U.S. patent application number 11/935351 was filed with the patent office on 2009-04-16 for genetically modified cells and methods for converting (r)-methylmalonyl coa to (s)-methylmalonyl coa.
Invention is credited to Linda Dayem, James Kealey, Larry Peck, Daniel SANTI.
Application Number | 20090098615 11/935351 |
Document ID | / |
Family ID | 22582342 |
Filed Date | 2009-04-16 |
United States Patent
Application |
20090098615 |
Kind Code |
A1 |
SANTI; Daniel ; et
al. |
April 16, 2009 |
GENETICALLY MODIFIED CELLS AND METHODS FOR CONVERTING
(R)-METHYLMALONYL COA TO (S)-METHYLMALONYL COA
Abstract
Recombinant Escherichia coli host cells that comprise
recombinant DNA expression vectors that drive expression of
methylmalonyl CoA mutase from Propionibacterium shermanii or
Streptomyces cinnamonensis as well as Propionibacterium shermanii
epimerase can produce S-methylmalonyl CoA, a required substrate for
the production of polyketides by most modular polyketide synthases
not present in wild-type E. coli host cells.
Inventors: |
SANTI; Daniel; (San
Francisco, CA) ; Peck; Larry; (San Carlos, CA)
; Dayem; Linda; (San Anselmo, CA) ; Kealey;
James; (San Anselmo, CA) |
Correspondence
Address: |
MORRISON & FOERSTER LLP
12531 HIGH BLUFF DRIVE, SUITE 100
SAN DIEGO
CA
92130-2040
US
|
Family ID: |
22582342 |
Appl. No.: |
11/935351 |
Filed: |
November 5, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10829897 |
Apr 21, 2004 |
7291486 |
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11935351 |
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09942407 |
Aug 29, 2001 |
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10829897 |
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09699136 |
Oct 27, 2000 |
7011959 |
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09942407 |
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60161703 |
Oct 27, 1999 |
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Current U.S.
Class: |
435/92 |
Current CPC
Class: |
C12N 9/90 20130101; C12P
11/00 20130101; C12P 19/62 20130101 |
Class at
Publication: |
435/92 |
International
Class: |
C12P 19/32 20060101
C12P019/32 |
Goverment Interests
STATEMENT OF GOVERNMENT INTEREST
[0002] This invention was supported in part by SBIR Grant No.
GM56575. The United States government has certain rights in this
invention.
Claims
1. A method for converting (R)-methylmalonyl CoA to
(S)-methylmalonyl CoA in a host cell, the method comprising
culturing the host cell of claim 22 under conditions such that said
epimerase is expressed, and (R)-methylmalonyl CoA is converted into
(S)-methylmalonyl CoA.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent Ser. No.
10/829,897, filed Apr. 21, 2004, issuing on Nov. 6, 2007 as U.S.
Pat. No. 7,291,486, which is a continuation of U.S. patent
application Ser. No. 09/942,407, filed Aug. 29, 2001, now
abandoned, which is a Divisional of U.S. patent Ser. No.
09/699,136, filed Oct. 27, 2000, which claims priority under 35
U.S.C. .sctn. 1.119(e) to U.S. Provisional Patent Application Ser.
No. 60/161,703, filed Oct. 27, 1999, and these applications are
expressly incorporated herein by reference in their entirety.
REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB
[0003] This application is being filed electronically via the USPTO
EFS-WEB server, as authorized and set forth in MPEP .sctn.1730
II.B.2(a)(A), and this electronic filing includes an electronically
submitted sequence (SEQ ID) listing. The entire content of this
sequence listing is herein incorporated by reference for all
purposes. The sequence listing is identified on the electronically
filed .txt file as follows:
TABLE-US-00001 File Name Date of Creation Size (bytes)
300622004902Seqlist.txt Nov. 5, 2007 3,844 bytes
FIELD OF THE INVENTION
[0004] The present invention provides recombinant methods and
materials for producing polyketides by recombinant DNA technology.
The invention relates to the fields of agriculture, animal
husbandry, chemistry, medicinal chemistry, medicine, molecular
biology, pharmacology, and veterinary technology.
BACKGROUND OF THE INVENTION
[0005] Polyketides represent a large family of diverse compounds
synthesized from 2-carbon units through a series of condensations
and subsequent modifications. Polyketides occur in many types of
organisms, including fungi and mycelial bacteria, in particular,
the actinomycetes. There are a wide variety of polyketide
structures, and the class of polyketides encompasses numerous
compounds with diverse activities. Erythromycin, FK-506, FK-520,
megalomicin, narbomycin, oleandomycin, picromycin, rapamycin,
spinocyn, and tylosin are examples of such compounds. Given the
difficulty in producing polyketide compounds by traditional
chemical methodology, and the typically low production of
polyketides in wild-type cells, there has been considerable
interest in finding improved or alternate means to produce
polyketide compounds. See PCT publication Nos. WO 93/13663; WO
95/08548; WO 96/40968; 97/02358; and 98/27203; U.S. Pat. Nos.
4,874,748; 5,063,155; 5,098,837; 5,149,639; 5,672,491; 5,712,146;
and 5,962,290; and Fu et al., 1994, Biochemistry 33: 9321-9326;
McDaniel et al., 1993, Science 262: 1546-1550; and Rohr, 1995,
Angew. Chem. Int. Ed. Engl. 34(8): 881-888, each of which is
incorporated herein by reference.
[0006] Polyketides are synthesized in nature by polyketide synthase
(PKS) enzymes. These enzymes, which are complexes of multiple large
proteins, are similar to the synthases that catalyze condensation
of 2-carbon units in the biosynthesis of fatty acids. PKS enzymes
are encoded by PKS genes that usually consist of three or more open
reading frames (ORFs). Two major types of PKS enzymes are known;
these differ in their composition and mode of synthesis. These two
major types of PKS enzymes are commonly referred to as Type I or
"modular" and Type II "iterative" PKS enzymes. A third type of PKS
found primarily in fungal cells has features of both the Type I and
Type II enzymes and is referred to as a "fungal" PKS enzymes.
[0007] Modular PKSs are responsible for producing a large number of
12-, 14-, and 16-membered macrolide antibiotics including
erythromycin, megalomicin, methymycin, narbomycin, oleandomycin,
picromycin, and tylosin. Each ORF of a modular PKS can comprise
one, two, or more "modules" of ketosynthase activity, each module
of which consists of at least two (if a loading module) and more
typically three (for the simplest extender module) or more
enzymatic activities or "domains." These large multifunctional
enzymes (>300,000 kDa) catalyze the biosynthesis of polyketide
macrolactones through multistep pathways involving decarboxylative
condensations between acyl thioesters followed by cycles of varying
.beta.-carbon processing activities (see O'Hagan, D. The polyketide
metabolites; E. Horwood: New York, 1991, incorporated herein by
reference).
[0008] During the past half decade, the study of modular PKS
function and specificity has been greatly facilitated by the
plasmid-based Streptomyces coelicolor expression system developed
with the 6-deoxyerythronolide B (6-dEB) synthase (DEBS) genes (see
Kao et al., 1994, Science, 265: 509-512, McDaniel et al., 1993,
Science 262: 1546-1557, and U.S. Pat. Nos. 5,672,491 and 5,712,146,
each of which is incorporated herein by reference). The advantages
to this plasmid-based genetic system for DEBS are that it overcomes
the tedious and limited techniques for manipulating the natural
DEBS host organism, Saccharopolyspora erythraea, allows more facile
construction of recombinant PKSs, and reduces the complexity of PKS
analysis by providing a "clean" host background. This system also
expedited construction of the first combinatorial modular
polyketide library in Streptomyces (see PCT publication Nos. WO
98/49315 and 00/024907, each of which is incorporated herein by
reference).
[0009] The ability to control aspects of polyketide biosynthesis,
such as monomer selection and degree of .beta.-carbon processing,
by genetic manipulation of PKSs has stimulated great interest in
the combinatorial engineering of novel antibiotics (see Hutchinson,
1998, Curr. Opin. Microbiol. 1: 319-329; Carreras and Santi, 1998,
Curr. Opin. Biotech. 9: 403-411; and U.S. Pat. Nos. 5,712,146 and
5,672,491, each of which is incorporated herein by reference). This
interest has resulted in the cloning, analysis, and manipulation by
recombinant DNA technology of genes that encode PKS enzymes. The
resulting technology allows one to manipulate a known PKS gene
cluster either to produce the polyketide synthesized by that PKS at
higher levels than occur in nature or in hosts that otherwise do
not produce the polyketide. The technology also allows one to
produce molecules that are structurally related to, but distinct
from, the polyketides produced from known PKS gene clusters.
[0010] There has been a great deal of interest in expressing
polyketides produced by Type I and Type II PKS enzymes in host
cells that do not normally express such enzymes. For example, the
production of the fungal polyketide 6-methylsalicylic acid (6-MSA)
in heterologous E. coli, yeast, and plant cells has been reported.
See Kealey et al., January 1998, Production of a polyketide natural
product in nonpolyketide-producing prokaryotic and eukaryotic host,
Proc. Natl. Acad. Sci. USA 95:505-9, U.S. Pat. No. 6,033,883, and
PCT Patent Publication Nos. 98/27203 and 99/02669, each of which is
incorporated herein by reference. Heterologous production of 6-MSA
required or was considerably increased by co-expression of a
heterologous acyl carrier protein synthase (ACPS) and that, for E.
coli, media supplements were helpful in increasing the level of the
malonyl CoA substrate utilized in 6-MSA biosynthesis. See also, PCT
Patent Publication No. 97/13845, incorporated herein by
reference.
[0011] The biosynthesis of other polyketides requires substrates
other than or in addition to malonyl CoA. Such substrates include,
for example, propionyl CoA, 2-methylmalonyl CoA, 2-hydroxymalonyl
CoA, and 2-ethylmalonyl CoA. Of the myriad host cells possible for
utilization as polyketide producing hosts, many do not naturally
produce such substrates. Given the potential for making valuable
and useful polyketides in large quantities in heterologous host
cells, there is a need for host cells capable of making the
substrates required for polyketide biosynthesis. The present
invention helps meet that need by providing recombinant host cells,
expression vectors, and methods for making polyketides in diverse
host cells.
SUMMARY OF THE INVENTION
[0012] The present invention provides recombinant host cells and
expression vectors for making products in host cells that are
otherwise unable to make those products due to the lack of a
biosynthetic pathway to produce a precursor required for
biosynthesis of the product. The present invention also provides
methods for increasing the amounts of a product produced in a host
cell by providing recombinant biosynthetic pathways for production
of a precursor utilized in the biosynthesis of a product.
[0013] In one embodiment, the host cell does not produce the
precursor, and the host cell is modified by introduction of a
recombinant expression vector so that it can produce the precursor.
In another embodiment, the precursor is produced in the host cell
in small amounts, and the host cell is modified by introduction of
a recombinant expression vector so that it can produce the
precursor in larger amounts. In a preferred embodiment, the
precursor is a primary metabolite that is produced in first cell
but not in a second heterologous cell. In accordance with the
methods of the invention, the genes that encode the enzymes that
produce the primary metabolite in the first cell are transferred to
the second cell. The transfer is accomplished using an expression
vector of the invention. The expression vector drives expression of
the genes and production of the metabolite in the second cell.
[0014] In a preferred embodiment, the product is a polyketide. The
polyketide is a polyketide synthesized by either a modular,
iterative, or fungal PKS. The precursor is selected from the group
consisting of malonyl CoA, propionyl CoA, methylmalonyl CoA,
ethylmalonyl CoA, and hydroxymalonyl or methoxymalonyl CoA. In an
especially preferred embodiment, the polyketide utilizes
methylmalonyl CoA in its biosynthesis. In one preferred embodiment,
the polyketide is synthesized by a modular PKS that requires
methylmalonyl CoA to synthesize the polyketide.
[0015] In one embodiment, the host cell is either a procaryotic or
eukaryotic host cell. In one embodiment, the host cell is an E.
coli host cell. In another embodiment, the host cell is a yeast
host cell. In another embodiment, the host cell is an Actinomycetes
host cell, including but not limited to a Streptomyces host cell.
In another embodiment, the host cell is a plant host cell. In a
preferred embodiment, the host cell is either an E. coli or yeast
host cell, the product is a polyketide, and the precursor is
methylmalonyl CoA.
[0016] In one embodiment, the invention provides a recombinant
expression vector that comprises a promoter positioned to drive
expression of one or more genes that encode the enzymes required
for biosynthesis of a precursor. In a preferred embodiment, the
promoter is derived from a PKS gene. In a related embodiment, the
invention provides recombinant host cells comprising one or more
expression vectors that drive expression of the enzymes that
produce the precursor.
[0017] In another embodiment, the invention provides a recombinant
host cell that comprises not only an expression vector of the
invention but also an expression vector that comprises a promoter
positioned to drive expression of a PKS. In a related embodiment,
the invention provides recombinant host cells comprising the vector
that produces the PKS and its corresponding polyketide. In a
preferred embodiment, the host cell is an E. coli or yeast host
cell.
[0018] These and other embodiments of the invention are described
in more detail in the following description, the examples, and
claims set forth below.
BRIEF DESCRIPTION OF THE FIGURES
[0019] FIG. 1 shows the modules and domains of DEBS and the
biosynthesis of 6-dEB from propionyl CoA and methylmalonyl CoA.
[0020] FIG. 2 shows the construction of pSK-MUT, in which four PCR
fragments were sequenced and pieced together to form the complete
mutase gene in pSK-bluescript.
[0021] FIG. 3 shows the acyl-CoA analysis in BL21(DE3) panD strains
in vivo.
[0022] FIG. 4 shows the results of CoA analysis of E. coli
overexpressing methylmalonyl-CoA mutase. The levels of .sup.3H
detected in fractions collected from HPLC of cell-free extracts
from .sup.3H P-alanine-fed E. coli harboring either the pET control
vector grown without hydroxocobalamin (solid trace), pET grown with
hydroxocobalamin (dash-dotted trace), pET overexpressing the mutase
and grown without hydroxocobalamin (dotted trace), or pET
overexpressing the mutase and grown with hydroxocobalamin (dashed
trace) are shown.
[0023] FIG. 5 shows the three routes or biosynthetic pathways for
the synthesis of methylmalonyl-CoA that can be engineered into
yeast.
[0024] FIG. 6 shows acyl-CoA analysis of E. coli overexpressing
methylmalonyl-CoA mutase. The levels of .sup.3H detected in
fractions collected from HPLC of cell-free extracts from .sup.3H
P-alanine-fed E. coli harboring either the pET control vector
(solid trace) or pET overexpressing the mutase (dashed trace) is
shown.
[0025] FIG. 7 shows acyl-CoA analysis in S. cerevisiae. The levels
of .sup.3H detected in fractions collected from HPLC of cell-free
extracts from .sup.3H P-alanine-fed S. cerevisiae after growth ofr
24 hours (solid trace), 48 hours (dashed trace) and 66 hours
(dotted trace) is shown.
[0026] FIG. 8 shows the Common Cloning Cassette.
[0027] FIG. 9 shows a general method for cloning genes into yeast
expression vectors.
DETAILED DESCRIPTION OF THE INVENTION
[0028] The present invention provides recombinant host cells and
expression vectors for making products in host cells, which are
otherwise unable to make those products due to the lack of a
biosynthetic pathway to produce a precursor required for
biosynthesis of the product. As used herein, the term recombinant
refers to a cell, compound, or composition produced at least in
part by human intervention, particularly by modification of the
genetic material of a cell. The present invention also provides
methods for increasing the amounts of a product produced in a host
cell by providing recombinant biosynthetic pathways for production
of a precursor utilized in the biosynthesis of a product.
[0029] In one embodiment, the host cell does not produce the
precursor, and the host cell is modified by introduction of a
recombinant expression vector so that it can produce the precursor.
In another embodiment, the precursor is produced in the host cell
in small amounts, and the host cell is modified by introduction of
a recombinant expression vector so that it can produce the
precursor in larger amounts. In a preferred embodiment, the
precursor is a primary metabolite that is produced in first cell
but not in a second heterologous cell. In accordance with the
methods of the invention, the genes that encode the enzymes that
produce the primary metabolite in the first cell are transferred to
the second cell. The transfer is accomplished using an expression
vector of the invention. The expression vector drives expression of
the genes and production of the metabolite in the second cell.
[0030] The invention, in its most general form, concerns the
introduction, in whole or in part, of a metabolic pathway from one
cell into a heterologous host cell. The invention also encompasses
the modification of an existing metabolic pathway, in whole or in
part, in a cell, through the introduction of heterologous genetic
material into the cell. In all embodiments, the resulting cell is
different with regard to its cellular physiology and biochemistry
in a manner such that the bio-synthesis, bio-degradation,
transport, biochemical modification, or levels of intracellular
metabolites allow production or improve expression of desired
products. The invention is exemplified by increasing the level of
polyketides produced in a heterologous host and by restricting the
chemical composition of products to the desired structures.
[0031] Thus, in a preferred embodiment, the product produced by the
cell is a polyketide. The polyketide is a polyketide synthesized by
either a modular, iterative, or fungal PKS. The precursor is
selected from the group consisting of malonyl CoA, propionyl CoA,
methylmalonyl CoA, ethylmalonyl CoA, and hydroxymalonyl or
methoxymalonyl CoA. In an especially preferred embodiment, the
polyketide utilizes methylmalonyl CoA in its biosynthesis. In one
preferred embodiment, the polyketide is synthesized by a modular
PKS that requires methylmalonyl CoA to synthesize the
polyketide.
[0032] The polyketide class of natural products includes members
having diverse structural and pharmacological properties (see
Monaghan and Tkacz, 1990, Annu. Rev. Microbiol. 44: 271,
incorporated herein by reference). Polyketides are assembled by
polyketide synthases through successive condensations of activated
coenzyme-A thioester monomers derived from small organic acids such
as acetate, propionate, and butyrate. Active sites required for
condensation include an acyltransferase (AT), acyl carrier protein
(ACP), and beta-ketoacylsynthase (KS). Each condensation cycle
results in a .beta.-keto group that undergoes all, some, or none of
a series of processing activities. Active sites that perform these
reactions include a ketoreductase (KR), dehydratase (DH), and
enoylreductase (ER). Thus, the absence of any beta-keto processing
domain results in the presence of a ketone, a KR alone gives rise
to a hydroxyl, a KR and DH result in an alkene, while a KR, DH, and
ER combination leads to complete reduction to an alkane. After
assembly of the polyketide chain, the molecule typically undergoes
cyclization(s) and post-PKS modification (e.g. glycosylation,
oxidation, acylation) to achieve the final active compound.
[0033] Macrolides such as erythromycin and megalomicin are
synthesized by modular PKSs (see Cane et al., 1998, Science 282:
63, incorporated herein by reference). For illustrative purposes,
the PKS that produces the erythromycin polyketide
(6-deoxyerythronolide B synthase or DEBS; see U.S. Pat. No.
5,824,513, incorporated herein by reference) is shown in FIG. 1.
DEBS is the most characterized and extensively used modular PKS
system. DEBS synthesizes the polyketide 6-deoxyerythronolide B
(6-dEB) from propionyl CoA and methylmalonyl CoA. In modular PKS
enzymes such as DEBS, the enzymatic steps for each round of
condensation and reduction are encoded within a single "module" of
the polypeptide (i.e., one distinct module for every condensation
cycle). DEBS consists of a loading module and 6 extender modules
and a chain terminating thioesterase (TE) domain within three
extremely large polypeptides encoded by three open reading frames
(ORFs, designated eryAI, eryAII, and eryAIII).
[0034] Each of the three polypeptide subunits of DEBS (DEBSI,
DEBSII, and DEBSIII) contains 2 extender modules, DEBSI
additionally contains the loading module. Collectively, these
proteins catalyze the condensation and appropriate reduction of 1
propionyl CoA starter unit and 6 methylmalonyl CoA extender units.
Modules 1, 2, 5, and 6 contain KR domains; module 4 contains a
complete set, KR/DHIER, of reductive and dehydratase domains; and
module 3 contains no functional reductive domain. Following the
condensation and appropriate dehydration and reduction reactions,
the enzyme bound intermediate is lactonized by the TE at the end of
extender module 6 to form 6-dEB.
[0035] More particularly, the loading module of DEBS consists of
two domains, an acyl-transferase (AT) domain and an acyl carrier
protein (ACP) domain. In other PKS enzymes, the loading module is
not composed of an AT and an ACP but instead utilizes a partially
inactivated KS, an AT, and an ACP. This partially inactivated KS is
in most instances called KS.sup.Q, where the superscript letter is
the abbreviation for the amino acid, glutamine, that is present
instead of the active site cysteine required for full activity. The
AT domain of the loading module recognizes a particular acyl CoA
(propionyl for DEBS, which can also accept acetyl) and transfers it
as a thiol ester to the ACP of the loading module. Concurrently,
the AT on each of the extender modules recognizes a particular
extender-CoA (methylmalonyl for DEBS) and transfers it to the ACP
of that module to form a thioester. Once the PKS is primed with
acyl- and malonyl-ACPs, the acyl group of the loading module
migrates to form a thiol ester (trans-esterification) at the KS of
the first extender module; at this stage, extender module 1
possesses an acyl-KS and a methylmalonyl ACP. The acyl group
derived from the loading module is then covalently attached to the
alpha-carbon of the malonyl group to form a carbon-carbon bond,
driven by concomitant decarboxylation, and generating a new
acyl-ACP that has a backbone two carbons longer than the loading
unit (elongation or extension). The growing polyketide chain is
transferred from the ACP to the KS of the next module, and the
process continues.
[0036] The polyketide chain, growing by two carbons each module, is
sequentially passed as a covalently bound thiol ester from module
to module, in an assembly line-like process. The carbon chain
produced by this process alone would possess a ketone at every
other carbon atom, producing a poyketone, from which the name
polyketide arises. Commonly, however, the beta keto group of each
two-carbon unit is modified just after it has been added to the
growing polyketide chain but before it is transferred to the next
module by either a KR, a KR plus a DH, or a KR, a DH, and an ER. As
noted above, modules may contain additional enzymatic activities as
well.
