5)
activity for the production of fatty esters. In another embodiment,
the host cell has OleA activity for the production of ketones. In
another embodiment, the host cell has OleBCD activity for the
production of internal olefins. In another embodiment, the host
cell has acyl-ACP reductase (AAR) (E.C. 1.2.1.80) activity and
alcohol dehydrogenase activity (E.C. 1.1.1.1.) for the production
of fatty alcohols. In another embodiment, the host cell has
thioesterase (E.C. 3.1.2.* or E.C. 3.1. 2.14 or E.C. 3.1.1.5)
activity and decarboxylase activity for making terminal olefins.
The expression of enzymatic activities in microorganisms and
microbial cells is taught by U.S. Pat. Nos. 8,097,439; 8,110,093;
8,110,670; 8,183,028; 8,268,599; 8,283,143; 8,232,924; 8,372,610;
and 8,530,221, which are incorporated herein by reference. In other
embodiments, the host cells or microorganisms that are used to
express ACP and other biosynthetic enzymes will include certain
native enzyme activities that are upregulated or overexpressed in
order to produce one or more particular fatty acid derivative(s)
such as fatty aldehydes and/or fatty alcohols. In one embodiment,
the host cell has a native thioesterase (E.C. 3.1.2.* or E.C. 3.1.
2.14 or E.C. 3.1.1.5) activity for the production of fatty acids
which can be increased by overexpressing the thioesterase gene.
[0124] The present disclosure includes host strains or
microorganisms that express genes that code for AAR and other
biosynthetic enzymes (supra). The recombinant host cells produce
fatty acid derivatives such as fatty aldehydes and fatty alcohols
and compositions and blends thereof. The fatty acid derivatives are
typically recovered from the culture medium and/or are isolated
from the host cells. In one embodiment, the fatty acid derivatives
such as fatty aldehydes and fatty alcohols are recovered from the
culture medium (extracellular). In another embodiment, the fatty
acid derivatives such as fatty aldehydes and fatty alcohols are
isolated from the host cells (intracellular). In another
embodiment, the fatty acid derivatives such as fatty aldehydes and
fatty alcohols are recovered from the culture medium and isolated
from the host cells. The fatty acid derivatives composition
produced by a host cell can be analyzed using methods known in the
art, for example, GC-FID, in order to determine the distribution of
particular fatty acid derivatives as well as chain lengths and
degree of saturation of the components of the fatty acid derivative
composition such as fatty aldehyde and fatty alcohol
compositions.
[0125] Examples of host cells that function as microorganisms
(e.g., microbial cells), include but are not limited to cells from
the genus Escherichia, Bacillus, Lactobacillus, Zymomonas,
Rhodococcus, Pseudomonas, Aspergillus, Trichoderma, Neurospora,
Fusarium, Humicola, Rhizomucor, Kluyveromyces, Pichia, Mucor,
Myceliophtora, Penicillium, Phanerochaete, Pleurotus, Trametes,
Chrysosporium, Saccharomyces, Stenotrophamonas,
Schizosaccharomyces, Yarrowia, or Streptomyces. In some
embodiments, the host cell is a Gram-positive bacterial cell. In
other embodiments, the host cell is a Gram-negative bacterial cell.
In some embodiments, the host cell is an E. coli cell. In some
embodiment, the host cell is an E. coli B cell, an E. coli C cell,
an E. coli K cell, or an E. coli W cell. In other embodiments, the
host cell is a Bacillus lentus cell, a Bacillus brevis cell, a
Bacillus stearothermophilus cell, a Bacillus licheniformis cell, a
Bacillus alkalophilus cell, a Bacillus coagulans cell, a Bacillus
circulans cell, a Bacillus pumilis cell, a Bacillus thuringiensis
cell, a Bacillus clausii cell, a Bacillus megaterium cell, a
Bacillus subtilis cell, or a Bacillus amyloliquefaciens cell. In
still other embodiments, the host cell is a Trichoderma koningii
cell, a Trichoderma viride cell, a Trichoderma reesei cell, a
Trichoderma longibrachiatum cell, an Aspergillus awamori cell, an
Aspergillus fumigates cell, an Aspergillus foetidus cell, an
Aspergillus nidulans cell, an Aspergillus niger cell, an
Aspergillus oryzae cell, a Humicola insolens cell, a Humicola
lanuginose cell, a Rhodococcus opacus cell, a Rhizomucor miehei
cell, or a Mucor michei cell. In yet other embodiments, the host
cell is a Streptomyces lividans cell or a Streptomyces murinus
cell. In yet other embodiments, the host cell is an Actinomycetes
cell. In some embodiments, the host cell is a Saccharomyces
cerevisiae cell. In other embodiments, the host cell is a cell from
a eukaryotic plant, algae, cyanobacterium, green-sulfur bacterium,
green non-sulfur bacterium, purple sulfur bacterium, purple
non-sulfur bacterium, extremophile, yeast, fungus, an engineered
organism thereof, or a synthetic organism. In some embodiments, the
host cell is light-dependent or fixes carbon. In some embodiments,
the host cell has autotrophic activity. In some embodiments, the
host cell has photoautotrophic activity, such as in the presence of
light. In some embodiments, the host cell is heterotrophic or
mixotrophic in the absence of light. In certain embodiments, the
host cell is a cell from Arabidopsis thaliana, Panicum virgatum,
Miscanthus giganteus, Zea mays, Botryococcuse braunii,
Chlamydomonas reinhardtii, Dunaliela salina, Synechococcus Sp. FCC
7002, Synechococcus Sp. PCC 7942, Synechocystis Sp. PCC 6803,
Thermosynechococcus elongates BP-1, Chlorobium tepidum,
Chlorojlexus auranticus, Chromatiumm vinosum, Rhodospirillum
rubrum, Rhodobacter capsulatus, Rhodopseudomonas palusris,
Clostridium ljungdahlii, Clostridium thermocellum, Penicillium
chrysogenum, Pichia pastoris, Saccharomyces cerevisiae,
Schizosaccharomyces pombe, Pseudomonas fluorescens, or Zymomonas
mobilis. In one particular embodiment, the microbial cell is from a
cyanobacteria including, but not limited to, Prochlorococcus,
Synechococcus, Synechocystis, Cyanothece, and Nostoc punctiforme.
In another embodiment, the microbial cell is from a specific
cyanobacterial species including, but not limited to, Synechococcus
elongatus PCC7942, Synechocystis sp. PCC6803, and Synechococcus sp.
PCC7001.
[0126] Culture of Recombinant Host Cells and Fermentation
[0127] As used herein, the term fermentation broadly refers to the
conversion of organic materials into target substances by host
cells, for example, the conversion of a carbon source by
recombinant host cells into fatty acids or derivatives thereof by
propagating a culture of the recombinant host cells in a media
comprising the carbon source. The conditions permissive for the
production refer to any conditions that allow a host cell to
produce a desired product, such as a fatty aldehyde or a fatty
alcohol. Similarly, the condition or conditions in which the
polynucleotide sequence of a vector is expressed means any
conditions that allow a host cell to synthesize a polypeptide.
Suitable conditions include, for example, fermentation conditions.
Fermentation conditions can include many parameters including, but
not limited to, temperature ranges, levels of aeration, feed rates
and media composition. Each of these conditions, individually and
in combination, allows the host cell to grow. Fermentation can be
aerobic, anaerobic, or variations thereof (such as micro-aerobic).
Exemplary culture media include broths or gels. Generally, the
medium includes a carbon source that can be metabolized by a host
cell directly. In addition, enzymes can be used in the medium to
facilitate the mobilization (e.g., the depolymerization of starch
or cellulose to fermentable sugars) and subsequent metabolism of
the carbon source.
[0128] For small scale production, the engineered host cells can be
grown in batches of, for example, about 100 .mu.L, 200 .mu.L, 300
.mu.L, 400 .mu.L, 500 .mu.L, 1 mL, 5 mL, 10 mL, 15 mL, 25 mL, 50
mL, 75 mL, 100 mL, 500 mL, 1 L, 2 L, 5 L, or 10 L; fermented; and
induced to express a desired polynucleotide sequence, such as a
polynucleotide sequence encoding an ACP and/or biosynthetic
polypeptide. For large scale production, the engineered host cells
can be grown in batches of about 10 L, 100 L, 1000 L, 10,000 L,
100,000 L, and 1,000,000 L or larger; fermented; and induced to
express a desired polynucleotide sequence. Alternatively, large
scale fed-batch fermentation may be carried out. The fatty acid
derivative compositions described herein are found in the
extracellular environment of the recombinant host cell culture and
can be readily isolated from the culture medium. A fatty acid
derivative such as a fatty aldehyde or fatty alcohol may be
secreted by the recombinant host cell, transported into the
extracellular environment or passively transferred into the
extracellular environment of the recombinant host cell culture. The
fatty acid derivative is isolated from a recombinant host cell
culture using routine methods known in the art.
[0129] Products Derived from Recombinant Host Cells
[0130] As used herein, the fraction of modern carbon or fM has the
same meaning as defined by National Institute of Standards and
Technology (NIST) Standard Reference Materials (SRMs4990B and
4990C, known as oxalic acids standards HOxI and HOxII,
respectively. The fundamental definition relates to 0.95 times the
.sup.14C/.sup.12C isotope ratio HOxI (referenced to AD 1950). This
is roughly equivalent to decay-corrected pre-Industrial Revolution
wood. For the current living biosphere (plant material), fM is
approximately 1.1. Bioproducts (e.g., the fatty acid derivatives
including fatty aldehydes and/or fatty alcohols produced in
accordance with the present disclosure) include biologically
produced organic compounds. In particular, the fatty acid
derivatives produced using the fatty acid biosynthetic pathway
herein, have not been produced from renewable sources and, as such,
are new compositions of matter. These new bioproducts can be
distinguished from organic compounds derived from petrochemical
carbon on the basis of dual carbon-isotopic fingerprinting or
.sup.14C dating. Additionally, the specific source of biosourced
carbon (e.g., glucose vs. glycerol) can be determined by dual
carbon-isotopic fingerprinting (see, e.g., U.S. Pat. No.
7,169,588). The ability to distinguish bioproducts from petroleum
based organic compounds is beneficial in tracking these materials
in commerce. For example, organic compounds or chemicals including
both biologically based and petroleum based carbon isotope profiles
may be distinguished from organic compounds and chemicals made only
of petroleum based materials. Hence, the bioproducts herein can be
followed or tracked in commerce on the basis of their unique carbon
isotope profile. Bioproducts can be distinguished from petroleum
based organic compounds by comparing the stable carbon isotope
ratio (.sup.13C/.sup.12C) in each sample. The .sup.13C/.sup.12C
ratio in a given bioproduct is a consequence of the
.sup.13C/.sup.12C ratio in atmospheric carbon dioxide at the time
the carbon dioxide is fixed. It also reflects the precise metabolic
pathway. Regional variations also occur. Petroleum, C3 plants (the
broadleaf), C4 plants (the grasses), and marine carbonates all show
significant differences in .sup.13C/.sup.12C and the corresponding
.delta..sup.13C values. Furthermore, lipid matter of C3 and C4
plants analyze differently than materials derived from the
carbohydrate components of the same plants as a consequence of the
metabolic pathway. Within the precision of measurement, .sup.13C
shows large variations due to isotopic fractionation effects, the
most significant of which for bioproducts is the photosynthetic
mechanism. The major cause of differences in the carbon isotope
ratio in plants is closely associated with differences in the
pathway of photosynthetic carbon metabolism in the plants,
particularly the reaction occurring during the primary
carboxylation (i.e., the initial fixation of atmospheric CO.sub.2).
Two large classes of vegetation are those that incorporate the C3
(or Calvin-Benson) photosynthetic cycle and those that incorporate
the C4 (or Hatch-Slack) photosynthetic cycle. In C3 plants, the
primary CO.sub.2 fixation or carboxylation reaction involves the
enzyme ribulose-1,5-diphosphate carboxylase, and the first stable
product is a 3-carbon compound. C3 plants, such as hardwoods and
conifers, are dominant in the temperate climate zones. In C4
plants, an additional carboxylation reaction involving another
enzyme, phosphoenol-pyruvate carboxylase, is the primary
carboxylation reaction. The first stable carbon compound is a
4-carbon acid that is subsequently decarboxylated. The CO.sub.2
thus released is refixed by the C3 cycle. Examples of C4 plants are
tropical grasses, corn, and sugar cane. Both C4 and C3 plants
exhibit a range of .sup.13C/.sup.12C isotopic ratios, but typical
values are about -7 to about -13 per mil for C4 plants and about
-19 to about -27 per mil for C3 plants (see, e.g., Stuiver et al.
(1977) Radiocarbon 19:355). Coal and petroleum fall generally in
this latter range. The .sup.13C measurement scale was originally
defined by a zero set by Pee Dee Belemnite (PDB) limestone, where
values are given in parts per thousand deviations from this
material. The 813C values are expressed in parts per thousand (per
mil), abbreviated, .Salinity., and are calculated as follows:
.delta..sup.13C(.Salinity.)=[(.sup.13C/.sup.12C)
sample-(.sup.13C/.sup.12C) standard]/(.sup.13C/.sup.12C)
standard.times.1000
[0131] Since the PDB reference material (RM) has been exhausted, a
series of alternative RMs have been developed in cooperation with
the IAEA, USGS, NIST, and other selected international isotope
laboratories. Notations for the per mil deviations from PDB is
.delta..sup.13C. Measurements are made on CO.sub.2 by high
precision stable ratio mass spectrometry (IRMS) on molecular ions
of masses 44, 45, and 46. The compositions described herein include
bioproducts produced by any of the methods described herein,
including, for example, fatty acid derivative products.
Specifically, the bioproduct can have a .delta..sup.13C of about
-28 or greater, about -27 or greater, -20 or greater, -18 or
greater, -15 or greater, -13 or greater, -10 or greater, or -8 or
greater. For example, the bioproduct can have a .delta..sup.13C of
about -30 to about -15, about -27 to about -19, about -25 to about
-21, about -15 to about -5, about -13 to about -7, or about -13 to
about -10. In other instances, the bioproduct can have a
.delta..sup.13C of about -10, -11, -12, or -12.3. Bioproducts
produced in accordance with the disclosure herein, can also be
distinguished from petroleum based organic compounds by comparing
the amount of .sup.14C in each compound. Because .sup.14C has a
nuclear half-life of 5730 years, petroleum based fuels containing
older carbon can be distinguished from bioproducts which contain
newer carbon (see, e.g., Currie, Source Apportionment of
Atmospheric Particles, Characterization of Environmental Particles,
J. Buffle and H. P. van Leeuwen, Eds., 1 of Vol. I of the IUPAC
Environmental Analytical Chemistry Series (Lewis Publishers, Inc.)
3-74, (1992)). The basic assumption in radiocarbon dating is that
the constancy of .sup.14C concentration in the atmosphere leads to
the constancy of .sup.14C in living organisms. However, because of
atmospheric nuclear testing since 1950 and the burning of fossil
fuel since 1850, .sup.14C has acquired a second, geochemical time
characteristic. Its concentration in atmospheric CO.sub.2, and
hence in the living biosphere, approximately doubled at the peak of
nuclear testing, in the mid-1960s. It has since been gradually
returning to the steady-state cosmogenic (atmospheric) baseline
isotope rate (.sup.14C/.sup.12C) of about 1.2.times.10.sup.-12,
with an approximate relaxation "half-life" of 7-10 years. This
latter half-life must not be taken literally; rather, one must use
the detailed atmospheric nuclear input/decay function to trace the
variation of atmospheric and biospheric .sup.14C since the onset of
the nuclear age. It is this latter biospheric .sup.14C time
characteristic that holds out the promise of annual dating of
recent biospheric carbon. .sup.14C can be measured by accelerator
mass spectrometry (AMS), with results given in units of fraction of
modern carbon (fM). fM is defined by National Institute of
Standards and Technology (NIST) Standard Reference Materials (SRMs)
4990B and 4990C. As used herein, fraction of modern carbon or fM
has the same meaning as defined by National Institute of Standards
and Technology (NIST) Standard Reference Materials (SRMs) 4990B and
4990C, known as oxalic acids standards HOxI and HOxII,
respectively. The fundamental definition relates to 0.95 times the
.sup.14C/.sup.12C isotope ratio HOxI (referenced to AD 1950). This
is roughly equivalent to decay-corrected pre-Industrial Revolution
wood. For the current living biosphere (plant material), fM is
approximately 1.1. The compositions described herein include
bioproducts that can have an fM.sup.14C of at least about 1. For
example, the bioproduct of the disclosure can have an fM.sup.14C of
at least about 1.01, an fM.sup.14C of about 1 to about 1.5, an
fM.sup.14C of about 1.04 to about 1.18, or an fM.sup.14C of about
1.111 to about 1.124.
[0132] Another measurement of .sup.14C is known as the percent of
modern carbon (pMC). For an archaeologist or geologist using
.sup.14C dates, AD 1950 equals zero years old. This also represents
100 pMC. Bomb carbon in the atmosphere reached almost twice the
normal level in 1963 at the peak of thermo-nuclear weapons. Its
distribution within the atmosphere has been approximated since its
appearance, showing values that are greater than 100 pMC for plants
and animals living since AD 1950. It has gradually decreased over
time with today's value being near 107.5 pMC. This means that a
fresh biomass material, such as corn, would give a .sup.14C
signature near 107.5 pMC. Petroleum based compounds will have a pMC
value of zero. Combining fossil carbon with present day carbon will
result in a dilution of the present day pMC content. By presuming
107.5 pMC represents the .sup.14C content of present day biomass
materials and 0 pMC represents the .sup.14C content of petroleum
based products, the measured pMC value for that material will
reflect the proportions of the two component types. For example, a
material derived 100% from present day soybeans would give a
radiocarbon signature near 107.5 pMC. If that material was diluted
50% with petroleum based products, it would give a radiocarbon
signature of approximately 54 pMC. A biologically based carbon
content is derived by assigning 100% equal to 107.5 pMC and 0%
equal to 0 pMC. For example, a sample measuring 99 pMC will give an
equivalent biologically based carbon content of 93%. This value is
referred to as the mean biologically based carbon result and
assumes all the components within the analyzed material originated
either from present day biological material or petroleum based
material. A bioproduct comprising one or more fatty acid
derivatives as described herein can have a pMC of at least about
50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100. In other
instances, a fatty acid derivative described herein can have a pMC
of between about 50 and about 100; about 60 and about 100; about 70
and about 100; about 80 and about 100; about 85 and about 100;
about 87 and about 98; or about 90 and about 95. In yet other
instances, a fatty acid derivative described herein can have a pMC
of about 90, 91, 92, 93, 94, or 94.2.
[0133] Fatty Aldehyde and Fatty Alcohol Compositions and their
Use
[0134] Aldehydes are used to produce many specialty chemicals. For
example, aldehydes are used to produce polymers, resins (e.g.,
BAKELITE resin), dyes, flavorings, plasticizers, perfumes,
pharmaceuticals, and other chemicals, some of which may be used as
solvents, preservatives, or disinfectants. In addition, certain
natural and synthetic compounds, such as vitamins and hormones, are
aldehydes, and many sugars contain aldehyde groups. Fatty aldehydes
can be converted to fatty alcohols by chemical or enzymatic
reduction. Fatty alcohols also have multiple commercial uses.
Worldwide annual sales of fatty alcohols and their derivatives are
in excess of U.S. $1 billion. The shorter chain fatty alcohols are
used in the cosmetic and food industries as emulsifiers,
emollients, and thickeners. Due to their amphiphilic nature, fatty
alcohols behave as nonionic surfactants, which are useful in
personal care and household products, such as, for example,
detergents. In addition, fatty alcohols are used in waxes, gums,
resins, pharmaceutical salves and lotions, lubricating oil
additives, textile antistatic and finishing agents, plasticizers,
cosmetics, industrial solvents, and solvents for fats.
[0135] The disclosure also provides a surfactant composition or a
detergent composition including a fatty alcohol produced by any of
the methods described herein. One of ordinary skill in the art will
appreciate that, depending upon the intended purpose of the
surfactant or detergent composition, different fatty alcohols can
be produced and used. For example, when the fatty alcohols
described herein are used as a feedstock for surfactant or
detergent production, one of ordinary skill in the art will
appreciate that the characteristics of the fatty alcohol feedstock
will affect the characteristics of the surfactant or detergent
composition produced. Hence, the characteristics of the surfactant
or detergent composition can be selected for by producing
particular fatty alcohols for use as a feedstock. A fatty
alcohol-based surfactant and/or detergent composition described
herein can be mixed with other surfactants and/or detergents well
known in the art. In some embodiments, the mixture can include at
least about 10%, at least about 15%, at least about 20%, at least
about 30%, at least about 40%, at least about 50%, at least about
60%, or a range bounded by any two of the foregoing values, by
weight of the fatty alcohol. In other examples, a surfactant or
detergent composition can be made that includes at least about 5%,
at least about 10%, at least about 20%, at least about 30%, at
least about 40%, at least about 50%, at least about 60%, at least
about 70%, at least about 80%, at least about 85%, at least about
90%, at least about 95%, or a range bounded by any two of the
foregoing values, by weight of a fatty alcohol that includes a
carbon chain that is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, or 22 carbons in length. Such surfactant or detergent
compositions also can include at least one additive, such as a
microemulsion or a surfactant or detergent from nonmicrobial
sources such as plant oils or petroleum, which can be present in
the amount of at least about 5%, at least about 10%, at least about
15%, at least about 20%, at least about 30%, at least about 40%, at
least about 50%, at least about 60%, at least about 70%, at least
about 80%, at least about 85%, at least about 90%, at least about
95%, or a range bounded by any two of the foregoing values, by
weight of the fatty alcohol.
EXAMPLES
[0136] The following specific examples are intended to illustrate
the disclosure and should not be construed as limiting the scope of
the claims.
[0137] Protocols and Methods
[0138] Screening a Library
[0139] All protocols described herein rely on a 96 well
plate-master block-2 mL system (Greiner Bio-One, Monroe, N.C. or
Corning, Amsterdam, The Netherlands) for growing cultures, and
plates (Costar, Inc.) for extracting fatty acid species from the
culture broth. The protocols provided below are examples of
fermentation conditions. Alternative protocols can be used to
evaluate fatty acid species production.
[0140] 32.degree. C. Culture Protocol (4NBT)
[0141] 204 LB culture (from an LB culture growing in a 96 well
plate) was used to inoculate 400 .mu.L 2NBT media (Table 2), which
was then incubated for approximately 16 hours at 32.degree. C.
shaking. 20 .mu.L of the overnight seed was used to inoculate 400
.mu.L 4NBT with either 1 or 2 g/L nitrogen (NBT_1N or NBT_2N).
After growing at 32.degree. C. for 6 hours, the cultures were
induced with IPTG (final concentration 1 mM) (Table 2). The
cultures were then incubated at 32.degree. C. with shaking for 18
hours, after which they were extracted following the standard
extraction protocol detailed below.
TABLE-US-00002 TABLE 2 Media Names and Formulations Media Name
Formulation 2NBT 1 X 5 .times. Salt Soln. with NH4Cl 1 g/L 100 g/L
NH4Cl 1 mg/L 10 mg/mL Thiamine 1 mM 1M MgSO4 0.1 mM 1M CaCl2 20 g/L
500 g/L glucose 1 X 1000 .times. TM2 10 mg/L 10 g/L Fe Citrate 100
.mu.g/mL 100 mg/ml spectinomycin 100 mM 2M BisTris (pH 7.0) 4NBT_1N
1 X 5 .times. Salt Soln. with NH4Cl 1 mg/L 10 mg/mL Thiamine 1 mM
1M MgSO4 0.1 mM 1M CaCl2 40 g/L 500 g/L glucose 1 X 1000 .times.
TM2 10 mg/L 10 g/L Fe Citrate 100 .mu.g/mL 100 mg/ml spectinomycin
100 mM 2M BisTris (pH 7.0) 4NBT_2N 1 X 5 .times. Salt Soln. with
NH4Cl 1 g/L 100 g/L NH4Cl 1 mg/L 10 mg/mL Thiamine 1 mM 1M MgSO4
0.1 mM 1M CaC12 40 g/L 500 g/L glucose 1 X 1000 .times. TM2 10 mg/L
10 g/L Fe Citrate 100 .mu.g/mL 100 mg/ml spectinomycin 100 mM 2M
BisTris (pH 7.0)
[0142] Fatty Acid Species Standard Extraction Protocol
[0143] To each well to be extracted 40 .mu.L of 1M HCl, followed by
300 .mu.L of butyl acetate (with 500 mg/L C11-FAME as internal
standard) was added. The 96 well plates were then heat-sealed using
a plate sealer (ALPS-300 heater; Abgene, ThermoScientific,
Rockford, Ill.), and shaken for 15 minutes at 2000 rpm using
MIXMATE mixer (Eppendorf, Hamburg, Germany). After shaking, the
plates were centrifuged for 10 minutes at 4500 rpm at room
temperature (Allegra X-15R, rotor SX4750A, Beckman Coulter, Brea,
Calif.) to separate the aqueous and organic layers. 504, of the
organic layer was transferred to a 96 well plate (polypropylene,
Corning, Amsterdam, The Netherlands), which was subsequently heat
sealed and stored at -20.degree. C. until evaluated by GC-FID using
the FALC_Broth.met method. The FALC_Broth.met method was carried
out as follows: 1 .mu.L of sample was injected onto an analytical
column (DB-1, 10 m.times.180 .mu.m.times.0.2 .mu.M film thickness,
available from JW 121-101A) in an Agilent 7890A GC Ultra device
(Agilent, Santa Clara, Calif.) with a flame ionization detector
(FID). The instrument was set up to detect and quantify C6 to C18
fatty alcohols. The protocol detailed above represents standard
conditions, which may be modified as necessary to optimize the
analytical results.
[0144] Fatty Acid Species--Standard Nile Red Assay Protocol
[0145] After 24 hours of fermentation, a Nile Red assay was
performed by adding 70 .mu.L of fermentation broth to 130 .mu.L of
1.54 .mu.g/mL Nile Red in 84.6% water and 15.4% acetonitrile
solution for a final assay concentration of 1 .mu.g/mL Nile Red in
a Greiner MicrolonFluotrac 200 plate, and mixed by pipetting up and
down. Relative fluorescence units were measured at excitation of
540 nm and emission of 630 nm using the SpectraMax M2 unit.
[0146] Building Error Prone Libraries
[0147] Standard techniques known to those of skill in the art were
used to prepare error prone libraries. In one example, the vector
backbone was prepared using restriction endonucleases in the
vector, while the creation of diversity in the DNA insert was
generated by PCR amplification from a DNA template under conditions
favoring the incorporation of mismatched nucleotides. In one
approach, the cloning of the vector backbone and a DNA insert with
diversity was performed using the INFUSION Cloning System (Clontech
Laboratories, Inc., Mountain View, Calif.), according to the
manufacturer's protocol.
[0148] Building Saturation Libraries
[0149] Standard techniques known to those of skill in the art were
used to prepare saturation libraries. In one example, the vector
backbone was prepared using restriction endonucleases in the
vector, while the creation of diversity in the DNA insert was
generated using degenerate primers. In one approach, the cloning of
the vector backbone and a DNA insert with diversity was performed
using INFUSION Cloning System (Clontech Laboratories, Inc.,
Mountain View, Calif.) according to the manufacturer's
protocol.
[0150] Building Combination Libraries
[0151] Mutations identified as beneficial were combined to provide
AAR variants with further improvements in the production of fatty
alcohol species. Standard techniques known to those of skill in the
art were used to prepare the combination libraries. In one example,
the vector backbone was prepared using restriction endonucleases in
the vector, while the creation of diversity in the DNA insert was
generated using primers to introduce the desired mutations. As
described above, in one approach, the cloning of the vector
backbone and a DNA insert with diversity was performed using
INFUSION Cloning System (Clontech Laboratories, Inc., Mountain
View, Calif.), according to manufacturer's protocol. Combination
libraries can be generated using the transfer PCR (tPCR) protocol
(Erijman et al. (2011) J. Structural Bio. 175:171-177).