[0037] Once a polyketide chain traverses the final extender module
of a PKS, it encounters the releasing domain or thioesterase found
at the carboxyl end of most PKSs. Here, the polyketide is cleaved
from the enzyme and typically cyclyzed. The resulting polyketide
can be modified further by tailoring or modification enzymes; these
enzymes add carbohydrate groups or methyl groups, or make other
modifications, i.e., oxidation or reduction, on the polyketide core
molecule. For example, the final steps in conversion of 6-dEB to
erythromycin A include the actions of a number of modification
enzymes, such as: C-6 hydroxylation, attachment of mycarose and
desosamine sugars, C-12 hydroxylation (which produces erythromycin
C), and conversion of mycarose to cladinose via O-methylation.
[0038] With this overview of PKS and post-PKS modification enzymes
and their substrates, one can better appreciate the benefits
provided by the present invention. DEBS is produced naturally in
Saccharopolyspora erythraea and has been transferred to a variety
of Streptomyces species, such as S. coelicolor CH999 and S.
lividans K4-114 and K4-155, in which it functions without further
modification of the host cell to produce 6-dEB. Thus, S. erythraea,
S. coelicolor, and S. lividans make the required precursors for
6-dEB synthesis. However, many other non-Saccharopolyspora,
non-Streptomyces host cells do not make all of the required
precursors or make them only at levels sufficient to support only
very small amounts of polyketide biosynthesis.
[0039] The present invention provides recombinant DNA expression
vectors and methods for making a polyketide and its required
precursors in any host cell. In one embodiment, the host cell is
either a procaryotic or eukaryotic host cell. In a preferred
embodiment, the host cell is an E. coli host cell. In another
preferred embodiment, the host cell is a yeast host cell. In
another embodiment, the host cell is a plant host cell. In a
preferred embodiment, the host cell is either an E. coli or yeast
host cell, the product is a polyketide, and the precursor is
methylmalonyl CoA.
[0040] The recombinant expression vectors of the invention comprise
a promoter positioned to drive expression of one or more genes that
encode the enzymes required for biosynthesis of a precursor. In a
preferred embodiment, the promoter is derived from a PKS gene. In
another preferred embodiment, the promoter is one derived from a
host cell gene or from a virus or phage that normally infects the
host cell and is heterologous to the gene that encodes the
biosynthetic enzyme.
[0041] In another embodiment, the invention provides a recombinant
host cell that comprises not only an expression vector of the
invention but also an expression vector that comprises a promoter
positioned to drive expression of a PKS. In a related embodiment,
the invention provides recombinant host cells comprising the vector
that produces the PKS and its corresponding polyketide. In a
preferred embodiment, the host cell is an E. coli or yeast host
cell.
[0042] Neither E. coli nor yeast makes sufficient methylmalonyl CoA
to support biosynthesis of large amounts of polyketides that
require methylmalonyl CoA in their biosynthesis, and most species
do not produce the methylmalonyl CoA substrate at all. In one
embodiment, the present invention provides E. coli, yeast, and
other host cells that produce methylmalonyl CoA in amounts
sufficient to support polyketide biosynthesis. In preferred
embodiments, the cells produce sufficient amounts of methylmalonyl
CoA to support biosynthesis of polyketides requiring methylmalonyl
CoA for their biosynthesis at levels ranging from 1 .mu.g/L, to 1
mg/L, to 10 mg/L, to 100 mg/L, to 1 g/L, to 10 g/L.
[0043] In one embodiment, the host cells of the invention have been
modified to express a heterologous methylmalonyl CoA mutase. This
enzyme, which converts succinyl CoA to methylmalonyl CoA (although
the reverse reaction is 20 times more favored) has been expressed
in E. coli using a gene cloned from propionibacteria but was
inactive due to the lack of vitamin B12. In accordance with the
methods of the present invention, this enzyme can be made in an
active form in E. coli and other host cells by either expressing
(constitutively or otherwise) a B12 transporter gene, such as the
endogenous E. coli gene and/or by utilizing a media that
facilitates B 12 uptake (as used herein, B12 can refer to the
precursor hydroxocobalamin, which is converted to B12). While
certain methylmalonyl CoA mutases make the R-isomer, including the
methylmalonyl CoA mutases derived from the propionibacteria, the
R-isomer can be converted to the S-isomer using an epimerase. For
example, epimerase genes from propionibacteria or Streptomyces can
be employed for this purpose.
[0044] In another embodiment, the host cells of the invention have
been modified to express a heterologous propionyl CoA carboxylase
that converts propionyl CoA to methylmalonyl CoA. In this
embodiment, one can further increase the amount of methylmalonyl
CoA precursor by culturing the cells in a media supplemented with
propionate. In a preferred embodiment, the host cells are E. coli
host cells.
[0045] Thus, in accordance with the methods of the invention, the
heterologous production of certain polyketides in E. coli, yeast,
and other host organisms require both the heterologous expression
of a desired PKS and also the enzymes that produce at least some of
the substrate molecules required by the PKS. These substrate
molecules, called precursors, are not normally found as
intracelluar metabolites in the host organism or are present in low
abundance. The present invention provides a method to produce or
modify the composition or quantities of intracellular metabolites
within a host organism where such metabolites are not naturally
present or are present in non-optimal amounts.
[0046] A specific embodiment of the present invention concerns the
introduction and modification of biochemical pathways for
methylmalonyl CoA biosynthesis. Methylmalonyl CoA, as noted above,
is a substrate utilized for the synthesis of polyketides by many
polyketide synthases. Some of the known biochemical pathways for
the intracellular production of methylmalonyl CoA employ enzymes
and their corresponding genes found in certain organisms. These
enzymes and genes have not been found, or are otherwise
non-optimal, in other organisms. These other organisms include
those that could otherwise be very useful as heterologous hosts for
the production of polyketides. The present invention provides
methods to engineer a host organism so that it contains a new or
modified ability to produce methylmalonyl CoA and/or to increase or
decrease the levels of methylmalonyl CoA in the host.
[0047] As noted above, two biochemical pathways involving
methylmalonyl CoA are particularly relevant to this aspect of the
present invention. These pathways are the methylmalonyl CoA mutase
pathway, hereafter referred to as the MUT pathway, and the
propionyl CoA carboxylase pathway, hereafter referred to as the PCC
pathway.
[0048] The MUT pathway includes the enzymes methylmalonyl CoA
mutase (5.4.99.2, using the numbering system devised by the
Nomenclature Committee of the International Union of biochemistry
and Molecular Biology), methylmalonyl CoA epimerase (5.1.99.1), and
malonyl CoA decarboxylase (4.1.1.9). The biochemical pathway
includes the conversion of succinyl CoA to (R)-methylmalonyl CoA
through the action of methylmalonyl CoA mutase (5.4.99.2) followed
by the conversion of (R)-methylmalonyl CoA to (S)-methylmalonyl CoA
through the action of methylmalonyl CoA epimerase (5.1.99.1).
(S)-methylmalonyl CoA is a substrate utilized by several polyketide
synthases. The enzyme malonyl CoA decarboxylase (4.1.1.9) catalyzes
the decarboxylation of malonyl CoA but is also reported to catalyze
the decarboxylation of (R)-methylmalonyl CoA to form propionyl CoA.
Propionyl CoA is a substrate utilized by some polyketide
synthases.
[0049] The PCC pathway includes the enzymes propionyl CoA
carboxylase (6.4.1.3) and propionyl CoA synthetase (6.2.1.17). The
biochemical pathway includes the conversion of propionate to
propionyl CoA through the action of propionyl CoA synthetase
(6.2.1.17) followed by the conversion of propionyl CoA to
(S)-methylmalonyl CoA through the action of propionyl CoA
carboxylase (6.4.1.3). (S)-methylmalonyl CoA is the substrate
utilized by many polyketide synthases.
[0050] An illustrative embodiment of the present invention employs
specific enzymes from these pathways. As those skilled in the art
will recognize upon contemplation of this description of the
invention, the invention can also be practiced using additional
and/or alternative enzymes involved in the MUT and PCC pathways.
Moreover, the invention can be practiced using additional and
alternative pathways for methylmalonyl CoA and other intracelluar
metabolites.
[0051] The methods of the invention involve the introduction of
genetic material into a host strain of choice to modify or alter
the cellular physiology and biochemistry of the host. Through the
introduction of genetic material, the host strain acquires new
properties, e.g. the ability to produce a new, or greater
quantities of, an intracellular metabolite. In an illustrative
embodiment of the invention, the introduction of genetic material
into the host strain results in a new or modified ability to
produce methylmalonyl CoA. The genetic material introduced into the
host strain contains gene(s), or parts of genes, coding for one or
more of the enzymes involved in the bio-synthesis/bio-degradation
of methylmalonyl CoA and may also include additional elements for
the expression and/or regulation of expression of these genes, e.g.
promoter sequences. Specific gene sequences coding for enzymes
involved in the bio-synthesis/bio-degradation of methylmalonyl CoA
are listed below.
[0052] A suitable methylmalonyl CoA mutase (5.4.99.2) gene can be
isolated from Streptomyces cinnamonensis. See Birch et al., 1993,
J. Bacteriol. 175: 3511-3519, entitled "Cloning, sequencing, and
expression of the gene encoding methylmalonyl-coenzyme A mutase
from Streptomyces cinnamonensis." This enzyme is a two subunit
enzyme; the A and B subunit coding sequences are available under
Genbank accession L10064. Another suitable methylmalonyl CoA mutase
gene can be isolated from Propionibacterium shermanii. See Marsh et
al., 1989, Biochem. J. 260: 345-352, entitled "Cloning and
structural characterization of the genes coding for
adenosylcobalamin-dependent methylmalonyl CoA mutase from
Propionibacterium shermanii." Alternatively, a suitable
methylmalonyl CoA mutase gene can be isolated from Porphyromonas
gingivalis. See Jackson et al., 1995, Gene 167: 127-132, entitled
"Cloning, expression and sequence analysis of the genes encoding
the heterodimeric methylmalonyl CoA mutase of Porphyromonas
gingivalis W50." Alternatively, suitable methylmalonyl CoA mutase
genes can be isolated from any of the sources noted in the
following table of a partial BLAST search report or from additional
BLAST analyses.
TABLE-US-00002 Results of BLAST Search of NCBI Database for
Methylmalonyl CoA Mutase mutA gb|L10064|STMMUTA Streptomyces
cinnamonensis 931 0.0 (query sequence) gb|AD000015|MSGY175
Mycobacterium tuberculosis sequence 300 7e-80 emb|Z79701|MTCY277
Mycobacterium tuberculosis H37Rv 300 7e-80 gb|AD000001|MSGY456
Mycobacterium tuberculosis sequence 238 8e-76 emb|X14965|PSMUTAB
Propionibacterium shermanii mutA 268 5e-70 gb|L30136|POYMCMAB
Porphyromonas gingivalis 137 9e-31 gb|AE000375|AE000375 Escherichia
coli K-12 MG1655 134 1e-29 gb|U28377|ECU28377 Escherichia coli K-12
genome; 134 1e-29 emb|X66836|ECSERAICI E. coli serA, iciA, sbm
genes 133 1e-29 gb|AF080073|SMPCAS2 Sinorhizobium meliloti 130
2e-28 ref|NM_000255.1|MUT| Homo sapiens 113 2e-23
dbj|AP000006|AP000006 Pyrococcus horikoshii OT3 110 2e-22
emb|AJ248285.1|CNSPAX03 Pyrococcus abyssi 109 3e-22
emb|X51941|MMMMCOAM Mouse mRNA 109 3e-22 gb|AE000952|AE000952
Archaeoglobus fulgidus section 155 104 9e-21
emb|AJ237976.1|SCO237976 Streptomyces coelicolor icmA gene 103
2e-20 dbj|AP000062.1|AP000062 Aeropyrum pernix genomic DNA 102
3e-20 gb|U67612|SCU67612 Streptomyces cinnamonensis coenzyme B12 98
7e-19 gb|AE001015|AE001015 Archaeoglobus fulgidus section 92 97
1e-18 emb|X59424|BFOF4 Bacillus firmus OF4 genes for ATP binding 82
7e-14 mutB gb|L10064|STMMUTA Streptomyces cinnamonensis 1379 0.0
(query sequence) gb|AD000001|MSGY456 Mycobacterium tuberculosis
1018 0.0 emb|Z79701|MTCY277 Mycobacterium tuberculosis H37Rv 1017
0.0 gb|AD000015|MSGY175 Mycobacterium tuberculosis sequence 1017
0.0 emb|X14965|PSMUTAB Propionibacterium shermanii 996 0.0
gb|L30136|POYMCMAB Porphyromonas gingivalis methylmalonyl 882 0.0
ref|NM_000255.1|MUT| Homo sapiens methylmalonyl Coenzyme A 855 0.0
emb|X51941|MMMMCOAM Mouse mRNA 32 0.0 gb|U28377|ECU28377
Escherichia coli K-12 genome 798 0.0 gb|AE000375|AE000375
Escherichia coli K-12 MG1655 798 0.0 emb|X66836|ECSERAICI E. coli
serA, iciA, sbm genes 797 0.0 gb|AF080073|SMPCAS2 Sinorhizobium
meliloti 782 0.0 gb|AE001015|AE001015 Archaeoglobus fulgidus 516
e-145 dbj|AP000062.1|AP000062 Aeropyrum pernix genomic DNA 408
e-139 emb|AJ248285.1|CNSPAX03 Pyrococcus abyssi complete genome 486
e-135 dbj|AP000006|AP000006 Pyrococcus horikoshii OT3 genomic DNA
480 e-133 gb|AE000952|AE000952 Archaeoglobus fulgidus section 155
467 e-130 emb|Z35604.1|CEZK1058 Caenorhabditis elegans cosmid
ZK1058 316 e-109 emb|AJ237976.1|SCO237976 Streptomyces coelicolor
icmA 377 e-103 gb|U67612|SCU67612 Streptomyces cinnamonensis
coenzyme 372 e-101 emb|AL035161|SC9C7 Streptomyces coelicolor
cosmid 9C7 359 2e-97 gb|U28335|MEU28335 Methylobacterium extorquens
351 4e-95 gb|AF008569|AF008569 Streptomyces collinus coenzyme 337
8e-91 gb|U65074|ECU65074 Escherichia coli chromosome 275 3e-72
gb|M37500|HUMMUT03 Human methylmalonyl CoA mutase 202 3e-50
gb|AF178673.1|AF178673 Streptomyces cinnamonensis 183 1e-44
emb|Z49936.1|CEF13B10 Caenorhabditis elegans cosmid F13B10 138
2e-41 gb|M37499|HUMMUT02 Human methylmalonyl CoA mutase 112 4e-23
dbj|AP000001.1|AP000001 Pyrococcus horikoshii OT3 genomic 106 2e-21
emb|AJ248283.1|CNSPAX01 Pyrococcus abyssi complete genome 106 2e-21
gb|M37503|HUMMUT06 Human methylmalonyl CoA mutase 101 7e-20
gb|M37508|HUMMUT11 Human methylmalonyl CoA mutase 86 3e-15
gb|M37509|HUMMUT12 Human methylmalonyl CoA mutase 80 3e-13
gb|M37501|HUMMUT04 Human methylmalonyl CoA mutase 77 2e-12
[0053] Methylmalonyl CoA mutase requires vitamin B12
(adenosylcobalamin) as an essential cofactor for activity. One of
the difficulties in expressing active methylmalonyl CoA mutase in a
heterologous host is that the host organism may not provide
sufficient, if any, amounts of this cofactor. Work on the
expression of methionine synthase, a cobalamin-dependent enzyme, in
E. coli, a host that does not synthesize cobalamin, has shown that
it is possible to express an active cobalamin-dependent enzyme by
increasing the rate of cobalamin transport. See Amaratunga et al.,
1996, Biochemistry 35: 2453-2463, entitled "A synthetic module for
the metH gene permits facile mutagenesis of the cobalamin-binding
region of Escherichia coli methionine synthase: initial
characterization of seven mutant proteins," incorporated herein by
reference.
[0054] The methods of the present invention include the step of
increasing the availability of cobalamin for the heterologous
expression of active methylmalonyl CoA mutase in certain hosts,
e.g. E. coli. In particular, these methods incorporate growing
cells in a media that contains hydroxocobalamin and/or other
nutrients, as described in Amaratunga et al., supra. Additional
methods for increasing the availability of cobalamin include
constitutive and/or over-expression of vitamin B12 transporter
proteins and/or their regulators.
[0055] A suitable methylmalonyl CoA epimerase (5.1.99.1) gene for
purposes of the present invention can be isolated from Streptomyces
coelicolor as reported in GenBank locus SC5F2A as gene SC5F2A. 13
(referred to here as EP5) or from S. coelicolor as reported in
GenBank locus SC6A5 as gene SC6A5.34 (referred to here as EP6). See
Redenbach et al., 1996, Mol. Microbiol. 21 (1), 77-96, entitled "A
set of ordered cosmids and a detailed genetic and physical map for
the 8 Mb Streptomyces coelicolor A3(2) chromosome," incorporated
herein by reference. To date, no biochemical characterization of
the proteins encoded by the genes EP5 and EP6 has been carried out;
thus, the present invention provides a method for using these genes
to provide methylmalonyl CoA epimerase activity to a host. That
these genes encode proteins with methylmalonyl CoA epimerase
activity is supported by their homology to the sequence of a
2-arylpropionyl CoA epimerase from rat. See Reichel et al., 1997,
Mol. Pharmacol. 51: 576-582, entitled "Molecular cloning and
expression of a 2-arylpropionyl-coenzyme A epimerase: a key enzyme
in the inversion metabolism of ibuprofen," and Shieh & Chen,
1993, J. Biol. Chem. 268: 3487-3493, entitled "Purification and
characterization of novel `2-arylpropionyl CoA epimerases` from rat
liver cytosol and mitochondria." Both rat 2-arylpropionyl CoA
epimerase and methylmalonyl CoA epimerase catalyze the same
stereoisomeric inversion, but with different chemical groups
attached.
[0056] Biochemical characterization of a methylmalonyl CoA
epimerase enzyme purified from Propionibacterium shermanii has been
completed. See Leadlay, 1981, Biochem. J. 197: 413-419, entitled
"Purification and characterization of methylmalonyl CoA epimerase
from Propionibacterium shermanii," Leadlay & Fuller, 1983,
Biochem. J. 213: 635-642, entitled "Proton transfer in
methylmalonyl CoA epimerase from Propionibacterium shermanii:
Studies with specifically tritiated (2R)-methylmalonyl CoA as
substrate; Fuller & Leadlay, 1983, Biochem. J. 213: 643-650,
entitled "Proton transfer in methylmalonyl CoA epimerase from
Propionibacterium shermanii: The reaction of (2R)-methylmalonyl CoA
in tritiated water." The DNA sequence of the gene coding for this
enzyme from Propionibacterium shermanii is provided by the present
invention as SEQ ID NO: 1 in isolated and recombinant form and is
incorporated into expression vectors and host cells of the
invention. Suitable methylmalonyl CoA epimerase genes can be
isolated from a BLAST search using the P. shermanii sequence
provided in Example 1, below. Preferred epimerases in addition to
the P. shermanii epimerase include gene identified by homology with
the P. shermanii sequence located on cosmid 8F4 from the S.
coelicolor genome sequencing project and the B. subtilis epimerase
described by Haller et al., 2000, Biochemistry 39 (16): 4622-4629,
incorporated herein by reference.
[0057] One can also make S-methylmalonyl CoA from R-methylmalonyl
CoA utilizing an activity of malonyl CoA decarboxylase A, which
converts R-methylmalonyl CoA to propionyl CoA. As described above,
propionyl CoA can then be converted to S-methylmalonyl CoA by
propionyl CoA carboxylase. A suitable malonyl CoA decarboxylase
(4.1.1.9) gene for purposes of the present invention can be
isolated from Saccharopolyspora erythraea as reported in Hsieh
& Kolattukudy, 1994, J. Bacteriol. 176: 714-724, entitled
"Inhibition of erythromycin synthesis by disruption of
malonyl-coenzyme A decarboxylase gene eryM in Saccharopolyspora
erythraea." Alternatively, suitable malonyl CoA decarboxylase genes
can be isolated from any of the sources noted in the following
table of BLAST search reports or by additional BLAST searches.
TABLE-US-00003 Results of BLAST Search of NCBI Database for Malonyl
CoA Decarboxylase Malonyl CoA decarboxylase (DC)
gb|L05192|SERMALCOAD S. erythraea malonyl 664 0.0 (query sequence)
emb|AL022268|SC4H2 Streptomyces coelicolor cosmid 4H2 128 3e-28
emb|Z75555|MTCY02B10 Mycobacterium tuberculosis H37Rv 109 1e-22
gb|AD000018|MSGY151 Mycobacterium tuberculosis sequence 109 1e-22
gb|AF141323.1|AF141323 Shigella flexneri SHI-2 95 5e-18
emb|X76100|ECIUC E. coli plasmid iucA, iucB and iucC genes 92 3e-17
emb|AL116808.1|CNS01DGW Botrytis cinerea strain T4 cDNA 88 5e-16
gb|AF110737.1|AF110737 Sinorhizobium meliloti strain 2011 84 9e-15
emb|AL109846.1|SPBC17G9 S. pombe chromosome II cosmid c17G9 71
7e-11 gb|L06163|PSEAAC Pseudomonas fluorescens aminoglycoside 70
1e-10
[0058] A suitable propionyl CoA carboxylase (6.4.1.3) gene for
purposes of the present invention can be isolated from Streptomyces
coelicolor as reported in GenBank locus AF113605 (pccB), AF113604
(accA2) and AF113603 (accA1) by H. C. Gramajo and colleagues. The
propionyl CoA carboxylase gene product requires biotin for
activity. If the host cell does not make biotin, then the genes for
biotin tranport can be transferred to the host cell. Even if the
host cell makes or transports biotin, the endogenous biotin
transferase enzyme may not have sufficient activity (whether due to
specificity constraints or other reasons) to biotinylate the
propionyl CoA carboxylase at the rate required for high level
precursor synthesis. In this event, one can simply provide the host
cell with a sufficiently active biotin transferase enzyme gene, or
if there is an endogenous transferase gene, such as the birA gene
in E. coli, one can simply overexpress that gene by recombinant
methods. Many additional genes coding for propionyl CoA
carboxylases, or acetyl CoA carboxylases with relaxed substrate
specificity that includes propionate, have been reported and can be
used as sources for this gene, as shown in the following table.
TABLE-US-00004 Results of BLAST Search of NCBI Database for
Propionyl CoA Carboxylase Propionyl CoA Carboxylase (pccB)
gb|AF113605.1|AF113605 S. coelicolor propionyl 1035 0.0 (query
sequence) emb|X92557|SEPCCBBCP S. erythraea pccB, bcpA2, and orfX
800 0.0 emb|Z92771|MTCY71 Mycobacterium tuberculosis H37Rv 691 0.0
dbj|AB018531|AB018531 Corynebacterium glutamicum dtsR1 686 0.0
gb|U00012|U00012 Mycobacterium leprae cosmid B1308 686 0.0
dbj|AB018530|AB018530 Corynebacterium glutamicum dtsR gene 612
e-174 gb|AE001742.1|AE001742 Thermotoga maritima section 54 610
e-173 emb|AJ002015|PMAJ2015 Propionigenium modestum mmdD 589 e-167
dbj|AB007000|AB007000 Myxococcus xanthus MxppcB gene 588 e-166
gb|L48340|MTBKATA Methylobacterium extorquens catalase 588 e-166
gb|AE000952|AE000952 Archaeoglobus fulgidus section 155 572 e-162
dbj|AP000005|AP000005 Pyrococcus horikoshii OT3 genomic 570 e-161
emb|AJ248285.1|CNSPAX03 Pyrococcus abyssi complete genome 570 e-161
emb|AL031124|SC1C2 Streptomyces coelicolor cosmid 1C2 563 e-159
gb|L22208|VEIMCDC Veillonella parvula methylmalonyl CoA 558 e-157
gb|AF080235|AF080235 Streptomyces cyanogenus landomycin 552 e-155
emb|AJ235272|RPXX03 Rickettsia prowazekii strain Madrid E 545 e-153
dbj|AB000886|AB000886 Sus scrofa mRNA for Propionyl CoA 539 e-152
ref|NM_000532.1|PCCB| Homo sapiens propionyl Coenzyme A 538 e-151
emb|X73424|HSPCCBA Homo sapiens gene for propionyl CoA 538 e-151
gb|M14634|RATPCCB Rat mitochondrial propionyl CoA 535 e-150
gb|S67325|S67325 propionyl CoA carboxylase beta subunit 531 e-149
gb|U56964|CELF52E4 Caenorhabditis elegans cosmid F52E4 367 e-143
emb|Z99116|BSUB0013 Bacillus subtilis complete genome 494 e-138
dbj|D84432|BACJH642 Bacillus subtilis DNA, 283 Kb region 494 e-138
gb|AF042099|AF042099 Sulfolobus metallicus putative 486 e-136
emb|AL022076.1|MTV026 Mycobacterium tuberculosis H37Rv 483 e-135
gb|L04196|PRSTRANSC Propionibacterium shermanii 383 e-104
emb|AL023635.1|MLCB1243 Mycobacterium leprae cosmid B1243 356 1e-96
emb|Z70692.1|MTCY427 Mycobacterium tuberculosis H37Rv 353 1e-95
gb|L78825|MSGB1723CS Mycobacterium leprae cosmid B1723 DNA 319
4e-93 gb|M95713|RERCOABETA Rhodococcus erythropolis 340 5e-92
emb|Z99113|BSUB0010 Bacillus subtilis complete genome 325 2e-87
gb|U94697|CCU94697 Caulobacter crescentus DNA topoisomerase 270
6e-71 emb|Z95556|MTCY07A7 Mycobacterium tuberculosis H37Rv 253
9e-66 emb|Y07660|MTACCBC M. tuberculosis accBC gene 231 6e-59
emb|Z79700|MTCY10D7 Mycobacterium tuberculosis H37Rv 229 2e-58
dbj|AB018557.1|AB018557 Streptomyces griseus cyaA gene 228 5e-58
gb|U46844|MSU46844 Mycobacterium smegmatis catalase 209 2e-52
emb|Z19555.1|CEF02A9 Caenorhabditis elegans cosmid F02A9 105 9e-51
gb|M13573|HUMPCCB Human propionyl CoA carboxylase beta 194 5e-48
gb|AF030576|AF030576 Acidaminococcus fermentans 170 9e-41
emb|Y13917|BSY13917 Bacillus subtilis ppsE, yngL, yngK 149 2e-34
emb|X69435|AFGCDA A. fermentans GCDA gene for 107 1e-21
emb|Z82368|RPZ82368 R. prowazekii genomic DNA fragment 93 2e-17
gb|AF025469|CELW09B6 Caenorhabditis elegans cosmid W09B6 78 5e-13
gb|U87980|MRU87980 Malonomonas rubra putative IS-element 78 7e-13
gb|AE001518|AE001518 Helicobacter pylori, strain J99 75 6e-12
gb|AE000604.1|AE000604 Helicobacter pylori 26695 section 82 75
8e-12 gb|U89347|ACU89347 Acinetobacter calcoaceticus malonate 74
1e-11 emb|AL021961|ATF28A23 Arabidopsis thaliana DNA 61 2e-11
gb|AE001591|AE001591 Chlamydia pneumoniae section 7 73 2e-11
emb|Z46886|UMACCGEN U. maydis ACC gene for acetyl coa 71 1e-10
gb|U86128|SSPCCB1 Sus scrofa propionyl CoA carboxylase B 70 2e-10
emb|AJ006497|HSA006497 Homo sapiens PCCB gene, exons 11 70 2e-10
gb|AE001301|AE001301 Chlamydia trachomatis section 28 69 5e-10
gb|U32724|U32724 Haemophilus influenzae Rd section 39 68 8e-10
gb|U04358|PSU04358 Pseudomonas syringae pv. syringae Y30 68 8e-10
Propionyl CoA carboxylase (accA2) gb|AF113604.1|AF113604 S.
coelicolor putative 1101 0.0 (query sequence)
gb|AF113603.1|AF113603 Streptomyces coelicolor putative 1090 0.0
gb|AF126429.1|AF126429 Streptomyces venezuelae JadJ 967 0.0
emb|Z92771|MTCY71 Mycobacterium tuberculosis H37Rv 758 0.0
emb|X92557|SEPCCBBCP S. erythraea pccB, bcpA2, and orfX genes 753
0.0 emb|X92556|SEHGTABCP S. erythraea hgtA, bcpA1, and orf122 753
0.0 gb|U00012|U00012 Mycobacterium leprae cosmid B1308 746 0.0
emb|X63470|MLBCCPG M. leprae gene for biotin carboxyl 743 0.0
gb|U35023|CGU35023 Corynebacterium glutamicum thiosulfate 695 0.0
gb|U24659|SVU24659 Streptomyces venezuelae glucose 599 e-170
gb|AE000742|AE000742 Aquifex aeolicus section 74 413 e-113
gb|U67563|U67563 Methanococcus jannaschii section 105 405 e-111
gb|L36530|MQSPYRCARB Aedes aegypti pyruvate carboxylase 400 e-110
gb|AF132152.1|AF132152 Drosophila melanogaster clone 396 e-108
gb|L09192|MUSMPYR Mus musculus pyruvate carboxylase 393 e-107
gb|U36585|RNU36585 Rattus norvegicus pyruvate carboxylase 391 e-107
gb|U32314|RNU32314 Rattus norvegicus pyruvate carboxylase 391 e-107
gb|L14862|ANAACCC Anabaena sp. (PCC 7120) 49.1 kDa biotin 388 e-106
gb|U59234|SPU59234 Synechococcus PCC7942 biotin 387 e-106
gb|U04641|HSU04641 Human pyruvate carboxylase (PC) mRNA 387 e-106
ref|NM_000920.1|PC| Homo sapiens pyruvate carboxylase (PC) 386
e-105 gb|AE001090|AE001090 Archaeoglobus fulgidus section 17 383
e-104 dbj|D84432|BACJH642 Bacillus subtilis DNA, 283 Kb region 382
e-104 emb|Z99116|BSUB0013 Bacillus subtilis complete genome 382
e-104 gb|AE000942|AE000942 Methanobacterium thermoautotrophicum 382
e-104 gb|S72370|S72370 pyruvate carboxylase human, kidney 380 e-104
dbj|D64001|SYCCPNC Synechocystis sp. PCC6803 complete 379 e-103
gb|L14612|PSEACCBC Pseudomonas aeruginosa biotin carboxyl 376 e-103
gb|U32778|U32778 Haemophilus influenzae Rd section 93 375 e-102
emb|Z36087|SCYBR218C S. cerevisiae chromosome II 374 e-102
gb|U35647|SCU35647 Saccharomyces cerevisiae pyruvate 374 e-102
gb|J03889|YSCPCB Yeast (S. cerevisiae) pyruvate carboxylase 374
e-102 gb|U90879|ATU90879 Arabidopsis thaliana biotin carboxylase
374 e-102 emb|Z72584|SCYGL062W S. cerevisiae chromosome VII 374
e-102 emb|X59890|SCPYC2G S. cerivisiae PYC2 gene for pyruvate 373
e-102 gb|AE000749|AE000749 Aquifex aeolicus section 81 371 e-101
gb|AE001286|AE001286 Chlamydia trachomatis section 13 370 e-101
gb|AE001604|AE001604 Chlamydia pneumoniae section 20 369 e-100
gb|AF007100|AF007100 Glycine max biotin carboxylase 368 e-100
emb|Z95556|MTCY07A7 Mycobacterium tuberculosis H37Rv 367 e-100
emb|Z19549|MTBCARBCP M. tuberculosis gene for biotin 367 e-100
gb|AF068249|AF068249 Glycine max biotin carboxylase 366 1e-99
gb|L38260|TOBBCSO Nicotiana tabacum acetyl CoA 363 7e-99
gb|U36245|BSU36245 Bacillus subtilis biotin carboxyl 362 2e-98
gb|AF097728|AF097728 Aspergillus terreus pyruvate 361 3e-98
emb|AJ235272|RPXX03 Rickettsia prowazekii strain Madrid E 360 1e-97
dbj|D83706|D83706 Bacillus stearothermophilus DNA 360 1e-97
gb|AE000744|AE000744 Aquifex aeolicus section 76 358 3e-97
emb|AL109846.1|SPBC17G9 S. pombe chromosome II 356 1e-96
dbj|D78170|D78170 Yeast DNA for pyruvate carboxylase 353 1e-95
gb|M79446|ECOFABG Escherichia coli biotin carboxylase gene 352
2e-95 gb|M83198|ECOFABEGF Escherichia coli biotin carboxyl 352
2e-95 gb|AE00404|AE000404 Escherichia coli K-12 MG1655 352 2e-95
gb|U18997.1|ECOUW67 Escherichia coli K-12 chromosomal 352 2e-95
gb|M80458|ECOACOAC E. coli biotin carboxylase and biotin 352 2e-95
gb|U51439|REU51439 Rhizobium etli pyruvate carboxylase 351 5e-95
emb|Y13917|BSY13917 Bacillus subtilis ppsE, yngL, yngK 348 3e-94
emb|Z99113|BSUB0010 Bacillus subtilis complete genome 348 3e-94
gb|AE001274.1|AE001274 Leishmania major chromosome 1 347 6e-94
gb|AF042099|AF042099 Sulfolobus metallicus putative 346 1e-93
emb|Z81052.1|CED2023 Caenorhabditis elegans cosmid D2023 162 3e-92
emb|Z79700|MTCY10D7 Mycobacterium tuberculosis H37Rv 341 4e-92
emb|Z99111|BSUB0008 Bacillus subtilis complete genome 340 1e-91
gb|U12536|ATU12536 Arabidopsis thaliana 3-methylcrotonyl 338 4e-91
emb|Y11106|PPPYC1 P. pastoris PYC1 gene 338 4e-91
gb|AE001529|AE001529 Helicobacter pylori, strain J99 334 5e-90
gb|AE000553.1|AE000553 Helicobacter pylori 26695 333 7e-90
emb|Y09548|CGPYC Corynebacterium glutamicum pyc gene 333 1e-89
gb|AF038548|AF038548 Corynebacterium glutamicum pyruvate 333 1e-89
ref|NM_000282.1|PCCA| Homo sapiens Propionyl Coenzyme 333 1e-89
gb|M22631|RATPCOA Rat alpha-propionyl CoA carboxylase 332 2e-89
gb|U08469|GMU08469 Glycine max 3-methylcrotonyl CoA 328 3e-88
emb|Z83018|MTCY349 Mycobacterium tuberculosis H37Rv 318 4e-85
emb|AJ243652.1|PFL243652 Pseudomonas fluorescens uahA gene 316
1e-84 emb|Z36077|SCYBR208C S. cerevisiae chromosome II 312 2e-83
gb|M64926|YSCUAMD Yeast urea amidolyase (DUR1.2) gene 311 5e-83
emb|Z97025|BSZ97025 Bacillus subtilis nprE, yla[A, B, C, D, E, F
300 1e-79 emb|Z81074.1|CEF32B6 Caenorhabditis elegans cosmid F32B6
131 7e-78 gb|U00024|MTU00024 Mycobacterium tuberculosis cosmid tbc2
284 7e-75 gb|AD000009|MSGY2 Mycobacterium tuberculosis sequence 284
7e-75 gb|U34393|GMU34393 Glycine max acetyl CoA carboxylase 259
2e-67 gb|U49829|CELF27D9 Caenorhabditis elegans cosmid F27D9 186
4e-59 emb|AJ010111.1|BCE010111 Bacillus cereus pycA, ctaA, ctaB 208
5e-52 gb|U19183|ZMU19183 Zea mays acetyl-coenzyme A carboxylase 208
5e-52 gb|U10187|TAU10187 Triticum aestivum Tam 107 206 2e-51
gb|AF029895|AF029895 Triticum aestivum acetyl-coenzyme A 205 5e-51
gb|J03808|RATACACA Rat acetyl-coenzyme A carboxylase mRNA 204 8e-51
emb|X80045|OAACOAC O. aries mRNA for acetyl CoA carboxylase 203
1e-50 emb|X68968|HSACOAC H. sapiens mRNA for acetyl CoA 203 2e-50
emb|AJ132890.1|BTA132890 Bos taurus mRNA for acetyl 202 2e-50
gb|J03541|CHKCOACA Chicken acetyl CoA carboxylase mRNA 202 3e-50
dbj|D34630|ATHACCRNA Arabidopsis thaliana mRNA 199 2e-49
gb|L25042|ALFACCASE Medicago sativa acetyl CoA carboxylase 198
5e-49 emb|Z71631|SCYNR016C S. cerevisiae chromosome XIV 193 2e-47
gb|M92156|YSCFAS3A Saccharomyces cerevisiae acetyl CoA 193 2e-47
emb|Z49809|SC8261X S. cerevisiae chromosome XIII cosmid 8261 192
3e-47 emb|Z22558|SCHFA1GN S. cerevisiae HFA1 gene 192 3e-47
dbj|D78165|D78165 Saccharomyces cerevisiae DNA 192 3e-47
emb|Z46886|UMACCGEN U. maydis ACC gene for acetyl coa 190 1e-46
ref|NM_001093.1|ACACB| Homo sapiens acetyl Coenzyme A 181 5e-44
Propionyl CoA carboxylase (accA1) gb|AF113603.1|AF113603 S.
coelicolor putative 1101 0.0 (query sequence)
gb|AF113604.1|AF113604 Streptomyces coelicolor putative 1090 0.0
gb|AF126429.1|AF126429 Streptomyces venezuelae JadJ (jadJ) 967 0.0
emb|Z92771|MTCY71 Mycobacterium tuberculosis H37Rv 758 0.0
emb|X92557|SEPCCBBCP S. erythraea pccB, bcpA2, and orfX genes 753
0.0 emb|X92556|SEHGTABCP S. erythraea hgtA, bcpA1, and orf122 753
0.0 gb|U00012|U00012 Mycobacterium leprae cosmid B1308 745 0.0
emb|X63470|MLBCCPG M. leprae gene for biotin carboxyl 742 0.0
gb|U35023|CGU35023 Corynebacterium glutamicum thiosulfate 694 0.0
gb|U24659|SVU24659 Streptomyces venezuelae glucose 596 e-169
gb|AE000742|AE000742 Aquifex aeolicus section 74 417 e-115
gb|U67563|U67563 Methanococcus jannaschii section 105 413 e-114
gb|L36530|MQSPYRCARB Aedes aegypti pyruvate carboxylase 404 e-111
gb|AF132152.1|AF132152 Drosophila melanogaster clone 400 e-110
gb|L09192|MUSMPYR Mus musculus pyruvate carboxylase 397 e-109
gb|U36585|RNU36585 Rattus norvegicus pyruvate carboxylase 395 e-108
gb|U32314|RNU32314 Rattus norvegicus pyruvate carboxylase 395 e-108
gb|L14862|ANAACCC Anabaena sp. (PCC 7120) 49.1 kDa biotin 394 e-108
gb|U04641|HSU04641 Human pyruvate carboxylase (PC) mRNA 391 e-107
gb|U59234|SPU59234 Synechococcus PCC7942 biotin carboxylase 391
e-107 ref|NM_000920.1|PC| Homo sapiens pyruvate carboxylase (PC)
390 e-107 gb|AE001090|AE001090 Archaeoglobus fulgidus section 17
389 e-106 gb|AE000942|AE000942 Methanobacterium thermoautotrophicum
386 e-105 gb|S72370|S72370 pyruvate carboxylase human, kidney 384
e-105 dbj|D84432|BACJH642 Bacillus subtilis DNA, 283 Kb region 383
e-105 emb|Z99116|BSUB0013 Bacillus subtilis complete genome 383
e-105 dbj|D64001|SYCCPNC Synechocystis sp. PCC6803 383 e-104
gb|U35647|SCU35647 Saccharomyces cerevisiae pyruvate 382 e-104
emb|Z36087|SCYBR218C S. cerevisiae chromosome II 382 e-104
emb|Z72584|SCYGL062W S. cerevisiae chromosome VII 381 e-104
gb|J03889|YSCPCB Yeast (S. cerevisiae) pyruvate carboxylase 381
e-104 gb|L14612|PSEACCBC Pseudomonas aeruginosa biotin carboxyl 381
e-104 emb|X59890|SCPYC2G S. cerivisiae PYC2 gene for pyruvate 381
e-104 gb|U32778|U32778 Haemophilus influenzae Rd section 93 380
e-104 gb|U90879|ATU90879 Arabidopsis thaliana biotin carboxylase
377 e-103 gb|AE000749|AE000749 Aquifex aeolicus section 81 of 109
377 e-103 gb|AE001286|AE001286 Chlamydia trachomatis section 13 375
e-102 gb|AE001604|AE001604 Chlamydia pneumoniae section 20 374
e-102 gb|AF007100|AF007100 Glycine max biotin carboxylase 372 e-101
emb|Z95556|MTCY07A7 Mycobacterium tuberculosis H37Rv 369 e-100
emb|Z19549|MTBCARBCP M. tuberculosis gene for biotin 369 e-100
gb|AF068249|AF068249 Glycine max biotin carboxylase 369 e-100
gb|L38260|TOBBCSO Nicotiana tabacum acetyl CoA 367 e-100
gb|AF097728|AF097728 Aspergillus terreus pyruvate 366 1e-99
gb|AE000744|AE000744 Aquifex aeolicus section 76 of 109 364 4e-99
dbj|D83706|D83706 Bacillus stearothermophilus DNA 363 7e-99
gb|U36245|BSU36245 Bacillus subtilis biotin carboxyl 363 7e-99
emb|AL109846.1|SPBC17G9 S. pombe chromosome II 362 2e-98
emb|AJ235272|RPXX03 Rickettsia prowazekii strain Madrid E 361 3e-98
dbj|D78170|D78170 Yeast DNA for pyruvate carboxylase 359 2e-97
gb|M80458|ECOACOAC E. coli biotin carboxylase and biotin 358 3e-97
gb|M79446|ECOFABG Escherichia coli biotin carboxylase gene 358
3e-97 gb|M83198|ECOFABEGF Escherichia coli biotin carboxyl 358
3e-97 gb|AE000404|AE000404 Escherichia coli K-12 MG1655 358 3e-97
gb|U18997.1|ECOUW67 Escherichia coli K-12 chromosomal 358 3e-97
gb|U51439|REU51439 Rhizobium etli pyruvate carboxylase 355 3e-96
emb|Y13917|BSY13917 Bacillus subtilis ppsE, yngL, yngK, 354 4e-96
emb|Z99113|BSUB0010 Bacillus subtilis complete genome 354 4e-96
gb|AE001274.1|AE001274 Leishmania major chromosome 1 351 3e-95
gb|AF042099|AF042099 Sulfolobus metallicus putative 350 9e-95
emb|Z79700|MTCY10D7 Mycobacterium tuberculosis H37Rv 347 6e-94
emb|Z81052.1|CED2023 Caenorhabditis elegans cosmid D2023 168 1e-93
emb|Y11106|PPPYC1 P. pastoris PYC1 gene 345 2e-93
emb|Z99111|BSUB0008 Bacillus subtilis complete genome 343 8e-93
ref|NM_000282.1|PCCA| Homo sapiens Propionyl Coenzyme 340 6e-92
gb|M22631|RATPCOA Rat alpha-propionyl CoA carboxylase 340 1e-91
gb|U12536|ATU12536 Arabidopsis thaliana 3-methylcrotonyl 339 2e-9
emb|Y09548|CGPYC Corynebacterium glutamicum pyc gene 338 4e-91
gb|AF038548|AF038548 Corynebacterium glutamicum pyruvate 338 4e-91
gb|AE001529|AE001529 Helicobacter pylori, strain J99 337 8e-91
gb|AE000553.1|AE000553 Helicobacter pylori 26695 336 1e-90
gb|U08469|GMU08469 Glycine max 3-methylcrotonyl CoA 329 2e-88
emb|AJ243652.1|PFL243652 Pseudomonas fluorescens uahA gene 323
1e-86 emb|Z83018|MTCY349 Mycobacterium tuberculosis H37Rv 321 3e-86
emb|Z36077|SCYBR208C S. cerevisiae chromosome II 314 5e-84
gb|M64926|YSCUAMD Yeast urea amidolyase (DUR1.2) gene 312 2e-83
emb|Z97025|BSZ97025 Bacillus subtilis nprE, yla[A, B, C, D, E, 303
1e-80 emb|Z81074.1|CEF32B6 Caenorhabditis elegans cosmid F32B6 130
1e-78 gb|U00024|MTU00024 Mycobacterium tuberculosis cosmid tbc2 287
6e-76 gb|AD000009|MSGY2 Mycobacterium tuberculosis sequence 287
6e-76 gb|U34393|GMU34393 Glycine max acetyl CoA carboxylase 262
3e-68 gb|U49829|CELF27D9 Caenorhabditis elegans cosmid F27D9 190
2e-61 gb|U10187|TAU10187 Triticum aestivum Tam 107 213 2e-53
gb|U19183|ZMU19183 Zea mays acetyl-coenzyme A carboxylase 212 3e-53
emb|AJ010111.1|BCE010111 Bacillus cereus pycA, ctaA, ctaB 212 4e-53
gb|AF029895|AF029895 Triticum aestivum acetyl-coenzyme 209 2e-52
gb|J03808|RATACACA Rat acetyl-coenzyme A carboxylase 205 4e-51
emb|X80045|OAACOAC O. aries mRNA for acetyl CoA 205 5e-51
emb|X68968|HSACOAC H. sapiens mRNA for acetyl CoA 204 8e-51
dbj|D34630|ATHACCRNA Arabidopsis thaliana mRNA 203 1e-50
emb|AJ132890.1|BTA132890 Bos taurus mRNA for acetyl CoA 203 1e-50
gb|J03541|CHKCOACA Chicken acetyl CoA carboxylase mRNA 203 1e-50
gb|L25042|ALFACCASE Medicago sativa acetyl CoA carboxylase 202
2e-50 emb|Z71631|SCYNR016C S. cerevisiae chromosome XIV 196 1e-48
gb|M92156|YSCFAS3A Saccharomyces cerevisiae acetyl CoA 196 1e-48
emb|Z49809|SC8261X S. cerevisiae chromosome XIII cosmid 8261 195
4e-48 emb|Z22558|SCHFA1GN S. cerevisiae HFA1 gene 195 4e-48
dbj|D78165|D78165 Saccharomyces cerevisiae DNA 195 4e-48
emb|Z46886|UMACCGEN U. maydis ACC gene for acetyl coa 188 5e-46
gb|L20784|CCXACOAC Cyclotella cryptica acetyl CoA 182 2e-44
[0059] Those of skill in the art will recognize that, due to the
degenerate nature of the genetic code, a variety of DNA compounds
differing in their nucleotide sequences can be used to encode a
given amino acid sequence of the invention. The native DNA sequence
encoding the biosynthetic enzymes in the tables above are
referenced herein merely to illustrate a preferred embodiment of
the invention, and the invention includes DNA compounds of any
sequence that encode the amino acid sequences of the polypeptides
and proteins of the enzymes utilized in the methods of the
invention. In similar fashion, a polypeptide can typically tolerate
one or more amino acid substitutions, deletions, and insertions in
its amino acid sequence without loss or significant loss of a
desired activity. The present invention includes such polypeptides
with alternate amino acid sequences, and the amino acid sequences
encoded by the DNA sequences shown herein merely illustrate
preferred embodiments of the invention.