[0152] Library Screening
[0153] Once the library diversity was generated in an error-prone,
saturation library or combination library, it was screened using
one of the methods described above. Two types of hits were
identified: (1) increased amount of fatty alcohol (FALC titer);
and/or (2) increased amount of medium-chain FALC such as dodecanol
(C12) or tetradecanol (C14). Also identified were hexadecanol (C16)
and octadecanol (C18). The mutations in the AAR variants within
each hit were identified by sequencing using standard techniques
routinely employed by those of skill in the art. Tables 4, 5 and 6
list the mutations (hits) identified as beneficial in saturation
libraries and combination libraries.
Example 1: Improved Fatty Alcohol Production Using AAR_7942 by Acyl
Carrier Protein (ACP) Mediated Increased Flux Through the Fatty
Acid Synthesis Pathway
[0154] When terminal pathway enzymes from sources other than E.
coli are expressed in E. coli as the heterologous host to convert
fatty acyl-ACPs to products, limitations may exist in the
recognition, affinity and/or turnover of the recombinant pathway
enzyme towards the E. coli fatty acyl-ACPs. Although ACP proteins
are conserved to some extent in all organisms, their primary
sequence can differ even within a given species. In order to test
this hypothesis the acp genes from several cyanobacteria were
cloned downstream from the Synechococcus elongatus PCC7942 acyl-ACP
reductase (AAR_7942) present in plasmid pLS9-185, which is a
pCL1920 derivative (3-5 copies/cell). In addition, the sfp gene
(Accession no. X63158; SEQ ID NO: 11) from Bacillus subtilis,
encoding a phosphopantetheinyl transferase with broad substrate
specificity, was cloned downstream of the respective acp genes.
This enzyme is involved in conversion of the inactive apo-ACP to
the active holo-ACP. The plasmids constructed are described in
Table 3.
TABLE-US-00003 TABLE 3 Plasmids Coexpressing Cyanobacterial ACP
with and without B. subtilis sfp Downstream from S. elongatus
PCC7942 AAR Base ACP-SEQ ID Without Plasmid ACP Source NO. (NA/AA*)
sfp With sfp pLS9-185 Synechococcus 7/8 pDS168 pDS168S elongatus
7942 pLS9-185 Synechocystis sp. 3/4 pDS169 not available 6803
pLS9-185 Prochlorococcus 5/6 pDS170 pDS170S marinus MED4 pLS9-185
Nostoc 1/2 pDS171 pDS171S punctiforme 73102 pLS9-185 Nostoc sp.
7120 9/10 pDS172 pDS172S *NA = nucleic acid sequence; AA = amino
acid sequence/polypeptide sequence
[0155] All the acp genes were cloned with a synthetic RBS into the
EcoRI site immediately downstream of the aar gene in pLS9-185 using
INFUSION technology. The EcoRI site was reconstructed downstream of
the acp gene. Similarly, the B. subtilis sfp gene was INFUSION
cloned into this EcoRI site along with a synthetic RBS. All
plasmids were transformed into E. coli MG1655 DV2. The control for
these experiments was the expression of AAR alone (pLS9-185).
[0156] The results from standard shake flask fermentation
experiments are shown in FIG. 5. Significant improvement in fatty
alcohol titers were observed in strains containing the plasmids
pDS171S, pDS172 S, pDS168 and pDS169, demonstrating that ACP
overexpression can be beneficial for fatty alcohol production.
While not wishing to be bound by theory, it is hypothesized that
ACP overexpression can be beneficial for fatty alcohol production
by aiding in the recognition, affinity and/or turnover of acyl-ACPs
by the heterologous terminal pathway enzyme (see Table 3 (supra)
for the source of the ACPs and presence or absence of sfp.)
Example 2: Improved Fatty Alcohol Production Using AAR_7942 by
Acetyl-CoA Carboxylase (ACC) Mediated Increased Flux Through the
Fatty Acid Synthesis
[0157] The main precursors for fatty acid biosynthesis are
malonyl-CoA and acetyl-CoA. It has been suggested that these
precursors limit the rate of fatty acid biosynthesis in E. coli. In
this example, a synthetic acetyl-CoA carboxylase, acc, operon
[Corynebacterium glutamicum accDCAB+birA; SEQ ID NOs: 45 or 46, 48,
and 50 also referred to as D+] was expressed alongside acyl-ACP
reductase (AAR) from Synechococcus elongatus PCC7942 (AAR_7942).
The accD+operon was cloned downstream of the AAR_7942 gene in
plasmid pLS9-185. The resulting plasmid and the pLS9-185 control
plasmid were transformed into E. coli DV2. The strains were
evaluated for fatty alcohol production in a standard shake flask
protocol. As shown in FIG. 6, the coexpression of the synthetic
Corynebacterium glutamicum acc operon led to increased fatty
alcohol production.
Example 3: Error Prone Library, Combination and Limited Saturation
Libraries Prepared Using AAR_7942 as a Template
[0158] A. Error Prone Library
[0159] An error prone library of the acyl-ACP reductase from
Synechococcus elongates PCC7942 (AAR_7942) was built and screened
for variants that showed improvements over the wild type AAR_7942.
The plasmid used to make the error prone library was designated
"pDS171S" (see Table 3). The error prone library was screened using
one of the standard protocols described above. The improvements
were classified as either improving titer or increasing the
fraction of C10-C14 fatty alcohols produced without significantly
affecting titer (results not shown).
[0160] B. Combination and Limited Saturation Libraries
[0161] Standard techniques known to those of skill in the art were
used to prepare combination libraries and saturation libraries
based on positions 17, 18 and 19. The mutations tested in the
combination libraries and saturation libraries (Tables 4A and 4B)
were originally identified in the error prone library of AAR_7942
described above. Plasmids, strains and screening protocols used
were the same as described in Example 1. The results from screening
the error prone library are shown in Tables 4A and 4B. Table 4A
shows AAR_7942 mutations that led to increased fatty alcohol titers
and Table 4B shows mutations that led to increased fraction of C14
fatty alcohol without significantly affecting overall fatty alcohol
titer.
TABLE-US-00004 TABLE 4A Mutations From an AAR 7942 Combination and
Limited Saturation Libraries Correlated with Improved Fatty Alcohol
Titer Combo .alpha. Library Normalized Normalized FALC FALC C14
Titer C14-FALC (% over (% over Mutations (total*) (% fraction)
control) control) S18T, C63Y, S96T, 2473 48% 251% 294% L177H, A281V
S18T, C63Y, S96T, 2202 50% 200% 229% L177H, A281V E284K S18T, C63Y,
S96T, 2088 43% 189% 197% L177H S18T, C63Y 1798 39% 182% 241% C63H,
S967T, L177H, 2045 53% 180% 237% A281V S18T, C63Y, A281V, 1696 45%
172% 278% E283K S18T, C63Y 1640 43% 166% 260% S18T, L177H, A281V,
1618 44% 164% 270% E283K S18T, L177H, A281V, 1617 43% 164% 264%
E283K S18T, S96T, L177H, 1814 43% 160% 194% E283K S18T, C63H, L177H
1811 39% 160% 173% S18T, L177H, A281V, 1503 39% 152% 237% E283K
L65F, S96T, A281V, 1638 43% 144% 191% E283K C63Y, A281V, A282T 1622
38% 143% 168% L11F, C63Y, S96T, 1579 41% 143% 186% L177H, A281V
A282T, E283K L65F, S96T, L177H, 1585 42% 140% 189% A281V, E283K
G22S, C63H, S96T, 1229 39% 125% 237% L177H, E283K C63H, L65F, S96T,
1178 41% 119% 252% A281V S18T, L65F, S96T, 1136 33% 115% 203%
L177H, A281V C63R, L177H, A281V, 1275 44% 112% 196% E283K
Saturation Libraries Normalized Normalized FALC FALC C14% Tite
C14-FALC (% over over Mutations (total*) % fraction) control)
control) S18M 2809 40% 243% 212% S18F 2564 54% 222% 285% S18Y 2550
45% 221% 236% S18W 2530 55% 227% 314% S18M 2495 41% 224% 231% S18Y
2039 44% 188% 252% S18Y 1997 44% 179% 251% Saturation Libraries
S18T 1729 31% 155% 177% S18T 1690 31% 152% 176% S18T 1683 31% 151%
176% S18T 1599 30% 143% 172% S18L 1568 48% 141% 271% S18C 1381 28%
120% 149% S18C 1372 26% 123% 147% S18C 1368 28% 118% 148% S18L 1359
47% 122% 266% S18C 1355 26% 121% 148% S18V 1344 30% 121% 170% S18C
1340 26% 120% 147% S18L 1326 47% 119% 268% S18L 1315 47% 118% 266%
S18C 1313 26% 118% 149% S18V 1306 30% 121% 170% S18C 1305 26% 117%
148% S18L 1302 47% 117% 267% V17L 1188 23% 110% 130% V17L 1185 33%
103% 173% V17L 1175 32% 102% 171% Combo .beta. Library Normalized
Normalized FALC FALC C14% Tite C14-FALC (% over over (total*) %
fraction) control) control) S18W, S96T, E283K, 3437 49% 246% 255%
R286Q, A324V S18W 3286 53% 235% 275% S18W, S96T, E283K 3199 46%
229% 237% S18W, C63Y, S96T 3186 46% 228% 241% S18W, C68Y, E2834K
2949 50% 211% 260% C63Y, E283K 2680 36% 192% 189% S18W, C63Y 2675
54% 191% 280% C63Y, S96T 2583 38% 185% 196% C63Y, E283K, Q316K 2420
36% 173% 188% S96T 1605 23% 115% 121% E283K 1532 25% 110% 131%
TABLE-US-00005 TABLE 4B Mutations From AAR_7942 Combination and
Limited Saturation Libraries Conelated with Increased C14 Fatty
Alcohol Fraction Combo .alpha. Library Normalized Normalized FALC
FALC C14% Tite C14-FALC (% over over (total*) % fraction) control)
control) C63H, S96T, L177H, 2045.36 53% 180% 237% A281V G22S, C63H,
F64V, 152.47 51% 14% 233% S96T, L177H A281V S18T, C63H, S96T,
2202.46 50% 200% 229% L177H, A281V E283K S18T, C63Y, S96T, 2472.80
48% 251% 294% L177H, A281V S18T, C63Y, A281V, 1695.59 45% 172% 278%
E283K S18T, L177H, A281V, 1618.20 44% 164% 270% E283K C63R, L177H,
A281V, 1275.38 44% 112% 196% E283K S18T, C63H, L65F, 912.50 44% 83%
199% S96T, L177H A281V, E283K S18T, S96T, L177H, 1814.34 43% 160%
194% E283K S18T, L179H, A281V, 1616.51 43% 164% 264% E283K S18T,
C63H, S96T, 2088.12 43% 189% 197% L177H L65F, S96T, A281V, 1638.28
43% 144% 191% E283K C63H, L65F, A281V, 632.66 43% 56% 190% E283K
S18T, C63Y 1640.33 43% 166% 260% L65F, S96T, L177H, 1585.32 42%
140% 189% A281V, E283K C63Y, L65F, L177H 639.46 41% 56% 184% C63H,
L65F, S96T, 1177.76 41% 119% 252% A281V L11F, C63Y, S96T, 1578.54
41% 143% 186% L177H, A281V A282T, E283K S18T, C63Y 1797.99 39% 182%
241% S18T, L178H, A282V, 1502.59 39% 152% 237% E284K G23S, C63H,
S96T, 1229.34 39% 125% 237% L177H, E283K S18T, C63H, L177H 1811.40
39% 160% 173% C63Y, A281V, A282T 1622.23 38% 143% 168% S18T, C63Y,
L65F, 454.42 35% 41% 162% S96T, L177H A281V, E283K, M284I S18T,
L65F, S96T, 1135.95 33% 115% 203% L177H, A281V L65F, L178H, G180S,
151.40 33% 13% 146% E283K L66F, L177H, G180S, 143.83 31% 13% 138%
E283K S18T, C63H, L65F, 103.21 30% 9% 133% L177H Saturation
Libraries Normalized Normalized FALC FALC C14% Tite C14-FALC (%
over over (total*) % fraction) control) control) S18W 2530 55% 227%
314% S18F 2564 54% 222% 285% V17N 571 49% 49% 260% S18L 1568 48%
141% 271% S18L 1024 47% 92% 268% S18L 1326 47% 119% 268% S18L 1302
47% 117% 267% S18L 1106 47% 99% 267% S18L 1315 47% 118% 266% S18L
1359 47% 122% 266% S18Y 2550 45% 221% 236% S18Y 2039 44% 188% 252%
S18Y 1997 44% 179% 251% S18M 2495 41% 224% 231% S18M 2809 40% 243%
212% V17L 1127 33% 97% 175% V17L 1185 33% 103% 173% V17L 1091 32%
94% 171% V17L 1175 32% 102% 171% S18T 1729 31% 155% 177% S18T 1690
31% 152% 176% S18T 1683 31% 151% 176% S18T 1599 30% 143% 172% S18V
1344 30% 121% 170% S18V 1306 30% 121% 170% S18V 1015 30% 91% 169%
S18C 1381 28% 120% 149% S18C 1368 28% 118% 148% R19H, R58S 873 27%
81% 15.2% V17C 927 27% 80% 140% V17C 882 26% 76% 139% S18C 1313 26%
118% 149% S18C 1355 26% 121% 148% S18C 1305 26% 117% 148% S18C 1372
26% 123% 147% S18C 1340 26% 120% 147% V17W 849 25% 73% 134% V17W
815 25% 71% 133% V17W 831 25% 72% 132% V17W 840 25% 73% 131% R19V
844 24% 78% 139% R19V 813 24% 75% 136% R19V 821 24% 76% 135% R19V
862 24% 80% 135% R19V 814 24% 75% 135% R19K, V117L 1188 23% 110%
130% R19S 803 22% 74% 128% R19T 735 22% 68% 127% R19S 783 22% 72%
127% R19S 764 22% 75% 129% R19I 862 22% 80% 123% R19A 901 21% 83%
122% R19A 705 21% 65% 119% R19M 937 20% 86% 114% Combo .beta.
Library Normalized Normalized FALC FALC C14% Tite C14-FALC % over
over (total*) % fraction) control) control) S18W 3184 57% 228% 296%
S18W, C63Y 2675 54% 191% 280% S18W, C63Y, S95T 2832 54% 203% 279%
S18W, S96T, E283K 2874 54% 206% 278% S18W, C63Y, E283K 2915 51%
209% 263% S18W, S96T, E283K, 3437 49% 246% 255% R286Q, A324V C63Y,
S96T 2583 38% 185% 196% C63Y, E283K 2176 38% 156% 195% C63Y, E283K,
Q316K 2420 36% 173% 188% E283K 1474 25% 105% 132% S96T 1605 23%
115% 121% Combo 3 Library Normalized Normalized FALC FALC C14% Tite
C14-FALC (% over over (total*) % fraction) control) control) S18W
2853 217% 51% 274% Y23N 954 72% 35% 191% Y23N, Q238H 892 68% 35%
190% G150D, I274N 766 62% 30% 162% I274N 1359 107% 29% 161% I274N,
A285V 1317 103% 28% 155% P38T, I274N, A285V 587 46% 26% 143% Q238H,
M291V 1383 109% 22% 119% M291V 1618 123% 22% 118% G151D, M291V 1302
105% 21% 114% T135M, M291V 1265 100% 18% 101%
Example 4: Saturation Library Prepared Using AAR (S18W) as a
Template
[0162] A full saturation library of an acyl-ACP reductase variant
from Synechococcus elongatus PCC7942 ("`AAR(S18W)_7942"), was built
and screened for variants that showed improvements over AAR (S18W),
identified as a significantly improved AAR variant in the first
round of screening (Examples 2 and 3). The selection criteria was
an increase in FALC titer or an increase in the percent fraction of
C12. Engineering efforts were focused on relieving the dependence
of AAR on ACP overexpression in order to yield high titers. While
not wishing to be bound by theory, the advantage observed in
strains overexpressing ACP was hypothesized to result from a higher
concentration of fatty acid biosynthesis intermediates available
for cleavage by AAR. By screening saturation and combination
libraries built on AAR in a strain lacking ACP overexpression
(having a lower concentration of FAS intermediates), variants with
higher affinities for FAS intermediates could be selected.
[0163] The plasmid that used to make the full saturation library
was designated pAAR-1. It is a derivative of pLS9-185 harboring the
AAR gene encoding the (S18W) variant followed by the aldehyde
reductase gene, AlrA, from Acinetobacter baylyi (SEQ ID NO: 54).
AlrA was added to fully reduce fatty aldehyde intermediates
generated by AAR and not fully reduced by E. coli's endogenous
fatty aldehyde reductases. The full saturation library was screened
in strain Shu.002. Strain Shu.002 is DV2 PT5-ifab138 PT5_ifadR
(Table 7, infra). For ifab138 see SEQ ID NO: 55 in Table 10. The
libraries were screened using one of the standard protocols
described above. The improvements were classified as either
improving titer or increasing the fraction of C12 fatty alcohols
produced by the acyl-ACP reductase without significantly affecting
titer. The results from screening saturation libraries are shown in
Table 5 below.
TABLE-US-00006 TABLE 5 Mutations From an AAR (S18W) 7942 Full
Saturation Library Correlated with Increased Fatty Alcohol Titer
and/or Increased C12 Fatty Alcohol Fraction AAR mutations FALC
Titer* C12 (in addition to S18W) Total* FIOC** Fraction* FIOC**
T148V 747 1.21 7.9% 0.99 A159V 702 1.14 7.0% 0.87 T157V 674 1.10
7.2% 0.90 A135S 803 1.31 7.8% 0.98 A328S 712 1.16 8.1% 1.02 Q191A
780 1.27 7.7% 0.96 A285V, M291V 837 1.36 8.0% 1.00 Q277V 854 1.39
7.9% 0.99 Q155C 626 1.02 8.3% 1.04 E210Y 676 1.10 8.5% 1.06 T120S
623 1.01 8.1% 1.02 T236C 691 1.12 7.6% 0.95 Q335N 763 1.24 6.8%
0.86 C172L 700 1.14 7.7% 0.97 E283S 858 1.39 8.0% 1.00 L209R 837
1.36 7.6% 0.96 I153P 606 0.99 8.0% 1.00 A211W 797 1.30 6.9% 0.86
A324T 909 1.48 4.0% 0.50 W34F 817 1.22 7.5% 0.98 T187V 825 1.23
6.9% 0.91 D24E 737 1.10 7.1% 0.93 T188H 862 1.29 8.6% 1.13 V18A 798
1.19 6.4% 0.84 V18A, L338W 732 1.27 7.6% 0.97 T188V 775 1.34 7.4%
0.95 I168V 731 1.27 8.1% 1.03 W35F 666 1.15 7.5% 0.96 T148V 836
1.45 6.2% 0.80 Q155L 740 1.28 8.5% 1.08 T148C 731 1.26 8.6% 1.10
T148E 694 1.20 8.3% 1.06 A50Q, L337V 800 1.38 7.2% 0.92 R118Q 724
1.25 7.3% 0.94 L31V 901 1.56 6.8% 0.87 A135S 775 1.34 8.7% 1.12
D24Y 890 0.93 19.1% 2.05 C63A 895 0.94 16.1% 1.73 S113K 812 0.85
13.3% 1.43 L31V 645 0.68 15.7% 1.69 E283G 917 0.96 14.3% 1.54 A112R
918 0.96 10.7% 1.15 D43E 1002 1.05 13.0% 1.39 Q116G 894 0.94 14.0%
1.50 S86G 849 0.89 13.4% 1.44 *Average from 4 replicates **FIOC =
fold increase over AAR (S18W) control
Example 5: Combination Libraries Prepared Using AAR (S18W) as a
Template
[0164] Standard techniques known to those of skill in the art were
used to prepare combination libraries. The mutations tested in
combination libraries (Tables 6A, 6B and 6C) were originally
identified in the full saturation library (Example 4). The
combination libraries were constructed in the same plasmid and
screened in the same strain as described in Example 3. The
libraries were screened using one of the standard protocols
described above. The improvements were classified as either
improving titer or increasing the fraction of C12 fatty alcohols
produced by the acyl-ACP reductase without significantly affecting
titer. The results from screening AAR combination libraries are
shown in Tables 6A, 6B and 6C below.
TABLE-US-00007 TABLE 6A Mutations from the 1.sup.st AAR(S18W)_7942
Combination Library Correlated with Increased Fatty Alcohol Titer
AAR mutations FALC Titer* C12 (in addition to S18W) mg/L* FIOC**
Fraction* FIOC** A281L 1478 1.29 14.1% 1.11 A281Y 1527 1.28 12.1%
0.96 T154A, A281L 1550 1.28 14.3% 1.13 T154A, A281Y 1384 1.19 13.3%
1.05 C63G, A281F 1472 1.24 12.9% 1.02 C63G, A281L 1432 1.19 18.2%
1.44 N83G, A281Y 1428 1.23 10.7% 0.84 C63G, A281Y 1419 1.21 15.6%
1.24 S10G, A281L 1419 1.20 12.6% 1.00 S113K, Q155G 1305 1.15 12.3%
0.97 D43E, A50Q, A281L 1517 1.32 9.1% 0.72 M21L, N83G, A281Y 1513
1.29 10.2% 0.81 M21L, S113K, A281L 1392 1.20 20.4% 1.61 C63G,
T154A, A281F 1369 1.24 12.4% 0.98 C63G, N83G, A281Y 1330 1.32 14.6%
1.16 Q155G, A281L, E283G 1327 1.16 22.2% 1.76 A50Q, C63G, A281F
1286 1.15 17.9% 1.41 S18W, D43E, A50Q, A281L 1635 1.32 9.4% 0.77
M21L, C63G, S113K, T154A 1518 1.24 28.3% 2.23 A281L M21L, L31V,
N83G, Q116G 1353 1.18 15.3% 1.21 A281L *Average of 4 replicates
**FIOC = fold increase over (S18W) control
TABLE-US-00008 TABLE 6B Mutations from the 2.sup.nd AAR(S18W)_7942
Combination Library Correlated with Increased Fatty Alcohol Titer
AAR mutations FALC Titer* C12 (in addition to S18W) mg/L* FIOC**
Fraction* FIOC** S18W (control) 1034 1.00 10.0% 1.00 M21L, C63G,
S113K, T154A, 1179 1.14 29.4% 2.94 A281L D16L, M21L, C63G, S113K,
1221 1.18 19.4% 1.94 T154A, A281L L8A, D24V, C63G, S113K, 1414 1.37
10.4% 1.04 Q155L, A281L L8A, M21L, C63G, A77A***, 1310 1.27 14.2%
1.42 S113K, T154A A281L D24P, L31M, C63G, S113K, 1437 1.39 13.0%
1.30 T154A, A281L L8A, D16L, D24V, C63G, 1342 1.30 12.0% 1.20
S113K, T154A A281L D24E, C63G, S113K, T154A, 1425 1.38 14.3% 1.43
A281L *Average from 4 replicates **FIOC = fold increase over (S18W)
control ***A77A mutation is a gcc to gca silent codon mutation
TABLE-US-00009 TABLE 6C Mutations from the AAR (S18W)_7942
Combination Libraries Correlated with Increased Fraction of C12
Fatty Alcohol AAR mutations C12 FALC Titer* (in addition to S18W)
Fraction* FIOC** FIOC** S18W (control) 11.0% 1.00 1.00 M21L, C63G,
S113K, T154A, A281L 28.3% 2.57 1.24 M21L, Q155G, A281L 24.8% 2.25
1.10 Q155G, A281L, E283G 22.2% 2.02 1.16 M21L, S113K, A281L 20.4%
1.85 1.20 C63G, A281L 18.1% 1.65 1.18 A50Q, C63G, A281F 17.9% 1.63
1.15 C63G 17.7% 1.61 1.00 S10G, C63G, Q155G, S253N, A281F 16.1%
1.46 1.03 D43E, C63G, S113K, A281L 16.0% 1.46 0.98 A281L 15.6% 1.42
1.20 M21L, L31V, N83G, Q116G, A281L 15.3% 1.39 1.18 C63G, N83G,
A281Y 14.6% 1.33 1.32 A281L 14.1% 1.28 1.29 *Average from 4
replicates **FIOC = fold increase over (S18W) control
Example 6: Increased Flux Through the Fatty Acid Synthesis
Pathway--iFab and iFadR Mediated Fatty Alcohol Production Using
AAR
[0165] In this example, improved fatty alcohol production using AAR
was shown by increasing flux through the fatty acid biosynthesis
pathway mediated by the overexpression of a synthetic operon
comprising several FAB proteins (ifab138) and/or overexpression of
FadR protein (ifad), a regulator of fatty acid metabolism. iFAB138
(SEQ ID NO:55) includes in the following order the genes fabV from
Vibrio cholerae, FabH, fabD, fabG and fabA from Salmonella
typhimurium, and FabF from Clostridium acetobutylicum, and it is
integrated as a synthetic operon controlled either by the PlacUV5
or PT5 promoter. iFadR includes the fadR gene from Escherichia coli
(SEQ ID NO:56) controlled by the T5 promoter. The components
present in the E. coli strains evaluated in this example are shown
in Table 7, below.
TABLE-US-00010 TABLE 7 E. coli Strains with iFAB138 or fadR
Components DV2 BD061 BD064 Shu002 i(PlacUV5-fab138) - + + -
i(PT5-fab138) - - - + i(PT5-fadR) - - + +
[0166] AAR (S18W) was expressed from plasmid pDS311, a variant of
plasmid pDS171S, in which AAR codon 18 specified a tryptophan
instead of a serine (pCL-AAR(S18W)+ACP-sfp) and the aldehyde
reductase gene, alrA, from Acinetobacter baylyi (SEQ ID NO: 54) was
cloned downstream of the sfp gene from B. subtilis. pDS311 was
transformed into strains DV2, BD061, BD064 and Shu002. The strains
were evaluated in the 4NBT 2N protocol (see above). As shown in
FIG. 7, fatty alcohol production significantly increased as a
result of the presence of ifab138 and ifadR, with strain Shu002
showing the highest fatty alcohol titer.
Example 7: Improved AAR Variants in a Strain with Increased Flux
Through the Fatty Acid Synthesis Pathway
[0167] In this example, improved fatty alcohol production in
recombinant host cells transformed with AAR variants from
combination libraries was demonstrated in Shu2, an E. coli strain
with increased flux through the fatty acid biosynthesis pathway
mediated by the overexpression of the ifab138 and overexpression of
FadR protein (supra). Four AAR variants from the second combination
library (Table 6B) were evaluated. These variants harbor the
following mutations: Com2a: S18W, D24P, L31M, C63G, S113K, T154A,
A281L; Comb2b: S18W, D16L, M21L, C63G, S113K, T154A, A281L; Com2c:
S18W, L8A, M21L, C63G, A77A, S113K, T154A, A281L; Com2d: D24E,
C63G, S113K, T154A, A281L (see also FIG. 8). These variants were
cloned into the backbone of plasmid pJL104. pJL104 was created by
cloning the synthetic accD+operon from C. glutamicum (as described
in Example 2, supra) downstream of the alrA gene in pDS311. The
resulting plasmids were transformed into strain Shu002 and the
strains were evaluated in the 4NBT_1N protocol (supra). As shown in
FIG. 8, elevated fatty alcohol production was observed in all
strains. Strain BD064 harboring plasmid pDS311, which expresses the
S18W variant of AAR_7942, was also evaluated in tank fermentations.
The strain produced fatty alcohols with a maximum titer of 42 g/L,
a yield of 12.6% (on glucose) and a productivity of 0.62 g/L/h as
shown in FIG. 9. The chain length distribution of the fatty
alcohols was as follows: 1.2% C8, 7.7% C10, 26.9% C12, 44.7% C14,
16.0% C16 and 1.9% C18. The fraction of saturated and unsaturated
fatty alcohols was 72.6% and 27.4%, respectively.