[0060] Thus, in an especially preferred embodiment, the present
invention provides DNA molecules in the form of recombinant DNA
expression vectors or plasmids, as described in more detail below,
that encode one or more precursor biosynthetic enzymes. Generally,
such vectors can either replicate in the cytoplasm of the host cell
or integrate into the chromosomal DNA of the host cell. In either
case, the vector can be a stable vector (i.e., the vector remains
present over many cell divisions, even if only with selective
pressure) or a transient vector (i.e., the vector is gradually lost
by host cells with increasing numbers of cell divisions). The
invention provides DNA molecules in isolated (i.e., not pure, but
existing in a preparation in an abundance and/or concentration not
found in nature) and purified (i.e., substantially free of
contaminating materials or substantially free of materials with
which the corresponding DNA would be found in nature) form.
[0061] In one important embodiment, the invention provides methods
for the heterologous expression of one or more of the biosynthetic
genes involved in S-methylmalonyl CoA biosynthesis and recombinant
DNA expression vectors useful in the method. Thus, included within
the scope of the invention are recombinant expression vectors that
include such nucleic acids. The term expression vector refers to a
nucleic acid that can be introduced into a host cell or cell-free
transcription and translation system. An expression vector can be
maintained permanently or transiently in a cell, whether as part of
the chromosomal or other DNA in the cell or in any cellular
compartment, such as a replicating vector in the cytoplasm. An
expression vector also comprises a promoter that drives expression
of an RNA, which typically is translated into a polypeptide in the
cell or cell extract. For efficient translation of RNA into
protein, the expression vector also typically contains a
ribosome-binding site sequence positioned upstream of the start
codon of the coding sequence of the gene to be expressed. Other
elements, such as enhancers, secretion signal sequences,
transcription termination sequences, and one or more marker genes
by which host cells containing the vector can be identified and/or
selected, may also be present in an expression vector. Selectable
markers, i.e., genes that confer antibiotic resistance or
sensitivity, are preferred and confer a selectable phenotype on
transformed cells when the cells are grown in an appropriate
selective medium.
[0062] The various components of an expression vector can vary
widely, depending on the intended use of the vector and the host
cell(s) in which the vector is intended to replicate or drive
expression. Expression vector components suitable for the
expression of genes and maintenance of vectors in E. coli, yeast,
Streptomyces, and other commonly used cells are widely known and
commercially available. For example, suitable promoters for
inclusion in the expression vectors of the invention include those
that function in eucaryotic or procaryotic host cells. Promoters
can comprise regulatory sequences that allow for regulation of
expression relative to the growth of the host cell or that cause
the expression of a gene to be turned on or off in response to a
chemical or physical stimulus. For E. coli and certain other
bacterial host cells, promoters derived from genes for biosynthetic
enzymes, antibiotic-resistance conferring enzymes, and phage
proteins can be used and include, for example, the galactose,
lactose (lac), maltose, tryptophan (trp), beta-lactamase (bla),
bacteriophage lambda PL, and T5 promoters. In addition, synthetic
promoters, such as the tac promoter (U.S. Pat. No. 4,551,433), can
also be used. For E. coli expression vectors, it is useful to
include an E. coli origin of replication, such as from pUC, p1P,
p1I, and pBR.
[0063] Thus, recombinant expression vectors contain at least one
expression system, which, in turn, is composed of at least a
portion of PKS and/or other biosynthetic gene coding sequences
operably linked to a promoter and optionally termination sequences
that operate to effect expression of the coding sequence in
compatible host cells. The host cells are modified by
transformation with the recombinant DNA expression vectors of the
invention to contain the expression system sequences either as
extrachromosomal elements or integrated into the chromosome. The
resulting host cells of the invention are useful in methods to
produce PKS enzymes as well as polyketides and antibiotics and
other useful compounds derived therefrom.
[0064] Preferred host cells for purposes of selecting vector
components for expression vectors of the present invention include
fungal host cells such as yeast and procaryotic host cells such as
E. coli, but mammalian host cells can also be used. In hosts such
as yeasts, plants, or mammalian cells that ordinarily do not
produce polyketides, it may be necessary to provide, also typically
by recombinant means, suitable holo-ACP synthases to convert the
recombinantly produced PKS to functionality. Provision of such
enzymes is described, for example, in PCT publication Nos. WO
97/13845 and 98/27203, each of which is incorporated herein by
reference.
[0065] The recombinant host cells of the invention can express all
of the polyketide biosynthetic genes or only a subset of the same.
For example, if only the genes for a PKS are expressed in a host
cell that otherwise does not produce polyketide modifying enzymes
(such as hydroxylation, epoxidation, or glycosylation enzymes) that
can act on the polyketide produced, then the host cell produces
unmodified polyketides, called macrolide aglycones. Such macrolide
aglycones can be hydroxylated and glycosylated by adding them to
the fermentation of a strain such as, for example, Streptomyces
antibioticus or Saccharopolyspora erythraea, that contains the
requisite modification enzymes.
[0066] There are a wide variety of diverse organisms that can
modify macrolide aglycones to provide compounds with, or that can
be readily modified to have, useful activities. For example,
Saccharopolyspora eythraea can convert 6-dEB to a variety of useful
compounds. The erythronolide 6-dEB is converted by the eryF gene
product to erythronolide B, which is, in turn, glycosylated by the
eryB gene product to obtain 3-O-mycarosylerythronolide B, which
contains L-mycarose at C-3. The enzyme eryC gene product then
converts this compound to erythromycin D by glycosylation with
D-desosamine at C-5. Erythromycin D, therefore, differs from 6-dEB
through glycosylation and by the addition of a hydroxyl group at
C-6. Erythromycin D can be converted to erythromycin B in a
reaction catalyzed by the eryG gene product by methylating the
L-mycarose residue at C-3. Erythromycin D is converted to
erythromycin C by the addition of a hydroxyl group at C-12 in a
reaction catalyzed by the eryK gene product. Erythromycin A is
obtained from erythromycin C by methylation of the mycarose residue
in a reaction catalyzed by the eryG gene product. The unmodified
polyketides provided by the present invention, such as, for
example, 6-dEB produced in E. coli, can be provided to cultures of
S. erythraea and converted to the corresponding derivatives of
erythromycins A, B, C, and D in accordance with the procedure
provided in the examples below. To ensure that only the desired
compound is produced, one can use an S. erythraea eryA mutant that
is unable to produce 6-dEB but can still carry out the desired
conversions (Weber et al., 1985, J. Bacteriol. 164(1): 425-433).
Also, one can employ other mutant strains, such as eryB, eryC,
eryG, and/or eryK mutants, or mutant strains having mutations in
multiple genes, to accumulate a preferred compound. The conversion
can also be carried out in large fermentors for commercial
production.
[0067] Moreover, there are other useful organisms that can be
employed to hydroxylate and/or glycosylate the compounds of the
invention. As described above, the organisms can be mutants unable
to produce the polyketide normally produced in that organism, the
fermentation can be carried out on plates or in large fermentors,
and the compounds produced can be chemically altered after
fermentation. Thus, Streptomyces venezuelae, which produces
picromycin, contains enzymes that can transfer a desosaminyl group
to the C-5 hydroxyl and a hydroxyl group to the C-12 position. In
addition, S. venezuelae contains a glucosylation activity that
glucosylates the 2'-hydroxyl group of the desosamine sugar. This
latter modification reduces antibiotic activity, but the glucosyl
residue is removed by enzymatic action prior to release of the
polyketide from the cell. Another organism, S. narbonensis,
contains the same modification enzymes as S. venezuelae, except the
C-12 hydroxylase. Thus, the present invention provides the
compounds produced by hydroxylation and glycosylation of the
macrolide aglycones of the invention by action of the enzymes
endogenous to S. narbonensis and S. venezuelae.
[0068] Other organisms suitable for making compounds of the
invention include Micromonospora megalomicea, Streptomyces
antibioticus, S. fradiae, and S. thermotolerans. M. megalomicea
glycosylates the C-3 hydroxyl with mycarose, the C-5 hydroxyl with
desosamine, and the C-6 hydroxyl with megosamine, and hydroxylates
the C-6 position. S. antibioticus produces oleandomycin and
contains enzymes that hydroxylate the C-6 and C-12 positions,
glycosylate the C-3 hydroxyl with oleandrose and the C-5 hydroxyl
with desosamine, and form an epoxide at C-8-C-8a. S. fradiae
contains enzymes that glycosylate the C-5 hydroxyl with mycaminose
and then the 4'-hydroxyl of mycaminose with mycarose, forming a
disaccharide. S. thermotolerans contains the same activities as S.
fradiae, as well as acylation activities. Thus, the present
invention provides the compounds produced by hydroxylation and
glycosylation of the macrolide aglycones of the invention by action
of the enzymes endogenous to M. megalomicea, S. antibioticus, S.
fradiae, and S. thermotolerans.
[0069] The present invention also provides methods and genetic
constructs for producing the glycosylated and/or hydroxylated
compounds of the invention directly in the host cell of interest.
Thus, the genes that encode polyketide modification enzymes can be
included in the host cells of the invention. Lack of adequate
resistance to a polyketide can be overcome by providing the host
cell with an MLS resistance gene (ermE and mgt/lrm, for example),
which confer resistance to several 14-membered macrolides (see
Cundliffe, 1989, Annu. Rev. Microbiol. 43:207-33; Jenkins and
Cundliffe, 1991, Gene 108:55-62; and Cundliffe, 1992, Gene,
115:75-84, each of which is incorporated herein by reference).
[0070] The recombinant host cells of the invention can be used to
produce polyketides (both macrolide aglycones and their modified
derivatives) that are naturally occurring or produced by
recombinant DNA technology. In one important embodiment, the
recombinant host cells of the invention are used to produce hybrid
PKS enzymes. For purposes of the invention, a hybrid PKS is a
recombinant PKS that comprises all or part of one or more extender
modules, loading module, and/or thioesterase/cyclase domain of a
first PKS and all or part of one or more extender modules, loading
module, and/or thioesterase/cyclase domain of a second PKS.
[0071] Those of skill in the art will recognize that all or part of
either the first or second PKS in a hybrid PKS of the invention
need not be isolated from a naturally occurring source. For
example, only a small portion of an AT domain determines its
specificity. See PCT patent application No. WO US99/15047 (PCT
publication No. WO 00/08138), and Lau et al., infra, incorporated
herein by reference. The state of the art in DNA synthesis allows
the artisan to construct de novo DNA compounds of size sufficient
to construct a useful portion of a PKS module or domain. Thus, the
desired derivative coding sequences can be synthesized using
standard solid phase synthesis methods such as those described by
Jaye et al., 1984, J. Biol. Chem. 259: 6331, and instruments for
automated synthesis are available commercially from, for example,
Applied Biosystems, Inc. For purposes of the invention, such
synthetic DNA compounds are deemed to be a portion of a PKS.
[0072] A hybrid PKS for purposes of the present invention can
result not only:
[0073] (i) from fusions of heterologous domain (where heterologous
means the domains in a module are derived from at least two
different naturally occurring modules) coding sequences to produce
a hybrid module coding sequence contained in a PKS gene whose
product is incorporated into a PKS,
but also:
[0074] (ii) from fusions of heterologous module (where heterologous
module means two modules are adjacent to one another that are not
adjacent to one another in naturally occurring PKS enzymes) coding
sequences to produce a hybrid coding sequence contained in a PKS
gene whose product is incorporated into a PKS,
[0075] (iii) from expression of one or more PKS genes from a first
PKS gene cluster with one or more PKS genes from a second PKS gene
cluster, and
[0076] (iv) from combinations of the foregoing.
Various hybrid PKSs of the invention illustrating these various
alternatives are described herein.
[0077] Recombinant methods for manipulating modular PKS genes to
make hybrid PKS enzymes are described in U.S. Pat. Nos. 5,672,491;
5,843,718; 5,830,750; and 5,712,146; and in PCT publication Nos.
98/49315 and 97/02358, each of which is incorporated herein by
reference. A number of genetic engineering strategies have been
used with DEBS to demonstrate that the structures of polyketides
can be manipulated to produce novel natural products, primarily
analogs of the erythromycins (see the patent publications
referenced supra and Hutchinson, 1998, Curr Opin Microbiol.
1:319-329, and Baltz, 1998, Trends Microbiol. 6:76-83, incorporated
herein by reference).
[0078] These techniques include: (i) deletion or insertion of
modules to control chain length, (ii) inactivation of
reduction/dehydration domains to bypass beta-carbon processing
steps, (iii) substitution of AT domains to alter starter and
extender units, (iv) addition of reduction/dehydration domains to
introduce catalytic activities, and (v) substitution of
ketoreductase KR domains to control hydroxyl stereochemistry. In
addition, engineered blocked mutants of DEBS have been used for
precursor directed biosynthesis of analogs that incorporate
synthetically derived starter units. For example, more than 100
novel polyketides were produced by engineering single and
combinatorial changes in multiple modules of DEBS. Hybrid PKS
enzymes based on DEBS with up to three catalytic domain
substitutions were constructed by cassette mutagenesis, in which
various DEBS domains were replaced with domains from the rapamycin
PKS (see Schweke et al., 1995, Proc. Nat. Acad. Sci. USA 92,
7839-7843, incorporated herein by reference) or one more of the
DEBS KR domains was deleted. Functional single domain replacements
or deletions were combined to generate DEBS enzymes with double and
triple catalytic domain substitutions (see McDaniel et al., 1999,
Proc. Nat. Acad. Sci. USA 96, 1846-1851, incorporated herein by
reference).
[0079] Methods for generating libraries of polyketides have been
greatly improved by cloning PKS genes as a set of three or more
mutually selectable plasmids, each carrying a different wild-type
or mutant PKS gene, then introducing all possible combinations of
the plasmids with wild-type, mutant, and hybrid PKS coding
sequences into the same host (see U.S. patent application Ser. No.
60/129,731, filed 16 Apr. 1999, and PCT Pub. No. WO 98/27203, each
of which is incorporated herein by reference). This method can also
incorporate the use of a KS1.degree. mutant, which by mutational
biosynthesis can produce polyketides made from diketide starter
units (see Jacobsen et al., 1997, Science 277, 367-369,
incorporated herein by reference), as well as the use of a
truncated gene that leads to 12-membered macrolides or an elongated
gene that leads to 16-membered ketolides. Moreover, by utilizing in
addition one or more vectors that encode glycosyl biosynthesis and
transfer genes, such as those of the present invention for
megosamine, desosamine, oleandrose, cladinose, and/or mycarose (in
any combination), a large collection of glycosylated polyketides
can be prepared.
[0080] The following table lists references describing illustrative
PKS genes and corresponding enzymes that can be utilized in the
construction of the recombinant hybrid PKSs and the corresponding
DNA compounds that encode them. Also presented are various
references describing tailoring enzymes and corresponding genes
that can be employed in accordance with the methods of the
invention.
Avermectin
[0081] U.S. Pat. No. 5,252,474 to Merck. [0082] MacNeil et al.,
1993, Industrial Microorganisms: Basic and Applied Molecular
Genetics, Baltz, Hegeman, & Skatrud, eds. (ASM), pp. 245-256, A
Comparison of the Genes Encoding the Polyketide Synthases for
Avermectin, Erythromycin, and Nemadectin.
[0083] MacNeil et al., 1992, Gene 115: 119-125, Complex
Organization of the Streptomyces avermitilis genes encoding the
avermectin polyketide synthase.
Candicidin (FR008)
[0084] Hu et al., 1994, Mol. Microbiol. 14: 163-172.
Epothilone
[0084] [0085] PCT Pat. Pub. No. WO 00/031247 to Kosan.
Erythromycin
[0085] [0086] PCT Pub. No. 93/13663 to Abbott. [0087] U.S. Pat. No.
5,824,513 to Abbott. [0088] Donadio et al., 1991, Science
252:675-9. [0089] Cortes et al., 8 Nov. 1990, Nature 348:176-8, An
unusually large multifunctional polypeptide in the erythromycin
producing polyketide synthase of Saccharopolyspora erythraea.
Glycosylation Enzymes
[0089] [0090] PCT Pat. App. Pub. No. 97/23630 to Abbott.
FK-506
[0090] [0091] Motamedi et al., 1998, The biosynthetic gene cluster
for the macrolactone ring of the immunosuppressant FK506, Eur. J.
biochem. 256: 528-534. [0092] Motamedi et al., 1997, Structural
organization of a multifunctional polyketide synthase involved in
the biosynthesis of the macrolide immunosuppressant FK506, Eur. J.
Biochem. 244: 74-80.
Methyltransferase
[0092] [0093] U.S. Pat. No. 5,264,355, issued 23 Nov. 1993,
Methylating enzyme from Streptomyces MA6858. 31-O-desmethyl-FK506
methyltransferase. [0094] Motamedi et al., 1996, Characterization
of methyltransferase and hydroxylase genes involved in the
biosynthesis of the immunosuppressants FK506 and FK520, J.
Bacteriol. 178: 5243-5248.
FK-520
[0094] [0095] PCT Pat. Pub. No. WO 00/020601 to Kosan. See also
Nielsen et al., 1991, Biochem. 30:5789-96 (enzymology of pipecolate
incorporation).
Lovastatin
[0095] [0096] U.S. Pat. No. 5,744,350 to Merck.
Narbomycin (and Picromycin)
[0096] [0097] PCT Pat. Pub. No. WO 99/61599 to Kosan.
Nemadectin
[0097] [0098] MacNeil et al., 1993, supra.
Niddamycin
[0099] Kakavas et al., 1997, Identification and characterization of
the niddamycin polyketide synthase genes from Streptomyces
caelestis, J. Bacteriol. 179: 7515-7522.
Oleandomycin
[0100] Swan et al., 1994, Characterisation of a Streptomyces
antibioticus gene encoding a type I polyketide synthase which has
an unusual coding sequence, Mol. Gen. Genet. 242: 358-362. [0101]
PCT Pat. Pub. No. WO 00/026349 to Kosan. [0102] Olano et al., 1998,
Analysis of a Streptomyces antibioticus chromosomal region involved
in oleandomycin biosynthesis, which encodes two
glycosyltransferases responsible for glycosylation of the
macrolactone ring, Mol. Gen. Genet. 259(3): 299-308.