Example 8: Increased MED4 Acyl Aldehyde Reductase (AAR) Activity
and Redistribution of Chain Length Selectivity for C14 Fatty
Alcohol Production
[0168] AAR is one of the components essential for cyanobacterial
alkane biosynthesis, another essential component is an aldehyde
decarbonylase (ADC). The inventors discovered that the
Prochlorococcus marinus MED4_AAR is catalytically inactive without
the presence of MED4_ADC, i.e., when only MED4_AAR is expressed in
E. coli no products are detected, and when MED4_AAR and ADC are
coexpressed in E. coli the only products detected are alkanes. The
inventors also discovered that when MED4_AAR is coexpressed in E.
coli with an apparently catalytically inactive variant of MED4_ADC
in which histidine 156 is replaced by arginine (referred to
subsequently as MED4_ADC (H165R)), fatty alcohols, and no alkanes
are detected (see FIG. 10). It was concluded from this data that
MED4_AAR requires physical interaction with MED4_ADC to be
catalytically active. The inventors used this system to identify
MED4_AAR variants with increased activity and/or altered substrate
specificity for purposes of FALC production.
[0169] MED4_ADC(H156R) was expressed together with a full
saturation library of MED4_AAR. The MED4_AAR saturation library was
prepared in a plasmid pCL1920-derivative (pLS9-195) and introduced
into a production strain carrying plasmid pGLAK-043 (which is
plasmid pACYC-Ptrc-MED4_ADC harboring the H156R mutation in the ADC
gene). The clones were induced and AAR variants were selected based
upon production of more fatty alcohol than the wild type AAR enzyme
or the ability to produce fatty alcohols with an altered chain
length profile, e.g., an increased fraction of C14 fatty alcohol.
Selected clones were then re-tested in a validation round. All
variants that showed consistent FALC titers amongst the primary and
secondary fermentations were re-grown, plasmid DNA was isolated,
sequenced and re-introduced into the parental production strain for
further testing. These new transformants were then subjected to
another confirmatory fermentation and analysis. Table 8 below shows
representative data from 16 AAR variants that produced the highest
FALC titers (ordered from top to bottom in descending activity).
The variants ranged from 1.4-fold to 2.2 fold over wild type. These
variants showed the ability to increase MED4_AAR activity using
directed evolution techniques and furthermore form the basis for
further improvements.
TABLE-US-00011 TABLE 8 FALC Productivity of MED4 AAR Mutants
Relative to the Wild Type (WT) MED4 AAR Fold FALC increase AAR
Mutation over Wild Type V346P 2.2 Q40V 2.2 A345R 2.1 L344S 2.1 D61E
2.1 V346G 2.0 L344D 1.9 G52V 1.9 A345* 1.9 L344T 1.8 K303G 1.8
L344A 1.8 H340P 1.6 S588V 1.5 K339L 1.4 G273E 1.4 *Truncated
variant missing the last two amino acids.
[0170] The data set was also scanned for AAR variants which
displayed altered chain length profiles. The most common species
produced by the wild type AAR has a chain length of C16. An
increased proportion of FALC species with chain lengths shorter
than C16 is of interest. Two variant clones were identified that
showed an approximately 3-fold increase in the quantity of C14 FALC
(FIG. 10). The sequencing of these clones revealed that they were
identical D61E mutants with the same nucleotide codon sequence. The
plasmid DNA for the D61E variant was reintroduced into the parental
strain containing the H156R ADC. The results show that expression
of the D61E variant of AAR in a recombinant host cell skews the
chain length distribution of FALC species toward shorter carbon
chains. Table 9 below illustrates the FALC chain length
distribution produced by recombinant host cells expressing the D61E
variant of MED4_AAR compared to wild type (WT) MED4_AAR and the
V346P variant of MED4_AAR which did not produce any products with
altered chain lengths.
TABLE-US-00012 TABLE 9 Chain length distribution for AAR variants
and wild type AAR Fatty Alcohol MED4 AAR/ADC C14% C16% C18% AAR
WT/ADC(H156R) 6 92 2 AAR(D61E)/ADC(H156R) 14* 85 0
AAR(V346P)/ADC(H156R) 4 92 4 AAR WT/ADC WT ND ND ND AAR(D61E)/ADC
WT ND ND ND AAR(V346P)/ADC WT ND ND ND ND = not detected *1%
variance attributable to deviations from averaging
[0171] These variants can be further recombined and screened for
improvements in the MED4_ADC (H156R) background. The MED4_AAR
(D61E) variant and further mutated progeny may be useful to
decrease the average chain length of both fatty alcohols and
alkanes. The MED4_AAR (D61E) variant demonstrates that MED4_AAR
chain-length specificity is malleable and presents the possibility
for further improvement of this activity through additional protein
engineering efforts. All variants described were sequenced from
progeny of pLS9-195 and contained codon mutations corresponding to
the listed amino acid substitutions.
TABLE-US-00013 TABLE 10 Names Related to Sequence Listing SEQ ID NO
Type Name 1 nucleic acid seq. Nostoc punctiforme PCC 73102_acp
Accession# YP_001867863 2 amino acid seq. Nostoc punctiforme PCC
73102_acp Accession# YP_001867863 3 nucleic acid seq. Synechocystis
sp. PCC 6803_acp Accession # NP_440632.1 4 amino acid seq.
Synechocystis sp. PCC 6803_acp Accession # NP_440632.1 5 nucleic
acid seq. Prochlorococcus marinas subsp. pastoris str. CCMP1986_acp
Accession# NP_893725.1 6 amino acid seq. Prochlorococcus marinas
subsp. pastoris str. CCMP1986_acp Accession# NP_893725.1 7 nucleic
acid seq. Synechococcus elongatus PCC 7942_acp Accession# YP_399555
8 amino acid seq. Synechococcus elongatus PCC 7942_acp Accession#
YP_399555 9 nucleic acid seq. Nostoc sp. PCC 7120_acp Accession#
NP_487382.1 10 amino acid seq. Nostoc sp. PCC 7120_acp Accession#
NP_487382.1 11 nucleic acid seq. B. subtilis sfp (synthesized) as
in accession# X63158.1 12 amino acid seq. B. subtilis sfp
(synthesized) as in accession# X63158.1 13 primer seq. 168IFF 14
primer seq. 168IFR 15 primer seq. 169IFF 16 primer seq. 169IFR 17
primer seq. 170IFF 18 primer seq. 170IFR 19 primer seq. 171IFF 20
primer seq. 171IFR 21 primer seq. 172IFF 22 primer seq. 172IFR 23
primer seq. 168SIFF 24 primer seq. 170S1FF 25 primer seq. 171SIFF
26 primer seq. 168SIFR 27 nucleic acid seq. Synechococcus elongatus
PCC7942 YP_400611 (Synpcc7942_1594) Acyl-CoA Reductase (AAR) 28
nucleic acid seq. Synechococcus elongatus PCC7942 YP_400611
(Synpcc7942_1594) Acyl-CoA Reductase (AAR) 29 nucleic acid seq.
Synechocystis sp. PCC6803 sll0209 (NP_442146) AAR 30 amino acid
seq. Synechocystis sp. PCC6803 sll0209 (NP_442146) AAR 31 nucleic
acid seq. Cyanothece sp. ATCC51142 cce_1430 (YP_001802846) AAR 32
amino acid seq. Cyanothece sp. ATCC51142 cce_1430 (YP_001802846)
AAR 33 nucleic acid seq. Prochlorococcus marinus CCMP1986 PMM0533
(NP_892651) AAR 34 amino acid seq. Prochlorococcus marinus CCMP1986
PMM0533 (NP_892651) AAR 35 nucleic acid seq. Gloeobacter violaceus
PCC7421 NP_96091 (gll3145) AAR 36 amino acid seq. Gloeobacter
violaceus PCC7421 NP_96091 (gll3145) AAR 37 nucleic acid seq.
Nostoc punctiforme PCC73102 ZP_00108837 (Npun02004176) AAR 38 amino
acid seq. Nostoe punctiforme PCC73102 ZP_00108837 (Npun02004176)
AAR 39 nucleic acid seq. Anabaena variabilis ATCC29413 YP_323044
(Ava_2534) AAR 40 amino acid seq. Anabaena variabilis ATCC29413
YP_323044 (Ava_2534) AAR 41 nucleic acid seq. Synechococcus
elongatus PCC6301 YP_170761 (syc0051_d) AAR 42 amino acid seq.
Synechococcus elongatus PCC6301 YP_170761 (syc0051_d) AAR 43
nucleic acid seq. Nostoc sp. PCC7120 alr5284 (NP_489324) AAR 44
amino acid seq. Nostoc sp. PCC7120 alr5284 (NP_489324) AAR 45
nucleic acid seq. birA from Corynebacterium glutamicum (YP_224991)
46 synthetic DNA birA from Corynebacterium glutamicum (YP_224991)
47 amino acid seq. birA from Corynebacterium glutamicum (YP_224991)
48 nucleic acid seq. accDA1 (dtsR) from Corynebacteritun glutamicum
(YP_224991) 49 amino acid seq. accDA1 (dtsR) from Corynebacterium
glutamicum (YP_224991) 50 nucleic acid seq. accCB from
Corynebacterium glutamicum (YP_224991) 51 amino acid seq. accCB
from Corynebacterium glutamicum (YP_224991) 52 amino acid seq. AlrA
Acinetobacter sp. M-1 53 amino acid seq. AlrAadp1 54 amino acid
seq. alrAadp1 Acinetobacter baylyi ADP1-WT Protein 55 nucleic acid
seq. iFAB138 56 nucleic acid seq. FadR from E.coli MG1655
(NP_415705) 57 amino acid seq. AAR Mutant with mutation S18W (made
from Synechococcus elongatus PCC7942 YP_400611 (Synpcc7942_1594)
Acyl-CoA Reductase (AAR))
[0172] As is apparent to one with skill in the art, various
modifications and variations of the above aspects and embodiments
can be made without departing from the spirit and scope of this
disclosure. Such modifications and variations are within the scope
of this disclosure.
Sequence CWU 1
1
801255DNANostoc punctiforme 1atgagccaaa cggaactttt tgaaaaggtc
aagaaaatcg tcatcgaaca actgagtgtt 60gaagatgctt ccaaaatcac tccacaagct
aagtttatgg aagatttagg agctgattcc 120ctggatactg ttgaactcgt
gatggctttg gaagaagaat ttgatatcga aattcccgac 180gaagctgccg
agcagattgt atcggttcaa gacgcagtag attacatcaa taacaaagtt
240gctgcatcag cttaa 255284PRTNostoc punctiforme 2Met Ser Gln Thr
Glu Leu Phe Glu Lys Val Lys Lys Ile Val Ile Glu1 5 10 15Gln Leu Ser
Val Glu Asp Ala Ser Lys Ile Thr Pro Gln Ala Lys Phe 20 25 30Met Glu
Asp Leu Gly Ala Asp Ser Leu Asp Thr Val Glu Leu Val Met 35 40 45Ala
Leu Glu Glu Glu Phe Asp Ile Glu Ile Pro Asp Glu Ala Ala Glu 50 55
60Gln Ile Val Ser Val Gln Asp Ala Val Asp Tyr Ile Asn Asn Lys Val65
70 75 80Ala Ala Ser Ala3234DNASynechocystis sp. 3atgaatcagg
aaatttttga aaaagtaaaa aaaatcgtcg tggaacagtt ggaagtggat 60cctgacaaag
tgacccccga tgccaccttt gccgaagatt taggggctga ttccctcgat
120acagtggaat tggtcatggc cctggaagaa gagtttgata ttgaaattcc
cgatgaagtg 180gcggaaacca ttgataccgt gggcaaagcc gttgagcata
tcgaaagtaa ataa 234477PRTSynechocystis sp. 4Met Asn Gln Glu Ile Phe
Glu Lys Val Lys Lys Ile Val Val Glu Gln1 5 10 15Leu Glu Val Asp Pro
Asp Lys Val Thr Pro Asp Ala Thr Phe Ala Glu 20 25 30Asp Leu Gly Ala
Asp Ser Leu Asp Thr Val Glu Leu Val Met Ala Leu 35 40 45Glu Glu Glu
Phe Asp Ile Glu Ile Pro Asp Glu Val Ala Glu Thr Ile 50 55 60Asp Thr
Val Gly Lys Ala Val Glu His Ile Glu Ser Lys65 70
755243DNAProchlorococcus marinus 5atgtcacaag aagaaatcct tcaaaaagta
tgctctattg tttctgagca actaagtgtt 60gaatcagccg aagtaaaatc tgattcaaac
tttcaaaatg atttaggtgc agactcccta 120gacaccgtag agctagttat
ggctcttgaa gaagcatttg atatcgagat acctgatgaa 180gcagctgaag
gtatcgcaac agtaggagat gctgttaaat tcatcgaaga aaaaaaaggt 240taa
243680PRTProchlorococcus marinus 6Met Ser Gln Glu Glu Ile Leu Gln
Lys Val Cys Ser Ile Val Ser Glu1 5 10 15Gln Leu Ser Val Glu Ser Ala
Glu Val Lys Ser Asp Ser Asn Phe Gln 20 25 30Asn Asp Leu Gly Ala Asp
Ser Leu Asp Thr Val Glu Leu Val Met Ala 35 40 45Leu Glu Glu Ala Phe
Asp Ile Glu Ile Pro Asp Glu Ala Ala Glu Gly 50 55 60Ile Ala Thr Val
Gly Asp Ala Val Lys Phe Ile Glu Glu Lys Lys Gly65 70 75
807243DNASynechococcus elongatus 7atgagccaag aagacatctt cagcaaagtc
aaagacattg tggctgagca gctgagtgtg 60gatgtggctg aagtcaagcc agaatccagc
ttccaaaacg atctgggagc ggactcgctg 120gacaccgtgg aactggtgat
ggctctggaa gaggctttcg atatcgaaat ccccgatgaa 180gccgctgaag
gcattgcgac cgttcaagac gccgtcgatt tcatcgctag caaagctgcc 240tag
243880PRTSynechococcus elongatus 8Met Ser Gln Glu Asp Ile Phe Ser
Lys Val Lys Asp Ile Val Ala Glu1 5 10 15Gln Leu Ser Val Asp Val Ala
Glu Val Lys Pro Glu Ser Ser Phe Gln 20 25 30Asn Asp Leu Gly Ala Asp
Ser Leu Asp Thr Val Glu Leu Val Met Ala 35 40 45Leu Glu Glu Ala Phe
Asp Ile Glu Ile Pro Asp Glu Ala Ala Glu Gly 50 55 60Ile Ala Thr Val
Gln Asp Ala Val Asp Phe Ile Ala Ser Lys Ala Ala65 70 75
809255DNANostoc sp. 9atgagccaat cagaaacttt tgaaaaagtc aaaaaaattg
ttatcgaaca actaagtgtg 60gagaaccctg acacagtaac tccagaagct agttttgcca
acgatttaca ggctgattcc 120ctcgatacag tagaactagt aatggctttg
gaagaagaat ttgatatcga aattcccgat 180gaagccgcag agaaaattac
cactgttcaa gaagcggtgg attacatcaa taaccaagtt 240gccgcatcag cttaa
2551084PRTNostoc sp. 10Met Ser Gln Ser Glu Thr Phe Glu Lys Val Lys
Lys Ile Val Ile Glu1 5 10 15Gln Leu Ser Val Glu Asn Pro Asp Thr Val
Thr Pro Glu Ala Ser Phe 20 25 30Ala Asn Asp Leu Gln Ala Asp Ser Leu
Asp Thr Val Glu Leu Val Met 35 40 45Ala Leu Glu Glu Glu Phe Asp Ile
Glu Ile Pro Asp Glu Ala Ala Glu 50 55 60Lys Ile Thr Thr Val Gln Glu
Ala Val Asp Tyr Ile Asn Asn Gln Val65 70 75 80Ala Ala Ser
Ala11674DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 11atgaagattt acggaattta tatggaccgc
ccgctttcac aggaagaaaa tgaacggttc 60atgactttca tatcacctga aaaacgggag
aaatgccgga gattttatca taaagaagat 120gctcaccgca ccctgctggg
agatgtgctc gttcgctcag tcataagcag gcagtatcag 180ttggacaaat
ccgatatccg ctttagcacg caggaatacg ggaagccgtg catccctgat
240cttcccgacg ctcatttcaa catttctcac tccggccgct gggtcattgg
tgcgtttgat 300tcacagccga tcggcataga tatcgaaaaa acgaaaccga
tcagccttga gatcgccaag 360cgcttctttt caaaaacaga gtacagcgac
cttttagcaa aagacaagga cgagcagaca 420gactattttt atcatctatg
gtcaatgaaa gaaagcttta tcaaacagga aggcaaaggc 480ttatcgcttc
cgcttgattc cttttcagtg cgcctgcatc aggacggaca agtatccatt
540gagcttccgg acagccattc cccatgctat atcaaaacgt atgaggtcga
tcccggctac 600aaaatggctg tatgcgccgc acaccctgtt tccccgagga
tatcacaatg gtctcgtacg 660aagagctttt ataa 67412224PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
12Met Lys Ile Tyr Gly Ile Tyr Met Asp Arg Pro Leu Ser Gln Glu Glu1
5 10 15Asn Glu Arg Phe Met Thr Phe Ile Ser Pro Glu Lys Arg Glu Lys
Cys 20 25 30Arg Arg Phe Tyr His Lys Glu Asp Ala His Arg Thr Leu Leu
Gly Asp 35 40 45Val Leu Val Arg Ser Val Ile Ser Arg Gln Tyr Gln Leu
Asp Lys Ser 50 55 60Asp Ile Arg Phe Ser Thr Gln Glu Tyr Gly Lys Pro
Cys Ile Pro Asp65 70 75 80Leu Pro Asp Ala His Phe Asn Ile Ser His
Ser Gly Arg Trp Val Ile 85 90 95Gly Ala Phe Asp Ser Gln Pro Ile Gly
Ile Asp Ile Glu Lys Thr Lys 100 105 110Pro Ile Ser Leu Glu Ile Ala
Lys Arg Phe Phe Ser Lys Thr Glu Tyr 115 120 125Ser Asp Leu Leu Ala
Lys Asp Lys Asp Glu Gln Thr Asp Tyr Phe Tyr 130 135 140His Leu Trp
Ser Met Lys Glu Ser Phe Ile Lys Gln Glu Gly Lys Gly145 150 155
160Leu Ser Leu Pro Leu Asp Ser Phe Ser Val Arg Leu His Gln Asp Gly
165 170 175Gln Val Ser Ile Glu Leu Pro Asp Ser His Ser Pro Cys Tyr
Ile Lys 180 185 190Thr Tyr Glu Val Asp Pro Gly Tyr Lys Met Ala Val
Cys Ala Ala His 195 200 205Pro Asp Phe Pro Glu Asp Ile Thr Met Val
Ser Tyr Glu Glu Leu Leu 210 215 2201352DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
13ggcaatttga gaatttaagg aggaaaacaa aatgagccaa gaagacatct tc
521433DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 14cccaagcttc gaattcctag gcagctttgc tag
331556DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 15ggcaatttga gaatttaagg aggaaaacaa aatgaatcag
gaaatttttg aaaaag 561641DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 16cccaagcttc gaattcttat
ttactttcga tatgctcaac g 411753DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 17ggcaatttga gaatttaagg
aggaaaacaa aatgtcacaa gaagaaatcc ttc 531846DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
18cccaagcttc gaattcttaa cctttttttt cttcgatgaa tttaac
461953DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 19ggcaatttga gaatttaagg aggaaaacaa aatgagccaa
acggaacttt ttg 532038DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 20cccaagcttc gaattcttaa
gctgatgcag caactttg 382153DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 21ggcaatttga gaatttaagg
aggaaaacaa aatgagccaa tcagaaactt ttg 532234DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
22cccaagcttc gaattcttaa gctgatgcgg caac 342343DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
23agctgcctag gaatttaagg aggaataaac catgaagatt tac
432443DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 24aaaaggttaa gaatttaagg aggaataaac catgaagatt tac
432543DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 25atcagcttaa gaatttaagg aggaataaac catgaagatt tac
432641DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 26cccaagcttc gaattcttat aaaagctctt cgtacgagac c
41271029DNASynechococcus elongatus 27atggcattcg gtcttatcgg
tcatctcacc agtttggagc aggcccgcga cgtttctcgc 60aggatgggct acgacgaata
cgccgatcaa ggattggagt tttggagtag cgctcctcct 120caaatcgttg
atgaaatcac agtcaccagt gccacaggca aggtgattca cggtcgctac
180atcgaatcgt gtttcttgcc ggaaatgctg gcggcgcgcc gcttcaaaac
agccacgcgc 240aaagttctca atgccatgtc ccatgcccaa aaacacggca
tcgacatctc ggccttgggg 300ggctttacct cgattatttt cgagaatttc
gatttggcca gtttgcggca agtgcgcgac 360actaccttgg agtttgaacg
gttcaccacc ggcaatactc acacggccta cgtaatctgt 420agacaggtgg
aagccgctgc taaaacgctg ggcatcgaca ttacccaagc gacagtagcg
480gttgtcggcg cgactggcga tatcggtagc gctgtctgcc gctggctcga
cctcaaactg 540ggtgtcggtg atttgatcct gacggcgcgc aatcaggagc
gtttggataa cctgcaggct 600gaactcggcc ggggcaagat tctgcccttg
gaagccgctc tgccggaagc tgactttatc 660gtgtgggtcg ccagtatgcc
tcagggcgta gtgatcgacc cagcaaccct gaagcaaccc 720tgcgtcctaa
tcgacggggg ctaccccaaa aacttgggca gcaaagtcca aggtgagggc
780atctatgtcc tcaatggcgg ggtagttgaa cattgcttcg acatcgactg
gcagatcatg 840tccgctgcag agatggcgcg gcccgagcgc cagatgtttg
cctgctttgc cgaggcgatg 900ctcttggaat ttgaaggctg gcatactaac
ttctcctggg gccgcaacca aatcacgatc 960gagaagatgg aagcgatcgg
tgaggcatcg gtgcgccacg gcttccaacc cttggcattg 1020gcaatttga
102928342PRTSynechococcus elongatus 28Met Ala Phe Gly Leu Ile Gly
His Leu Thr Ser Leu Glu Gln Ala Arg1 5 10 15Asp Val Ser Arg Arg Met
Gly Tyr Asp Glu Tyr Ala Asp Gln Gly Leu 20 25 30Glu Phe Trp Ser Ser
Ala Pro Pro Gln Ile Val Asp Glu Ile Thr Val 35 40 45Thr Ser Ala Thr
Gly Lys Val Ile His Gly Arg Tyr Ile Glu Ser Cys 50 55 60Phe Leu Pro
Glu Met Leu Ala Ala Arg Arg Phe Lys Thr Ala Thr Arg65 70 75 80Lys
Val Leu Asn Ala Met Ser His Ala Gln Lys His Gly Ile Asp Ile 85 90
95Ser Ala Leu Gly Gly Phe Thr Ser Ile Ile Phe Glu Asn Phe Asp Leu
100 105 110Ala Ser Leu Arg Gln Val Arg Asp Thr Thr Leu Glu Phe Glu
Arg Phe 115 120 125Thr Thr Gly Asn Thr His Thr Ala Tyr Val Ile Cys
Arg Gln Val Glu 130 135 140Ala Ala Ala Lys Thr Leu Gly Ile Asp Ile
Thr Gln Ala Thr Val Ala145 150 155 160Val Val Gly Ala Thr Gly Asp
Ile Gly Ser Ala Val Cys Arg Trp Leu 165 170 175Asp Leu Lys Leu Gly
Val Gly Asp Leu Ile Leu Thr Ala Arg Asn Gln 180 185 190Glu Arg Leu
Asp Asn Leu Gln Ala Glu Leu Gly Arg Gly Lys Ile Leu 195 200 205Pro
Leu Glu Ala Ala Leu Pro Glu Ala Asp Phe Ile Val Trp Val Ala 210 215
220Ser Met Pro Gln Gly Val Val Ile Asp Pro Ala Thr Leu Lys Gln
Pro225 230 235 240Cys Val Leu Ile Asp Gly Gly Tyr Pro Lys Asn Leu
Gly Ser Lys Val 245 250 255Gln Gly Glu Gly Ile Tyr Val Leu Asn Gly
Gly Val Val Glu His Cys 260 265 270Phe Asp Ile Asp Trp Gln Ile Met
Ser Ala Ala Glu Met Ala Arg Pro 275 280 285Glu Arg Gln Met Phe Ala
Cys Phe Ala Glu Ala Met Leu Leu Glu Phe 290 295 300Glu Gly Trp His
Thr Asn Phe Ser Trp Gly Arg Asn Gln Ile Thr Ile305 310 315 320Glu
Lys Met Glu Ala Ile Gly Glu Ala Ser Val Arg His Gly Phe Gln 325 330
335Pro Leu Ala Leu Ala Ile 340291023DNASynechocystis sp.