Platenolide
[0102] [0103] EP Pat. App. Pub. No. 791,656 to Lilly.
Rapamycin
[0103] [0104] Schwecke et al., August 1995, The biosynthetic gene
cluster for the polyketide rapamycin, Proc. Natl. Acad. Sci. USA
92:7839-7843. [0105] Aparicio et al., 1996, Organization of the
biosynthetic gene cluster for rapamycin in Streptomyces
hygroscopicus: analysis of the enzymatic domains in the modular
polyketide synthase, Gene 169: 9-16.
Rifamycin
[0106] August et al., 13 Feb. 1998, Biosynthesis of the ansamycin
antibiotic rifamycin: deductions from the molecular analysis of the
rif biosynthetic gene cluster of Amycolatopsis mediterranei S669,
Chemistry & Biology, 5(2): 69-79.
Soraphen
[0107] U.S. Pat. No. 5,716,849 to Novartis. [0108] Schupp et al.,
1995, J. Bacteriology 177: 3673-3679. A Sorangium cellulosum
(Myxobacterium) Gene Cluster for the Biosynthesis of the Macrolide
Antibiotic Soraphen A: Cloning, Characterization, and Homology to
Polyketide Synthase Genes from Actinomycetes.
Spiramycin
[0108] [0109] U.S. Pat. No. 5,098,837 to Lilly.
Activator Gene
[0109] [0110] U.S. Pat. No. 5,514,544 to Lilly.
Tylosin
[0110] [0111] EP Pub. No. 791,655 to Lilly. [0112] Kuhstoss et al.,
1996, Gene 183:231-6., Production of a novel polyketide through the
construction of a hybrid polyketide synthase. [0113] U.S. Pat. No.
5,876,991 to Lilly.
Tailoring Enzymes
[0113] [0114] Merson-Davies and Cundliffe, 1994, Mol. Microbiol.
13: 349-355. Analysis of five tylosin biosynthetic genes from the
tylBA region of the Streptomyces fradiae genome.
[0115] As the above Table illustrates, there are a wide variety of
PKS genes that serve as readily available sources of DNA and
sequence information for use in constructing the hybrid
PKS-encoding DNA compounds of the invention.
[0116] In constructing hybrid PKSs, certain general methods may be
helpful. For example, it is often beneficial to retain the
framework of the module to be altered to make the hybrid PKS. Thus,
if one desires to add DH and ER functionalities to a module, it is
often preferred to replace the KR domain of the original module
with a KR, DH, and ER domain-containing segment from another
module, instead of merely inserting DH and ER domains. One can
alter the stereochemical specificity of a module by replacement of
the KS domain with a KS domain from a module that specifies a
different stereochemistry. See Lau et al., 1999, "Dissecting the
role of acyltransferase domains of modular polyketide synthases in
the choice and stereochemical fate of extender units" Biochemistry
38(5): 1643-1651, incorporated herein by reference. One can alter
the specificity of an AT domain by changing only a small segment of
the domain. See Lau et al., supra. One can also take advantage of
known linker regions in PKS proteins to link modules from two
different PKSs to create a hybrid PKS. See Gokhale et al., 16 Apr.
1999, Dissecting and Exploiting Intermodular Communication in
Polyketide Synthases", Science 284: 482-485, incorporated herein by
reference.
[0117] The hybrid PKS-encoding DNA compounds can be and often are
hybrids of more than two PKS genes. Even where only two genes are
used, there are often two or more modules in the hybrid gene in
which all or part of the module is derived from a second (or third)
PKS gene.
[0118] The invention also provides libraries of PKS genes, PKS
proteins, and ultimately, of polyketides, that are constructed by
generating modifications in a PKS so that the protein complexes
produced have altered activities in one or more respects and thus
produce polyketides other than the natural product of the PKS.
Novel polyketides may thus be prepared, or polyketides in general
prepared more readily, using this method. By providing a large
number of different genes or gene clusters derived from a naturally
occurring PKS gene cluster, each of which has been modified in a
different way from the native cluster, an effectively combinatorial
library of polyketides can be produced as a result of the multiple
variations in these activities. As will be further described below,
the metes and bounds of this embodiment of the invention can be
described on the polyketide, protein, and the encoding nucleotide
sequence levels.
[0119] There are at least five degrees of freedom for constructing
a hybrid PKS in terms of the polyketide that will be produced.
First, the polyketide chain length is determined by the number of
extender modules in the PKS, and the present invention includes
hybrid PKSs that contain 6, as wells as fewer or more than 6,
extender modules. Second, the nature of the carbon skeleton of the
PKS is determined by the specificities of the acyl transferases
that determine the nature of the extender units at each position,
e.g., malonyl, methylmalonyl, ethylmalonyl, or other substituted
malonyl. Third, the loading module specificity also has an effect
on the resulting carbon skeleton of the polyketide. The loading
module may use a different starter unit, such as acetyl, butyryl,
and the like. As noted above, another method for varying loading
module specificity involves inactivating the KS activity in
extender module 1 (KS1) and providing alternative substrates,
called diketides, that are chemically synthesized analogs of
extender module 1 diketide products, for extender module 2. This
approach was illustrated in PCT publication Nos. 97/02358 and
99/03986, incorporated herein by reference, wherein the KS1
activity was inactivated through mutation. Fourth, the oxidation
state at various positions of the polyketide will be determined by
the dehydratase and reductase portions of the modules. This will
determine the presence and location of ketone and alcohol moieties
and C--C double bonds or C--C single bonds in the polyketide.
Finally, the stereochemistry of the resulting polyketide is a
function of three aspects of the synthase. The first aspect is
related to the AT/KS specificity associated with substituted
malonyls as extender units, which affects stereochemistry only when
the reductive cycle is missing or when it contains only a
ketoreductase, as the dehydratase would abolish chirality. Second,
the specificity of the ketoreductase may determine the chirality of
any beta-OH. Finally, the enoylreductase specificity for
substituted malonyls as extender units may influence the
stereochemistry when there is a complete KR/DH/ER available.
[0120] Thus, the modular PKS systems generally permit a wide range
of polyketides to be synthesized. As compared to the aromatic PKS
systems, the modular PKS systems accept a wider range of starter
units, including aliphatic monomers (acetyl, propionyl, butyryl,
isovaleryl, etc.), aromatics (aminohydroxybenzoyl), alicyclics
(cyclohexanoyl), and heterocyclics (thiazolyl). Certain modular
PKSs have relaxed specificity for their starter units (Kao et al.,
1994, Science, supra). Modular PKSs also exhibit considerable
variety with regard to the choice of extender units in each
condensation cycle. The degree of beta-ketoreduction following a
condensation reaction can be altered by genetic manipulation
(Donadio et al., 1991, Science, supra; Donadio et al., 1993, Proc.
Natl. Acad. Sci. USA 90: 7119-7123). Likewise, the size of the
polyketide product can be varied by designing mutants with the
appropriate number of modules (Kao et al., 1994, J. Am. Chem. Soc.
116:11612-11613). Lastly, modular PKS enzymes are particularly well
known for generating an impressive range of asymmetric centers in
their products in a highly controlled manner. The polyketides,
antibiotics, and other compounds produced by the methods of the
invention are typically single stereoisomeric forms. Although the
compounds of the invention can occur as mixtures of stereoisomers,
it may be beneficial in some instances to generate individual
stereoisomers. Thus, the combinatorial potential within modular PKS
pathways based on any naturally occurring modular PKS scaffold is
virtually unlimited.
[0121] While hybrid PKSs are most often produced by "mixing and
matching" portions of PKS coding sequences, mutations in DNA
encoding a PKS can also be used to introduce, alter, or delete an
activity in the encoded polypeptide. Mutations can be made to the
native sequences using conventional techniques. The substrates for
mutation can be an entire cluster of genes or only one or two of
them; the substrate for mutation may also be portions of one or
more of these genes. Techniques for mutation include preparing
synthetic oligonucleotides including the mutations and inserting
the mutated sequence into the gene encoding a PKS subunit using
restriction endonuclease digestion. See, e.g., Kunkel, 1985, Proc.
Natl. Acad. Sci. USA 82: 448; Geisselsoder et al., 1987,
BioTechniques 5:786. Alternatively, the mutations can be effected
using a mismatched primer (generally 10-20 nucleotides in length)
that hybridizes to the native nucleotide sequence, at a temperature
below the melting temperature of the mismatched duplex. The primer
can be made specific by keeping primer length and base composition
within relatively narrow limits and by keeping the mutant base
centrally located. See Zoller and Smith, 1983, Methods Enzymol.
100:468. Primer extension is effected using DNA polymerase, the
product cloned, and clones containing the mutated DNA, derived by
segregation of the primer extended strand, selected. Identification
can be accomplished using the mutant primer as a hybridization
probe. The technique is also applicable for generating multiple
point mutations. See, e.g., Dalbie-McFarland et al., 1982, Proc.
Natl. Acad. Sci. USA 79: 6409. PCR mutagenesis can also be used to
effect the desired mutations.
[0122] Random mutagenesis of selected portions of the nucleotide
sequences encoding enzymatic activities can also be accomplished by
several different techniques known in the art, e.g., by inserting
an oligonucleotide linker randomly into a plasmid, by irradiation
with X-rays or ultraviolet light, by incorporating incorrect
nucleotides during in vitro DNA synthesis, by error-prone PCR
mutagenesis, by preparing synthetic mutants, or by damaging plasmid
DNA in vitro with chemicals. Chemical mutagens include, for
example, sodium bisulfite, nitrous acid, nitrosoguanidine,
hydroxylamine, agents which damage or remove bases thereby
preventing normal base-pairing such as hydrazine or formic acid,
analogues of nucleotide precursors such as 5-bromouracil,
2-aminopurine, or acridine intercalating agents such as proflavine,
acriflavine, quinacrine, and the like. Generally, plasmid DNA or
DNA fragments are treated with chemical mutagens, transformed into
E. coli and propagated as a pool or library of mutant plasmids.
[0123] In constructing a hybrid PKS of the invention, regions
encoding enzymatic activity, i.e., regions encoding corresponding
activities from different PKS synthases or from different locations
in the same PKS, can be recovered, for example, using PCR
techniques with appropriate primers. By "corresponding" activity
encoding regions is meant those regions encoding the same general
type of activity. For example, a KR activity encoded at one
location of a gene cluster "corresponds" to a KR encoding activity
in another location in the gene cluster or in a different gene
cluster. Similarly, a complete reductase cycle could be considered
corresponding. For example, KR/DH/ER can correspond to a KR
alone.
[0124] If replacement of a particular target region in a host PKS
is to be made, this replacement can be conducted in vitro using
suitable restriction enzymes. The replacement can also be effected
in vivo using recombinant techniques involving homologous sequences
framing the replacement gene in a donor plasmid and a receptor
region in a recipient plasmid. Such systems, advantageously
involving plasmids of differing temperature sensitivities are
described, for example, in PCT publication No. WO 96/40968,
incorporated herein by reference. The vectors used to perform the
various operations to replace the enzymatic activity in the host
PKS genes or to support mutations in these regions of the host PKS
genes can be chosen to contain control sequences operably linked to
the resulting coding sequences in a manner such that expression of
the coding sequences can be effected in an appropriate host.
[0125] However, simple cloning vectors may be used as well. If the
cloning vectors employed to obtain PKS genes encoding derived PKS
lack control sequences for expression operably linked to the
encoding nucleotide sequences, the nucleotide sequences are
inserted into appropriate expression vectors. This need not be done
individually, but a pool of isolated encoding nucleotide sequences
can be inserted into expression vectors, the resulting vectors
transformed or transfected into host cells, and the resulting cells
plated out into individual colonies. The invention provides a
variety of recombinant DNA compounds in which the various coding
sequences for the domains and modules of the PKS are flanked by
non-naturally occurring restriction enzyme recognition sites.
[0126] The various PKS nucleotide sequences can be cloned into one
or more recombinant vectors as individual cassettes, with separate
control elements, or under the control of, e.g., a single promoter.
The PKS subunit encoding regions can include flanking restriction
sites to allow for the easy deletion and insertion of other PKS
subunit encoding sequences so that hybrid PKSs can be generated.
The design of such unique restriction sites is known to those of
skill in the art and can be accomplished using the techniques
described above, such as site-directed mutagenesis and PCR.
[0127] The expression vectors containing nucleotide sequences
encoding a variety of PKS enzymes for the production of different
polyketides are then transformed into the appropriate host cells to
construct the library. In one straightforward approach, a mixture
of such vectors is transformed into the selected host cells and the
resulting cells plated into individual colonies and selected to
identify successful transformants. Each individual colony has the
ability to produce a particular PKS synthase and ultimately a
particular polyketide. Typically, there will be duplications in
some, most, or all of the colonies; the subset of the transformed
colonies that contains a different PKS in each member colony can be
considered the library. Alternatively, the expression vectors can
be used individually to transform hosts, which transformed hosts
are then assembled into a library. A variety of strategies are
available to obtain a multiplicity of colonies each containing a
PKS gene cluster derived from the naturally occurring host gene
cluster so that each colony in the library produces a different PKS
and ultimately a different polyketide. The number of different
polyketides that are produced by the library is typically at least
four, more typically at least ten, and preferably at least 20, and
more preferably at least 50, reflecting similar numbers of
different altered PKS gene clusters and PKS gene products. The
number of members in the library is arbitrarily chosen; however,
the degrees of freedom outlined above with respect to the variation
of starter, extender units, stereochemistry, oxidation state, and
chain length enables the production of quite large libraries.
[0128] Methods for introducing the recombinant vectors of the
invention into suitable hosts are known to those of skill in the
art and typically include the use of CaCl.sub.2 or agents such as
other divalent cations, lipofection, DMSO, protoplast
transformation, infection, transfection, and electroporation. The
polyketide producing colonies can be identified and isolated using
known techniques and the produced polyketides further
characterized. The polyketides produced by these colonies can be
used collectively in a panel to represent a library or may be
assessed individually for activity.
[0129] The libraries of the invention can thus be considered at
four levels: (1) a multiplicity of colonies each with a different
PKS encoding sequence; (2) the proteins produced from the coding
sequences; (3) the polyketides produced from the proteins assembled
into a function PKS; and (4) antibiotics or compounds with other
desired activities derived from the polyketides.
[0130] Colonies in the library are induced to produce the relevant
synthases and thus to produce the relevant polyketides to obtain a
library of polyketides. The polyketides secreted into the media can
be screened for binding to desired targets, such as receptors,
signaling proteins, and the like. The supernatants per se can be
used for screening, or partial or complete purification of the
polyketides can first be effected. Typically, such screening
methods involve detecting the binding of each member of the library
to receptor or other target ligand. Binding can be detected either
directly or through a competition assay. Means to screen such
libraries for binding are well known in the art. Alternatively,
individual polyketide members of the library can be tested against
a desired target. In this event, screens wherein the biological
response of the target is measured can more readily be included.
Antibiotic activity can be verified using typical screening assays
such as those set forth in Lehrer et al., 1991, J. Immunol. Meth.
137: 167-173, incorporated herein by reference, and in the Examples
below.
[0131] The invention provides methods for the preparation of a
large number of polyketides. These polyketides are useful
intermediates in formation of compounds with antibiotic or other
activity through hydroxylation, epoxidation, and glycosylation
reactions as described above. In general, the polyketide products
of the PKS must be further modified, typically by hydroxylation and
glycosylation, to exhibit antibiotic activity. Hydroxylation
results in the novel polyketides of the invention that contain
hydroxyl groups at C-6, which can be accomplished using the
hydroxylase encoded by the eryF gene, and/or C-12, which can be
accomplished using the hydroxylase encoded by the picK or eryK
gene. Also, the oleP gene is available in recombinant form, which
can be used to express the oleP gene product in any host cell. A
host cell, such as a Streptomyces host cell or a Saccharopolyspora
erythraea host cell, modified to express the oleP gene thus can be
used to produce polyketides comprising the C-8-C-8a epoxide present
in oleandomycin. Thus the invention provides such modified
polyketides. The presence of hydroxyl groups at these positions can
enhance the antibiotic activity of the resulting compound relative
to its unhydroxylated counterpart.
[0132] Methods for glycosylating the polyketides are generally
known in the art; the glycosylation may be effected intracellularly
by providing the appropriate glycosylation enzymes or may be
effected in vitro using chemical synthetic means as described
herein and in PCT publication No. WO 98/49315, incorporated herein
by reference. Preferably, glycosylation with desosamine, mycarose,
and/or megosamine is effected in accordance with the methods of the
invention in recombinant host cells provided by the invention. In
general, the approaches to effecting glycosylation mirror those
described above with respect to hydroxylation. The purified
enzymes, isolated from native sources or recombinantly produced may
be used in vitro. Alternatively and as noted, glycosylation may be
effected intracellularly using endogenous or recombinantly produced
intracellular glycosylases. In addition, synthetic chemical methods
may be employed.
[0133] The antibiotic modular polyketides may contain any of a
number of different sugars, although D-desosamine, or a close
analog thereof, is most common. Erythromycin, picromycin,
megalomicin, narbomycin, and methymycin contain desosamine.
Erythromycin also contains L-cladinose (3-O-methyl mycarose).
Tylosin contains mycaminose (4-hydroxy desosamine), mycarose and
6-deoxy-D-allose. 2-acetyl-1-bromodesosamine has been used as a
donor to glycosylate polyketides by Masamune et al., 1975, J. Am.
Chem. Soc. 97: 3512-3513. Other, apparently more stable donors
include glycosyl fluorides, thioglycosides, and
trichloroacetimidates; see Woodward et al., 1981, J. Am. Chem. Soc.
103: 3215; Martin et al., 1997, J. Am. Chem. Soc. 119: 3193;
Toshima et al., 1995, J. Am. Chem. Soc. 117: 3717; Matsumoto et
al., 1988, Tetrahedron Lett. 29: 3575. Glycosylation can also be
effected using the polyketide aglycones as starting materials and
using Saccharopolyspora eythraea or Streptomyces venezuelae or
other host cell to make the conversion, preferably using mutants
unable to synthesize macrolides, as discussed above.
[0134] Thus, a wide variety of polyketides can be produced by the
hybrid PKS enzymes of the invention. These polyketides are useful
as antibiotics and as intermediates in the synthesis of other
useful compounds. In one important aspect, the invention provides
methods for making antibiotic compounds related in structure to
erythromycin, a potent antibiotic compound. The invention also
provides novel ketolide compounds, polyketide compounds with potent
antibiotic activity of significant interest due to activity against
antibiotic resistant strains of bacteria. See Griesgraber et al.,
1996, J. Antibiot. 49: 465-477, incorporated herein by reference.
Most if not all of the ketolides prepared to date are synthesized
using erythromycin A, a derivative of 6-dEB, as an intermediate.
See Griesgraber et al., supra; Agouridas et al., 1998, J. Med.
Chem. 41: 4080-4100, U.S. Pat. Nos. 5,770,579; 5,760,233;
5,750,510; 5,747,467; 5,747,466; 5,656,607; 5,635,485; 5,614,614;
5,556,118; 5,543,400; 5,527,780; 5,444,051; 5,439,890; 5,439,889;
and PCT publication Nos. WO 98/09978 and 98/28316, each of which is
incorporated herein by reference.
[0135] As noted above, the hybrid PKS genes of the invention can be
expressed in a host cell that contains the desosamine, megosamine,
and/or mycarose biosynthetic genes and corresponding transferase
genes as well as the required hydroxylase gene(s), which may be
either picK, megK, or eryK (for the C-12 position) and/or megF or
eryF (for the C-6 position). The resulting compounds have
antibiotic activity but can be further modified, as described in
the patent publications referenced above, to yield a desired
compound with improved or otherwise desired properties.
Alternatively, the aglycone compounds can be produced in the
recombinant host cell, and the desired glycosylation and
hydroxylation steps carried out in vitro or in vivo, in the latter
case by supplying the converting cell with the aglycone, as
described above.
[0136] As described above, there are a wide variety of diverse
organisms that can modify compounds such as those described herein
to provide compounds with or that can be readily modified to have
useful activities. For example, Saccharopolyspora erythraea can
convert 6-dEB to a variety of useful compounds. The compounds
provided by the present invention can be provided to cultures of
Saccharopolyspora erythraea and converted to the corresponding
derivatives of erythromycins A, B, C, and D in accordance with the
procedure provided in the Examples, below. To ensure that only the
desired compound is produced, one can use an S. erythraea eryA
mutant that is unable to produce 6-dEB but can still carry out the
desired conversions (Weber et al., 1985, J. Bacteriol. 164(1):
425-433). Also, one can employ other mutant strains, such as eryB,
eryC, eryG, and/or eryK mutants, or mutant strains having mutations
in multiple genes, to accumulate a preferred compound. The
conversion can also be carried out in large fermentors for
commercial production. Each of the erythromycins A, B, C, and D has
antibiotic activity, although erythromycin A has the highest
antibiotic activity. Moreover, each of these compounds can form,
under treatment with mild acid, a C-6 to C-9 hemiketal with
motilide activity. For formation of hemiketals with motilide
activity, erythromycins B, C, and D, are preferred, as the presence
of a C-12 hydroxyl allows the formation of an inactive compound
that has a hemiketal formed between C-9 and C-12.
[0137] Thus, the present invention provides the compounds produced
by hydroxylation and glycosylation of the compounds of the
invention by action of the enzymes endogenous to Saccharopolyspora
erythraea and mutant strains of S. erythraea. Such compounds are
useful as antibiotics or as motilides directly or after chemical
modification. For use as antibiotics, the compounds of the
invention can be used directly without further chemical
modification. Erythromycins A, B, C, and D all have antibiotic
activity, and the corresponding compounds of the invention that
result from the compounds being modified by Saccharopolyspora
erythraea also have antibiotic activity. These compounds can be
chemically modified, however, to provide other compounds of the
invention with potent antibiotic activity. For example, alkylation
of erythromycin at the C-6 hydroxyl can be used to produce potent
antibiotics (clarithromycin is C-6-O-methyl), and other useful
modifications are described in, for example, Griesgraber et al.,
1996, J. Antibiot. 49: 465-477, Agouridas et al., 1998, J. Med.
Chem. 41: 4080-4100, U.S. Pat. Nos. 5,770,579; 5,760,233;
5,750,510; 5,747,467; 5,747,466; 5,656,607; 5,635,485; 5,614,614;
5,556,118; 5,543,400; 5,527,780; 5,444,051; 5,439,890; and
5,439,889; and PCT publication Nos. WO 98/09978 and 98/28316, each
of which is incorporated herein by reference.
[0138] For use as motilides, the compounds of the invention can be
used directly without further chemical modification. Erythromycin
and certain erythromycin analogs are potent agonists of the motilin
receptor that can be used clinically as prokinetic agents to induce
phase III of migrating motor complexes, to increase esophageal
peristalsis and LES pressure in patients with GERD, to accelerate
gastric emptying in patients with gastric paresis, and to stimulate
gall bladder contractions in patients after gallstone removal and
in diabetics with autonomic neuropathy. See Omura et al., 1987,
Macrolides with gastrointestinal motor stimulating activity, J.
Med. Chem. 30: 1941-3). The corresponding compounds of the
invention that result from the compounds of the invention being
modified by Saccharopolyspora erythraea also have motilide
activity, particularly after conversion, which can also occur in
vivo, to the C-6 to C-9 hemiketal by treatment with mild acid.
Compounds lacking the C-12 hydroxyl are especially preferred for
use as motilin agonists. These compounds can also be further
chemically modified, however, to provide other compounds of the
invention with potent motilide activity.
[0139] Moreover, and also as noted above, there are other useful
organisms that can be employed to hydroxylate and/or glycosylate
the compounds of the invention. As described above, the organisms
can be mutants unable to produce the polyketide normally produced
in that organism, the fermentation can be carried out on plates or
in large fermentors, and the compounds produced can be chemically
altered after fermentation. In addition to Saccharopolyspora
erythraea, Streptomyces venezuelae, S. narbonensis, S.
antibioticus, Micromonospora megalomicea, S. fradiae, and S.
thermotolerans can also be used. In addition to antibiotic
activity, compounds of the invention produced by treatment with M.
megalomicea enzymes can have antiparasitic activity as well. Thus,
the present invention provides the compounds produced by
hydroxylation and glycosylation by action of the enzymes endogenous
to S. erythraea, S. venezuelae, S. narbonensis, S. antibioticus, M.
megalomicea, S. fradiae, and S. thermotolerans.
[0140] The compounds of the invention can be isolated from the
fermentation broths of these cultured cells and purified by
standard procedures. The compounds can be readily formulated to
provide the pharmaceutical compositions of the invention. The
pharmaceutical compositions of the invention can be used in the
form of a pharmaceutical preparation, for example, in solid,
semisolid, or liquid form. This preparation will contain one or
more of the compounds of the invention as an active ingredient in
admixture with an organic or inorganic carrier or excipient
suitable for external, enteral, or parenteral application. The
active ingredient may be compounded, for example, with the usual
non-toxic, pharmaceutically acceptable carriers for tablets,
pellets, capsules, suppositories, solutions, emulsions,
suspensions, and any other form suitable for use.
[0141] The carriers which can be used include water, glucose,
lactose, gum acacia, gelatin, mannitol, starch paste, magnesium
trisilicate, talc, corn starch, keratin, colloidal silica, potato
starch, urea, and other carriers suitable for use in manufacturing
preparations, in solid, semi-solid, or liquified form. In addition,
auxiliary stabilizing, thickening, and coloring agents and perfumes
may be used. For example, the compounds of the invention may be
utilized with hydroxypropyl methylcellulose essentially as
described in U.S. Pat. No. 4,916,138, incorporated herein by
reference, or with a surfactant essentially as described in EPO
patent publication No. 428,169, incorporated herein by
reference.
[0142] Oral dosage forms may be prepared essentially as described
by Hondo et al., 1987, Transplantation Proceedings XIX, Supp. 6:
17-22, incorporated herein by reference. Dosage forms for external
application may be prepared essentially as described in EPO patent
publication No. 423,714, incorporated herein by reference. The
active compound is included in the pharmaceutical composition in an
amount sufficient to produce the desired effect upon the disease
process or condition.
[0143] For the treatment of conditions and diseases caused by
infection, a compound of the invention may be administered orally,
topically, parenterally, by inhalation spray, or rectally in dosage
unit formulations containing conventional non-toxic
pharmaceutically acceptable carriers, adjuvant, and vehicles. The
term parenteral, as used herein, includes subcutaneous injections,
and intravenous, intramuscular, and intrasternal injection or
infusion techniques.
[0144] Dosage levels of the compounds of the invention are of the
order from about 0.01 mg to about 50 mg per kilogram of body weight
per day, preferably from about 0.1 mg to about 10 mg per kilogram
of body weight per day. The dosage levels are useful in the
treatment of the above-indicated conditions (from about 0.7 mg to
about 3.5 mg per patient per day, assuming a 70 kg patient). In
addition, the compounds of the invention may be administered on an
intermittent basis, i.e., at semi-weekly, weekly, semi-monthly, or
monthly intervals.
[0145] The amount of active ingredient that may be combined with
the carrier materials to produce a single dosage form will vary
depending upon the host treated and the particular mode of
administration. For example, a formulation intended for oral
administration to humans may contain from 0.5 mg to 5 gm of active
agent compounded with an appropriate and convenient amount of
carrier material, which may vary from about 5 percent to about 95
percent of the total composition. Dosage unit forms will generally
contain from about 0.5 mg to about 500 mg of active ingredient. For
external administration, the compounds of the invention may be
formulated within the range of, for example, 0.00001% to 60% by
weight, preferably from 0.001% to 10% by weight, and most
preferably from about 0.005% to 0.8% by weight.
[0146] It will be understood, however, that the specific dose level
for any particular patient will depend on a variety of factors.
These factors include the activity of the specific compound
employed; the age, body weight, general health, sex, and diet of
the subject; the time and route of administration and the rate of
excretion of the drug; whether a drug combination is employed in
the treatment; and the severity of the particular disease or
condition for which therapy is sought.
[0147] A detailed description of the invention having been provided
above, the following examples are given for the purpose of
illustrating the invention and shall not be construed as being a
limitation on the scope of the invention or claims.
EXAMPLE 1
Production of Methylmalonyl-CoA in E. Coli
[0148] This example describes, in part A, the cloning and
expression of methylmalonyl-CoA mutase, and in part B, the cloning
and expression of methylmalonyl-CoA epimerase, in E. coli.
A. Cloning and Expression of Methylmalonyl-CoA Mutase
[0149] Methylmalonyl-CoA mutase was cloned from Propionibacterium
shermanii and expressed in E. coli. The holoenzyme mm-CoA mutase
was obtained by growing cells in the presence of hydroxocobalamin
and was shown to be active without addition of vitamin B12.
Methylmalonyl-CoA was produced in vivo, as seen by CoA analysis
using a panD strain of BL21 (DE3).
[0150] To support modular polyketide production in E. coli, the
invention provides methods and reagents to produce
(S)-methylmalonyl-CoA, which is not naturally present in E. coli,
by overexpressing mm-CoA mutase and mm-CoA epimerase in E. coli. An
active, FLAG-tagged version of the mm-CoA mutase from S.
cinnamonensis was expressed in XL1Blue cells, which were grown in
the presence of hydroxocobalamin in a synthetic, vitamin-free media
to produce active holoenzyme. The CoA levels in the cells were
analyzed by feeding labeled .beta.-alanine; for this purpose it is
beneficial to have a panD strain, which is a .beta.-alanine
auxotroph. The mutase DNA rearranged in the panD strain of SJ16, a
recA.sup.+ strain, such that the CoA analysis had to be carried out
without the panD. This resulted in a lower signal to noise ratio,
but elevated mm-CoA levels could still be detected. As an
alternative to the S. cinnamonensis genes, the invention provides a
mm-CoA mutase from P. shermanii cloned into an E. coli expression
vector, which is active without addition of vitamin B12, and which
elevates mm-CoA levels in E. coli in a panD strain compatible with
the mutase DNA.
[0151] Propionibacterium freudenreichii subsp. shermanii was
obtained as a stab in tomato juice agar from derived from a
freeze-dried specimen from NCIMB, Scotland (NCIMB # 9885). E. coli
strain gg3, a panD version of BL21 (DE3) was used for the CoA
analysis. E. coli strains gg1 and gg2, recA.sup.- versions of the
SJ16 panD strain, were also used. The vector pKK** is a version of
pKK223-3 in which the cloning region is altered to range from Nde1
to EcoR1 and an extra Nde1 site is deleted. Growth of P. shermanii
and preparation of genomic DNA was conducted as described in the
literature.
[0152] Subcloning of methylmalonyl-CoA mutase from P. shermanii
into E. coli was conducted as follows. The gene for mm-CoA mutase
consists of two subunits, mutA and mutB, which were amplified by
PCR from P. shermanii genomic DNA in a total of four fragments.
Naturally occurring restriction sites were used to piece the gene
together. Unique restriction sites were introduced at both ends of
the gene for cloning purposes, and the start codon for the mutB
gene was changed from GTG to ATG. As illustrated below, these four
fragments were cloned into a Bluescript.TM. (Stratagene) vector,
sequenced, and then pieced together to form the complete mutase
gene. The gene was then cloned into expression vectors pET22b and
pKK** between the restriction sites Nde1 and HindIII, to form
pET-MUT and pKK**-MUT.
[0153] The pET-MUT was transformed into competent cells BL21(DE3)
and later into cells gg3, which are apanD version of BL21(DE3). The
pKK**-MUT was transformed into SJ16 panD and into XL1Blue. The DNA
was tested by screening several colonies with Nde1 and HindIII, to
determine if the mutase gene was still present or if it had
rearranged.
[0154] For SDS-PAGE analysis, cells of strain BL21(DE3) containing
pET-MUT (and pET alone, as a control) were grown aerobically at
27.degree. C. in MUT media with 100 .mu.g/ml carbenicillin (carb)
(MUT media is M9 salts, glucose, thiamine, trace elements and amino
acids, as previously described for the expression of methionine
synthase (Amaratunga, M., et al., A synthetic module for the metH
gene permits facile mutagenesis of the cobalam in-binding region of
Escherichia coli methionine synthase: initial characterization of
seven mutant proteins. Biochemistry, 1996. 35(7): p. 2453-63).
Overnight cultures (250 .mu.l) were used to inoculate 25 mL of MUT
media (carb), which were grown at 27.degree. C. to an OD.sub.600 of
approximately 0.5. The cultures were then induced with IPTG to 1 mM
final concentration. Two cultures were left at 27.degree. C. for
three hours while duplicate cultures were grown at 37.degree. C.
for two hours. The cells were collected by centrifugation and the
pellets were stored at -80.degree. C. prior to analysis. The cells
were lysed by sonication and both the soluble and insoluble phases
were examined by SDS/PAGE. This procedure was repeated for cells of
strain XL1Blue containing pKK**-MUT.
[0155] For expression of active mm-CoA mutase (with
hydroxocobalamin), cells of strain gg3 containing pET-MUT (and pET
alone, as a control) were grown in MUT media (carb) and 5 .mu.M
beta-alanine for approximately 20 hours at 27.degree. C. The
following operations were performed in a dark room with a red
safelight: 125-mL flasks, each containing 25 mL of MUT media with
carb and 5 .mu.M .beta.-alanine and wrapped in aluminum foil, were
inoculated with 5 .mu.M hydroxocobalamin and then with 250 .mu.L
from the respective starter cultures. After shaking overnight at
27.degree. C., the cultures were induced with IPTG to 1 mM final
concentration and grown for an additional 4:45 hours, at which
point they were collected (in Falcon tubes wrapped in aluminum
foil) by centrifugation at 4000 rpm for 10 minutes. The pellets
were stored in the dark at -80.degree. C. prior to assaying.
[0156] The mutase assay was performed as follows. All operations
were performed in the dark or under a red safelight. The pellet
from 25 mL of culture was thawed, washed in buffer C (50 mM
potassium phosphate pH 7.4, 5 mM EDTA, 10% glycerol), and
resuspended in 0.5 mL of buffer C containing protease inhibitors (1
tablet per 10 mL of buffer). Following sonication on ice, the
extract was clarified by centrifugation at 4.degree. C. for 10
minutes at maximum speed in an Eppendorf microfuge; the supernatent
was assayed. Enzyme assays contained, in a final volume of 100
.mu.L, 0.2 mM (2R,2S)-methylmalonyl-CoA, mutase extract, and buffer
C containing protease inhibitors. Reactions for assays with vitamin
B12 were as above but contained 0.01 mM vitamin B12, in which case
the mutase extract was incubated with the vitamin B12 in a total
volume of 75 .mu.L for 5 minutes at 30.degree. C. prior to
initiation of reaction with methylmalonyl-CoA. After the desired
length of incubation at 30.degree. C., the reaction was stopped by
the addition of 50 .mu.L of 10% trichloroacetic acid (TCA) and
placed on ice for approximately 10 minutes. Cellular debris and
precipitated protein were removed by centrifugation for 5 minutes
in an Eppendorf microfuge at 4.degree. C. An aliquot (100 .mu.L) of
the supernatant was injected onto the HPLC to quantify conversion
of methylmalonyl-CoA to succinyl-CoA. One time point was taken
after 20 minutes of incubation at 30.degree. C., and the sample was
assayed for conversion of mm-CoA to succinyl-CoA. All operations
were performed exclusively under a red safelight until the reaction
was stopped by addition of TCA.
[0157] The CoA analysis was performed as described in the
literature, except that 5 .mu.M of hydroxocobalamin were added at
the time of IPTG induction, and the tubes were wrapped in aluminum
foil and grown at 27.degree. C. instead of 30.degree. C. The CoA
peaks, which eluted in approximately one minute each, were
collected manually, as well as approximately one minute of sample
both before and after each peak. In some tests, fractions were
collected every 30 seconds. All samples were counted in the
scintillation counter.
[0158] The two subunits of the gene encoding methylmalonyl-CoA
mutase are translationally coupled--the GTG start codon of the
downstream subunit mutB overlaps with the ATG codon of mutA. The
GTG valine start was mutated to an ATG methionine start (which does
not alter any other amino acids), because E. coli utilizes the
methionine start more efficiently. Sequencing the mm-CoA mutase
gene revealed a discrepancy between the sequence observed and the
published sequence. A "GC" instead of a "CG" changed two amino
acids from Asp, Val to Glu, Leu. The crystal structure of mm-CoA
mutase from P. shermanii showed that the two amino acids are indeed
Glu, Leu, so the published sequence is in error. The mm-CoA mutase
gene was subcloned into two different E. coli expression systems:
pET, which is under control of the strong T7 promoter, and pKK,
which uses the leaky tac promoter. First it was necessary to find
strains in which the mutase DNA did not rearrange. It was
previously observed that a FLAG-tagged version of the mutase from
S. cinnamonensis rearranged in SJ16 panD and in BL21(DE3), which
are both recA.sup.+ strains, but not in XL1Blue, which is
recA.sup.-. This mutase DNA (P. shermanii) also rearranged in the
SJ16 cells but not in the BL21(DE3) cells. Thus a panD version of
BL21(DE3) was created (gg3) for use with the pET vector. A
recA.sup.- version of SJ16 was also created (gg1, gg2) for use with
the pKK system; however, the mutase DNA rearranged in this strain
as well.
[0159] Different growth conditions were tested to find conditions
in which the two subunits of the mutase were expressed in the
soluble phase in approximately equal molar ratios. In general, it
seemed that the higher temperature of 37.degree. C. caused the
mutase to appear predominantly in the insoluble form. Growth
exclusively at 27.degree. C. resulted in soluble protein with an
approximately equal subunit ratio.
[0160] FIG. 3 shows the comparison of in vivo acyl-CoA levels in
BL21(DE3) panDstrains with and without mm-CoA mutase. For each CoA,
the ratio of the amount in the strain containing the mutase to the
amount in the control strain was determined. Interestingly,
malonyl-CoA was increased about 25-fold and succinyl-CoA about
3-fold. Acetyl-CoA and CoA were increased just slightly, and
propionyl-CoA was not detected in either case.
[0161] To express active mutase in vivo, it was necessary to grow
cells in a defined media (MUT media) that allows uptake of the
vitamin B12 precursor hydroxocobalamin; this is similar to an
established protocol for expression of active methionine synthase,
which also requires B12. Cell extracts overexpressing the mutase
were shown to convert mm-CoA to succinyl CoA without the addition
of vitamin B12. Only one time point (at 20 minutes) was assayed to
confirm activity; the specific activity of the mutase was not
determined.
[0162] Thus, methylmalonyl-CoA mutase was expressed as the active
holoenzyme in E. coli, and methylmalonyl-CoA was produced in vivo.
Because a slow, spontaneous chemical epimerization between (R)- and
(S)-mm-CoA does exist (approximately 3% in 15 minutes), it may be
helpful to determine the relative amounts of these diastereomers in
cells overexpressing the mutase. Enough (S)-mm-CoA may be present
to support polyketide production in some cells without addition of
an epimerase. To facilitate the eventual production of polyketides
in E. coli, the mutase gene can be incorporated into the chromosome
of the BL21 panD cell or other host cell.
[0163] FIG. 2 shows the construction of pSK-MUT, in which four PCR
fragments were sequenced and pieced together to form the complete
mutase gene in pSK-bluescript.
[0164] In follow-up experiments, the specific activity of the
mutase was determined and an in-depth CoA analysis was completed.
As shown in FIG. 3, the CoA levels in the cells were again analyzed
using a panD strain, which is a .beta.-alanine auxotroph.
.sup.3H-.beta.-alanine was fed to the cells and incorporated into
the acyl-CoAs, which were separated via HPLC and counted. The CoA
pools for cell extracts with and without the mutase, as well as
with and without hydroxocobalamin, were examined.
[0165] To test whether acyl-CoAs degrade in TCA, the following
tests were conducted. The CoA mix consisted of 1.6 mM each of
malonyl-, methylmalonyl-, succinyl-, acetyl-, and propionyl-CoA,
plus 0.5 mM CoA. An aliquot (10 .mu.L) of this mix was added to 100
.mu.L 10% TCA, 50 .mu.L were immediately injected to the HPLC for
CoA analysis, and the remainder was promptly frozen on dry ice. The
frozen portion was then thawed and loaded immediately to the HPLC.
Again, 10 .mu.L of the CoA mix were added to 100 .mu.L 10% TCA, 50
.mu.L were left on ice for 15 minutes and then injected to the
HPLC, the remainder was left at 4.degree. C. overnight and injected
to the HPLC the next morning. The area under each CoA peak was
noted. This same procedure was followed but using a mixture of TCA
and buffer A from the mutase assay.
[0166] The CoA analysis described here is carried out on cells
which are lysed in 10% TCA. Thus, determining whether the CoAs
degrade significantly in TCA and in a mixture of TCA and buffer A
from the mutase assay is important. The tests showed that the
percent of each CoA relative to the total CoA pool, as well as the
overall amount of CoA, remained constant after freeze/thawing,
after leaving on ice for 15 minutes, and after leaving the sample
overnight at 4.degree. C. Thus, the CoAs are stable in TCA and in
the mutase assay buffer after the cells are lysed or after the
assays are completed, and prior to HPLC analysis.
[0167] Although the CoAs are stable in TCA and buffer at 4.degree.
C., they degraded at 30.degree. C., the temperature at which the
mutase assay was performed. In five minutes under the assay
conditions, about 4% of the methylmalonyl-CoA hydrolyzed to CoA.
The succinyl-CoA hydrolyzed at a comparable rate. Thus, the mutase
assay is suboptimal for extremely quantitative results.
[0168] When 0.2 mM methylmalonyl-CoA was incubated with a crude
lysate from cell extracts overexpressing the mutase, succinyl-CoA
was produced. No succinyl-CoA was observed when methylmalonyl-CoA
was incubated with lysates from the control strain (containing the
plasmid vector but lacking the mutase genes). Under these
expression and assay conditions, a specific activity of
approximately 0.04 U/mg was observed in the crude extracts. When
cells overexpressing the mutase were grown in MUT media without
hydroxocobalamin, no mutase activity was observed; however, mutase
activity could be detected by addition of vitamin B12 in vitro.
Adding vitamin B12 to extracts that were grown in the presence of
hydroxocobalamin resulted in increased mutase activity, suggesting
that a significant amount of expressed mutase is present as the
apo-enzyme. This might have occurred because the enzyme was
expressed faster than the hydroxocobalamin could be transported
into the cell, or because the vitamin B12 cofactor was lost during
preparation of the extract.
[0169] FIG. 4 shows the comparison of in vivo acyl-CoA levels with
and without the mutase and with and without hydroxocobalamin. In
the cells overexpressing the mutase and grown with
hydroxocobalamin, methylmalonyl-CoA comprised 13% of the overall
CoA pool, whereas in the other cells no methylmalonyl-CoA was
detectable. The background level of counts is about 0.25% of the
overall number of counts in the CoAs, suggesting that any
methymalonyl-CoA present in E. coli strains not overexpressing the
mutase would comprise at most 0.25% of the overall CoA pool, or 2%
of the amount of methylmalonyl-CoA observed in the strain
overexpressing the mutase. The composition of the CoA pool observed
for the E. coli panD strain is consistent with that observed
previously for E. coli panD mutants grown on glucose.
[0170] Thus, the methylmalonyl-CoA mutase from P. shermanii has
been overexpressed as the active holoenzyme in E. coli and shown to
produce (2R)-methylmalonyl-CoA in vivo. Conversion of (2R)- to
(2S)-methylmalonyl-CoA via methylmalonyl-CoA epimerase should
provide an adequate supply of the correct isomer of
methylmalonyl-CoA to support heterologous production of complex
polyketides E. coli.