29atgtttggtc ttattggtca tctcacgagt ttagaacacg cccaagcggt tgctgaagat
60ttaggctatc ctgagtacgc caaccaaggc ctggattttt ggtgttcggc tcctccccaa
120gtggttgata attttcaggt gaaaagtgtg acggggcagg tgattgaagg
caaatatgtg 180gagtcttgct ttttgccgga aatgttaacc caacggcgga
tcaaagcggc cattcgtaaa 240atcctcaatg ctatggccct ggcccaaaag
gtgggcttgg atattacggc cctgggaggc 300ttttcttcaa tcgtatttga
agaatttaac ctcaagcaaa ataatcaagt ccgcaatgtg 360gaactagatt
ttcagcggtt caccactggt aatacccaca ccgcttatgt gatctgccgt
420caggtcgagt ctggagctaa acagttgggt attgatctaa gtcaggcaac
ggtagcggtt 480tgtggcgcca cgggagatat tggtagcgcc gtatgtcgtt
ggttagatag caaacatcaa 540gttaaggaat tattgctaat tgcccgtaac
cgccaaagat tggaaaatct ccaagaggaa 600ttgggtcggg gcaaaattat
ggatttggaa acagccctgc cccaggcaga tattattgtt 660tgggtggcta
gtatgcccaa gggggtagaa attgcggggg aaatgctgaa aaagccctgt
720ttgattgtgg atgggggcta tcccaagaat ttagacacca gggtgaaagc
ggatggggtg 780catattctca agggggggat tgtagaacat tcccttgata
ttacctggga aattatgaag 840attgtggaga tggatattcc ctcccggcaa
atgttcgcct gttttgcgga ggccattttg 900ctagagtttg agggctggcg
cactaatttt tcctggggcc gcaaccaaat ttccgttaat 960aaaatggagg
cgattggtga agcttctgtc aagcatggct tttgcccttt agtagctctt 1020tag
102330340PRTSynechocystis sp. 30Met Phe Gly Leu Ile Gly His Leu Thr
Ser Leu Glu His Ala Gln Ala1 5 10 15Val Ala Glu Asp Leu Gly Tyr Pro
Glu Tyr Ala Asn Gln Gly Leu Asp 20 25 30Phe Trp Cys Ser Ala Pro Pro
Gln Val Val Asp Asn Phe Gln Val Lys 35 40 45Ser Val Thr Gly Gln Val
Ile Glu Gly Lys Tyr Val Glu Ser Cys Phe 50 55 60Leu Pro Glu Met Leu
Thr Gln Arg Arg Ile Lys Ala Ala Ile Arg Lys65 70 75 80Ile Leu Asn
Ala Met Ala Leu Ala Gln Lys Val Gly Leu Asp Ile Thr 85 90 95Ala Leu
Gly Gly Phe Ser Ser Ile Val Phe Glu Glu Phe Asn Leu Lys 100 105
110Gln Asn Asn Gln Val Arg Asn Val Glu Leu Asp Phe Gln Arg Phe Thr
115 120 125Thr Gly Asn Thr His Thr Ala Tyr Val Ile Cys Arg Gln Val
Glu Ser 130 135 140Gly Ala Lys Gln Leu Gly Ile Asp Leu Ser Gln Ala
Thr Val Ala Val145 150 155 160Cys Gly Ala Thr Gly Asp Ile Gly Ser
Ala Val Cys Arg Trp Leu Asp 165 170 175Ser Lys His Gln Val Lys Glu
Leu Leu Leu Ile Ala Arg Asn Arg Gln 180 185 190Arg Leu Glu Asn Leu
Gln Glu Glu Leu Gly Arg Gly Lys Ile Met Asp 195 200 205Leu Glu Thr
Ala Leu Pro Gln Ala Asp Ile Ile Val Trp Val Ala Ser 210 215 220Met
Pro Lys Gly Val Glu Ile Ala Gly Glu Met Leu Lys Lys Pro Cys225 230
235 240Leu Ile Val Asp Gly Gly Tyr Pro Lys Asn Leu Asp Thr Arg Val
Lys 245 250 255Ala Asp Gly Val His Ile Leu Lys Gly Gly Ile Val Glu
His Ser Leu 260 265 270Asp Ile Thr Trp Glu Ile Met Lys Ile Val Glu
Met Asp Ile Pro Ser 275 280 285Arg Gln Met Phe Ala Cys Phe Ala Glu
Ala Ile Leu Leu Glu Phe Glu 290 295 300Gly Trp Arg Thr Asn Phe Ser
Trp Gly Arg Asn Gln Ile Ser Val Asn305 310 315 320Lys Met Glu Ala
Ile Gly Glu Ala Ser Val Lys His Gly Phe Cys Pro 325 330 335Leu Val
Ala Leu 340311023DNACyanothece sp. 31atgtttggtt taattggtca
tcttacaagt ttagaacacg cccactccgt tgctgatgcc 60tttggctatg gcccatacgc
cactcaggga cttgatttgt ggtgttctgc tccaccccaa 120ttcgtcgagc
attttcatgt tactagcatc acaggacaaa ccatcgaagg aaagtatata
180gaatccgctt tcttaccaga aatgctgata aagcgacgga ttaaagcagc
aattcgcaaa 240atactgaatg cgatggcctt tgctcagaaa aataacctta
acatcacagc attagggggc 300ttttcttcga ttatttttga agaatttaat
ctcaaagaga atagacaagt tcgtaatgtc 360tctttagagt ttgatcgctt
caccaccgga aacacccata ctgcttatat catttgtcgt 420caagttgaac
aggcatccgc taaactaggg attgacttat cccaagcaac ggttgctatt
480tgcggggcaa ccggagatat tggcagtgca gtgtgtcgtt ggttagatag
aaaaaccgat 540acccaggaac tattcttaat tgctcgcaat aaagaacgat
tacaacgact gcaagatgag 600ttgggacggg gtaaaattat gggattggag
gaggctttac ccgaagcaga tattatcgtt 660tgggtggcga gtatgcccaa
aggagtggaa attaatgccg aaactctcaa aaaaccctgt 720ttaattatcg
atggtggtta tcctaagaat ttagacacaa aaattaaaca tcctgatgtc
780catatcctga aagggggaat tgtagaacat tctctagata ttgactggaa
gattatggaa 840actgtcaata tggatgttcc ttctcgtcaa atgtttgctt
gttttgccga agccatttta 900ttagagtttg aacaatggca cactaatttt
tcttggggac gcaatcaaat tacagtgact 960aaaatggaac aaataggaga
agcttctgtc aaacatgggt tacaaccgtt gttgagttgg 1020taa
102332340PRTCyanothece sp. 32Met Phe Gly Leu Ile Gly His Leu Thr
Ser Leu Glu His Ala His Ser1 5 10 15Val Ala Asp Ala Phe Gly Tyr Gly
Pro Tyr Ala Thr Gln Gly Leu Asp 20 25 30Leu Trp Cys Ser Ala Pro Pro
Gln Phe Val Glu His Phe His Val Thr 35 40 45Ser Ile Thr Gly Gln Thr
Ile Glu Gly Lys Tyr Ile Glu Ser Ala Phe 50 55 60Leu Pro Glu Met Leu
Ile Lys Arg Arg Ile Lys Ala Ala Ile Arg Lys65 70 75 80Ile Leu Asn
Ala Met Ala Phe Ala Gln Lys Asn Asn Leu Asn Ile Thr 85 90 95Ala Leu
Gly Gly Phe Ser Ser Ile Ile Phe Glu Glu Phe Asn Leu Lys 100 105
110Glu Asn Arg Gln Val Arg Asn Val Ser Leu Glu Phe Asp Arg Phe Thr
115 120 125Thr Gly Asn Thr His Thr Ala Tyr Ile Ile Cys Arg Gln Val
Glu Gln 130 135 140Ala Ser Ala Lys Leu Gly Ile Asp Leu Ser Gln Ala
Thr Val Ala Ile145 150 155 160Cys Gly Ala Thr Gly Asp Ile Gly Ser
Ala Val Cys Arg Trp Leu Asp 165 170 175Arg Lys Thr Asp Thr Gln Glu
Leu Phe Leu Ile Ala Arg Asn Lys Glu 180 185 190Arg Leu Gln Arg Leu
Gln Asp Glu Leu Gly Arg Gly Lys Ile Met Gly 195 200 205Leu Glu Glu
Ala Leu Pro Glu Ala Asp Ile Ile Val Trp Val Ala Ser 210 215 220Met
Pro Lys Gly Val Glu Ile Asn Ala Glu Thr Leu Lys Lys Pro Cys225 230
235 240Leu Ile Ile Asp Gly Gly Tyr Pro Lys Asn Leu Asp Thr Lys Ile
Lys 245 250 255His Pro Asp Val His Ile Leu Lys Gly Gly Ile Val Glu
His Ser Leu 260 265 270Asp Ile Asp Trp Lys Ile Met Glu Thr Val Asn
Met Asp Val Pro Ser 275 280 285Arg Gln Met Phe Ala Cys Phe Ala Glu
Ala Ile Leu Leu Glu Phe Glu 290 295 300Gln Trp His Thr Asn Phe Ser
Trp Gly Arg Asn Gln Ile Thr Val Thr305 310 315 320Lys Met Glu Gln
Ile Gly Glu Ala Ser Val Lys His Gly Leu Gln Pro 325 330 335Leu Leu
Ser Trp 340331044DNAProchlorococcus marinus 33atggcatttg ggcttatagg
tcattcaact agttttgaag atgcaaaaag aaaggcttca 60ttattgggct ttgatcatat
tgcggatggt gatttagatg tttggtgcac agctccacct 120caactagttg
aaaatgtaga ggttaaaagt gctataggta tatcaattga aggttcttat
180attgattcat gtttcgttcc tgaaatgctt tcaagattta aaacggcaag
aagaaaagta 240ttaaatgcaa tggaattagc tcaaaaaaaa ggtattaata
ttaccgcttt gggggggttc 300acttctatca tctttgaaaa ttttaatctc
cttcaacata agcagattag aaacacttca 360ctagagtggg aaaggtttac
aactggtaat actcatactg cgtgggttat ttgcaggcaa 420ttagagatga
atgctcctaa aataggtatt gatcttaaaa gcgcaacagt tgctgtagtt
480ggtgctactg gagatatagg cagtgctgtt tgtcgatggt taatcaataa
aacaggtatt 540ggggaacttc ttttggtagc taggcaaaag gaacccttgg
attctttgca aaaggaatta 600gatggtggaa ctatcaaaaa tctagatgaa
gcattgcctg aagcagatat tgttgtatgg 660gtagcaagta tgccaaagac
aatggaaatc gatgctaata atcttaaaca accatgttta 720atgattgatg
gaggttatcc aaagaatcta gatgaaaaat ttcaaggaaa taatatacat
780gttgtaaaag gaggtatagt aagattcttc aatgatatag gttggaatat
gatggaacta 840gctgaaatgc aaaatcccca gagagaaatg tttgcatgct
ttgcagaagc aatgatttta 900gaatttgaaa aatgtcatac aaactttagc
tggggaagaa ataatatatc tctcgagaaa 960atggagttta ttggagctgc
ttctgtaaag catggcttct ctgcaattgg cctagataag 1020catccaaaag
tactagcagt ttga 104434347PRTProchlorococcus marinus 34Met Ala Phe
Gly Leu Ile Gly His Ser Thr Ser Phe Glu Asp Ala Lys1 5 10 15Arg Lys
Ala Ser Leu Leu Gly Phe Asp His Ile Ala Asp Gly Asp Leu 20 25 30Asp
Val Trp Cys Thr Ala Pro Pro Gln Leu Val Glu Asn Val Glu Val 35 40
45Lys Ser Ala Ile Gly Ile Ser Ile Glu Gly Ser Tyr Ile Asp Ser Cys
50 55 60Phe Val Pro Glu Met Leu Ser Arg Phe Lys Thr Ala Arg Arg Lys
Val65 70 75 80Leu Asn Ala Met Glu Leu Ala Gln Lys Lys Gly Ile Asn
Ile Thr Ala 85 90 95Leu Gly Gly Phe Thr Ser Ile Ile Phe Glu Asn Phe
Asn Leu Leu Gln 100 105 110His Lys Gln Ile Arg Asn Thr Ser Leu Glu
Trp Glu Arg Phe Thr Thr 115 120 125Gly Asn Thr His Thr Ala Trp Val
Ile Cys Arg Gln Leu Glu Met Asn 130 135 140Ala Pro Lys Ile Gly Ile
Asp Leu Lys Ser Ala Thr Val Ala Val Val145 150 155 160Gly Ala Thr
Gly Asp Ile Gly Ser Ala Val Cys Arg Trp Leu Ile Asn 165 170 175Lys
Thr Gly Ile Gly Glu Leu Leu Leu Val Ala Arg Gln Lys Glu Pro 180 185
190Leu Asp Ser Leu Gln Lys Glu Leu Asp Gly Gly Thr Ile Lys Asn Leu
195 200 205Asp Glu Ala Leu Pro Glu Ala Asp Ile Val Val Trp Val Ala
Ser Met 210 215 220Pro Lys Thr Met Glu Ile Asp Ala Asn Asn Leu Lys
Gln Pro Cys Leu225 230 235 240Met Ile Asp Gly Gly Tyr Pro Lys Asn
Leu Asp Glu Lys Phe Gln Gly 245 250 255Asn Asn Ile His Val Val Lys
Gly Gly Ile Val Arg Phe Phe Asn Asp 260 265 270Ile Gly Trp Asn Met
Met Glu Leu Ala Glu Met Gln Asn Pro Gln Arg 275 280 285Glu Met Phe
Ala Cys Phe Ala Glu Ala Met Ile Leu Glu Phe Glu Lys 290 295 300Cys
His Thr Asn Phe Ser Trp Gly Arg Asn Asn Ile Ser Leu Glu Lys305 310
315 320Met Glu Phe Ile Gly Ala Ala Ser Val Lys His Gly Phe Ser Ala
Ile 325 330 335Gly Leu Asp Lys His Pro Lys Val Leu Ala Val 340
345351053DNAGloeobacter violaceus 35atgtttggcc tgatcggaca
cttgaccaat ctttcccatg cccagcgggt cgcccgcgac 60ctgggctacg acgagtatgc
aagccacgac ctcgaattct ggtgcatggc ccctccccag 120gcggtcgatg
aaatcacgat caccagcgtc accggtcagg tgatccacgg tcagtacgtc
180gaatcgtgct ttctgccgga gatgctcgcc cagggccgct tcaagaccgc
catgcgcaag 240atcctcaatg ccatggccct ggtccagaag cgcggcatcg
acattacggc cctgggaggc 300ttctcgtcga tcatcttcga gaatttcagc
ctcgataaat tgctcaacgt ccgcgacatc 360accctcgaca tccagcgctt
caccaccggc aacacccaca cggcctacat cctttgtcag 420caggtcgagc
agggtgcggt acgctacggc atcgatccgg ccaaagcgac cgtggcggta
480gtcggggcca ccggcgacat cggtagcgcc gtctgccgat ggctcaccga
ccgcgccggc 540atccacgaac tcttgctggt ggcccgcgac gccgaaaggc
tcgaccggct gcagcaggaa 600ctcggcaccg gtcggatcct gccggtcgaa
gaagcacttc ccaaagccga catcgtcgtc 660tgggtcgcct cgatgaacca
gggcatggcc atcgaccccg ccggcctgcg caccccctgc 720ctgctcatcg
acggcggcta ccccaagaac atggccggca ccctgcagcg cccgggcatc
780catatcctcg acggcggcat ggtcgagcac tcgctcgaca tcgactggca
gatcatgtcg 840tttctaaatg tgcccaaccc cgcccgccag ttcttcgcct
gcttcgccga gtcgatgctg 900ctggaattcg aagggcttca cttcaatttt
tcctggggcc gcaaccacat caccgtcgag 960aagatggccc agatcggctc
gctgtctaaa aaacatggct ttcgtcccct gcttgaaccc 1020agtcagcgca
gcggcgaact cgtacacgga taa 105336350PRTGloeobacter violaceus 36Met
Phe Gly Leu Ile Gly His Leu Thr Asn Leu Ser His Ala Gln Arg1 5 10
15Val Ala Arg Asp Leu Gly Tyr Asp Glu Tyr Ala Ser His Asp Leu Glu
20 25 30Phe Trp Cys Met Ala Pro Pro Gln Ala Val Asp Glu Ile Thr Ile
Thr 35 40 45Ser Val Thr Gly Gln Val Ile His Gly Gln Tyr Val Glu Ser
Cys Phe 50 55 60Leu Pro Glu Met Leu Ala Gln Gly Arg Phe Lys Thr Ala
Met Arg Lys65 70 75 80Ile Leu Asn Ala Met Ala Leu Val Gln Lys Arg
Gly Ile Asp Ile Thr 85 90 95Ala Leu Gly Gly Phe Ser Ser Ile Ile Phe
Glu Asn Phe Ser Leu Asp 100 105 110Lys Leu Leu Asn Val Arg Asp Ile
Thr Leu Asp Ile Gln Arg Phe Thr 115 120 125Thr Gly Asn Thr His Thr
Ala Tyr Ile Leu Cys Gln Gln Val Glu Gln 130 135 140Gly Ala Val Arg
Tyr Gly Ile Asp Pro Ala Lys Ala Thr Val Ala Val145 150 155 160Val
Gly Ala Thr Gly Asp Ile Gly Ser Ala Val Cys Arg Trp Leu Thr 165 170
175Asp Arg Ala Gly Ile His Glu Leu Leu Leu Val Ala Arg Asp Ala Glu
180 185 190Arg Leu Asp Arg Leu Gln Gln Glu Leu Gly Thr Gly Arg Ile
Leu Pro 195 200 205Val Glu Glu Ala Leu Pro Lys Ala Asp Ile Val Val
Trp Val Ala Ser 210 215 220Met Asn Gln Gly Met Ala Ile Asp Pro Ala
Gly Leu Arg Thr Pro Cys225 230 235 240Leu Leu Ile Asp Gly Gly Tyr
Pro Lys Asn Met Ala Gly Thr Leu Gln 245 250 255Arg Pro Gly Ile His
Ile Leu Asp Gly Gly Met Val Glu His Ser Leu 260 265 270Asp Ile Asp
Trp Gln Ile Met Ser Phe Leu Asn Val Pro Asn Pro Ala 275 280 285Arg
Gln Phe Phe Ala Cys Phe Ala Glu Ser Met Leu Leu Glu Phe Glu 290 295
300Gly Leu His Phe Asn Phe Ser Trp Gly Arg Asn His Ile Thr Val
Glu305 310 315 320Lys Met Ala Gln Ile Gly Ser Leu Ser Lys Lys His
Gly Phe Arg Pro 325 330 335Leu Leu Glu Pro Ser Gln Arg Ser Gly Glu
Leu Val His Gly 340 345 350371020DNANostoc punctiforme 37atgtttggtc
taattggaca tctgactagt ttagaacacg ctcaagccgt agcccaagaa 60ttgggatacc
cagaatatgc cgatcaaggg ctagactttt ggtgcagcgc cccgccgcaa
120attgtcgata gtattattgt caccagtgtt actgggcaac aaattgaagg
acgatatgta 180gaatcttgct ttttgccgga aatgctagct agtcgccgca
tcaaagccgc aacacggaaa 240atcctcaacg ctatggccca tgcacagaag
cacggcatta acatcacagc tttaggcgga 300ttttcctcga ttatttttga
aaactttaag ttagagcagt ttagccaagt ccgaaatatc 360aagctagagt
ttgaacgctt caccacagga aacacgcata ctgcctacat tatttgtaag
420caggtggaag aagcatccaa acaactggga attaatctat caaacgcgac
tgttgcggta 480tgtggagcaa ctggggatat tggtagtgcc gttacacgct
ggctagatgc gagaacagat 540gtccaagaac tcctgctaat cgcccgcgat
caagaacgtc tcaaagagtt gcaaggcgaa 600ctggggcggg ggaaaatcat
gggtttgaca gaagcactac cccaagccga tgttgtagtt 660tgggttgcta
gtatgcccag aggcgtggaa attgacccca ccactttgaa acaaccctgt
720ttgttgattg atggtggcta tcctaaaaac ttagcaacaa aaattcaata
tcctggcgta 780cacgtgttaa atggtgggat tgtagagcat tccctggata
ttgactggaa aattatgaaa 840atagtcaata tggacgtgcc agcccgtcag
ttgtttgcct gttttgccga atcaatgcta 900ctggaatttg agaagttata
cacgaacttt tcgtggggac ggaatcagat taccgtagat 960aaaatggagc
agattggccg ggtgtcagta aaacatggat ttagaccgtt gttggtttag
102038339PRTNostoc punctiforme 38Met Phe Gly Leu Ile Gly His Leu
Thr Ser Leu Glu His Ala Gln Ala1 5 10 15Val Ala Gln Glu Leu Gly Tyr
Pro Glu Tyr Ala Asp Gln Gly Leu Asp 20 25 30Phe Trp Cys Ser Ala Pro
Pro Gln Ile Val Asp Ser Ile Ile Val Thr 35 40 45Ser Val Thr Gly Gln
Gln Ile Glu Gly Arg Tyr Val Glu Ser Cys Phe 50 55 60Leu Pro Glu Met
Leu Ala Ser Arg Arg Ile Lys Ala Ala Thr Arg Lys65 70 75 80Ile Leu
Asn Ala Met Ala His Ala Gln Lys His Gly Ile Asn Ile Thr 85 90 95Ala
Leu Gly Gly Phe Ser Ser Ile Ile Phe Glu Asn Phe Lys Leu Glu 100 105
110Gln Phe Ser Gln Val Arg Asn Ile Lys Leu Glu Phe Glu Arg Phe Thr
115 120 125Thr Gly Asn Thr His Thr Ala Tyr Ile Ile Cys Lys Gln Val
Glu Glu 130 135 140Ala Ser Lys Gln Leu Gly Ile Asn Leu Ser Asn Ala
Thr Val Ala Val145 150 155 160Cys Gly Ala Thr Gly Asp Ile Gly Ser
Ala Val Thr Arg Trp Leu Asp 165 170 175Ala Arg Thr Asp Val Gln Glu
Leu Leu Leu Ile Ala Arg Asp Gln Glu 180 185 190Arg Leu Lys Glu Leu
Gln Gly Glu Leu Gly Arg Gly Lys Ile Met Gly 195 200 205Leu Thr Glu
Ala Leu Pro Gln Ala Asp Val Val Val Trp Val Ala Ser 210 215 220Met
Pro Arg Gly Val Glu Ile Asp Pro Thr Thr Leu Lys Gln Pro Cys225 230
235 240Leu Leu Ile Asp Gly Gly Tyr Pro Lys Asn Leu Ala Thr Lys Ile
Gln 245 250 255Tyr Pro Gly Val His Val Leu Asn Gly Gly Ile Val Glu
His Ser Leu 260 265 270Asp Ile Asp Trp Lys Ile Met Lys Ile Val Asn
Met Asp Val Pro Ala 275 280 285Arg Gln Leu Phe Ala Cys Phe Ala Glu
Ser Met Leu Leu Glu Phe Glu 290 295 300Lys Leu Tyr Thr Asn Phe Ser
Trp Gly Arg Asn Gln Ile Thr Val Asp305 310 315 320Lys Met Glu Gln
Ile Gly Arg Val Ser Val Lys His Gly Phe Arg Pro 325 330 335Leu Leu
Val391020DNAAnabaena variabilis 39atgtttggtc taattggaca tctgacaagt
ttagaacacg ctcaagcggt agctcaagaa 60ctgggatacc cagaatacgc cgaccaaggg
ctagattttt ggtgcagcgc tccaccgcaa 120atagttgacc acattaaagt
tactagcatt actggtgaaa taattgaagg gaggtatgta 180gaatcttgct
ttttaccaga aatgctagcc agccgtagga ttaaagccgc aacccgcaaa
240gtcctcaatg ctatggctca tgctcaaaaa catggcattg acatcaccgc
tttgggtggt 300ttctcctcca ttatttttga aaacttcaaa ttggaacagt
ttagccaagt tcgtaatgtc 360acactagagt ttgaacgctt cactacaggc
aacactcaca cagcttatat catttgtcgg 420caggtagaac aagcatcaca
acaactcggc attgaactct cccaagcaac agtagctata 480tgtggggcta
ctggtgacat tggtagtgca gttactcgct ggctggatgc caaaacagac
540gtaaaagaat tactgttaat cgcccgtaat caagaacgtc tccaagagtt
gcaaagcgag 600ttgggacgcg gtaaaatcat gagcctagat gaagcattgc
ctcaagctga tattgtagtt 660tgggtagcta gtatgcctaa aggcgtggaa
attaatcctc aagttttgaa acaaccctgt 720ttattgattg atggtggtta
tccgaaaaac ttgggtacaa aagttcagta tcctggtgtt 780tatgtactga
acggaggtat cgtcgaacat tccctagata ttgactggaa aatcatgaaa
840atagtcaata tggatgtacc tgcacgccaa ttatttgctt gttttgcgga
atctatgctc 900ttggaatttg agaagttgta cacgaacttt tcttgggggc
gcaatcagat taccgtagac 960aaaatggagc agattggtca agcatcagtg
aaacatgggt ttagaccact gctggtttag 102040339PRTAnabaena variabilis
40Met Phe Gly Leu Ile Gly His Leu Thr Ser Leu Glu His Ala Gln Ala1
5 10 15Val Ala Gln Glu Leu Gly Tyr Pro Glu Tyr Ala Asp Gln Gly Leu
Asp 20 25 30Phe Trp Cys Ser Ala Pro Pro Gln Ile Val Asp His Ile Lys
Val Thr 35 40 45Ser Ile Thr Gly Glu Ile Ile Glu Gly Arg Tyr Val Glu
Ser Cys Phe 50 55 60Leu Pro Glu Met Leu Ala Ser Arg Arg Ile Lys Ala
Ala Thr Arg Lys65 70 75 80Val Leu Asn Ala Met Ala His Ala Gln Lys
His Gly Ile Asp Ile Thr 85 90 95Ala Leu Gly Gly Phe Ser Ser Ile Ile
Phe Glu Asn Phe Lys Leu Glu 100 105 110Gln Phe Ser Gln Val Arg Asn
Val Thr Leu Glu Phe Glu Arg Phe Thr 115 120 125Thr Gly Asn Thr His
Thr Ala Tyr Ile Ile Cys Arg Gln Val Glu Gln 130 135 140Ala Ser Gln
Gln Leu Gly Ile Glu Leu Ser Gln Ala Thr Val Ala Ile145 150 155
160Cys Gly Ala Thr Gly Asp Ile Gly Ser Ala Val Thr Arg Trp Leu Asp
165 170 175Ala Lys Thr Asp Val Lys Glu Leu Leu Leu Ile Ala Arg Asn
Gln Glu 180 185 190Arg Leu Gln Glu Leu Gln Ser Glu Leu Gly Arg Gly
Lys Ile Met Ser 195 200 205Leu Asp Glu Ala Leu Pro Gln Ala Asp Ile
Val Val Trp Val Ala Ser 210 215 220Met Pro Lys Gly Val Glu Ile Asn
Pro Gln Val Leu Lys Gln Pro Cys225 230 235 240Leu Leu Ile Asp Gly
Gly Tyr Pro Lys Asn Leu Gly Thr Lys Val Gln
245 250 255Tyr Pro Gly Val Tyr Val Leu Asn Gly Gly Ile Val Glu His
Ser Leu 260 265 270Asp Ile Asp Trp Lys Ile Met Lys Ile Val Asn Met
Asp Val Pro Ala 275 280 285Arg Gln Leu Phe Ala Cys Phe Ala Glu Ser
Met Leu Leu Glu Phe Glu 290 295 300Lys Leu Tyr Thr Asn Phe Ser Trp
Gly Arg Asn Gln Ile Thr Val Asp305 310 315 320Lys Met Glu Gln Ile
Gly Gln Ala Ser Val Lys His Gly Phe Arg Pro 325 330 335Leu Leu
Val411026DNASynechococcus elongatus 41atgttcggtc ttatcggtca
tctcaccagt ttggagcagg cccgcgacgt ttctcgcagg 60atgggctacg acgaatacgc
cgatcaagga ttggagtttt ggagtagcgc tcctcctcaa 120atcgttgatg
aaatcacagt caccagtgcc acaggcaagg tgattcacgg tcgctacatc
180gaatcgtgtt tcttgccgga aatgctggcg gcgcgccgct tcaaaacagc
cacgcgcaaa 240gttctcaatg ccatgtccca tgcccaaaaa cacggcatcg
acatctcggc cttggggggc 300tttacctcga ttattttcga gaatttcgat
ttggccagtt tgcggcaagt gcgcgacact 360accttggagt ttgaacggtt
caccaccggc aatactcaca cggcctacgt aatctgtaga 420caggtggaag
ccgctgctaa aacgctgggc atcgacatta cccaagcgac agtagcggtt
480gtcggcgcga ctggcgatat cggtagcgct gtctgccgct ggctcgacct
caaactgggt 540gtcggtgatt tgatcctgac ggcgcgcaat caggagcgtt
tggataacct gcaggctgaa 600ctcggccggg gcaagattct gcccttggaa
gccgctctgc cggaagctga ctttatcgtg 660tgggtcgcca gtatgcctca
gggcgtagtg atcgacccag caaccctgaa gcaaccctgc 720gtcctaatcg
acgggggcta ccccaaaaac ttgggcagca aagtccaagg tgagggcatc
780tatgtcctca atggcggggt agttgaacat tgcttcgaca tcgactggca
gatcatgtcc 840gctgcagaga tggcgcggcc cgagcgccag atgtttgcct
gctttgccga ggcgatgctc 900ttggaatttg aaggctggca tactaacttc
tcctggggcc gcaaccaaat cacgatcgag 960aagatggaag cgatcggtga
ggcatcggtg cgccacggct tccaaccctt ggcattggca 1020atttga
102642340PRTSynechococcus elongatus 42Met Phe Gly Leu Ile Gly His
Leu Thr Ser Leu Glu Gln Ala Arg Asp1 5 10 15Val Ser Arg Arg Met Gly
Tyr Asp Glu Tyr Ala Asp Gln Gly Leu Glu 20 25 30Phe Trp Ser Ser Ala
Pro Pro Gln Ile Val Asp Glu Ile Thr Val Thr 35 40 45Ser Ala Thr Gly
Lys Val Ile His Gly Arg Tyr Ile Glu Ser Cys Phe 50 55 60Leu Pro Glu
Met Leu Ala Ala Arg Arg Phe Lys Thr Ala Thr Arg Lys65 70 75 80Val
Leu Asn Ala Met Ser His Ala Gln Lys His Gly Ile Asp Ile Ser 85 90
95Ala Leu Gly Gly Phe Thr Ser Ile Ile Phe Glu Asn Phe Asp Leu Ala
100 105 110Ser Leu Arg Gln Val Arg Asp Thr Thr Leu Glu Phe Glu Arg
Phe Thr 115 120 125Thr Gly Asn Thr His Thr Ala Tyr Val Ile Cys Arg
Gln Val Glu Ala 130 135 140Ala Ala Lys Thr Leu Gly Ile Asp Ile Thr
Gln Ala Thr Val Ala Val145 150 155 160Val Gly Ala Thr Gly Asp Ile
Gly Ser Ala Val Cys Arg Trp Leu Asp 165 170 175Leu Lys Leu Gly Val
Gly Asp Leu Ile Leu Thr Ala Arg Asn Gln Glu 180 185 190Arg Leu Asp
Asn Leu Gln Ala Glu Leu Gly Arg Gly Lys Ile Leu Pro 195 200 205Leu
Glu Ala Ala Leu Pro Glu Ala Asp Phe Ile Val Trp Val Ala Ser 210 215
220Met Pro Gln Gly Val Val Ile Asp Pro Ala Thr Leu Lys Gln Pro
Cys225 230 235 240Val Leu Ile Asp Gly Gly Tyr Pro Lys Asn Leu Gly
Ser Lys Val Gln 245 250 255Gly Glu Gly Ile Tyr Val Leu Asn Gly Gly
Val Val Glu His Cys Phe 260 265 270Asp Ile Asp Trp Gln Ile Met Ser
Ala Ala Glu Met Ala Arg Pro Glu 275 280 285Arg Gln Met Phe Ala Cys
Phe Ala Glu Ala Met Leu Leu Glu Phe Glu 290 295 300Gly Trp His Thr
Asn Phe Ser Trp Gly Arg Asn Gln Ile Thr Ile Glu305 310 315 320Lys
Met Glu Ala Ile Gly Glu Ala Ser Val Arg His Gly Phe Gln Pro 325 330
335Leu Ala Leu Ala 340431020DNANostoc sp. 43atgtttggtc taattggaca
tctgacaagt ttagaacacg ctcaagcggt agctcaagaa 60ctgggatacc cagaatacgc
cgaccaaggg ctagattttt ggtgtagcgc tccaccgcaa 120atagttgacc
acattaaagt tactagtatt actggtgaaa taattgaagg gaggtatgta
180gaatcttgct ttttaccgga gatgctagcc agtcgtcgga ttaaagccgc
aacccgcaaa 240gtcctcaatg ctatggctca tgctcaaaag aatggcattg
atatcacagc tttgggtggt 300ttctcctcca ttatttttga aaactttaaa
ttggagcagt ttagccaagt tcgtaatgtg 360acactagagt ttgaacgctt
cactacaggc aacactcaca cagcatatat tatttgtcgg 420caggtagaac
aagcatcaca acaactcggc attgaactct cccaagcaac agtagctata
480tgtggggcta ctggtgatat tggtagtgca gttactcgct ggctggatgc
taaaacagac 540gtgaaagaat tgctgttaat cgcccgtaat caagaacgtc
tccaagagtt gcaaagcgag 600ctgggacgcg gtaaaatcat gagccttgat
gaagcactgc cccaagctga tatcgtagtt 660tgggtagcca gtatgcctaa
aggtgtggaa attaatcctc aagttttgaa gcaaccctgt 720ttgctgattg
atgggggtta tccgaaaaac ttgggtacaa aagttcagta tcctggtgtt
780tatgtactga acggcggtat cgtcgaacat tcgctggata ttgactggaa
aatcatgaaa 840atagtcaata tggatgtacc tgcacgccaa ttatttgctt
gttttgcgga atctatgctc 900ttggaatttg agaagttgta cacgaacttt
tcttgggggc gcaatcagat taccgtagac 960aaaatggagc agattggtca
agcatcagtg aaacatgggt ttagaccact gctggtttag 102044339PRTNostoc sp.
44Met Phe Gly Leu Ile Gly His Leu Thr Ser Leu Glu His Ala Gln Ala1
5 10 15Val Ala Gln Glu Leu Gly Tyr Pro Glu Tyr Ala Asp Gln Gly Leu
Asp 20 25 30Phe Trp Cys Ser Ala Pro Pro Gln Ile Val Asp His Ile Lys
Val Thr 35 40 45Ser Ile Thr Gly Glu Ile Ile Glu Gly Arg Tyr Val Glu
Ser Cys Phe 50 55 60Leu Pro Glu Met Leu Ala Ser Arg Arg Ile Lys Ala
Ala Thr Arg Lys65 70 75 80Val Leu Asn Ala Met Ala His Ala Gln Lys
Asn Gly Ile Asp Ile Thr 85 90 95Ala Leu Gly Gly Phe Ser Ser Ile Ile
Phe Glu Asn Phe Lys Leu Glu 100 105 110Gln Phe Ser Gln Val Arg Asn
Val Thr Leu Glu Phe Glu Arg Phe Thr 115 120 125Thr Gly Asn Thr His
Thr Ala Tyr Ile Ile Cys Arg Gln Val Glu Gln 130 135 140Ala Ser Gln
Gln Leu Gly Ile Glu Leu Ser Gln Ala Thr Val Ala Ile145 150 155
160Cys Gly Ala Thr Gly Asp Ile Gly Ser Ala Val Thr Arg Trp Leu Asp
165 170 175Ala Lys Thr Asp Val Lys Glu Leu Leu Leu Ile Ala Arg Asn
Gln Glu 180 185 190Arg Leu Gln Glu Leu Gln Ser Glu Leu Gly Arg Gly
Lys Ile Met Ser 195 200 205Leu Asp Glu Ala Leu Pro Gln Ala Asp Ile
Val Val Trp Val Ala Ser 210 215 220Met Pro Lys Gly Val Glu Ile Asn
Pro Gln Val Leu Lys Gln Pro Cys225 230 235 240Leu Leu Ile Asp Gly
Gly Tyr Pro Lys Asn Leu Gly Thr Lys Val Gln 245 250 255Tyr Pro Gly
Val Tyr Val Leu Asn Gly Gly Ile Val Glu His Ser Leu 260 265 270Asp
Ile Asp Trp Lys Ile Met Lys Ile Val Asn Met Asp Val Pro Ala 275 280
285Arg Gln Leu Phe Ala Cys Phe Ala Glu Ser Met Leu Leu Glu Phe Glu
290 295 300Lys Leu Tyr Thr Asn Phe Ser Trp Gly Arg Asn Gln Ile Thr
Val Asp305 310 315 320Lys Met Glu Gln Ile Gly Gln Ala Ser Val Lys
His Gly Phe Arg Pro 325 330 335Leu Leu Val45867DNACorynebacterium
glutamicum 45ttgggcgtgt cgcccttaaa gcgcgctttt cgacgcgacc ccactacatt
ggcttccatg 60aacgttgaca tttcacgatc cagagagccg ctaaacgttg agctcctgaa
ggaaaaattg 120ctccaaaacg gtgactttgg ccaggtcatt tacgaaaaag
tgacaggctc cactaatgct 180gacttgctgg cacttgcagg ttctggcgct
ccaaactgga cggtgaaaac tgtcgagttt 240caagatcatg cgcgtgggcg
actcggccgc ccgtggtctg cccctgaggg ttcccaaaca 300atcgtgtctg
tgctcgttca actatctatt gatcaagtgg accggattgg cactattcca
360ctcgcggcgg gactcgctgt catggatgcg ttgaatgacc tcggtgtgga
aggtgccgga 420ctgaaatggc ccaacgatgt tcaaatccac ggcaagaaac
tctgcggcat cctggtggaa 480gccaccggct ttgattccac cccaacagtt
gtcatcggtt ggggcactaa tatcagcctg 540actaaagagg agcttcctgt
tcctcatgca acttccctcg cattggaagg tgttgaagtc 600gacagaacca
cattccttat taatatgctc acacatctgc atactcgact ggaccagtgg
660cagggtccaa gtgtggattg gctcgatgat taccgtgcgg tatgttccag
tattggccaa 720gatgttcgag tgcttctacc tggggataaa gaactcttag
gtgaagcgat cggtgtcgcg 780actggcggag aaattcgtgt tcgcgatgct
tcgggcaccg ttcacaccct caacgccggt 840gaaattacgc accttcgcct gcagtaa
86746810DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 46atgaatgttg acattagccg ctctcgtgaa
ccgttgaacg tggaactgtt gaaagaaaaa 60ctgctgcaga acggtgattt cggtcaagtg
atctacgaga aggtcaccgg ctctaccaat 120gcggacctgc tggctctggc
gggcagcggc gctccaaact ggaccgtcaa gactgttgaa 180tttcaggacc
acgcccgtgg ccgtctgggt cgtccgtgga gcgcaccgga gggttcccaa
240accatcgtca gcgttctggt ccaactgagc attgatcagg tggaccgtat
tggtacgatc 300ccgctggccg caggcttggc tgttatggat gcgctgaatg
atctgggcgt ggagggtgca 360ggcctgaaat ggccgaacga tgttcagatc
cacggtaaga agttgtgcgg tattctggtt 420gaagcaaccg gcttcgactc
cactccgacc gtggttatcg gttggggtac gaatatctcg 480ttgacgaaag
aagagctgcc ggtcccgcac gcgaccagcc tggccctgga gggtgttgaa
540gttgaccgta cgacgttcct gattaacatg ctgacccatc tgcatacccg
tctggatcag 600tggcagggtc cgtctgtgga ctggctggat gactatcgcg
cggtttgtag cagcattggc 660caagatgtgc gtgtcctgct gcctggtgac
aaagagctgc tgggcgaggc gattggcgtg 720gcgaccggtg gtgagatccg
tgtgcgcgac gccagcggca cggtccacac gctgaatgcg 780ggtgaaatca
cgcatctgcg tttgcaataa 81047269PRTCorynebacterium glutamicum 47Met
Asn Val Asp Ile Ser Arg Ser Arg Glu Pro Leu Asn Val Glu Leu1 5 10
15Leu Lys Glu Lys Leu Leu Gln Asn Gly Asp Phe Gly Gln Val Ile Tyr
20 25 30Glu Lys Val Thr Gly Ser Thr Asn Ala Asp Leu Leu Ala Leu Ala
Gly 35 40 45Ser Gly Ala Pro Asn Trp Thr Val Lys Thr Val Glu Phe Gln
Asp His 50 55 60Ala Arg Gly Arg Leu Gly Arg Pro Trp Ser Ala Pro Glu
Gly Ser Gln65 70 75 80Thr Ile Val Ser Val Leu Val Gln Leu Ser Ile
Asp Gln Val Asp Arg 85 90 95Ile Gly Thr Ile Pro Leu Ala Ala Gly Leu
Ala Val Met Asp Ala Leu 100 105 110Asn Asp Leu Gly Val Glu Gly Ala
Gly Leu Lys Trp Pro Asn Asp Val 115 120 125Gln Ile His Gly Lys Lys
Leu Cys Gly Ile Leu Val Glu Ala Thr Gly 130 135 140Phe Asp Ser Thr
Pro Thr Val Val Ile Gly Trp Gly Thr Asn Ile Ser145 150 155 160Leu
Thr Lys Glu Glu Leu Pro Val Pro His Ala Thr Ser Leu Ala Leu 165 170
175Glu Gly Val Glu Val Asp Arg Thr Thr Phe Leu Ile Asn Met Leu Thr
180 185 190His Leu His Thr Arg Leu Asp Gln Trp Gln Gly Pro Ser Val
Asp Trp 195 200 205Leu Asp Asp Tyr Arg Ala Val Cys Ser Ser Ile Gly
Gln Asp Val Arg 210 215 220Val Leu Leu Pro Gly Asp Lys Glu Leu Leu
Gly Glu Ala Ile Gly Val225 230 235 240Ala Thr Gly Gly Glu Ile Arg
Val Arg Asp Ala Ser Gly Thr Val His 245 250 255Thr Leu Asn Ala Gly
Glu Ile Thr His Leu Arg Leu Gln 260 265481632DNACorynebacterium
glutamicum 48atgaccattt cctcaccttt gattgacgtc gccaaccttc cagacatcaa
caccactgcc 60ggcaagatcg ccgaccttaa ggctcgccgc gcggaagccc atttccccat
gggtgaaaag 120gcagtagaga aggtccacgc tgctggacgc ctcactgccc
gtgagcgctt ggattactta 180ctcgatgagg gctccttcat cgagaccgat
cagctggctc gccaccgcac caccgctttc 240ggcctgggcg ctaagcgtcc
tgcaaccgac ggcatcgtga ccggctgggg caccattgat 300ggacgcgaag
tctgcatctt ctcgcaggac ggcaccgtat tcggtggcgc gcttggtgag
360gtgtacggcg aaaagatgat caagatcatg gagctggcaa tcgacaccgg
ccgcccattg 420atcggtcttt acgaaggcgc tggcgctcgt attcaggacg
gcgctgtctc cctggacttc 480atttcccaga ccttctacca aaacattcag
gcttctggcg ttatcccaca gatctccgtc 540atcatgggcg catgtgcagg
tggcaacgct tacggcccag ctctgaccga cttcgtggtc 600atggtggaca
agacctccaa gatgttcgtt accggcccag acgtgatcaa gaccgtcacc
660ggcgaggaaa tcacccagga agagcttggc ggagcaacca cccacatggt
gaccgctggt 720aactcccact acaccgctgc gaccgatgag gaagcactgg
attgggtaca ggacctggtg 780tccttcctcc catccaacaa tcgctcctac
gcaccgatgg aagacttcga cgaggaagaa 840ggcggcgttg aagaaaacat
caccgctgac gatctgaagc tcgacgagat catcccagat 900tccgcgaccg
ttccttacga cgtccgcgat gtcatcgaat gcctcaccga cgatggcgaa
960tacctggaaa tccaggcaga ccgcgcagaa aacgttgtta ttgcattcgg
ccgcatcgaa 1020ggccagtccg ttggctttgt tgccaaccag ccaacccagt
tcgctggctg cctggacatc 1080gactcctctg agaaggcagc tcgcttcgtc
cgcacctgcg acgcgttcaa catcccaatc 1140gtcatgcttg tcgacgtccc
cggcttcctc ccaggcgcag gccaggagta cggtggcatt 1200ctgcgtcgtg
gcgcaaagct gctctacgca tacggcgaag caaccgttcc aaagatcacc
1260gtcaccatgc gtaaggctta cggcggagcg tactgcgtga tgggttccaa
gggcttgggc 1320tctgacatca accttgcatg gccaaccgca cagatcgccg
tcatgggcgc tgctggcgca 1380gttggattca tctaccgcaa ggagctcatg
gcagctgatg ccaagggcct cgataccgta 1440gctctggcta agtccttcga
gcgcgagtat gaagaccaca tgctcaaccc gtaccacgct 1500gcagaacgtg
gcctgatcga cgccgtgatc ctgccaagcg aaacccgcgg acagatttcc
1560cgcaaccttc gcctgctcaa gcacaagaac gtcactcgcc ctgctcgcaa
gcacggcaac 1620atgccactgt aa 163249543PRTCorynebacterium glutamicum
49Met Thr Ile Ser Ser Pro Leu Ile Asp Val Ala Asn Leu Pro Asp Ile1
5 10 15Asn Thr Thr Ala Gly Lys Ile Ala Asp Leu Lys Ala Arg Arg Ala
Glu 20 25 30Ala His Phe Pro Met Gly Glu Lys Ala Val Glu Lys Val His
Ala Ala 35 40 45Gly Arg Leu Thr Ala Arg Glu Arg Leu Asp Tyr Leu Leu
Asp Glu Gly 50 55 60Ser Phe Ile Glu Thr Asp Gln Leu Ala Arg His Arg
Thr Thr Ala Phe65 70 75 80Gly Leu Gly Ala Lys Arg Pro Ala Thr Asp
Gly Ile Val Thr Gly Trp 85 90 95Gly Thr Ile Asp Gly Arg Glu Val Cys
Ile Phe Ser Gln Asp Gly Thr 100 105 110Val Phe Gly Gly Ala Leu Gly
Glu Val Tyr Gly Glu Lys Met Ile Lys 115 120 125Ile Met Glu Leu Ala
Ile Asp Thr Gly Arg Pro Leu Ile Gly Leu Tyr 130 135 140Glu Gly Ala
Gly Ala Arg Ile Gln Asp Gly Ala Val Ser Leu Asp Phe145 150 155
160Ile Ser Gln Thr Phe Tyr Gln Asn Ile Gln Ala Ser Gly Val Ile Pro
165 170 175Gln Ile Ser Val Ile Met Gly Ala Cys Ala Gly Gly Asn Ala
Tyr Gly 180 185 190Pro Ala Leu Thr Asp Phe Val Val Met Val Asp Lys
Thr Ser Lys Met 195 200 205Phe Val Thr Gly Pro Asp Val Ile Lys Thr
Val Thr Gly Glu Glu Ile 210 215 220Thr Gln Glu Glu Leu Gly Gly Ala
Thr Thr His Met Val Thr Ala Gly225 230 235 240Asn Ser His Tyr Thr
Ala Ala Thr Asp Glu Glu Ala Leu Asp Trp Val 245 250 255Gln Asp Leu
Val Ser Phe Leu Pro Ser Asn Asn Arg Ser Tyr Ala Pro 260 265 270Met
Glu Asp Phe Asp Glu Glu Glu Gly Gly Val Glu Glu Asn Ile Thr 275 280
285Ala Asp Asp Leu Lys Leu Asp Glu Ile Ile Pro Asp Ser Ala Thr Val
290 295 300Pro Tyr Asp Val Arg Asp Val Ile Glu Cys Leu Thr Asp Asp
Gly Glu305 310 315 320Tyr Leu Glu Ile Gln Ala Asp Arg Ala Glu Asn
Val Val Ile Ala Phe 325 330 335Gly Arg Ile Glu Gly Gln Ser Val Gly
Phe Val Ala Asn Gln Pro Thr 340 345 350Gln Phe Ala Gly Cys Leu Asp
Ile Asp Ser Ser Glu Lys Ala Ala Arg 355 360 365Phe Val Arg Thr Cys
Asp Ala Phe Asn Ile Pro Ile Val Met Leu Val 370 375 380Asp Val Pro
Gly Phe Leu Pro Gly Ala Gly Gln Glu Tyr Gly Gly Ile385 390 395
400Leu Arg Arg Gly Ala Lys Leu Leu Tyr Ala Tyr Gly Glu Ala Thr Val
405 410 415Pro Lys Ile Thr Val Thr Met Arg Lys Ala Tyr Gly Gly Ala
Tyr Cys 420 425 430Val Met Gly Ser Lys Gly Leu Gly Ser Asp Ile Asn
Leu Ala Trp Pro 435 440 445Thr Ala Gln Ile Ala Val Met Gly Ala Ala
Gly Ala Val Gly Phe Ile 450 455 460Tyr Arg Lys Glu Leu Met Ala Ala
Asp Ala Lys Gly Leu Asp Thr Val465 470 475 480Ala Leu Ala Lys Ser
Phe Glu
Arg Glu Tyr Glu Asp His Met Leu Asn 485 490 495Pro Tyr His Ala Ala
Glu Arg Gly Leu Ile Asp Ala Val Ile Leu Pro 500 505 510Ser Glu Thr
Arg Gly Gln Ile Ser Arg Asn Leu Arg Leu Leu Lys His 515 520 525Lys
Asn Val Thr Arg Pro Ala Arg Lys His Gly Asn Met Pro Leu 530 535
540501776DNACorynebacterium glutamicum 50atgtcagtcg agactcgcaa
gatcaccaag gttcttgtcg ctaaccgtgg tgagattgca 60atccgcgtgt tccgtgcagc
tcgagatgaa ggcatcggat ctgtcgccgt ctacgcagag 120ccagatgcag
atgcaccatt cgtgtcatat gcagacgagg cttttgccct cggtggccaa
180acatccgctg agtcctacct tgtcattgac aagatcatcg atgcggcccg
caagtccggc 240gccgacgcca tccaccccgg ctacggcttc ctcgcagaaa
acgctgactt cgcagaagca 300gtcatcaacg aaggcctgat ctggattgga
ccttcacctg agtccatccg ctccctcggc 360gacaaggtca ccgctcgcca
catcgcagat accgccaagg ctccaatggc tcctggcacc 420aaggaaccag
taaaagacgc agcagaagtt gtggctttcg ctgaagaatt cggtctccca
480atcgccatca aggcagcttt cggtggcggc ggacgtggca tgaaggttgc
ctacaagatg 540gaagaagtcg ctgacctctt cgagtccgca acccgtgaag
caaccgcagc gttcggccgc 600ggcgagtgct tcgtggagcg ctacctggac
aaggcacgcc acgttgaggc tcaggtcatc 660gccgataagc acggcaacgt
tgttgtcgcc ggaacccgtg actgctccct gcagcgccgt 720ttccagaagc
tcgtcgaaga agcaccagca ccattcctca ccgatgacca gcgcgagcgt
780ctccactcct ccgcgaaggc tatctgtaag gaagctggct actacggtgc
aggcaccgtt 840gagtacctcg ttggctccga cggcctgatc tccttcctcg
aggtcaacac ccgcctccag 900gtggaacacc cagtcaccga agagaccacc
ggcatcgacc tggtccgcga aatgttccgc 960atcgcagaag gccacgagct
ctccatcaag gaagatccag ctccacgcgg ccacgcattc 1020gagttccgca
tcaacggcga agacgctggc tccaacttca tgcctgcacc aggcaagatc
1080accagctacc gcgagccaca gggcccaggc gtccgcatgg actccggtgt
cgttgaaggt 1140tccgaaatct ccggacagtt cgactccatg ctggcaaagc
tgatcgtttg gggcgacacc 1200cgcgagcagg ctctccagcg ctcccgccgt
gcacttgcag agtacgttgt cgagggcatg 1260ccaaccgtta tcccattcca
ccagcacatc gtggaaaacc cagcattcgt gggcaacgac 1320gaaggcttcg
agatctacac caagtggatc gaagaggttt gggataaccc aatcgcacct
1380tacgttgacg cttccgagct cgacgaagat gaggacaaga ccccagcaca
gaaggttgtt 1440gtggagatca acggccgtcg cgttgaggtt gcactcccag
gcgatctggc actcggtggc 1500accgctggtc ctaagaagaa ggccaagaag
cgtcgcgcag gtggtgcaaa ggctggcgta 1560tccggcgatg cagtggcagc
tccaatgcag ggcactgtca tcaaggtcaa cgtcgaagaa 1620ggcgctgaag
tcaacgaagg cgacaccgtt gttgtcctcg aggctatgaa gatggaaaac
1680cctgtgaagg ctcataagtc cggaaccgta accggcctta ctgtcgctgc
aggcgagggt 1740gtcaacaagg gcgttgttct cctcgagatc aagtaa
177651591PRTCorynebacterium glutamicum 51Met Ser Val Glu Thr Arg
Lys Ile Thr Lys Val Leu Val Ala Asn Arg1 5 10 15Gly Glu Ile Ala Ile
Arg Val Phe Arg Ala Ala Arg Asp Glu Gly Ile 20 25 30Gly Ser Val Ala
Val Tyr Ala Glu Pro Asp Ala Asp Ala Pro Phe Val 35 40 45Ser Tyr Ala
Asp Glu Ala Phe Ala Leu Gly Gly Gln Thr Ser Ala Glu 50 55 60Ser Tyr
Leu Val Ile Asp Lys Ile Ile Asp Ala Ala Arg Lys Ser Gly65 70 75
80Ala Asp Ala Ile His Pro Gly Tyr Gly Phe Leu Ala Glu Asn Ala Asp
85 90 95Phe Ala Glu Ala Val Ile Asn Glu Gly Leu Ile Trp Ile Gly Pro
Ser 100 105 110Pro Glu Ser Ile Arg Ser Leu Gly Asp Lys Val Thr Ala
Arg His Ile 115 120 125Ala Asp Thr Ala Lys Ala Pro Met Ala Pro Gly
Thr Lys Glu Pro Val 130 135 140Lys Asp Ala Ala Glu Val Val Ala Phe
Ala Glu Glu Phe Gly Leu Pro145 150 155 160Ile Ala Ile Lys Ala Ala
Phe Gly Gly Gly Gly Arg Gly Met Lys Val 165 170 175Ala Tyr Lys Met
Glu Glu Val Ala Asp Leu Phe Glu Ser Ala Thr Arg 180 185 190Glu Ala
Thr Ala Ala Phe Gly Arg Gly Glu Cys Phe Val Glu Arg Tyr 195 200
205Leu Asp Lys Ala Arg His Val Glu Ala Gln Val Ile Ala Asp Lys His
210 215 220Gly Asn Val Val Val Ala Gly Thr Arg Asp Cys Ser Leu Gln
Arg Arg225 230 235 240Phe Gln Lys Leu Val Glu Glu Ala Pro Ala Pro
Phe Leu Thr Asp Asp 245 250 255Gln Arg Glu Arg Leu His Ser Ser Ala
Lys Ala Ile Cys Lys Glu Ala 260 265 270Gly Tyr Tyr Gly Ala Gly Thr
Val Glu Tyr Leu Val Gly Ser Asp Gly 275 280 285Leu Ile Ser Phe Leu
Glu Val Asn Thr Arg Leu Gln Val Glu His Pro 290 295 300Val Thr Glu
Glu Thr Thr Gly Ile Asp Leu Val Arg Glu Met Phe Arg305 310 315
320Ile Ala Glu Gly His Glu Leu Ser Ile Lys Glu Asp Pro Ala Pro Arg
325 330 335Gly His Ala Phe Glu Phe Arg Ile Asn Gly Glu Asp Ala Gly
Ser Asn 340 345 350Phe Met Pro Ala Pro Gly Lys Ile Thr Ser Tyr Arg
Glu Pro Gln Gly 355 360 365Pro Gly Val Arg Met Asp Ser Gly Val Val
Glu Gly Ser Glu Ile Ser 370 375 380Gly Gln Phe Asp Ser Met Leu Ala
Lys Leu Ile Val Trp Gly Asp Thr385 390 395 400Arg Glu Gln Ala Leu
Gln Arg Ser Arg Arg Ala Leu Ala Glu Tyr Val 405 410 415Val Glu Gly
Met Pro Thr Val Ile Pro Phe His Gln His Ile Val Glu 420 425 430Asn
Pro Ala Phe Val Gly Asn Asp Glu Gly Phe Glu Ile Tyr Thr Lys 435 440
445Trp Ile Glu Glu Val Trp Asp Asn Pro Ile Ala Pro Tyr Val Asp Ala
450 455 460Ser Glu Leu Asp Glu Asp Glu Asp Lys Thr Pro Ala Gln Lys
Val Val465 470 475 480Val Glu Ile Asn Gly Arg Arg Val Glu Val Ala
Leu Pro Gly Asp Leu 485 490 495Ala Leu Gly Gly Thr Ala Gly Pro Lys
Lys Lys Ala Lys Lys Arg Arg 500 505 510Ala Gly Gly Ala Lys Ala Gly
Val Ser Gly Asp Ala Val Ala Ala Pro 515 520 525Met Gln Gly Thr Val
Ile Lys Val Asn Val Glu Glu Gly Ala Glu Val 530 535 540Asn Glu Gly
Asp Thr Val Val Val Leu Glu Ala Met Lys Met Glu Asn545 550 555
560Pro Val Lys Ala His Lys Ser Gly Thr Val Thr Gly Leu Thr Val Ala
565 570 575Ala Gly Glu Gly Val Asn Lys Gly Val Val Leu Leu Glu Ile
Lys 580 585 59052340PRTAcinetobacter sp. 52Met Ser Asn His Gln Ile
Arg Ala Tyr Ala Ala Met Gln Ala Gly Glu1 5 10 15Gln Val Val Pro Tyr
Gln Phe Asp Ala Gly Glu Leu Lys Ala His Gln 20 25 30Val Glu Val Lys
Val Glu Tyr Cys Gly Leu Cys His Ser Asp Leu Ser 35 40 45Val Ile Asn
Asn Glu Trp Gln Ser Ser Val Tyr Pro Ala Val Ala Gly 50 55 60His Glu
Ile Ile Gly Thr Ile Ile Ala Leu Gly Ser Glu Ala Lys Gly65 70 75
80Leu Lys Leu Gly Gln Arg Val Gly Ile Gly Trp Thr Ala Glu Thr Cys
85 90 95Gln Ala Cys Asp Pro Cys Ile Gly Gly Asn Gln Val Leu Cys Thr
Gly 100 105 110Glu Lys Lys Ala Thr Ile Ile Gly His Ala Gly Gly Phe
Ala Asp Lys 115 120 125Val Arg Ala Gly Trp Gln Trp Val Ile Pro Leu
Pro Asp Asp Leu Asp 130 135 140Pro Glu Ser Ala Gly Pro Leu Leu Cys
Gly Gly Ile Thr Val Leu Asp145 150 155 160Pro Leu Leu Lys His Lys
Ile Gln Ala Thr His His Val Gly Val Ile 165 170 175Gly Ile Gly Gly
Leu Gly His Ile Ala Ile Lys Leu Leu Lys Ala Trp 180 185 190Gly Cys
Glu Ile Thr Ala Phe Ser Ser Asn Pro Asp Lys Thr Glu Glu 195 200
205Leu Lys Ala Asn Gly Ala Asp Gln Val Val Asn Ser Arg Asp Ala Gln
210 215 220Ala Ile Lys Gly Thr Arg Trp Lys Leu Ile Ile Leu Ser Thr
Ala Asn225 230 235 240Gly Thr Leu Asn Val Lys Ala Tyr Leu Asn Thr
Leu Ala Pro Lys Gly 245 250 255Ser Leu His Phe Leu Gly Val Thr Leu
Glu Pro Ile Pro Val Ser Val 260 265 270Gly Ala Ile Met Gly Gly Ala
Lys Ser Val Thr Ser Ser Pro Thr Gly 275 280 285Ser Pro Leu Ala Leu
Arg Gln Leu Leu Gln Phe Ala Ala Arg Lys Asn 290 295 300Ile Ala Pro
Gln Val Glu Leu Phe Pro Met Ser Gln Leu Asn Glu Ala305 310 315
320Ile Glu Arg Leu His Ser Gly Gln Ala Arg Tyr Arg Ile Val Leu Lys
325 330 335Ala Asp Phe Asp 34053314PRTAcinetobacter baylyi 53Met
Ala Thr Thr Asn Val Ile His Ala Tyr Ala Ala Met Gln Ala Gly1 5 10
15Glu Ala Leu Val Pro Tyr Ser Phe Asp Ala Gly Glu Leu Gln Pro His
20 25 30Gln Val Glu Val Lys Val Glu Tyr Cys Gly Leu Cys His Ser Asp
Val 35 40 45Ser Val Leu Asn Asn Glu Trp His Ser Ser Val Tyr Pro Val
Val Ala 50 55 60Gly His Glu Val Ile Gly Thr Ile Thr Gln Leu Gly Ser
Glu Ala Lys65 70 75 80Gly Leu Lys Ile Gly Gln Arg Val Gly Ile Gly