[0171] FIG. 4 shows the results of CoA analysis of E. coli
overexpressing methylmalonyl-CoA mutase. The levels of .sup.3H
detected in fractions collected from HPLC of cell-free extracts
from .sup.3H .beta.-alanine-fed E. coli harboring either the pET
control vector grown without hydroxocobalamin (solid line), pET
grown with hydroxocobalamin (dash-dotted), pET overexpressing the
mutase and grown without hydroxocobalamin (dotted), or pET
overexpressing the mutase and grown with hydroxocobalamin (dashed)
are shown.
B. Cloning and Expression of Methylmalonyl-CoA Epimerase
[0172] The mm-CoA epimerase from Propionibacterium shermanii was
purified and used to obtain N-terminal protein sequence as well as
internal peptide sequence from LysC-generated peptides. The
epimerase gene was cloned using hybridization probes designed from
the peptide sequences.
[0173] Propionibacterium freudenreichii subsp. shermanii was
obtained and cultured as described in part A. Purification of
mm-CoA epimerase from P. shermanii was based on a modification of
the published procedure. The procedure utilized a 10 L culture,
which was lysed by sonication followed by column chromatography in
the order: DE-52, Hydroxyapatite, Phenylsepharose, MonoQ anion
exchange, and C-8 RP HPLC.
[0174] All operations were performed at 4.degree. C., except the
C-8 RP HPLC, which was performed at room temperature, and all
buffers contained 0.1 mM PMSF, unless otherwise stated. The
epimerase assay was performed essentially as described in the
literature. Protein concentration was determined using the method
of Bradford. The overall yield of epimerase activity was not
determined.
[0175] More specifically, cell paste (75 g) was resuspended in 50
mL buffer (50 mM Tris-HC1 pH 7.5, 0.1M KCl, 0.2 mM PMSF, 1 mM EDTA)
and sonicated using a macrotip with a diameter of 1.2 cm. With
pulses of 0.5 seconds ON and 0.3 seconds OFF, the cells were
sonicated twice for 30 seconds each at a power setting of 4,
followed by five times for 30 seconds each at a power setting of 6.
A clear, amber-colored supernatant (53.5 ml) was obtained after
spinning for 35 minutes at 12,000 rpm.
[0176] The crude extract from above was applied to a column
(diameter 2.5 cm, height 15 cm) of 73 mL of DE-52 resin
equilibrated with 50 mM Tris-HCl pH 7.5, 0.1M KCl. The column was
washed at 1 ml/min with three column volumes of the above buffer,
followed by a linear gradient to 50 mM Tris-HCl pH 7.5, 0.5 M KCl
over seven column volumes. Six mL fractions were collected and
assayed for epimerase activity. The epimerase was found
predominantly in the flow-through and in several early fractions.
The flow-through and active fractions were combined (325 mL) and
dialyzed against 4 liters of 50 mM Tris-HCl pH 7.5, 10% glycerol,
followed by 4 liters of 10 mM sodium phosphate pH 6.5, 10% glycerol
(final volume 250 mL).
[0177] A 7.5 mL hydroxyapatite biogel HTP gel column (diameter 1.5
cm, height 16 cm) was equilibrated with 10 mM sodium phosphate pH
6.5, 5% glycerol. After loading of the enzyme solution (using
repeated injections) and washing with three column volumes of the
above buffer, a gradient to 200 mM sodium phosphate pH 6.5, 5%
glycerol was effected over 20 column volumes at a flow rate of 1
ml/min. The 2 mL fractions were assayed for epimerase activity, and
fractions containing epimerase activity were pooled for a total of
99 ml.
[0178] To the 99 mL sample from above, solid ammonium sulfate to
1.5 M final concentration was added slowly and with stirring at
4.degree. C. over 30 minutes. This suspension (100 mL) was loaded,
by repeated injection, onto a 6.6 mL column (1 cm.times.height 8.5
cm) of phenyl-sepharose resin equilibrated in 20 mM sodium
phosphate buffer pH 6.5, 1.5 M ammonium sulfate. The column was
washed at 1 ml/min with three column volumes of this buffer,
followed by a linear gradient to 20 mM sodium phosphate buffer pH
6.5, 10% glycerol, over 24 column volumes. After assaying the 3 mL
fractions for epimerase activity, the fractions containing
epimerase activity were pooled and dialyzed against 50 mM Tris-HCl
pH 7.5.
[0179] A mono Q 5/5 prepacked column was equilibrated with 25 mM
Tris-HCl pH 7.5 at 0.5 mL/min. The sample from the previous step
was loaded onto the column, which was then washed with 5 column
volumes of the above buffer, followed by a linear gradient to 50 mM
Tris-HCl pH 7.5, 1 M NaCl, 5% glycerol, over 50 column volumes. The
1 mL fractions were assayed for epimerase activity. Several
fractions containing epimerase activity were stored separately; the
fraction with the most activity was used for the next purification
step.
[0180] A reverse-phase column was equilibrated with water
containing 0.1% trifluoroacetic acid; 120 .mu.L (concentrated from
0.5 mL of the active fraction from above, using an Amicon
microconcentrator) was injected onto the column at a flow rate of
0.2 mL/min and washed for five minutes with the above solvent
system. Then a linear gradient over 50 minutes to acetonitrile
containing 0.1% trifluoroacetic acid was implemented. The peaks
were collected manually and the peak corresponding to the epimerase
(as determined by SDS/PAGE) was dried to completeness, resuspended
in water and stored at -80.degree. C.
[0181] For Lys C mediated digestion of the HPLC-purified epimerase,
the epimerase fraction (11751rp2-B, 200 .mu.L) collected from
reverse phase HPLC was dried to completeness and resuspended in 40
.mu.L water. To 30 .mu.L of the sample was added 5 .mu.L of 1 M
Tris/HCl, pH 8, 1.5 .mu.L of 0.1 M DTT, 2 .mu.L of Lys C protease
(0.2 .mu.g). A control reaction contained all of the above
components except the epimerase. The reactions were incubated
overnight at 37.degree. C. An aliquot of the reaction (5 .mu.L) was
diluted to 60 .mu.L with water and loaded to the HPLC, using the
same HPLC program that was used to purify the epimerase. The
analytical HPLC showed that the Lys C digestion was not complete.
An additional aliquot of Lys C (0.2 .mu.g) was added to the
reactions and incubation was continued overnight at 37.degree. C.
Following overnight incubation, an aliquot of the reaction (5
.mu.L) was diluted to 60 .mu.L with water and subjected to the
HPLC. The HPLC showed that the digestion was complete. The
remainder of the reaction was loaded to the HPLC and individual
peaks were collected manually. HPLC of the control reaction showed
no significant peptide fragments arising from self-digestion of the
LysC.
[0182] An aliquot of the pure epimerase, as well as a peptide
collected from the procedure described above, were submitted for
N-terminal amino acid sequencing. Based on the amino acid sequences
from above, several degenerate primers were designed as described
below that introduced unique restriction sites to either end of the
eventual PCR product. These primers were used in PCR with P.
shermanii genomic DNA to obtain a 200 base-pair product, which was
cloned into a Bluescript.TM. (Stratagene) vector and submitted for
sequencing.
[0183] A cosmid library of P. shermanii was prepared, essentially
as described in the Stratagene cosmid manual. The titer of this
cosmid library was approximately 11 cfu (colony forming units) per
.mu.L, for a total yield of 5556 cfu. A plasmid library of P.
shermanii was prepared by digesting P. shermanii genomic DNA with
SacI and ligating the resulting mixture into a Bluescript.TM.
vector also cut with SacI. To determine the average insert size (2
kb), ten random clones were digested with SacI. The ligation
mixture was re-transformed 5 times, pooled and plated on one large
LB (carb) plate, resulting in a lawn of colonies that were scraped
together and resuspended in LB as the plasmid library. The titer of
this plasmid library was approximately 64,000 cfu per .mu.L.
[0184] Several degenerate primers based on the amino acid sequences
were prepared and used in PCR with P. shermanii genomic DNA to
obtain a 180 base-pair product, which was cloned into a
Bluescript.TM. vector and sequenced. Several different probes were
made. The first probe was made using the random priming method to
incorporate either .sup.32P or digoxigenin into the epimerase
fragment. A probe was made from the cloned fragment by
amplification of the fragment via PCR, using the digoxigenin
labeling method. The PCR product was gel isolated, quantified, and
used to probe the cosmid library. Colonies that hybridized to the
probe were restreaked from master plates, and five colonies from
the re-streaked plates were picked, cosmids were isolated, and the
insert sequences screened for the epimerase gene by PCR. Several
cosmids that were scored positive for epimerase DNA sequence by PCR
were subjected to DNA sequencing using epimerase-specific primers.
The cosmid designated 117-167-A7 contained the full epimerase
sequence.
[0185] The sequence of the putative epimerase gene contained in
cosmid 117-167-A7 was aligned to the N-terminal epimerase sequence
already known. The several hundred base pairs downstream of this
sequence were translated in all three frames and a stop codon in
one of the frames was found that yielded a protein of the expected
size. The entire sequence was used to search the protein database
via BLAST analysis, and the sequence showed high homology to the
sequence of a putative epimerase from S. coelicolor identified in
accordance with the methods of the invention. PCR primers were
designed based on the DNA sequence of the cloned P. shermanii
epimerase and the gene was amplified from P. shermanii genomic DNA
with NdeI and BamHI sites at the 5'-end, an internal NdeI site was
destroyed near the 5' end, and NheI and AvrII sites were introduced
at the 3'-end. Following PCR, the 447 bp product was cloned into a
Bluescript vector (143-6-11) and sequenced. Also, four additional
sequencing primers were designed to provide several-fold coverage
of the epimerase gene. The full epimerase gene sequence provided in
isolated and recombinant form by the present invention is provided
below as SEQ ID NO: 1, and the protein sequence of the epimerase is
provided below as SEQ ID NO: 2.
The epimerase gene was then cloned into a pET expression vector;
the construct was named pET-epsherm.
[0186] For the cloning of epimerase genes from B. subtilis
(described by Haller et al., supra) and S. coelicolor (from cosmid
8F4 in the S. coelicolor, genome sequencing project), primers were
designed to PCR these genes from their respective genomic DNAs and
to incorporate either a PacI or NdeI site at the 5' end, and an
NsiI site at the 3' end. The PCR products were cloned into a
Bluescript.TM. vector and sequenced. Mutation-free clones were
obtained for the S. coelicolor epimerase, but the B. subtilis
epimerase contained two point mutations in all three clones tested:
C to T at base pair 37, and G to A at base pair 158. When the PCR
for this epimerase gene was repeated and the product cloned and
sequenced, the same mutations were present, implying that the
original sequence was in error. The cloned epimerases from B.
subtilis and S. coelicolor were cloned as NdeI/NsiI fragments into
an intermediate vector 116-172a, a Bluescript.TM. pET plasmid
containing the T-7 promoter and terminator sequences. The cloned
epimerases from B. subtilis and S. coelicolor are pET-epsub and
pET-epcoel, respectively. The epimerase genes were also excised
along with the T7 promoter as PacI/NsiI fragments, as shown
schematically below. [0187] ---PacI---T7 promoter------epimerase
gene--------NsiI--- and cloned into the PacI/NsiI restricted vector
133-9b, to form a single operon with the epimerase gene located
downstream of the two mutase genes. The epimerase gene from P.
shermanii was cloned as above except that it was cloned into
116-172a as an NdeI/AvrII fragment, excised along with the T7
promoter as a PacI/NheI fragment, and cloned into 133-9b between
PacI and NheI sites. The constructs are pET-mutAB-T7-epsherm,
pET-mutAB-T7-epsub, and pET-mutAB-T7-epcoel.
[0188] As an alternative to the mutase from P. shermanii, S.
coelicolor, and B. subtilis, one can clone by PCR from E. coli
genomic DNA the single gene for Sbm (sleeping beauty mutase).
Genomic DNA of E. coli BL21(DE3)/PanD was prepared using a kit
purchased from Qiagen. The gene for Sbm (Sleeping beauty mutase, a
methylmalonyl-CoA mutase) was amplified by PCR from E. coli
BL21(DE3)/PanD genomic DNA. The PCR fragment was gel isolated,
cloned into PCRscript and sequenced to yield the mutation-free
clone 143-11-54. Excised as an NdeI/SacI fragment, sbm was cloned
into pET22b, thence as a NdeI/XhoI fragment into pET16b to
introduce an N-terminal His-Tag (143-49-2). Sbm was also cloned
between NdeI and SpeI into 116-95B.43, a pET22b vector that allows
subsequent cloning of the epimerase genes downstream of the sbm.
That construct was named 143-40-39.
[0189] Cells of strain BL21(DE3) containing pET-epsherm,
pET-epcoel, pET-epsub, or a control pET vector were grown overnight
at 37.degree. C. in 2 mL LB containing 100 .mu.g/ml carbenicillin.
The starter culture (250 .mu.L) was used to inoculate 25 mL LB
containing 100 .mu.g/ml carbenicillin. The cultures were grown at
37.degree. C. to an OD of approximately 0.4, then induced with IPTG
to 1 mM final concentration and grown for an additional 3 hours at
30.degree. C. The cells were collected by centrifugation at 4000
rpm for 10 minutes, and the pellets were stored at -80.degree. C.
prior to assay. The epimerase from P. shermanii expressed well in
E. coli; SDS gel analysis revealed an overexpressed protein at
approximately 22 kDa. The S. coelicolor epimerase also expressed
well, at a molecular weight of approximately 19 kDa, and the B.
subtilis epimerase was expressed, but mostly in inclusion bodies (a
faint band is present at approximately 19 kDa), which can be
overcome by use of alternate expression systems.
[0190] Epimerase activity was measured in crude extracts of E. coli
harboring either pET-epsherm, pET-epcoel, pET-epsub, or a control
pET vector. The epimerase assay couples transcarboxylase, which
converts (S)-methylmalonyl-CoA into propionyl-CoA, to malate
dehydrogenase, which converts NADH into NAD.sup.+, producing a
decrease in absorbance at 340 nm. The assay is initiated with a
racemic mixture of (R,S)-methylmalonyl-CoA; when the (S)-isomer is
consumed as described below, a steady background rate is observed
at about one-tenth of the initial rate. When an extract containing
epimerase is added to the assay, the (R)-isomer is converted to
(S)-, resulting in a further decrease in absorbance. In crude E.
coli extracts, however, a significant background rate is observed,
probably due to an endogenous NADH oxidase. Thus the epimerase must
be expressed at a sufficiently high level to conclude that it is
active. The assay was conducted as follows.
[0191] The pellet from approximately 20 mL of culture was thawed
and resuspended in 2 mL 1.times. assay buffer containing a protease
inhibitor cocktail tablet. The cells were disrupted by sonication
(two sonication cycles for 30 seconds each at a power setting of 2
[pulse ON 0.5 sec/pulse OFF 0.5 sec]). After spinning for 10
minutes at 13,000 rpm in an Eppendorf centrifuge, the supernatents
were saved for assay. Methylmalonyl-CoA epimerase activity was
assayed using a modification of the method of Leadlay et al.,
Biochem. J. 197: 413-419, "Purification and characterization of
methylmalonyl CoA epimerase from Propionibacterium shermanii"
(1981). The assays were performed at 30.degree. C. with a 1 cm path
length plastic cuvette, in a final volume of 1.5 mL. The reaction
mixtures contained 0.2 M potassium phosphate buffer pH 6.9, 0.1 M
ammonium sulfate, 5 mM sodium pyruvate, 0.08 mM
(2R,2S)-methylmalonyl-CoA, 0.05 units of partially purified
transcarboxylase, 0.16 mM NADH, and 2.5 units malate dehydrogenase.
The reaction was initiated with (2R,2S)-methylmalonyl-CoA and the
decrease in absorbance at 340 nm was monitored, reflecting the
disappearance of the 2S isomer. When the decrease in absorbance at
340 nm reached the basal level (usually around 10% of the initial
transcarboxylase rate), an extract containing epimerase was added
and a further decrease in absorbance was observed. The chemicals
and enzymes used in the epimerase assay were purchased from Sigma,
except for transcarboxylase, which was obtained as a crude
preparation from Case Western Reserve.
[0192] The crude extracts harboring both the P. shermanii and S.
coelicolor epimerases had specific activities (approximately 30
units/mg) at least 10 times higher than that of the control.
However, no activity above the background level was observed in the
extract harboring the B. subtilis epimerase, possibly because it
was not expressed at a high enough level, or as noted above, was
expressed as insoluble inclusion bodies. The pET-mutAB-T7-epsherm
construct was also expressed in E. coli. The resulting crude
extract contained epimerase activity that was significantly above
the background level; thus, the epimerase is functional in this
construct. The mutase did not interfere in the epimerase assay,
because these cells were grown without addition of
hydroxocobalamin, the cofactor for mutase activity. These results
show that one can express both active mutase and active epimerase
in an E. coli cell. These results also show that the
methylmalonyl-CoA epimerase from P. shermanii was cloned, expressed
in E. coli, and active, and that the putative epimerase from S.
coelicolor is a methylmalonyl-CoA epimerase. These genes can be
integrated into the chromosome of an E. coli PanD strain or other
strain and used for the production of polyketides built in whole or
in part from methylmalonyl CoA.
EXAMPLE 2
Production of Methylmalonyl CoA in Yeast
[0193] This example describes the construction of strains of
Saccharomyces cerevisiae optimized for polyketide overproduction.
In particular, this example describes the construction of yeast
host strains that (i) produce substrates and post-translational
modification enzymes necessary to express polyketides made by
modular polyketide synthases; (ii) have necessary nutritional
deficiencies to allow positive selection of at least three
compatible plasmids; and/or (iii) are suitable to permit
radioactive labeling of acyl-CoA pools and polyketide synthases and
demonstrates that such strains can express a modular PKS and
produce a complex polyketide at levels suitable for commercial
development. References are cited in this example by a number
corresponding to the numbered list of references below, each of
which is incorporated herein by reference.
[0194] With appropriate strain modifications, S. cerevisiae is an
ideal host for polyketide production. S. cerevisiae is capable of
producing very high levels of polyketides. Introduction of the gene
for the iterative PKS, 6-MSAS, along with the gene for Sfp, a
P-pant transferase from B. subtilis, led to the production of an
impressive 2 g/L 6-MSA in shake-flasks without optimization Kealey,
J. T., et al., Production of a polyketide natural product in
nonpolyketide-producing prokaryotic and eukaryotic hosts. Proc Natl
Acad Sci USA, 1998. 95(2): p. 505-9, incorporated herein by
reference. The genetics of yeast is very well understood. Genes can
readily be inserted into the chromosome, and the complete genome
sequence provides relevant knowledge regarding metabolic pathways
and neutral insertion sites. In addition, several strong,
controllable promoters are available. Proteins have less tendency
to form inclusion bodies in yeast, compared to E. coli. Yeast has a
relatively short doubling time in comparison to native polyketide
producing organisms. S. cerevisiae has a doubling time of 1 to 2 hr
compared to 4 to 24 hr for a typical polyketide producer, which has
obvious benefits in genetic development, process development, and
large-scale production.
[0195] The fact that yeast grow as single cells provides an
additional benefit over filamentous organisms (typical polyketide
producers). Mycelial fermentations are viscous and frequently
behave as non-Newtonian fluids. This fluid rheology provides a
significant obstacle to the process scientist both in terms of
uniform nutrient transport to the cells and in handling the
fermentation broth. Employing yeast as a host, even at high cell
densities, avoids such impediments. Because of the extensive
history of yeast in single cell protein production and the
expression of recombinant proteins, scalable fermentation protocols
for yeast have been developed. Yeast can be grown in fed-batch
fermentations to very high cell densities (>100 g/L biomass) as
compared to typical polyketide producers (10-20 g/L biomass). Thus,
comparing organisms with the same specific productivity (g
polyketide/g biomass/day), yeast would provide a higher volumetric
productivity (g polyketide/L/day). Finally, S. cerevisiae is
classified by the FDA as a "Generally Regarded As Safe" (GRAS)
organism. GRAS classification will facilitate approval of drugs
produced in yeast as compared to other host cells.
[0196] S. cerevisiae also has disadvantages as a host for
polyketide biosynthesis, most of which are related to the fact that
yeast did not evolve to produce polyketides. Yeast does not contain
methylmalonyl-CoA, a necessary precursor for biosynthesis of many
polyketides. Yeast does not have a suitable P-pant transferase
capable of the necessary post translational modification of ACP
domains of a PKS. Yeast codons are biased towards A+T, whereas most
polyketide producers have high G+C codons; thus, yeast may have low
amounts of some tRNAs needed for PKS gene expression. The
correction of these deficiencies is described in this example, and
the invention also provides modified yeast host cells useful to
facilitate analysis of success.
[0197] Other case-by-case potential issues with yeast include the
possibility that some polyketide products may be toxic or may
require additional modifications for maturation (e.g.
glycosylation, P450 hydroxylation). Several methods provided by the
invention may be taken to circumvent these issues should they
arise. For toxicity, production may be controlled to occur in
stationary phase growth (as with 6-MSA production); resistance
factors from the wild type host may be introduced into the yeast
host (e.g. methylation of ribosomes for some antibiotics); a
non-toxic-precursor to the polyketide may be produced and converted
ex vivo (e.g. produce 6-dEB in one strain and convert it to
erythromycin in another), and others. Additional modifications to
the polyketide may be accomplished by cloning and expressing
modification enzymes into the host strain, chemical or enzymic
transformation, and/or biosynthetic transformation in a second
strain (e.g. convert 6-dEB analogs to erythromycin analogs by
feeding 6-dEB to a Streptomyces or Saccharopolyspora strain capable
of glycosylation and P450 hydroxylation).
[0198] Most modular PKSs require either or both malonyl-CoA or
(2S)-methylmalonyl-CoA as a source of 2-carbon units for polyketide
biosynthesis. The malonyl-CoA pools in yeast are quite sufficient
for polyketide synthesis, as illustrated by the production of large
amounts of 6-MSA in yeast. However, S. cerevisiae does not produce
(2S)-methylmalonyl-CoA and does not possess biosynthetic pathways
for methylmalonyl-CoA biosynthesis. Hence, a heterologous
biosynthetic pathway must be introduced into S. cerevisiae to
support biosynthesis of polyketides that use (2S)-methylmalonyl-CoA
as a precursor.