Trp Thr Ala Glu Ser 85 90 95Cys Gln Ala Cys Asp Gln Cys Ile Ser Gly
Gln Gln Val Leu Cys Thr 100 105 110Gly Glu Asn Thr Ala Thr Ile Ile
Gly His Ala Gly Gly Phe Ala Asp 115 120 125Lys Val Arg Ala Gly Trp
Gln Trp Val Ile Pro Leu Pro Asp Glu Leu 130 135 140Asp Pro Thr Ser
Ala Gly Pro Leu Leu Cys Gly Gly Ile Thr Val Phe145 150 155 160Asp
Pro Ile Leu Lys His Gln Ile Gln Ala Ile His His Val Ala Val 165 170
175Ile Gly Ile Gly Gly Leu Gly His Met Ala Ile Lys Leu Leu Lys Ala
180 185 190Trp Gly Cys Glu Ile Thr Ala Phe Ser Ser Asn Pro Asn Lys
Thr Asp 195 200 205Glu Leu Lys Ala Met Gly Ala Asp His Val Val Asn
Ser Arg Asp Asp 210 215 220Ala Glu Ile Lys Ser Gln Gln Gly Lys Phe
Asp Leu Leu Leu Ser Thr225 230 235 240Val Asn Val Pro Leu Asn Trp
Asn Ala Tyr Leu Asn Thr Leu Ala Pro 245 250 255Asn Gly Thr Phe His
Phe Leu Gly Val Val Met Glu Pro Ile Pro Val 260 265 270Pro Val Gly
Ala Leu Leu Gly Gly Ala Lys Ser Leu Thr Ala Ser Pro 275 280 285Thr
Gly Ser Pro Ala Ala Leu Arg Lys Leu Leu Glu Phe Ala Ala Arg 290 295
300Lys Asn Ile Ala Pro Gln Ile Glu Met Tyr305
31054341PRTAcinetobacter baylyi 54Met Ala Thr Thr Asn Val Ile His
Ala Tyr Ala Ala Met Gln Ala Gly1 5 10 15Glu Ala Leu Val Pro Tyr Ser
Phe Asp Ala Gly Glu Leu Gln Pro His 20 25 30Gln Val Glu Val Lys Val
Glu Tyr Cys Gly Leu Cys His Ser Asp Val 35 40 45Ser Val Leu Asn Asn
Glu Trp His Ser Ser Val Tyr Pro Val Val Ala 50 55 60Gly His Glu Val
Ile Gly Thr Ile Thr Gln Leu Gly Ser Glu Ala Lys65 70 75 80Gly Leu
Lys Ile Gly Gln Arg Val Gly Ile Gly Trp Thr Ala Glu Ser 85 90 95Cys
Gln Ala Cys Asp Gln Cys Ile Ser Gly Gln Gln Val Leu Cys Thr 100 105
110Gly Glu Asn Thr Ala Thr Ile Ile Gly His Ala Gly Gly Phe Ala Asp
115 120 125Lys Val Arg Ala Gly Trp Gln Trp Val Ile Pro Leu Pro Asp
Glu Leu 130 135 140Asp Pro Thr Ser Ala Gly Pro Leu Leu Cys Gly Gly
Ile Thr Val Phe145 150 155 160Asp Pro Ile Leu Lys His Gln Ile Gln
Ala Ile His His Val Ala Val 165 170 175Ile Gly Ile Gly Gly Leu Gly
His Met Ala Ile Lys Leu Leu Lys Ala 180 185 190Trp Gly Cys Glu Ile
Thr Ala Phe Ser Ser Asn Pro Asn Lys Thr Asp 195 200 205Glu Leu Lys
Ala Met Gly Ala Asp His Val Val Asn Ser Arg Asp Asp 210 215 220Ala
Glu Ile Lys Ser Gln Gln Gly Lys Phe Asp Leu Leu Leu Ser Thr225 230
235 240Val Asn Val Pro Leu Asn Trp Asn Ala Tyr Leu Asn Thr Leu Ala
Pro 245 250 255Asn Gly Thr Phe His Phe Leu Gly Val Val Met Glu Pro
Ile Pro Val 260 265 270Pro Val Gly Ala Leu Leu Gly Gly Ala Lys Ser
Leu Thr Ala Ser Pro 275 280 285Thr Gly Ser Pro Ala Ala Leu Arg Lys
Leu Leu Glu Phe Ala Ala Arg 290 295 300Lys Asn Ile Ala Pro Gln Ile
Glu Met Tyr Pro Met Ser Glu Leu Asn305 310 315 320Glu Ala Ile Glu
Arg Leu His Ser Gly Gln Ala Arg Tyr Arg Ile Val 325 330 335Leu Lys
Ala Asp Phe 340554805DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 55tgtaggctgg
agctgcttcg aagttcctat actttctaga gaataggaac ttcggaatag 60gaacttcgaa
ctgcaggtcg acggatcccc ggaatattta aatcatttgt actttttgaa
120cagcagagtc gcattatggc caccgaagcc caggctgttg gacagaacgt
agttgacttc 180tgcattacgg ccctcgttag gaacgtaatc caggtcgcat
tccggatccg cctctttgta 240gccgatggtc ggcggaatga aaccctcttc
aatagctttg gcacagataa tcgcttcgac 300tgcaccgcca gcgcccagca
ggtggccggt catgctcttg gtgctagaca ccggcacttt 360gtaggcgtat
tcacccagga ccgtcttgat cgcttgggtt tcgaagctgt cattgtacgc
420cgtgctcgta ccgtgcgcgt tgatatagga aatgtcctct gggcggacat
tatcttcttc 480cattgccagt ttcattgcac gtgcaccacc ttcaccattc
ggcgctgggc tcgtgatatg 540atatgcgtcg caggtcgcac catagccaac
gatctcggca tagattttgg caccacgctt 600cagcgcgtgc tccaactctt
ccaagataac gataccgctg ccctcgccca tcacaaaacc 660gctgcgatcc
ttatcgaacg ggatgctggc gcgcttcggg tcctcagatt tggtcacggc
720cttcatcgag gcaaaacccg ccaggctcaa cggggtgata cctgcttcgc
taccaccaga 780gatcataacg tcgctataac caaacttaat gttacggaag
gactcaccaa tgctgttgtt 840cgcgctcgca catgcggtga caatggtcgt
gcaaatacct ttagcgccat aacgaatcgc 900cagattaccg cttgccatat
tcgcaatgat catcggaata gtcatagggc tcacacgacc 960cggacctttg
gtaatcagct tttcatcctg cttctcaatg gtgccgatgc cgccaatgcc
1020gctaccaaca atgacgccga aacgattctt atcaatcgac tccaggtcca
gtttgctgtc 1080cttgattgcc tcatccgccg caacgatcgc aaactggcta
aaacggtcca tacggttcgc 1140ctcacgcttg tcgataaagt cctccggggt
gaagtccttc acttcggcag ccagcttaac 1200tttgaaatcg gttgcgtcaa
acgctttgat cttgtcaatg ccacatttac cctctttgat 1260gctgcaccag
aagctatcag cgttgttacc caccggcgtc actgcaccaa tacccgtaat
1320gacaacgcgg cgattcattt tgttgcctcc ttttagaacg cggaagtatc
ctggaacaaa 1380ccgactttca aatcgtgtgc ggtatagatc aggcgaccat
ccaccagaac ctcaccgtcc 1440gccaggccca tgatcaggcg acggtttacg
atacgtttga aatgaatacg ataggtgact 1500ttcctggctg tcggcagaac
ctggccggta aatttcactt cgcccacgcc cagagcgcgg 1560cctttgcctt
cgccgcccaa ccagcccagg tagaatccca ccaattgcca catagcatcc
1620agacccagac aaccgggcat caccggatcg ccgataaagt ggcatccgaa
gaaccataga 1680tccggattga tatccagctc ggcttcgaca tagcctttgt
cgaaattgcc gcccgtttcg 1740gtcatcttaa cgacgcggtc catcatcagc
atgttcggtg cagggagttg cggcccttta 1800gcgccaaaca gttcaccacg
accagaggca agaaggtctt cttttgtata ggattcgcgt 1860ttatctacca
tgttttatgt aaaccttaaa attaaaccat gtacattccg ccgttgacgt
1920gcagagtctc accagtgatg taactcgctt cgtcagaggc taaaaatgca
accgcactgg 1980cgatttcctg agcgccgccg aggcgacccg caggcacctg
cgccaggata cccgcacgct 2040gatcgtcaga cagcgcacgc gtcatgtccg
tttcaataaa acccggagcc acaacattga 2100cagtaatacc acgggacgca
acttcacgcg ccagtgattt actgaaaccg atcaggcccg 2160ctttcgccgc
agcgtagttt gcctgacctg catttcccat ggtaccaacc acagaaccaa
2220tagtgataat gcgaccacaa cgctttttca tcatagcgcg cattaccgct
tttgacaggc 2280ggaaaacgga tgataagttg gtttcgataa tatcgttcca
ctcatcatct ttcattcgca 2340tcaacagatt atcacgagtg ataccggcat
tattaaccag gatatccact tcaccaaatt 2400ctgcgcgaat attttccaga
acagattcaa tagatgcagg atcggtcaca ttcaacatca 2460aacctttccc
gttagcacct aaatagtcgc taatgttctt cgcaccattt tcactggtcg
2520cagtcccgat aactttcgcg ccgcgggcaa cgagagtctc tgcaattgcg
cggcctatgc 2580cacggcttgc accagtcacc agcgcaatct ttccttcaaa
gctcatggtt ttcctctttt 2640attgcgtaag tgccgcagac agcgccgccg
gctcgttcag cgccgacgct gtcagggtgt 2700cgacaatacg tttcgtcaga
ccagtgagga ctttacctgg acccacttca taaagatgtt 2760caacgccctg
cgccgcgata aattccacgc tcttcgtcca ctgtaccgga ttgtacaact
2820ggcgaaccag cgcatcgcgg atagcggcgg catcggtttc acatttcacg
tcaacgttgt 2880tcactaccgg caccgttggc gcgctaaagg taattttggc
taattcaacc gccagcttat 2940ctgccgctgg tttcatcagc gcgcagtgcg
acggtacgct caccggcagc ggcagcgcgc 3000gtttcgcgcc agcggcttta
caggctgcgc
ccgcacgttc taccgcctct ttatgcccgg 3060cgataaccac ctgtcccggc
gagttaaagt taaccggcga aacaacctgc ccttcggcag 3120attcttcaca
ggctttagca atagaggcat catccagccc gatgatcgca gacatgccgc
3180cagtgccttc cggaaccgct tcctgcatga atttaccgcg catttccacc
agacgaacgg 3240catcagcaaa gttgatgacg ccagcgcaaa ccagcgcgga
atattcgccc aggctgtgac 3300ctgccattaa cgcaggcatt ttaccgccct
gctgctgcca aacgcgccaa agcgcgacgg 3360aagcggttaa taacgccggc
tgcgtctgcc aggttttatt cagttcttcc gctggacctt 3420gctgggtgag
cgcccacaga tcatatccca gagccgcaga agcttcagca aacgtttctt
3480ctacgatagg gtaatttgcc gccatctcgg ccaacatccc aacgctctga
gaaccctgac 3540cggggaacac aaatgcaaat tgcgtcatgt ttaaatcctt
atactagaaa cgaatcagcg 3600cggagcccca ggtgaatcca cccccgaagg
cttcaagcaa taccagctga ccggctttaa 3660ttcgcccgtc acgcacggct
tcatccagcg cgcacggcac agaagccgcg gaggtattgc 3720cgtgcctgtc
cagcgtgacg acgacattgt ccatcgacat gccgagtttt ttcgctgtcg
3780cgctaatgat acgcaggtta gcctgatgcg gcaccagcca atcgagttct
gagcgatcca 3840ggttattagc cgccagcgtc tcatcgacaa tatgcgccag
ttcagtgacc gccactttaa 3900agacttcatt gcccgccatt gtcaggtaaa
tcgggttatc cggatttacg cgatcggcat 3960tcggcagggt cagtaattca
ccgtaacggc catcggcatg aagatgagtg gagataatac 4020ccggttcttc
agaagcgctc agtacggccg cgcctgcgcc atcgccgaaa ataatgatcg
4080taccgcgatc gccaggatcg caagtgcggg ctaatacatc ggaaccgacc
accagcgcgt 4140gtttaaccgc gccggattta acgtactggt cggcgatgct
taacgcgtag gtgaaacctg 4200cgcacgctgc cgcgacatca aacgccgggc
aacctttaat accgagcata ctttgaatct 4260gacatgccgc gcttggaaat
gcatgcgttg ctgatgtggt agccaccaca atcaagccaa 4320tttggtcttt
atcgatcccc gccatctcaa tcgcgcgatt cgcagcggta aagcccatcg
4380tcgcgacagt ttcattcggc gcggcgatat ggcgtttacg aatacctgta
cgagtgacaa 4440tccactcgtc agaggtctca accatttttt ccagatcggc
gttagtccgc acttgttcgg 4500gcagatagct gccagtacca ataatcttcg
tatacatgta cgctcagtca ctaaattact 4560cgatatcaat cacatcaaat
tcgacttctg gattgacgtc agcatcgtaa tcaatgcctt 4620caatgccaaa
gccaaacagc ttgatgaact cttctttgta catgtcgtaa tcggtcagct
4680cacgcaggtt ctctgtggtg atttgtggcc acagatcacg gcagtgctgc
tgaatgtcat 4740cacgcagttc ccagtcatcc aaacgcagac gattgtgatc
atccacttcc ggcgctgaac 4800catct 480556239PRTEscherichia coli 56Met
Val Ile Lys Ala Gln Ser Pro Ala Gly Phe Ala Glu Glu Tyr Ile1 5 10
15Ile Glu Ser Ile Trp Asn Asn Arg Phe Pro Pro Gly Thr Ile Leu Pro
20 25 30Ala Glu Arg Glu Leu Ser Glu Leu Ile Gly Val Thr Arg Thr Thr
Leu 35 40 45Arg Glu Val Leu Gln Arg Leu Ala Arg Asp Gly Trp Leu Thr
Ile Gln 50 55 60His Gly Lys Pro Thr Lys Val Asn Asn Phe Trp Glu Thr
Ser Gly Leu65 70 75 80Asn Ile Leu Glu Thr Leu Ala Arg Leu Asp His
Glu Ser Val Pro Gln 85 90 95Leu Ile Asp Asn Leu Leu Ser Val Arg Thr
Asn Ile Ser Thr Ile Phe 100 105 110Ile Arg Thr Ala Phe Arg Gln His
Pro Asp Lys Ala Gln Glu Val Leu 115 120 125Ala Thr Ala Asn Glu Val
Ala Asp His Ala Asp Ala Phe Ala Glu Leu 130 135 140Asp Tyr Asn Ile
Phe Arg Gly Leu Ala Phe Ala Ser Gly Asn Pro Ile145 150 155 160Tyr
Gly Leu Ile Leu Asn Gly Met Lys Gly Leu Tyr Thr Arg Ile Gly 165 170
175Arg His Tyr Phe Ala Asn Pro Glu Ala Arg Ser Leu Ala Leu Gly Phe
180 185 190Tyr His Lys Leu Ser Ala Leu Cys Ser Glu Gly Ala His Asp
Gln Val 195 200 205Tyr Glu Thr Val Arg Arg Tyr Gly His Glu Ser Gly
Glu Ile Trp His 210 215 220Arg Met Gln Lys Asn Leu Pro Gly Asp Leu
Ala Ile Gln Gly Arg225 230 23557342PRTSynechococcus elongatus 57Met
Ala Phe Gly Leu Ile Gly His Leu Thr Ser Leu Glu Gln Ala Arg1 5 10
15Asp Val Trp Arg Arg Met Gly Tyr Asp Glu Tyr Ala Asp Gln Gly Leu
20 25 30Glu Phe Trp Ser Ser Ala Pro Pro Gln Ile Val Asp Glu Ile Thr
Val 35 40 45Thr Ser Ala Thr Gly Lys Val Ile His Gly Arg Tyr Ile Glu
Ser Cys 50 55 60Phe Leu Pro Glu Met Leu Ala Ala Arg Arg Phe Lys Thr
Ala Thr Arg65 70 75 80Lys Val Leu Asn Ala Met Ser His Ala Gln Lys
His Gly Ile Asp Ile 85 90 95Ser Ala Leu Gly Gly Phe Thr Ser Ile Ile
Phe Glu Asn Phe Asp Leu 100 105 110Ala Ser Leu Arg Gln Val Arg Asp
Thr Thr Leu Glu Phe Glu Arg Phe 115 120 125Thr Thr Gly Asn Thr His
Thr Ala Tyr Val Ile Cys Arg Gln Val Glu 130 135 140Ala Ala Ala Lys
Thr Leu Gly Ile Asp Ile Thr Gln Ala Thr Val Ala145 150 155 160Val
Val Gly Ala Thr Gly Asp Ile Gly Ser Ala Val Cys Arg Trp Leu 165 170
175Asp Leu Lys Leu Gly Val Gly Asp Leu Ile Leu Thr Ala Arg Asn Gln
180 185 190Glu Arg Leu Asp Asn Leu Gln Ala Glu Leu Gly Arg Gly Lys
Ile Leu 195 200 205Pro Leu Glu Ala Ala Leu Pro Glu Ala Asp Phe Ile
Val Trp Val Ala 210 215 220Ser Met Pro Gln Gly Val Val Ile Asp Pro
Ala Thr Leu Lys Gln Pro225 230 235 240Cys Val Leu Ile Asp Gly Gly
Tyr Pro Lys Asn Leu Gly Ser Lys Val 245 250 255Gln Gly Glu Gly Ile
Tyr Val Leu Asn Gly Gly Val Val Glu His Cys 260 265 270Phe Asp Ile
Asp Trp Gln Ile Met Ser Ala Ala Glu Met Ala Arg Pro 275 280 285Glu
Arg Gln Met Phe Ala Cys Phe Ala Glu Ala Met Leu Leu Glu Phe 290 295
300Glu Gly Trp His Thr Asn Phe Ser Trp Gly Arg Asn Gln Ile Thr
Ile305 310 315 320Glu Lys Met Glu Ala Ile Gly Glu Ala Ser Val Arg
His Gly Phe Gln 325 330 335Pro Leu Ala Leu Ala Ile
34058342PRTSynechococcus elongatus 58Met Ala Phe Gly Leu Ile Gly
His Leu Thr Ser Leu Glu Gln Ala Arg1 5 10 15Asp Val Trp Arg Arg Leu
Gly Tyr Asp Glu Tyr Ala Asp Gln Gly Leu 20 25 30Glu Phe Trp Ser Ser
Ala Pro Pro Gln Ile Val Asp Glu Ile Thr Val 35 40 45Thr Ser Ala Thr
Gly Lys Val Ile His Gly Arg Tyr Ile Glu Ser Gly 50 55 60Phe Leu Pro
Glu Met Leu Ala Ala Arg Arg Phe Lys Thr Ala Thr Arg65 70 75 80Lys
Val Leu Asn Ala Met Ser His Ala Gln Lys His Gly Ile Asp Ile 85 90
95Ser Ala Leu Gly Gly Phe Thr Ser Ile Ile Phe Glu Asn Phe Asp Leu
100 105 110Ala Lys Leu Arg Gln Val Arg Asp Thr Thr Leu Glu Phe Glu
Arg Phe 115 120 125Thr Thr Gly Asn Thr His Thr Ala Tyr Val Ile Cys
Arg Gln Val Glu 130 135 140Ala Ala Ala Lys Thr Leu Gly Ile Asp Ile
Ala Gln Ala Thr Val Ala145 150 155 160Val Val Gly Ala Thr Gly Asp
Ile Gly Ser Ala Val Cys Arg Trp Leu 165 170 175Asp Leu Lys Leu Gly
Val Gly Asp Leu Ile Leu Thr Ala Arg Asn Gln 180 185 190Glu Arg Leu
Asp Asn Leu Gln Ala Glu Leu Gly Arg Gly Lys Ile Leu 195 200 205Pro
Leu Glu Ala Ala Leu Pro Glu Ala Asp Phe Ile Val Trp Val Ala 210 215
220Ser Met Pro Gln Gly Val Val Ile Asp Pro Ala Thr Leu Lys Gln
Pro225 230 235 240Cys Val Leu Ile Asp Gly Gly Tyr Pro Lys Asn Leu
Gly Ser Lys Val 245 250 255Gln Gly Glu Gly Ile Tyr Val Leu Asn Gly
Gly Val Val Glu His Cys 260 265 270Phe Asp Ile Asp Trp Gln Ile Met
Ser Leu Ala Glu Met Ala Arg Pro 275 280 285Glu Arg Gln Met Phe Ala
Cys Phe Ala Glu Ala Met Leu Leu Glu Phe 290 295 300Glu Gly Trp His
Thr Asn Phe Ser Trp Gly Arg Asn Gln Ile Thr Ile305 310 315 320Glu
Lys Met Glu Ala Ile Gly Glu Ala Ser Val Arg His Gly Phe Gln 325 330
335Pro Leu Ala Leu Ala Ile 34059342PRTSynechococcus elongatus 59Met
Ala Phe Gly Leu Ile Gly His Ala Thr Ser Leu Glu Gln Ala Arg1 5 10
15Asp Val Trp Arg Arg Leu Gly Tyr Asp Glu Tyr Ala Asp Gln Gly Leu
20 25 30Glu Phe Trp Ser Ser Ala Pro Pro Gln Ile Val Asp Glu Ile Thr
Val 35 40 45Thr Ser Ala Thr Gly Lys Val Ile His Gly Arg Tyr Ile Glu
Ser Gly 50 55 60Phe Leu Pro Glu Met Leu Ala Ala Arg Arg Phe Lys Thr
Ala Thr Arg65 70 75 80Lys Val Leu Asn Ala Met Ser His Ala Gln Lys
His Gly Ile Asp Ile 85 90 95Ser Ala Leu Gly Gly Phe Thr Ser Ile Ile
Phe Glu Asn Phe Asp Leu 100 105 110Ala Lys Leu Arg Gln Val Arg Asp
Thr Thr Leu Glu Phe Glu Arg Phe 115 120 125Thr Thr Gly Asn Thr His
Thr Ala Tyr Val Ile Cys Arg Gln Val Glu 130 135 140Ala Ala Ala Lys
Thr Leu Gly Ile Asp Ile Ala Gln Ala Thr Val Ala145 150 155 160Val
Val Gly Ala Thr Gly Asp Ile Gly Ser Ala Val Cys Arg Trp Leu 165 170
175Asp Leu Lys Leu Gly Val Gly Asp Leu Ile Leu Thr Ala Arg Asn Gln
180 185 190Glu Arg Leu Asp Asn Leu Gln Ala Glu Leu Gly Arg Gly Lys
Ile Leu 195 200 205Pro Leu Glu Ala Ala Leu Pro Glu Ala Asp Phe Ile
Val Trp Val Ala 210 215 220Ser Met Pro Gln Gly Val Val Ile Asp Pro
Ala Thr Leu Lys Gln Pro225 230 235 240Cys Val Leu Ile Asp Gly Gly
Tyr Pro Lys Asn Leu Gly Ser Lys Val 245 250 255Gln Gly Glu Gly Ile
Tyr Val Leu Asn Gly Gly Val Val Glu His Cys 260 265 270Phe Asp Ile
Asp Trp Gln Ile Met Ser Leu Ala Glu Met Ala Arg Pro 275 280 285Glu
Arg Gln Met Phe Ala Cys Phe Ala Glu Ala Met Leu Leu Glu Phe 290 295
300Glu Gly Trp His Thr Asn Phe Ser Trp Gly Arg Asn Gln Ile Thr
Ile305 310 315 320Glu Lys Met Glu Ala Ile Gly Glu Ala Ser Val Arg
His Gly Phe Gln 325 330 335Pro Leu Ala Leu Ala Ile
34060342PRTSynechococcus elongatus 60Met Ala Phe Gly Leu Ile Gly
His Leu Thr Ser Leu Glu Gln Ala Arg1 5 10 15Leu Val Trp Arg Arg Leu
Gly Tyr Asp Glu Tyr Ala Asp Gln Gly Leu 20 25 30Glu Phe Trp Ser Ser
Ala Pro Pro Gln Ile Val Asp Glu Ile Thr Val 35 40 45Thr Ser Ala Thr
Gly Lys Val Ile His Gly Arg Tyr Ile Glu Ser Gly 50 55 60Phe Leu Pro
Glu Met Leu Ala Ala Arg Arg Phe Lys Thr Ala Thr Arg65 70 75 80Lys
Val Leu Asn Ala Met Ser His Ala Gln Lys His Gly Ile Asp Ile 85 90
95Ser Ala Leu Gly Gly Phe Thr Ser Ile Ile Phe Glu Asn Phe Asp Leu
100 105 110Ala Lys Leu Arg Gln Val Arg Asp Thr Thr Leu Glu Phe Glu
Arg Phe 115 120 125Thr Thr Gly Asn Thr His Thr Ala Tyr Val Ile Cys
Arg Gln Val Glu 130 135 140Ala Ala Ala Lys Thr Leu Gly Ile Asp Ile
Ala Gln Ala Thr Val Ala145 150 155 160Val Val Gly Ala Thr Gly Asp
Ile Gly Ser Ala Val Cys Arg Trp Leu 165 170 175Asp Leu Lys Leu Gly
Val Gly Asp Leu Ile Leu Thr Ala Arg Asn Gln 180 185 190Glu Arg Leu
Asp Asn Leu Gln Ala Glu Leu Gly Arg Gly Lys Ile Leu 195 200 205Pro
Leu Glu Ala Ala Leu Pro Glu Ala Asp Phe Ile Val Trp Val Ala 210 215
220Ser Met Pro Gln Gly Val Val Ile Asp Pro Ala Thr Leu Lys Gln
Pro225 230 235 240Cys Val Leu Ile Asp Gly Gly Tyr Pro Lys Asn Leu
Gly Ser Lys Val 245 250 255Gln Gly Glu Gly Ile Tyr Val Leu Asn Gly
Gly Val Val Glu His Cys 260 265 270Phe Asp Ile Asp Trp Gln Ile Met
Ser Leu Ala Glu Met Ala Arg Pro 275 280 285Glu Arg Gln Met Phe Ala
Cys Phe Ala Glu Ala Met Leu Leu Glu Phe 290 295 300Glu Gly Trp His
Thr Asn Phe Ser Trp Gly Arg Asn Gln Ile Thr Ile305 310 315 320Glu
Lys Met Glu Ala Ile Gly Glu Ala Ser Val Arg His Gly Phe Gln 325 330
335Pro Leu Ala Leu Ala Ile 34061342PRTSynechococcus elongatus 61Met
Ala Phe Gly Leu Ile Gly His Ala Thr Ser Leu Glu Gln Ala Arg1 5 10
15Asp Val Trp Arg Arg Met Gly Tyr Val Glu Tyr Ala Asp Gln Gly Leu
20 25 30Glu Phe Trp Ser Ser Ala Pro Pro Gln Ile Val Asp Glu Ile Thr
Val 35 40 45Thr Ser Ala Thr Gly Lys Val Ile His Gly Arg Tyr Ile Glu
Ser Gly 50 55 60Phe Leu Pro Glu Met Leu Ala Ala Arg Arg Phe Lys Thr
Ala Thr Arg65 70 75 80Lys Val Leu Asn Ala Met Ser His Ala Gln Lys
His Gly Ile Asp Ile 85 90 95Ser Ala Leu Gly Gly Phe Thr Ser Ile Ile
Phe Glu Asn Phe Asp Leu 100 105 110Ala Lys Leu Arg Gln Val Arg Asp
Thr Thr Leu Glu Phe Glu Arg Phe 115 120 125Thr Thr Gly Asn Thr His
Thr Ala Tyr Val Ile Cys Arg Gln Val Glu 130 135 140Ala Ala Ala Lys
Thr Leu Gly Ile Asp Ile Thr Leu Ala Thr Val Ala145 150 155 160Val
Val Gly Ala Thr Gly Asp Ile Gly Ser Ala Val Cys Arg Trp Leu 165 170
175Asp Leu Lys Leu Gly Val Gly Asp Leu Ile Leu Thr Ala Arg Asn Gln
180 185 190Glu Arg Leu Asp Asn Leu Gln Ala Glu Leu Gly Arg Gly Lys
Ile Leu 195 200 205Pro Leu Glu Ala Ala Leu Pro Glu Ala Asp Phe Ile
Val Trp Val Ala 210 215 220Ser Met Pro Gln Gly Val Val Ile Asp Pro
Ala Thr Leu Lys Gln Pro225 230 235 240Cys Val Leu Ile Asp Gly Gly
Tyr Pro Lys Asn Leu Gly Ser Lys Val 245 250 255Gln Gly Glu Gly Ile
Tyr Val Leu Asn Gly Gly Val Val Glu His Cys 260 265 270Phe Asp Ile
Asp Trp Gln Ile Met Ser Leu Ala Glu Met Ala Arg Pro 275 280 285Glu
Arg Gln Met Phe Ala Cys Phe Ala Glu Ala Met Leu Leu Glu Phe 290 295
300Glu Gly Trp His Thr Asn Phe Ser Trp Gly Arg Asn Gln Ile Thr
Ile305 310 315 320Glu Lys Met Glu Ala Ile Gly Glu Ala Ser Val Arg
His Gly Phe Gln 325 330 335Pro Leu Ala Leu Ala Ile
34062342PRTSynechococcus elongatus 62Met Ala Phe Gly Leu Ile Gly
His Leu Thr Ser Leu Glu Gln Ala Arg1 5 10 15Asp Val Trp Arg Arg Met
Gly Tyr Pro Glu Tyr Ala Asp Gln Gly Met 20 25 30Glu Phe Trp Ser Ser
Ala Pro Pro Gln Ile Val Asp Glu Ile Thr Val 35 40 45Thr Ser Ala Thr
Gly Lys Val Ile His Gly Arg Tyr Ile Glu Ser Gly 50 55 60Phe Leu Pro
Glu Met Leu Ala Ala Arg Arg Phe Lys Thr Ala Thr Arg65 70 75 80Lys
Val Leu Asn Ala Met Ser His Ala Gln Lys His Gly Ile Asp Ile 85 90
95Ser Ala Leu Gly Gly Phe Thr Ser Ile Ile Phe Glu Asn Phe Asp Leu
100 105 110Ala Lys Leu Arg Gln Val Arg Asp Thr Thr Leu Glu Phe Glu
Arg Phe 115 120 125Thr Thr Gly Asn Thr His Thr Ala Tyr Val Ile Cys
Arg Gln Val Glu 130 135 140Ala Ala Ala Lys Thr Leu Gly Ile Asp Ile
Ala Gln Ala Thr Val Ala145 150 155 160Val Val Gly Ala Thr Gly Asp
Ile Gly Ser Ala Val Cys Arg Trp Leu 165 170 175Asp Leu Lys Leu Gly
Val Gly Asp Leu Ile Leu Thr Ala Arg Asn Gln 180 185 190Glu Arg Leu
Asp Asn Leu Gln Ala Glu Leu Gly Arg Gly Lys Ile Leu 195
200 205Pro Leu Glu Ala Ala Leu Pro Glu Ala Asp Phe Ile Val Trp Val
Ala 210 215 220Ser Met Pro Gln Gly Val Val Ile Asp Pro Ala Thr Leu
Lys Gln Pro225 230 235 240Cys Val Leu Ile Asp Gly Gly Tyr Pro Lys
Asn Leu Gly Ser Lys Val 245 250 255Gln Gly Glu Gly Ile Tyr Val Leu
Asn Gly Gly Val Val Glu His Cys 260 265 270Phe Asp Ile Asp Trp Gln
Ile Met Ser Leu Ala Glu Met Ala Arg Pro 275 280 285Glu Arg Gln Met
Phe Ala Cys Phe Ala Glu Ala Met Leu Leu Glu Phe 290 295 300Glu Gly
Trp His Thr Asn Phe Ser Trp Gly Arg Asn Gln Ile Thr Ile305 310 315
320Glu Lys Met Glu Ala Ile Gly Glu Ala Ser Val Arg His Gly Phe Gln
325 330 335Pro Leu Ala Leu Ala Ile 34063342PRTSynechococcus
elongatus 63Met Ala Phe Gly Leu Ile Gly His Ala Thr Ser Leu Glu Gln
Ala Arg1 5 10 15Leu Val Trp Arg Arg Met Gly Tyr Val Glu Tyr Ala Asp
Gln Gly Leu 20 25 30Glu Phe Trp Ser Ser Ala Pro Pro Gln Ile Val Asp
Glu Ile Thr Val 35 40 45Thr Ser Ala Thr Gly Lys Val Ile His Gly Arg
Tyr Ile Glu Ser Gly 50 55 60Phe Leu Pro Glu Met Leu Ala Ala Arg Arg
Phe Lys Thr Ala Thr Arg65 70 75 80Lys Val Leu Asn Ala Met Ser His
Ala Gln Lys His Gly Ile Asp Ile 85 90 95Ser Ala Leu Gly Gly Phe Thr
Ser Ile Ile Phe Glu Asn Phe Asp Leu 100 105 110Ala Lys Leu Arg Gln
Val Arg Asp Thr Thr Leu Glu Phe Glu Arg Phe 115 120 125Thr Thr Gly
Asn Thr His Thr Ala Tyr Val Ile Cys Arg Gln Val Glu 130 135 140Ala
Ala Ala Lys Thr Leu Gly Ile Asp Ile Ala Gln Ala Thr Val Ala145 150
155 160Val Val Gly Ala Thr Gly Asp Ile Gly Ser Ala Val Cys Arg Trp
Leu 165 170 175Asp Leu Lys Leu Gly Val Gly Asp Leu Ile Leu Thr Ala
Arg Asn Gln 180 185 190Glu Arg Leu Asp Asn Leu Gln Ala Glu Leu Gly
Arg Gly Lys Ile Leu 195 200 205Pro Leu Glu Ala Ala Leu Pro Glu Ala
Asp Phe Ile Val Trp Val Ala 210 215 220Ser Met Pro Gln Gly Val Val
Ile Asp Pro Ala Thr Leu Lys Gln Pro225 230 235 240Cys Val Leu Ile
Asp Gly Gly Tyr Pro Lys Asn Leu Gly Ser Lys Val 245 250 255Gln Gly
Glu Gly Ile Tyr Val Leu Asn Gly Gly Val Val Glu His Cys 260 265
270Phe Asp Ile Asp Trp Gln Ile Met Ser Leu Ala Glu Met Ala Arg Pro
275 280 285Glu Arg Gln Met Phe Ala Cys Phe Ala Glu Ala Met Leu Leu
Glu Phe 290 295 300Glu Gly Trp His Thr Asn Phe Ser Trp Gly Arg Asn
Gln Ile Thr Ile305 310 315 320Glu Lys Met Glu Ala Ile Gly Glu Ala
Ser Val Arg His Gly Phe Gln 325 330 335Pro Leu Ala Leu Ala Ile
34064342PRTSynechococcus elongatus 64Met Ala Phe Gly Leu Ile Gly
His Leu Thr Ser Leu Glu Gln Ala Arg1 5 10 15Asp Val Trp Arg Arg Met
Gly Tyr Glu Glu Tyr Ala Asp Gln Gly Leu 20 25 30Glu Phe Trp Ser Ser
Ala Pro Pro Gln Ile Val Asp Glu Ile Thr Val 35 40 45Thr Ser Ala Thr
Gly Lys Val Ile His Gly Arg Tyr Ile Glu Ser Gly 50 55 60Phe Leu Pro
Glu Met Leu Ala Ala Arg Arg Phe Lys Thr Ala Thr Arg65 70 75 80Lys
Val Leu Asn Ala Met Ser His Ala Gln Lys His Gly Ile Asp Ile 85 90
95Ser Ala Leu Gly Gly Phe Thr Ser Ile Ile Phe Glu Asn Phe Asp Leu
100 105 110Ala Lys Leu Arg Gln Val Arg Asp Thr Thr Leu Glu Phe Glu
Arg Phe 115 120 125Thr Thr Gly Asn Thr His Thr Ala Tyr Val Ile Cys
Arg Gln Val Glu 130 135 140Ala Ala Ala Lys Thr Leu Gly Ile Asp Ile
Ala Gln Ala Thr Val Ala145 150 155 160Val Val Gly Ala Thr Gly Asp
Ile Gly Ser Ala Val Cys Arg Trp Leu 165 170 175Asp Leu Lys Leu Gly
Val Gly Asp Leu Ile Leu Thr Ala Arg Asn Gln 180 185 190Glu Arg Leu
Asp Asn Leu Gln Ala Glu Leu Gly Arg Gly Lys Ile Leu 195 200 205Pro
Leu Glu Ala Ala Leu Pro Glu Ala Asp Phe Ile Val Trp Val Ala 210 215
220Ser Met Pro Gln Gly Val Val Ile Asp Pro Ala Thr Leu Lys Gln
Pro225 230 235 240Cys Val Leu Ile Asp Gly Gly Tyr Pro Lys Asn Leu
Gly Ser Lys Val 245 250 255Gln Gly Glu Gly Ile Tyr Val Leu Asn Gly
Gly Val Val Glu His Cys 260 265 270Phe Asp Ile Asp Trp Gln Ile Met
Ser Leu Ala Glu Met Ala Arg Pro 275 280 285Glu Arg Gln Met Phe Ala
Cys Phe Ala Glu Ala Met Leu Leu Glu Phe 290 295 300Glu Gly Trp His
Thr Asn Phe Ser Trp Gly Arg Asn Gln Ile Thr Ile305 310 315 320Glu
Lys Met Glu Ala Ile Gly Glu Ala Ser Val Arg His Gly Phe Gln 325 330
335Pro Leu Ala Leu Ala Ile 34065347PRTProchlorococcus marinus 65Met
Ala Phe Gly Leu Ile Gly His Ser Thr Ser Phe Glu Asp Ala Lys1 5 10
15Arg Lys Ala Ser Leu Leu Gly Phe Asp His Ile Ala Asp Gly Asp Leu
20 25 30Asp Val Trp Cys Thr Ala Pro Pro Gln Leu Val Glu Asn Val Glu
Val 35 40 45Lys Ser Ala Ile Gly Ile Ser Ile Glu Gly Ser Tyr Ile Glu
Ser Cys 50 55 60Phe Val Pro Glu Met Leu Ser Arg Phe Lys Thr Ala Arg
Arg Lys Val65 70 75 80Leu Asn Ala Met Glu Leu Ala Gln Lys Lys Gly
Ile Asn Ile Thr Ala 85 90 95Leu Gly Gly Phe Thr Ser Ile Ile Phe Glu
Asn Phe Asn Leu Leu Gln 100 105 110His Lys Gln Ile Arg Asn Thr Ser
Leu Glu Trp Glu Arg Phe Thr Thr 115 120 125Gly Asn Thr His Thr Ala
Trp Val Ile Cys Arg Gln Leu Glu Met Asn 130 135 140Ala Pro Lys Ile
Gly Ile Asp Leu Lys Ser Ala Thr Val Ala Val Val145 150 155 160Gly
Ala Thr Gly Asp Ile Gly Ser Ala Val Cys Arg Trp Leu Ile Asn 165 170
175Lys Thr Gly Ile Gly Glu Leu Leu Leu Val Ala Arg Gln Lys Glu Pro
180 185 190Leu Asp Ser Leu Gln Lys Glu Leu Asp Gly Gly Thr Ile Lys
Asn Leu 195 200 205Asp Glu Ala Leu Pro Glu Ala Asp Ile Val Val Trp
Val Ala Ser Met 210 215 220Pro Lys Thr Met Glu Ile Asp Ala Asn Asn
Leu Lys Gln Pro Cys Leu225 230 235 240Met Ile Asp Gly Gly Tyr Pro
Lys Asn Leu Asp Glu Lys Phe Gln Gly 245 250 255Asn Asn Ile His Val
Val Lys Gly Gly Ile Val Arg Phe Phe Asn Asp 260 265 270Ile Gly Trp
Asn Met Met Glu Leu Ala Glu Met Gln Asn Pro Gln Arg 275 280 285Glu
Met Phe Ala Cys Phe Ala Glu Ala Met Ile Leu Glu Phe Glu Lys 290 295
300Cys His Thr Asn Phe Ser Trp Gly Arg Asn Asn Ile Ser Leu Glu
Lys305 310 315 320Met Glu Phe Ile Gly Ala Ala Ser Val Lys His Gly
Phe Ser Ala Ile 325 330 335Gly Leu Asp Lys His Pro Lys Val Leu Ala
Val 340 34566347PRTProchlorococcus marinus 66Met Ala Phe Gly Leu
Ile Gly His Ser Thr Ser Phe Glu Asp Ala Lys1 5 10 15Arg Lys Ala Ser
Leu Leu Gly Phe Asp His Ile Ala Asp Gly Asp Leu 20 25 30Asp Val Trp
Cys Thr Ala Pro Pro Gln Leu Val Glu Asn Val Glu Val 35 40 45Lys Ser
Ala Ile Gly Ile Ser Ile Glu Gly Ser Tyr Ile Asp Ser Cys 50 55 60Phe
Val Pro Glu Met Leu Ser Arg Phe Lys Thr Ala Arg Arg Lys Val65 70 75
80Leu Asn Ala Met Glu Leu Ala Gln Lys Lys Gly Ile Asn Ile Thr Ala
85 90 95Leu Gly Gly Phe Thr Ser Ile Ile Phe Glu Asn Phe Asn Leu Leu
Gln 100 105 110His Lys Gln Ile Arg Asn Thr Ser Leu Glu Trp Glu Arg
Phe Thr Thr 115 120 125Gly Asn Thr His Thr Ala Trp Val Ile Cys Arg
Gln Leu Glu Met Asn 130 135 140Ala Pro Lys Ile Gly Ile Asp Leu Lys
Ser Ala Thr Val Ala Val Val145 150 155 160Gly Ala Thr Gly Asp Ile
Gly Ser Ala Val Cys Arg Trp Leu Ile Asn 165 170 175Lys Thr Gly Ile
Gly Glu Leu Leu Leu Val Ala Arg Gln Lys Glu Pro 180 185 190Leu Asp
Ser Leu Gln Lys Glu Leu Asp Gly Gly Thr Ile Lys Asn Leu 195 200
205Asp Glu Ala Leu Pro Glu Ala Asp Ile Val Val Trp Val Ala Ser Met
210 215 220Pro Lys Thr Met Glu Ile Asp Ala Asn Asn Leu Lys Gln Pro
Cys Leu225 230 235 240Met Ile Asp Gly Gly Tyr Pro Lys Asn Leu Asp
Glu Lys Phe Gln Gly 245 250 255Asn Asn Ile His Val Val Lys Gly Gly
Ile Val Arg Phe Phe Asn Asp 260 265 270Ile Gly Trp Asn Met Met Glu
Leu Ala Glu Met Gln Asn Pro Gln Arg 275 280 285Glu Met Phe Ala Cys
Phe Ala Glu Ala Met Ile Leu Glu Phe Glu Lys 290 295 300Cys His Thr
Asn Phe Ser Trp Gly Arg Asn Asn Ile Ser Leu Glu Lys305 310 315
320Met Glu Phe Ile Gly Ala Ala Ser Val Lys His Gly Phe Ser Ala Ile
325 330 335Gly Leu Asp Lys His Pro Lys Val Leu Ala Pro 340
34567347PRTProchlorococcus marinus 67Met Ala Phe Gly Leu Ile Gly
His Ser Thr Ser Phe Glu Asp Ala Lys1 5 10 15Arg Lys Ala Ser Leu Leu
Gly Phe Asp His Ile Ala Asp Gly Asp Leu 20 25 30Asp Val Trp Cys Thr
Ala Pro Pro Val Leu Val Glu Asn Val Glu Val 35 40 45Lys Ser Ala Ile
Gly Ile Ser Ile Glu Gly Ser Tyr Ile Asp Ser Cys 50 55 60Phe Val Pro
Glu Met Leu Ser Arg Phe Lys Thr Ala Arg Arg Lys Val65 70 75 80Leu
Asn Ala Met Glu Leu Ala Gln Lys Lys Gly Ile Asn Ile Thr Ala 85 90
95Leu Gly Gly Phe Thr Ser Ile Ile Phe Glu Asn Phe Asn Leu Leu Gln
100 105 110His Lys Gln Ile Arg Asn Thr Ser Leu Glu Trp Glu Arg Phe
Thr Thr 115 120 125Gly Asn Thr His Thr Ala Trp Val Ile Cys Arg Gln
Leu Glu Met Asn 130 135 140Ala Pro Lys Ile Gly Ile Asp Leu Lys Ser
Ala Thr Val Ala Val Val145 150 155 160Gly Ala Thr Gly Asp Ile Gly
Ser Ala Val Cys Arg Trp Leu Ile Asn 165 170 175Lys Thr Gly Ile Gly
Glu Leu Leu Leu Val Ala Arg Gln Lys Glu Pro 180 185 190Leu Asp Ser
Leu Gln Lys Glu Leu Asp Gly Gly Thr Ile Lys Asn Leu 195 200 205Asp
Glu Ala Leu Pro Glu Ala Asp Ile Val Val Trp Val Ala Ser Met 210 215
220Pro Lys Thr Met Glu Ile Asp Ala Asn Asn Leu Lys Gln Pro Cys
Leu225 230 235 240Met Ile Asp Gly Gly Tyr Pro Lys Asn Leu Asp Glu
Lys Phe Gln Gly 245 250 255Asn Asn Ile His Val Val Lys Gly Gly Ile
Val Arg Phe Phe Asn Asp 260 265 270Ile Gly Trp Asn Met Met Glu Leu
Ala Glu Met Gln Asn Pro Gln Arg 275 280 285Glu Met Phe Ala Cys Phe
Ala Glu Ala Met Ile Leu Glu Phe Glu Lys 290 295 300Cys His Thr Asn
Phe Ser Trp Gly Arg Asn Asn Ile Ser Leu Glu Lys305 310 315 320Met
Glu Phe Ile Gly Ala Ala Ser Val Lys His Gly Phe Ser Ala Ile 325 330
335Gly Leu Asp Lys His Pro Lys Val Leu Ala Val 340
34568347PRTProchlorococcus marinus 68Met Ala Phe Gly Leu Ile Gly
His Ser Thr Ser Phe Glu Asp Ala Lys1 5 10 15Arg Lys Ala Ser Leu Leu
Gly Phe Asp His Ile Ala Asp Gly Asp Leu 20 25 30Asp Val Trp Cys Thr
Ala Pro Pro Gln Leu Val Glu Asn Val Glu Val 35 40 45Lys Ser Ala Ile
Gly Ile Ser Ile Glu Gly Ser Tyr Ile Asp Ser Cys 50 55 60Phe Val Pro
Glu Met Leu Ser Arg Phe Lys Thr Ala Arg Arg Lys Val65 70 75 80Leu
Asn Ala Met Glu Leu Ala Gln Lys Lys Gly Ile Asn Ile Thr Ala 85 90
95Leu Gly Gly Phe Thr Ser Ile Ile Phe Glu Asn Phe Asn Leu Leu Gln
100 105 110His Lys Gln Ile Arg Asn Thr Ser Leu Glu Trp Glu Arg Phe
Thr Thr 115 120 125Gly Asn Thr His Thr Ala Trp Val Ile Cys Arg Gln
Leu Glu Met Asn 130 135 140Ala Pro Lys Ile Gly Ile Asp Leu Lys Ser
Ala Thr Val Ala Val Val145 150 155 160Gly Ala Thr Gly Asp Ile Gly
Ser Ala Val Cys Arg Trp Leu Ile Asn 165 170 175Lys Thr Gly Ile Gly
Glu Leu Leu Leu Val Ala Arg Gln Lys Glu Pro 180 185 190Leu Asp Ser
Leu Gln Lys Glu Leu Asp Gly Gly Thr Ile Lys Asn Leu 195 200 205Asp
Glu Ala Leu Pro Glu Ala Asp Ile Val Val Trp Val Ala Ser Met 210 215
220Pro Lys Thr Met Glu Ile Asp Ala Asn Asn Leu Lys Gln Pro Cys
Leu225 230 235 240Met Ile Asp Gly Gly Tyr Pro Lys Asn Leu Asp Glu
Lys Phe Gln Gly 245 250 255Asn Asn Ile His Val Val Lys Gly Gly Ile
Val Arg Phe Phe Asn Asp 260 265 270Ile Gly Trp Asn Met Met Glu Leu
Ala Glu Met Gln Asn Pro Gln Arg 275 280 285Glu Met Phe Ala Cys Phe
Ala Glu Ala Met Ile Leu Glu Phe Glu Lys 290 295 300Cys His Thr Asn
Phe Ser Trp Gly Arg Asn Asn Ile Ser Leu Glu Lys305 310 315 320Met
Glu Phe Ile Gly Ala Ala Ser Val Lys His Gly Phe Ser Ala Ile 325 330
335Gly Leu Asp Lys His Pro Lys Val Leu Arg Val 340
34569347PRTProchlorococcus marinus 69Met Ala Phe Gly Leu Ile Gly
His Ser Thr Ser Phe Glu Asp Ala Lys1 5 10 15Arg Lys Ala Ser Leu Leu
Gly Phe Asp His Ile Ala Asp Gly Asp Leu 20 25 30Asp Val Trp Cys Thr
Ala Pro Pro Gln Leu Val Glu Asn Val Glu Val 35 40 45Lys Ser Ala Ile
Gly Ile Ser Ile Glu Gly Ser Tyr Ile Asp Ser Cys 50 55 60Phe Val Pro
Glu Met Leu Ser Arg Phe Lys Thr Ala Arg Arg Lys Val65 70 75 80Leu
Asn Ala Met Glu Leu Ala Gln Lys Lys Gly Ile Asn Ile Thr Ala 85 90
95Leu Gly Gly Phe Thr Ser Ile Ile Phe Glu Asn Phe Asn Leu Leu Gln
100 105 110His Lys Gln Ile Arg Asn Thr Ser Leu Glu Trp Glu Arg Phe
Thr Thr 115 120 125Gly Asn Thr His Thr Ala Trp Val Ile Cys Arg Gln
Leu Glu Met Asn 130 135 140Ala Pro Lys Ile Gly Ile Asp Leu Lys Ser
Ala Thr Val Ala Val Val145 150 155 160Gly Ala Thr Gly Asp Ile Gly
Ser Ala Val Cys Arg Trp Leu Ile Asn 165 170 175Lys Thr Gly Ile Gly
Glu Leu Leu Leu Val Ala Arg Gln Lys Glu Pro 180 185 190Leu Asp Ser
Leu Gln Lys Glu Leu Asp Gly Gly Thr Ile Lys Asn Leu 195 200 205Asp
Glu Ala Leu Pro Glu Ala Asp Ile Val Val Trp Val Ala Ser Met 210 215
220Pro Lys Thr Met Glu Ile Asp Ala Asn Asn Leu Lys Gln Pro Cys
Leu225 230
235 240Met Ile Asp Gly Gly Tyr Pro Lys Asn Leu Asp Glu Lys Phe Gln
Gly 245 250 255Asn Asn Ile His Val Val Lys Gly Gly Ile Val Arg Phe
Phe Asn Asp 260 265 270Ile Gly Trp Asn Met Met Glu Leu Ala Glu Met
Gln Asn Pro Gln Arg 275 280 285Glu Met Phe Ala Cys Phe Ala Glu Ala
Met Ile Leu Glu Phe Glu Lys 290 295 300Cys His Thr Asn Phe Ser Trp
Gly Arg Asn Asn Ile Ser Leu Glu Lys305 310 315 320Met Glu Phe Ile
Gly Ala Ala Ser Val Lys His Gly Phe Ser Ala Ile 325 330 335Gly Leu
Asp Lys His Pro Lys Val Ser Ala Val 340 34570347PRTProchlorococcus
marinus 70Met Ala Phe Gly Leu Ile Gly His Ser Thr Ser Phe Glu Asp
Ala Lys1 5 10 15Arg Lys Ala Ser Leu Leu Gly Phe Asp His Ile Ala Asp
Gly Asp Leu 20 25 30Asp Val Trp Cys Thr Ala Pro Pro Gln Leu Val Glu
Asn Val Glu Val 35 40 45Lys Ser Ala Ile Gly Ile Ser Ile Glu Gly Ser
Tyr Ile Asp Ser Cys 50 55 60Phe Val Pro Glu Met Leu Ser Arg Phe Lys
Thr Ala Arg Arg Lys Val65 70 75 80Leu Asn Ala Met Glu Leu Ala Gln
Lys Lys Gly Ile Asn Ile Thr Ala 85 90 95Leu Gly Gly Phe Thr Ser Ile
Ile Phe Glu Asn Phe Asn Leu Leu Gln 100 105 110His Lys Gln Ile Arg
Asn Thr Ser Leu Glu Trp Glu Arg Phe Thr Thr 115 120 125Gly Asn Thr
His Thr Ala Trp Val Ile Cys Arg Gln Leu Glu Met Asn 130 135 140Ala
Pro Lys Ile Gly Ile Asp Leu Lys Ser Ala Thr Val Ala Val Val145 150
155 160Gly Ala Thr Gly Asp Ile Gly Ser Ala Val Cys Arg Trp Leu Ile
Asn 165 170 175Lys Thr Gly Ile Gly Glu Leu Leu Leu Val Ala Arg Gln
Lys Glu Pro 180 185 190Leu Asp Ser Leu Gln Lys Glu Leu Asp Gly Gly
Thr Ile Lys Asn Leu 195 200 205Asp Glu Ala Leu Pro Glu Ala Asp Ile
Val Val Trp Val Ala Ser Met 210 215 220Pro Lys Thr Met Glu Ile Asp
Ala Asn Asn Leu Lys Gln Pro Cys Leu225 230 235 240Met Ile Asp Gly
Gly Tyr Pro Lys Asn Leu Asp Glu Lys Phe Gln Gly 245 250 255Asn Asn
Ile His Val Val Lys Gly Gly Ile Val Arg Phe Phe Asn Asp 260 265
270Ile Gly Trp Asn Met Met Glu Leu Ala Glu Met Gln Asn Pro Gln Arg
275 280 285Glu Met Phe Ala Cys Phe Ala Glu Ala Met Ile Leu Glu Phe
Glu Lys 290 295 300Cys His Thr Asn Phe Ser Trp Gly Arg Asn Asn Ile
Ser Leu Glu Lys305 310 315 320Met Glu Phe Ile Gly Ala Ala Ser Val
Lys His Gly Phe Ser Ala Ile 325 330 335Gly Leu Asp Lys His Pro Lys
Val Leu Ala Gly 340 34571347PRTProchlorococcus marinus 71Met Ala
Phe Gly Leu Ile Gly His Ser Thr Ser Phe Glu Asp Ala Lys1 5 10 15Arg
Lys Ala Ser Leu Leu Gly Phe Asp His Ile Ala Asp Gly Asp Leu 20 25
30Asp Val Trp Cys Thr Ala Pro Pro Gln Leu Val Glu Asn Val Glu Val
35 40 45Lys Ser Ala Ile Gly Ile Ser Ile Glu Gly Ser Tyr Ile Asp Ser
Cys 50 55 60Phe Val Pro Glu Met Leu Ser Arg Phe Lys Thr Ala Arg Arg
Lys Val65 70 75 80Leu Asn Ala Met Glu Leu Ala Gln Lys Lys Gly Ile
Asn Ile Thr Ala 85 90 95Leu Gly Gly Phe Thr Ser Ile Ile Phe Glu Asn
Phe Asn Leu Leu Gln 100 105 110His Lys Gln Ile Arg Asn Thr Ser Leu
Glu Trp Glu Arg Phe Thr Thr 115 120 125Gly Asn Thr His Thr Ala Trp
Val Ile Cys Arg Gln Leu Glu Met Asn 130 135 140Ala Pro Lys Ile Gly
Ile Asp Leu Lys Ser Ala Thr Val Ala Val Val145 150 155 160Gly Ala
Thr Gly Asp Ile Gly Ser Ala Val Cys Arg Trp Leu Ile Asn 165 170
175Lys Thr Gly Ile Gly Glu Leu Leu Leu Val Ala Arg Gln Lys Glu Pro
180 185 190Leu Asp Ser Leu Gln Lys Glu Leu Asp Gly Gly Thr Ile Lys
Asn Leu 195 200 205Asp Glu Ala Leu Pro Glu Ala Asp Ile Val Val Trp
Val Ala Ser Met 210 215 220Pro Lys Thr Met Glu Ile Asp Ala Asn Asn
Leu Lys Gln Pro Cys Leu225 230 235 240Met Ile Asp Gly Gly Tyr Pro
Lys Asn Leu Asp Glu Lys Phe Gln Gly 245 250 255Asn Asn Ile His Val
Val Lys Gly Gly Ile Val Arg Phe Phe Asn Asp 260 265 270Ile Gly Trp
Asn Met Met Glu Leu Ala Glu Met Gln Asn Pro Gln Arg 275 280 285Glu
Met Phe Ala Cys Phe Ala Glu Ala Met Ile Leu Glu Phe Glu Lys 290 295
300Cys His Thr Asn Phe Ser Trp Gly Arg Asn Asn Ile Ser Leu Glu
Lys305 310 315 320Met Glu Phe Ile Gly Ala Ala Ser Val Lys His Gly
Phe Ser Ala Ile 325 330 335Gly Leu Asp Lys His Pro Lys Val Asp Ala
Val 340 34572347PRTProchlorococcus marinus 72Met Ala Phe Gly Leu
Ile Gly His Ser Thr Ser Phe Glu Asp Ala Lys1 5 10 15Arg Lys Ala Ser
Leu Leu Gly Phe Asp His Ile Ala Asp Gly Asp Leu 20 25 30Asp Val Trp
Cys Thr Ala Pro Pro Gln Leu Val Glu Asn Val Glu Val 35 40 45Lys Ser
Ala Ile Val Ile Ser Ile Glu Gly Ser Tyr Ile Asp Ser Cys 50 55 60Phe
Val Pro Glu Met Leu Ser Arg Phe Lys Thr Ala Arg Arg Lys Val65 70 75
80Leu Asn Ala Met Glu Leu Ala Gln Lys Lys Gly Ile Asn Ile Thr Ala
85 90 95Leu Gly Gly Phe Thr Ser Ile Ile Phe Glu Asn Phe Asn Leu Leu
Gln 100 105 110His Lys Gln Ile Arg Asn Thr Ser Leu Glu Trp Glu Arg
Phe Thr Thr 115 120 125Gly Asn Thr His Thr Ala Trp Val Ile Cys Arg
Gln Leu Glu Met Asn 130 135 140Ala Pro Lys Ile Gly Ile Asp Leu Lys
Ser Ala Thr Val Ala Val Val145 150 155 160Gly Ala Thr Gly Asp Ile
Gly Ser Ala Val Cys Arg Trp Leu Ile Asn 165 170 175Lys Thr Gly Ile
Gly Glu Leu Leu Leu Val Ala Arg Gln Lys Glu Pro 180 185 190Leu Asp
Ser Leu Gln Lys Glu Leu Asp Gly Gly Thr Ile Lys Asn Leu 195 200
205Asp Glu Ala Leu Pro Glu Ala Asp Ile Val Val Trp Val Ala Ser Met
210 215 220Pro Lys Thr Met Glu Ile Asp Ala Asn Asn Leu Lys Gln Pro
Cys Leu225 230 235 240Met Ile Asp Gly Gly Tyr Pro Lys Asn Leu Asp
Glu Lys Phe Gln Gly 245 250 255Asn Asn Ile His Val Val Lys Gly Gly
Ile Val Arg Phe Phe Asn Asp 260 265 270Ile Gly Trp Asn Met Met Glu
Leu Ala Glu Met Gln Asn Pro Gln Arg 275 280 285Glu Met Phe Ala Cys
Phe Ala Glu Ala Met Ile Leu Glu Phe Glu Lys 290 295 300Cys His Thr
Asn Phe Ser Trp Gly Arg Asn Asn Ile Ser Leu Glu Lys305 310 315
320Met Glu Phe Ile Gly Ala Ala Ser Val Lys His Gly Phe Ser Ala Ile
325 330 335Gly Leu Asp Lys His Pro Lys Val Leu Ala Val 340
34573347PRTProchlorococcus marinus 73Met Ala Phe Gly Leu Ile Gly
His Ser Thr Ser Phe Glu Asp Ala Lys1 5 10 15Arg Lys Ala Ser Leu Leu
Gly Phe Asp His Ile Ala Asp Gly Asp Leu 20 25 30Asp Val Trp Cys Thr
Ala Pro Pro Gln Leu Val Glu Asn Val Glu Val 35 40 45Lys Ser Ala Ile
Gly Ile Ser Ile Glu Gly Ser Tyr Ile Asp Ser Cys 50 55 60Phe Val Pro
Glu Met Leu Ser Arg Phe Lys Thr Ala Arg Arg Lys Val65 70 75 80Leu
Asn Ala Met Glu Leu Ala Gln Lys Lys Gly Ile Asn Ile Thr Ala 85 90
95Leu Gly Gly Phe Thr Ser Ile Ile Phe Glu Asn Phe Asn Leu Leu Gln
100 105 110His Lys Gln Ile Arg Asn Thr Ser Leu Glu Trp Glu Arg Phe
Thr Thr 115 120 125Gly Asn Thr His Thr Ala Trp Val Ile Cys Arg Gln
Leu Glu Met Asn 130 135 140Ala Pro Lys Ile Gly Ile Asp Leu Lys Ser
Ala Thr Val Ala Val Val145 150 155 160Gly Ala Thr Gly Asp Ile Gly
Ser Ala Val Cys Arg Trp Leu Ile Asn 165 170 175Lys Thr Gly Ile Gly
Glu Leu Leu Leu Val Ala Arg Gln Lys Glu Pro 180 185 190Leu Asp Ser
Leu Gln Lys Glu Leu Asp Gly Gly Thr Ile Lys Asn Leu 195 200 205Asp
Glu Ala Leu Pro Glu Ala Asp Ile Val Val Trp Val Ala Ser Met 210 215
220Pro Lys Thr Met Glu Ile Asp Ala Asn Asn Leu Lys Gln Pro Cys
Leu225 230 235 240Met Ile Asp Gly Gly Tyr Pro Lys Asn Leu Asp Glu
Lys Phe Gln Gly 245 250 255Asn Asn Ile His Val Val Lys Gly Gly Ile
Val Arg Phe Phe Asn Asp 260 265 270Ile Gly Trp Asn Met Met Glu Leu
Ala Glu Met Gln Asn Pro Gln Arg 275 280 285Glu Met Phe Ala Cys Phe
Ala Glu Ala Met Ile Leu Glu Phe Glu Lys 290 295 300Cys His Thr Asn
Phe Ser Trp Gly Arg Asn Asn Ile Ser Leu Glu Lys305 310 315 320Met
Glu Phe Ile Gly Ala Ala Ser Val Lys His Gly Phe Ser Ala Ile 325 330
335Gly Leu Asp Lys His Pro Lys Val Thr Ala Val 340
34574347PRTProchlorococcus marinus 74Met Ala Phe Gly Leu Ile Gly
His Ser Thr Ser Phe Glu Asp Ala Lys1 5 10 15Arg Lys Ala Ser Leu Leu
Gly Phe Asp His Ile Ala Asp Gly Asp Leu 20 25 30Asp Val Trp Cys Thr
Ala Pro Pro Gln Leu Val Glu Asn Val Glu Val 35 40 45Lys Ser Ala Ile
Gly Ile Ser Ile Glu Gly Ser Tyr Ile Asp Ser Cys 50 55 60Phe Val Pro
Glu Met Leu Ser Arg Phe Lys Thr Ala Arg Arg Lys Val65 70 75 80Leu
Asn Ala Met Glu Leu Ala Gln Lys Lys Gly Ile Asn Ile Thr Ala 85 90
95Leu Gly Gly Phe Thr Ser Ile Ile Phe Glu Asn Phe Asn Leu Leu Gln
100 105 110His Lys Gln Ile Arg Asn Thr Ser Leu Glu Trp Glu Arg Phe
Thr Thr 115 120 125Gly Asn Thr His Thr Ala Trp Val Ile Cys Arg Gln
Leu Glu Met Asn 130 135 140Ala Pro Lys Ile Gly Ile Asp Leu Lys Ser
Ala Thr Val Ala Val Val145 150 155 160Gly Ala Thr Gly Asp Ile Gly
Ser Ala Val Cys Arg Trp Leu Ile Asn 165 170 175Lys Thr Gly Ile Gly
Glu Leu Leu Leu Val Ala Arg Gln Lys Glu Pro 180 185 190Leu Asp Ser
Leu Gln Lys Glu Leu Asp Gly Gly Thr Ile Lys Asn Leu 195 200 205Asp
Glu Ala Leu Pro Glu Ala Asp Ile Val Val Trp Val Ala Ser Met 210 215
220Pro Lys Thr Met Glu Ile Asp Ala Asn Asn Leu Lys Gln Pro Cys
Leu225 230 235 240Met Ile Asp Gly Gly Tyr Pro Lys Asn Leu Asp Glu
Lys Phe Gln Gly 245 250 255Asn Asn Ile His Val Val Lys Gly Gly Ile
Val Arg Phe Phe Asn Asp 260 265 270Ile Gly Trp Asn Met Met Glu Leu
Ala Glu Met Gln Asn Pro Gln Arg 275 280 285Glu Met Phe Ala Cys Phe
Ala Glu Ala Met Ile Leu Glu Phe Glu Gly 290 295 300Cys His Thr Asn
Phe Ser Trp Gly Arg Asn Asn Ile Ser Leu Glu Lys305 310 315 320Met
Glu Phe Ile Gly Ala Ala Ser Val Lys His Gly Phe Ser Ala Ile 325 330
335Gly Leu Asp Lys His Pro Lys Val Leu Ala Val 340
34575347PRTProchlorococcus marinus 75Met Ala Phe Gly Leu Ile Gly
His Ser Thr Ser Phe Glu Asp Ala Lys1 5 10 15Arg Lys Ala Ser Leu Leu
Gly Phe Asp His Ile Ala Asp Gly Asp Leu 20 25 30Asp Val Trp Cys Thr
Ala Pro Pro Gln Leu Val Glu Asn Val Glu Val 35 40 45Lys Ser Ala Ile
Gly Ile Ser Ile Glu Gly Ser Tyr Ile Asp Ser Cys 50 55 60Phe Val Pro
Glu Met Leu Ser Arg Phe Lys Thr Ala Arg Arg Lys Val65 70 75 80Leu
Asn Ala Met Glu Leu Ala Gln Lys Lys Gly Ile Asn Ile Thr Ala 85 90
95Leu Gly Gly Phe Thr Ser Ile Ile Phe Glu Asn Phe Asn Leu Leu Gln
100 105 110His Lys Gln Ile Arg Asn Thr Ser Leu Glu Trp Glu Arg Phe
Thr Thr 115 120 125Gly Asn Thr His Thr Ala Trp Val Ile Cys Arg Gln
Leu Glu Met Asn 130 135 140Ala Pro Lys Ile Gly Ile Asp Leu Lys Ser
Ala Thr Val Ala Val Val145 150 155 160Gly Ala Thr Gly Asp Ile Gly
Ser Ala Val Cys Arg Trp Leu Ile Asn 165 170 175Lys Thr Gly Ile Gly
Glu Leu Leu Leu Val Ala Arg Gln Lys Glu Pro 180 185 190Leu Asp Ser
Leu Gln Lys Glu Leu Asp Gly Gly Thr Ile Lys Asn Leu 195 200 205Asp
Glu Ala Leu Pro Glu Ala Asp Ile Val Val Trp Val Ala Ser Met 210 215
220Pro Lys Thr Met Glu Ile Asp Ala Asn Asn Leu Lys Gln Pro Cys
Leu225 230 235 240Met Ile Asp Gly Gly Tyr Pro Lys Asn Leu Asp Glu
Lys Phe Gln Gly 245 250 255Asn Asn Ile His Val Val Lys Gly Gly Ile
Val Arg Phe Phe Asn Asp 260 265 270Ile Gly Trp Asn Met Met Glu Leu
Ala Glu Met Gln Asn Pro Gln Arg 275 280 285Glu Met Phe Ala Cys Phe
Ala Glu Ala Met Ile Leu Glu Phe Glu Lys 290 295 300Cys His Thr Asn
Phe Ser Trp Gly Arg Asn Asn Ile Ser Leu Glu Lys305 310 315 320Met
Glu Phe Ile Gly Ala Ala Ser Val Lys His Gly Phe Ser Ala Ile 325 330
335Gly Leu Asp Lys His Pro Lys Val Ala Ala Val 340
34576347PRTProchlorococcus marinus 76Met Ala Phe Gly Leu Ile Gly
His Ser Thr Ser Phe Glu Asp Ala Lys1 5 10 15Arg Lys Ala Ser Leu Leu
Gly Phe Asp His Ile Ala Asp Gly Asp Leu 20 25 30Asp Val Trp Cys Thr
Ala Pro Pro Gln Leu Val Glu Asn Val Glu Val 35 40 45Lys Ser Ala Ile
Gly Ile Ser Ile Glu Gly Ser Tyr Ile Asp Ser Cys 50 55 60Phe Val Pro
Glu Met Leu Ser Arg Phe Lys Thr Ala Arg Arg Lys Val65 70 75 80Leu
Asn Ala Met Glu Leu Ala Gln Lys Lys Gly Ile Asn Ile Thr Ala 85 90
95Leu Gly Gly Phe Thr Ser Ile Ile Phe Glu Asn Phe Asn Leu Leu Gln
100 105 110His Lys Gln Ile Arg Asn Thr Ser Leu Glu Trp Glu Arg Phe
Thr Thr 115 120 125Gly Asn Thr His Thr Ala Trp Val Ile Cys Arg Gln
Leu Glu Met Asn 130 135 140Ala Pro Lys Ile Gly Ile Asp Leu Lys Ser
Ala Thr Val Ala Val Val145 150 155 160Gly Ala Thr Gly Asp Ile Gly
Ser Ala Val Cys Arg Trp Leu Ile Asn 165 170 175Lys Thr Gly Ile Gly
Glu Leu Leu Leu Val Ala Arg Gln Lys Glu Pro 180 185 190Leu Asp Ser
Leu Gln Lys Glu Leu Asp Gly Gly Thr Ile Lys Asn Leu 195 200 205Asp
Glu Ala Leu Pro Glu Ala Asp Ile Val Val Trp Val Ala Ser Met 210 215
220Pro Lys Thr Met Glu Ile Asp Ala Asn Asn Leu Lys Gln Pro Cys
Leu225 230 235 240Met Ile Asp Gly Gly Tyr Pro Lys Asn Leu Asp Glu
Lys Phe Gln Gly
245 250 255Asn Asn Ile His Val Val Lys Gly Gly Ile Val Arg Phe Phe
Asn Asp 260 265 270Ile Gly Trp Asn Met Met Glu Leu Ala Glu Met Gln
Asn Pro Gln Arg 275 280 285Glu Met Phe Ala Cys Phe Ala Glu Ala Met
Ile Leu Glu Phe Glu Lys 290 295 300Cys His Thr Asn Phe Ser Trp Gly
Arg Asn Asn Ile Ser Leu Glu Lys305 310 315 320Met Glu Phe Ile Gly
Ala Ala Ser Val Lys His Gly Phe Ser Ala Ile 325 330 335Gly Leu Asp
Lys Pro Pro Lys Val Leu Ala Val 340 34577347PRTProchlorococcus
marinus 77Met Ala Phe Gly Leu Ile Gly His Ser Thr Ser Phe Glu Asp
Ala Lys1 5 10 15Arg Lys Ala Ser Leu Leu Gly Phe Asp His Ile Ala Asp
Gly Asp Leu 20 25 30Asp Val Trp Cys Thr Ala Pro Pro Gln Leu Val Glu
Asn Val Glu Val 35 40 45Lys Ser Ala Ile Gly Ile Ser Ile Glu Gly Ser
Tyr Ile Asp Ser Cys 50 55 60Phe Val Pro Glu Met Leu Ser Arg Phe Lys
Thr Ala Arg Arg Lys Val65 70 75 80Leu Asn Ala Met Glu Leu Ala Gln
Lys Lys Gly Ile Asn Ile Thr Ala 85 90 95Leu Gly Gly Phe Thr Ser Ile
Ile Phe Glu Asn Phe Asn Leu Leu Gln 100 105 110His Lys Gln Ile Arg
Asn Thr Ser Leu Glu Trp Glu Arg Phe Thr Thr 115 120 125Gly Asn Thr
His Thr Ala Trp Val Ile Cys Arg Gln Leu Glu Met Asn 130 135 140Ala
Pro Lys Ile Gly Ile Asp Leu Lys Ser Ala Thr Val Ala Val Val145 150
155 160Gly Ala Thr Gly Asp Ile Gly Ser Ala Val Cys Arg Trp Leu Ile
Asn 165 170 175Lys Thr Gly Ile Gly Glu Leu Leu Leu Val Ala Arg Gln
Lys Glu Pro 180 185 190Leu Asp Ser Leu Gln Lys Glu Leu Asp Gly Gly
Thr Ile Lys Asn Leu 195 200 205Asp Glu Ala Leu Pro Glu Ala Asp Ile
Val Val Trp Val Ala Ser Met 210 215 220Pro Lys Thr Met Glu Ile Asp
Ala Asn Asn Leu Lys Gln Pro Cys Leu225 230 235 240Met Ile Asp Gly
Gly Tyr Pro Lys Asn Leu Asp Glu Lys Phe Gln Gly 245 250 255Asn Asn
Ile His Val Val Lys Gly Gly Ile Val Arg Phe Phe Asn Asp 260 265
270Ile Glu Trp Asn Met Met Glu Leu Ala Glu Met Gln Asn Pro Gln Arg
275 280 285Glu Met Phe Ala Cys Phe Ala Glu Ala Met Ile Leu Glu Phe
Glu Lys 290 295 300Cys His Thr Asn Phe Ser Trp Gly Arg Asn Asn Ile
Ser Leu Glu Lys305 310 315 320Met Glu Phe Ile Gly Ala Ala Ser Val
Lys His Gly Phe Ser Ala Ile 325 330 335Gly Leu Asp Lys His Pro Lys
Val Leu Ala Val 340 34578345PRTProchlorococcus marinus 78Met Ala
Phe Gly Leu Ile Gly His Ser Thr Ser Phe Glu Asp Ala Lys1 5 10 15Arg
Lys Ala Ser Leu Leu Gly Phe Asp His Ile Ala Asp Gly Asp Leu 20 25
30Asp Val Trp Cys Thr Ala Pro Pro Gln Leu Val Glu Asn Val Glu Val
35 40 45Lys Ser Ala Ile Gly Ile Ser Ile Glu Gly Ser Tyr Ile Asp Ser
Cys 50 55 60Phe Val Pro Glu Met Leu Ser Arg Phe Lys Thr Ala Arg Arg
Lys Val65 70 75 80Leu Asn Ala Met Glu Leu Ala Gln Lys Lys Gly Ile
Asn Ile Thr Ala 85 90 95Leu Gly Gly Phe Thr Ser Ile Ile Phe Glu Asn
Phe Asn Leu Leu Gln 100 105 110His Lys Gln Ile Arg Asn Thr Ser Leu
Glu Trp Glu Arg Phe Thr Thr 115 120 125Gly Asn Thr His Thr Ala Trp
Val Ile Cys Arg Gln Leu Glu Met Asn 130 135 140Ala Pro Lys Ile Gly
Ile Asp Leu Lys Ser Ala Thr Val Ala Val Val145 150 155 160Gly Ala
Thr Gly Asp Ile Gly Ser Ala Val Cys Arg Trp Leu Ile Asn 165 170
175Lys Thr Gly Ile Gly Glu Leu Leu Leu Val Ala Arg Gln Lys Glu Pro
180 185 190Leu Asp Ser Leu Gln Lys Glu Leu Asp Gly Gly Thr Ile Lys
Asn Leu 195 200 205Asp Glu Ala Leu Pro Glu Ala Asp Ile Val Val Trp
Val Ala Ser Met 210 215 220Pro Lys Thr Met Glu Ile Asp Ala Asn Asn
Leu Lys Gln Pro Cys Leu225 230 235 240Met Ile Asp Gly Gly Tyr Pro
Lys Asn Leu Asp Glu Lys Phe Gln Gly 245 250 255Asn Asn Ile His Val
Val Lys Gly Gly Ile Val Arg Phe Phe Asn Asp 260 265 270Ile Gly Trp
Asn Met Met Glu Leu Ala Glu Met Gln Asn Pro Gln Arg 275 280 285Glu
Met Phe Ala Cys Phe Ala Glu Ala Met Ile Leu Glu Phe Glu Lys 290 295
300Cys His Thr Asn Phe Ser Trp Gly Arg Asn Asn Ile Ser Leu Glu
Lys305 310 315 320Met Glu Phe Ile Gly Ala Ala Ser Val Lys His Gly
Phe Ser Ala Ile 325 330 335Gly Leu Asp Lys His Pro Lys Val Leu 340
34579341PRTSynechococcus elongatus 79Ala Phe Gly Leu Ile Gly His
Leu Thr Ser Leu Glu Gln Ala Arg Asp1 5 10 15Val Trp Arg Arg Met Gly
Tyr Asp Glu Tyr Ala Asp Gln Gly Leu Glu 20 25 30Phe Trp Ser Ser Ala
Pro Pro Gln Ile Val Asp Glu Ile Thr Val Thr 35 40 45Ser Ala Thr Gly
Lys Val Ile His Gly Arg Tyr Ile Glu Ser Cys Phe 50 55 60Leu Pro Glu
Met Leu Ala Ala Arg Arg Phe Lys Thr Ala Thr Arg Lys65 70 75 80Val
Leu Asn Ala Met Ser His Ala Gln Lys His Gly Ile Asp Ile Ser 85 90
95Ala Leu Gly Gly Phe Thr Ser Ile Ile Phe Glu Asn Phe Asp Leu Ala
100 105 110Ser Leu Arg Gln Val Arg Asp Thr Thr Leu Glu Phe Glu Arg
Phe Thr 115 120 125Thr Gly Asn Thr His Thr Ala Tyr Val Ile Cys Arg
Gln Val Glu Ala 130 135 140Ala Ala Lys Thr Leu Gly Ile Asp Ile Thr
Gln Ala Thr Val Ala Val145 150 155 160Val Gly Ala Thr Gly Asp Ile
Gly Ser Ala Val Cys Arg Trp Leu Asp 165 170 175Leu Lys Leu Gly Val
Gly Asp Leu Ile Leu Thr Ala Arg Asn Gln Glu 180 185 190Arg Leu Asp
Asn Leu Gln Ala Glu Leu Gly Arg Gly Lys Ile Leu Pro 195 200 205Leu
Glu Ala Ala Leu Pro Glu Ala Asp Phe Ile Val Trp Val Ala Ser 210 215
220Met Pro Gln Gly Val Val Ile Asp Pro Ala Thr Leu Lys Gln Pro
Cys225 230 235 240Val Leu Ile Asp Gly Gly Tyr Pro Lys Asn Leu Gly
Ser Lys Val Gln 245 250 255Gly Glu Gly Ile Tyr Val Leu Asn Gly Gly
Val Val Glu His Cys Phe 260 265 270Asp Ile Asp Trp Gln Ile Met Ser
Ala Ala Glu Met Ala Arg Pro Glu 275 280 285Arg Gln Met Phe Ala Cys
Phe Ala Glu Ala Met Leu Leu Glu Phe Glu 290 295 300Gly Trp His Thr
Asn Phe Ser Trp Gly Arg Asn Gln Ile Thr Ile Glu305 310 315 320Lys
Met Glu Ala Ile Gly Glu Ala Ser Val Arg His Gly Phe Gln Pro 325 330
335Leu Ala Leu Ala Ile 34080346PRTProchlorococcus marinus 80Ala Phe
Gly Leu Ile Gly His Ser Thr Ser Phe Glu Asp Ala Lys Arg1 5 10 15Lys
Ala Ser Leu Leu Gly Phe Asp His Ile Ala Asp Gly Asp Leu Asp 20 25
30Val Trp Cys Thr Ala Pro Pro Gln Leu Val Glu Asn Val Glu Val Lys
35 40 45Ser Ala Ile Gly Ile Ser Ile Glu Gly Ser Tyr Ile Glu Ser Cys
Phe 50 55 60Val Pro Glu Met Leu Ser Arg Phe Lys Thr Ala Arg Arg Lys
Val Leu65 70 75 80Asn Ala Met Glu Leu Ala Gln Lys Lys Gly Ile Asn
Ile Thr Ala Leu 85 90 95Gly Gly Phe Thr Ser Ile Ile Phe Glu Asn Phe
Asn Leu Leu Gln His 100 105 110Lys Gln Ile Arg Asn Thr Ser Leu Glu
Trp Glu Arg Phe Thr Thr Gly 115 120 125Asn Thr His Thr Ala Trp Val
Ile Cys Arg Gln Leu Glu Met Asn Ala 130 135 140Pro Lys Ile Gly Ile
Asp Leu Lys Ser Ala Thr Val Ala Val Val Gly145 150 155 160Ala Thr
Gly Asp Ile Gly Ser Ala Val Cys Arg Trp Leu Ile Asn Lys 165 170
175Thr Gly Ile Gly Glu Leu Leu Leu Val Ala Arg Gln Lys Glu Pro Leu
180 185 190Asp Ser Leu Gln Lys Glu Leu Asp Gly Gly Thr Ile Lys Asn
Leu Asp 195 200 205Glu Ala Leu Pro Glu Ala Asp Ile Val Val Trp Val
Ala Ser Met Pro 210 215 220Lys Thr Met Glu Ile Asp Ala Asn Asn Leu
Lys Gln Pro Cys Leu Met225 230 235 240Ile Asp Gly Gly Tyr Pro Lys
Asn Leu Asp Glu Lys Phe Gln Gly Asn 245 250 255Asn Ile His Val Val
Lys Gly Gly Ile Val Arg Phe Phe Asn Asp Ile 260 265 270Gly Trp Asn
Met Met Glu Leu Ala Glu Met Gln Asn Pro Gln Arg Glu 275 280 285Met
Phe Ala Cys Phe Ala Glu Ala Met Ile Leu Glu Phe Glu Lys Cys 290 295
300His Thr Asn Phe Ser Trp Gly Arg Asn Asn Ile Ser Leu Glu Lys
Met305 310 315 320Glu Phe Ile Gly Ala Ala Ser Val Lys His Gly Phe
Ser Ala Ile Gly 325 330 335Leu Asp Lys His Pro Lys Val Leu Ala Val
340 345
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