[0199] There are three routes or biosynthetic pathways for the
synthesis of methylmalonyl-CoA that can be engineered into yeast,
as shown in FIG. 5. These pathways have been shown to produce
methylmalonyl-CoA in E. coli and can be used to produce
methylmalonyl-CoA in yeast. This example describes the
identification of a system for methylmalonyl-CoA production in
yeast, and a method for introducing it into the yeast
chromosome.
[0200] The vitamin B12-dependent methylmalonyl-CoA mutase pathway
produces (2R)-methylmalonyl-CoA from succinyl-CoA. The
(2R)-methylmalonyl-CoA is converted to the (2S)-diastereomer via
methylmalonyl-CoA epimerase, as shown above. These enzymes are
present in a variety of organisms, but not yeast; BLAST searches of
the available genomic databases reveals at least 10
methylmalonyl-CoA mutases and 10 methylmalonyl-CoA epimerases in
various organisms. The Propionibacterium shermanii
methylmalonyl-CoA mutase has been expressed in E. coli as the
apo-enzyme, which requires addition of vitamin B12 for in vitro
activity (McKie, N., et al., Adenosylcobalamin-dependent
methylmalonyl-CoA mutasefrom Propionibacterium shermanii. Active
holoenzyme producedfrom Escherichia coli. Biochem J, 1990. 269(2):
p. 293-8; incorporated herein by reference). By use of a medium
that enables uptake of the vitamin B12 precursor hydroxocobalamin
(Amaratunga, M., et al., A synthetic module for the metH gene
permits facile mutagenesis of the cobalamin-binding region of
Escherichia coli methionine synthase: initial characterization of
seven mutant proteins. Biochemistry, 1996. 35(7): p. 2453-63;
incorporated herein by reference), and in accordance with the
methods of the invention, one can express active P. shermanii
methylmalonyl-CoA mutase holoenzyme in E. coli and produce
(2R)-methylmalonyl-CoA in such cells. In addition, one can employ
the single subunit methylmalonyl-CoA mutase from E. coli. The
present invention also provides the genes encoding
methylmalonyl-CoA epimerase from B. subtilis, P. shermanii and S.
coelicolor and methods for using them in converting
(2R)-methylmalonyl-CoA to the needed (2S)-diastereomer. A preferred
method is to express in yeast the methylmalonyl-CoA mutase from E.
coli, because it is a single ORF, and necessary codons are
plentiful in yeast. Alternatively, the P. shermanii enzyme can be
used.
[0201] PCC catalyzes the biotin-dependent carboxylation of
propionyl-CoA to produce (2S)-methylmalonyl-CoA, as shown above;
the pathway also includes a biotin carrier proteinibiotin
carboxylase. In S. coelicolor, Rodriguez and Gramajo identified
genes for PCC (pccB) and a biotin carrier proteinibiotin
carboxylase (accA1) (Rodriguez, E. and H. Gramajo, Genetic and
biochemical characterization of the alpha and beta components
ofapropionyl-CoA carboxylase complex of Streptomyces coelicolor
A3(2). Microbiology, 1999. 145(Pt 11)): p. 3109-19; incorporated
herein by reference). Introduction into E. coli of S. coelicolor
pccB and accA1 along with propionyl-CoA ligase (as a supply of
propionyl-CoA), results in the production of methylmalonyl-CoA in
that organism. A search of the genomic database reveals B. subtilis
as an additional source of the enzymes involved in the PCC
pathway.
[0202] In one embodiment of the invention, one can express the S.
coelicolor pccB and accA1 in yeast, because these are expressed and
the proteins are functional in E. coli. Should codon usage prove
suboptimal when expressing the S. coelicolor genes in yeast,
homologs from B. subtilis can be employed. Should the levels of
propionyl-CoA be suboptimal for PCC, one can co-express a
propionyl-CoA ligase in the yeast host. Intracellular propionyl-CoA
can be greatly increased in E. coli by expressing the Salmonella
propionyl-CoA ligase, PrpE, and supplementing the growth media with
propionate, as described below.
[0203] An additional method for the production of
(2S)-methylmalonyl-CoA provided by the present invention utilizes
the matB and matC genes from Rhizobium (An, J. H. and Y. S. Kim, A
gene cluster encoding malonyl-CoA decarboxylase (MatA), malonyl-CoA
synthetase (MatB) and a putative dicarboxylate carrier protein
(MatC) in Rhizobium trifolii--cloning, sequencing, and expression
of the enzymes in Escherichia coli. Eur J Biochem, 1998. 257(2): p.
395-402; incorporated herein by reference) or S. coelicolor (see
schematic above). The matABC genes code for a biosynthetic pathway
that converts malonate to acetyl-CoA through formation of
malonyl-CoA via MatB and subsequent decarboxylation by MatA. MatB,
the malonyl-CoA ligase, also accepts methylmalonate as a substrate
(An, J. H. and Y. S. Kim, A gene cluster encoding malonyl-CoA
decarboxylase (MatA), malonyl-CoA synthetase (MatB) and a putative
dicarboxylate carrier protein (MatC) in Rhizobium
trifolii--cloning, sequencing, and expression of the enzymes in
Escherichia coli. Eur J Biochem, 1998. 257(2): p. 395-402;
incorporated herein by reference) and catalyzes formation of
methylmalonyl-CoA. The substrates malonate or methylmalonate enter
the cell through a diacid transporter, the product of the matC
gene. Khosla et al. have shown that when E. coli containing the
Rhizobium matBC is fed (2R,2S)-mEthylmalonate,
(2R,2S)-methylmalonyl-CoA is produced. Furthermore, when an S.
coelicolor strain expressing the genes for the synthesis of the
polyketide aglycone, 6-deoxyeythronolide B (6-dEB), and containing
Rhizobium matBC, is fed methylmalonate, a 3-fold increase in
production of 6-dEB is observed. In accordance with the methods of
the invention, one can express the matB and matC genes from
Rhizobium in yeast, because these are expressed and the proteins
are functional in E. coli and S. coelicolor, or, alternatively the
matBC genes from S. coelicolor.
[0204] Active PKSs require post-translational
phosphopantetheinylation at each ACP of each module, but yeast does
not contain a P-pant transferase with the needed specificity
(Kealey, J. T., et al., Production of a polyketide natural product
in nonpolyketide-producing prokaryotic and eukaryotic hosts. Proc
Natl Acad Sci USA, 1998. 95(2): p. 505-9; incorporated herein by
reference). Previous work (Kealey, J. T., et al., Production of a
polyketide natural product in nonpolyketide-producing prokaryotic
and eukaryotic hosts. Proc Natl Acad Sci USA, 1998. 95(2): p.
505-9; incorporated herein by reference) has shown that
introduction of the B. subtilis P-pant transferase gene, sfp, into
yeast results in an expressed Sfp capable of modifying an iterative
PKS, 6-MSAS. Gokhale et al. demonstrated that the ACP domains in
the DEBS PKS are substrates for Sfp, so Sfp is a general modifying
enzyme for PKSs (Gokhale, R. S., et al., Dissecting and exploiting
intermodular communication in polyketide synthases. Science, 1999.
284(5413): p. 482-5; incorporated herein by reference). In
preferred yeast host cells of the invention, the sfp gene is
inserted into a neutral site of the yeast chromosome.
[0205] In developing a system to produce polyketides and optimize
fermentation procedures, the ability to measure intracellular
concentrations of substrates (i.e. acyl-CoAs) and of the PKS is
beneficial. In most cells, CoA esters are not present in sufficient
amounts to allow direct measurement by HPLC using ultraviolet
detection or other simple methods of detection. In E. coli, the
method of choice to quantify CoA pools is to feed [.sup.3H]
.beta.-alanine to a mutant deficient in aspartate decarboxylase
(PanD), which cannot produce endogenous .beta.-alanine (Jackowski,
S, and C. O. Rock, Regulation of coenzyme A biosynthesis. J
Bacteriol, 1981. 148(3): p. 926-32; incorporated herein by
reference). The PanD strain incorporates about ten-fold more
radioactivity into CoA pools than does wild type E. coli. Because
.beta.-alanine is a direct precursor of CoA, the radioactive label
enters the CoA pool without dilution, and acyl-CoAs can be
separated on HPLC and quantified by radioactivity measurement.
Because there is no radioisotope dilution, the radioactivity
measured reflects exact intracellular concentrations of the
acyl-CoAs.
[0206] BLAST searches did not reveal an E. coli PanD homolog in the
yeast genome; however, yeast may be a .beta.-alanine or
pantothenate auxotroph. Indeed, for CoA biosynthesis, yeast
requires either exogenous pantothenate, which enters the cell via
the Fen2p transporter, or exogenous .beta.-alanine, which enters
via the general amino acid permease (Gap1p) (Stolz, J. and N.
Sauer, The fenpropimorph resistance gene FEN2 from Saccharomyces
cerevisiae encodes a plasma membrane H+-pantothenate symporter. J
Biol Chem, 1999. 274(26): p. 18747-52; incorporated herein by
reference). [.sup.3H] .beta.-alanine is incorporated into CoA pools
of yeast (see below), but it is presently unknown whether isotope
dilution occurs due to endogenous .beta.-alanine production by some
unknown pathway. Thus, to enable quantitation, one can determine
the specific activity of CoA pools in yeast labeled with exogenous
[.sup.3H] .beta.-alanine. Cells producing polyketides generally
express low levels of high molecular weight PKSs that are barely
detectable on SDS-PAGE using protein stains. The ability to label
CoA with [.sup.3H] .beta.-alanine can also be used to quantify a
PKS expressed in the host cells because the phosphopantetheine
moiety of CoA containing .beta.-alanine is transferred to the ACP
domain in each module of a PKS. Thus, knowing the specific activity
of labeled intracellular CoAs, a PKS can be simply quantified by
radioactivity after SDS-PAGE.
[0207] The G+C content of most PKS genes is in the range of 60 to
70%, while that of yeast genes is 40%. Thus, some tRNAs needed to
translate PKS genes are scarce (but not absent) in yeast. However,
many genes with high G+C content have been expressed in yeast. As
examples, the large (1560 bp) DHFR-TS gene from Leishmania major
(63% G+C) is expressed well in yeast, despite the fact that it
contains several codons rarely used in yeast (Grumont, R., W.
Sirawaraporn, and D. V. Santi, Heterologous expression of the
bifunctional thymidylate synthase-dihydrofolate reductase from
Leishmania major. Biochemistry, 1988. 27(10): p. 3776-84;
incorporated herein by reference). Moreover, as mentioned below,
the PKS 6-MSAS (G+C=58%) is also expressed well in yeast (Kealey,
J. T., et al., Production of a polyketide natural product in
nonpolyketide-producing prokaryotic and eukaryotic hosts. Proc Natl
Acad Sci USA, 1998. 95(2): p. 505-9; incorporated herein by
reference). Thus, one can demonstrate the general applicability of
a yeast expression system without initial concern for potential
codon usage problems. Nevertheless, if a desired PKS does not
express well in yeast, the present invention provides several
methods to solve a "codon usage" problem observed with a particular
polyketide.
[0208] First, one can change the codons at the 5' end of the gene
to reflect those more frequently found in yeast genes. Batard et
al. (Batard, Y., et al., Increasing expression of P450 and
P450-reductase proteins from monocots in heterologous systems [In
Process Citation]. Arch Biochem Biophys, 2000. 379(1): p. 161-9;
incorporated herein by reference) successfully employed a similar
method to express in yeast wheat genes for a P450 and P450
reductase with high G+C content (56%) and strong bias of codon
usage unfavorable to yeast. Another method is to introduce yeast
tRNA genes with anti-codons modified to represent codons common in
PKS sequences. A similar method has been successfully used in E.
coli to enhance expression of high G+C genes (Carstens, C.-P., et
al., New BL21-CodonPlus.TM. Cells Correct Codon Bias in GC-Rich
Genomes. Strategies Newsletter from Stratagene Corp., 2000. 13(1):
p. 31-33; incorporated herein by reference), including PKS genes
from Actinomycetes. A third method is to synthesize chemically the
gene with codons optimized for expression in yeast. The cost for
contract synthesis of a 30,000 bp gene (e.g. .about.6-module PKS),
including sequence verification, is approximately $3 per base, or
about $100,000. For a valuable product (e.g. epothilone), the cost
is not prohibitive.
[0209] In an illustrative embodiment of the invention, a yeast
strain deficient in Ura, Trp, His and Leu biosynthesis is employed
as a host to allow selection of plasmids containing these markers.
This host is modified in accordance with the methods of the
invention by introducing genes that produce the needed
methylmalonyl-CoA substrate and P-pant transferase for
post-translational modifications of PKSs. These are preferably
integrated into the yeast chromosome, because they are necessary
for production of any polyketide. To validate functional expression
of the substrate genes, one can measure methylmalonyl-CoA pools.
For validation of P-pant transferase activity, one can coexpress
6-MSAS and measure [.sup.3H] phosphopantetheinylation of the enzyme
as well as 6-MSA production. Should either be deficient, one can
increase gene copy number.
[0210] For PKS gene expression, one can use replicating vectors
based on the 2 micron replicon, because plasmids may have to be
rescued for analysis should a problem arise. A typical modular PKS
gene cluster (eg. 3 ORFS, .about.10 kB each, as in erythromycin)
can be introduced on three or more vectors; such plasmids
(containing Ura, Trp and Leu markers) are available and similar to
those used in the studies of 6-MSAS expression in yeast. A PKS
consisting of three large proteins can be functionally
reconstituted from separately expressed genes (Xue, Q., et al., A
multiplasmid approach to preparing large libraries of polyketides.
Proc Natl Acad Sci USA, 1999. 96(21): p. 11740-5; incorporated
herein by reference). Once a system is established for a particular
PKS of interest, one can integrate the PKS genes into stable,
neutral sites of the chromosome.
[0211] Preferred promoters include the glucose repressible alcohol
dehydrogenase 2 (ADH2) promoter and the galactose-inducible (GAL1)
promoter. The former has been used to produce high amounts of the
polyketide 6-MSA in yeast, and the latter is highly controllable by
galactose in the medium.
[0212] A model modular PKS that one can use to optimize the yeast
host is the well studied DEBS1. In this model system, the first ORF
of the modular PKS for erythromycin biosynthesis (DEBS1) has been
fused to a thioesterase domain (TE) and produces a readily
detectable triketide lactone when expressed in S. coelicolor, and
more recently E. coli (Kao, C. M., et al., Engineered biosynthesis
of a triketide lactone from an incomplete modular polyketide
synthase. J. Am. Chem. Soc., 1994. 116(25): p. 11612-11613; Cortes,
J., et al., Repositioning of a domain in a modular polyketide
synthase to promote specific chain cleavage. Science, 1995.
268(5216): p. 1487-9; both incorporated herein by reference). The
gene contains 2 PKS modules, is about 12 kB, and produces a protein
that is 300 kDa. This model allows one to optimize the engineered
host for acyl-CoA levels and post-translational modifications, the
PKS for G+C content, and to develop the needed analytical methods.
Once optimized for DEBS1, one can express any given modular
PKS.
[0213] Previously, it has been shown that the fungal gene encoding
6-methylsalicylic acid synthase (6-MSAS) from Penicillium patulum
was expressed in S. cerevisiae and E. coli and the polyketide
6-methylsalicylic acid (6-MSA) was produced (Kealey, J. T., et al.,
Production of a polyketide natural product in
nonpolyketide-producing prokaryotic and eukaryotic hosts. Proc Natl
Acad Sci USA, 1998. 95(2): p. 505-9; incorporated herein by
reference). In both bacterial and yeast hosts, polyketide
production required co-expression of 6-MSAS and a heterologous
phosphopantetheinyl transferase (Sfp), which was required to
convert the expressed apo-PKS to the holo-enzyme. Production of
6-MSA in E. coli was both temperature- and glycerol-dependent and
levels of production (.about.60 mg/L) were lower than those of the
native host, P. patulum. In yeast, the 6-MSAS and sfp genes were
co-expressed from separate replicating plasmids, and gene
expression was driven by the glucose repressible alcohol
dehydrogenase 2 (ADH2) promoter. In a non-optimized shake flask
fermentation, the yeast system produced 6-MSA at levels of 2,000
mg/L. This was the first report of expression of an intact PKS gene
in yeast or E. coli, and demonstrated that extraordinarily high
levels of polyketides can be produced in yeast.
[0214] Previously, a two vector system was developed for
heterologous expression of the three genes comprising the DEBS
polyketide gene cluster (Ziermann, R., Betlach, M., A Two-vector
System for the Production of Recombinant Polyketides in
Streptomyces. J. Ind. Microbiol. Biotech., 2000, 24:46-50;
incorporated herein by reference). Individual DEBS genes and
pairwise combinations of two such genes were each cloned downstream
of the actinorhodin (acti) promoter in two compatible Streptomyces
vectors: the autonomously replicating vector, pKAO127'Kan', and the
integrating vector, pSET152. When the resulting plasmids were
either simultaneously or sequentially transformed into the
heterologous host, Streptomyces lividans K4-114, the polyketide
product, 6-dEB, was produced. This work showed that the DEBS genes
could be split apart and expressed on separate plasmids, and that
efficient trans-complementation of modular polyketide synthase
subunit proteins occurred in the heterologous host.
[0215] A three-plasmid system for heterologous expression of DEBS
has been developed to facilitate combinatorial biosynthesis of
polyketides made by type I modular PKSs (Xue, Q., et al., A
multiplasmid approach to preparing large libraries of polyketides.
Proc Natl Acad Sci USA, 1999. 96(21): p. 11740-5; incorporated
herein by reference). The eryA PKS genes encoding the three DEBS
subunits were individually cloned into three compatible
Streptomyces vectors carrying mutually selectable antibiotic
resistance markers. A strain of Streptomyces lividans transformed
with all three plasmids produced 6-dEB at a level similar to that
of a strain transformed with a single plasmid containing all three
genes. The utility of this system in combinatorial biosynthesis was
demonstrated through production of a large library of greater than
60 modified polyketide macrolactones, using versions of each
plasmid constructed to contain defined mutations. Combinations of
these vector sets were introduced into S. lividans, resulting in
strains producing a wide range of 6-dEB analogs. This method can be
extended to any modular PKS and has the potential to produce
thousands of novel natural products, including ones derived from
further modification of the PKS products by tailoring enzymes.
Moreover, the ability to express the modular PKSs (such as DEBS)
from three separate plasmids provides advantages in the
commercialization of polyketide production by heterologous
expression of modular PKSs in yeast and E. coli in accordance with
the methods of the present invention.
[0216] As described in Example 1, the translationally coupled
genes, mutA and mutB, encoding the .beta.- and .alpha.-subunits of
methylmalonyl-CoA mutase from Propionibacterium shermanii, were
amplified by PCR and inserted into an E. coli expression vector
containing a T-7 promoter. The naturally occurring GTG start codon
for mutB was changed to ATG to facilitate expression (Amaratunga,
M., et al., A synthetic module for the metH gene permits facile
mutagenesis of the cobalamin-binding region of Escherichia coli
methionine synthase: initial characterization of seven mutant
proteins. Biochemistry, 1996. 35(7): p. 2453-63; incorporated
herein by reference). Heterologous expression of the mutase genes
in media containing [.sup.3H] .beta.-alanine and the
adenosylcobalamin (coenzyme B.sub.12) precursor, hydroxocobalamin,
yielded active methylmalonyl-CoA mutase. HPLC analysis of extracts
from E. coli BL21(DE3)/panD harboring the mutase genes indicated
production of methylmalonyl-CoA, which comprised 13% of the
intracellular CoA pool (shown in FIG. 6). This work demonstrates
that one can introduce a biosynthetic pathway for an important PKS
substrate into a heterologous host, and that one can measure the
intracellular concentration of acyl-CoAs. In accordance with the
present inveniton, the methylmalonyl-CoA mutase gene (sbm) from E.
coli, which has codon usage closer to yeast and encodes a single
polypeptide (Haller, T., et al., Discovering new enzymes and
metabolic pathways: conversion of succinate to propionate by
Escherichia coli. Biochemistry, 2000. 39(16): p. 4622-9;
incorporated herein by reference), can also be employed.
[0217] FIG. 6 shows acyl-CoA analysis of E. coli overexpressing
methylmalonyl-CoA mutase. The level of .sup.3H detected in
fractions collected from HPLC of cell-free extracts from [.sup.3H]
.beta.-alanine-fed E. coli harboring either the pET control vector
(solid trace) or pET overexpressing the mutase (dashed trace) is
shown.
[0218] As described in Example 1, methylmalonyl-CoA epimerase was
purified from Propionibacterium shermanii and N-terminal and
internal protein sequence was obtained. Degenerate PCR primers
based on the amino acid sequences were designed and were used to
amplify a 180 bp PCR product from P. shermanii genomic DNA. The PCR
product was labeled and used to isolate the epimerase gene from P.
shermanii. The methylmalonyl-CoA epimerase genes from B. subtilis
(Haller, T., et al., Discovering new enzymes and metabolic
pathways: conversion of succinate to propionate by Escherichia
coli. Biochemistry, 2000. 39(16): p. 4622-9; incorporated herein by
reference), and S. coelicolor can also be employed in the methods
of the present invention.
[0219] Propionyl-CoA is not detected in E. coli SJ16 cells grown in
the presence of [.sup.3H] .beta.-alanine with or without the
addition of propionate in the growth media. When E. coli SJ16 cells
were transformed with a pACYC-derived plasmid containing the
Salmonella typhimurium propionyl-CoA ligase gene (prpE) under the
control of the lac promoter, a small amount of propionyl-CoA was
observed (.about.0.2% of total CoA pool) in cell extracts. When 5
mM sodium propionate was included in the culture medium, about
14-fold more propionyl-CoA was produced (.about.3% of the total CoA
pool).
[0220] FIG. 7 shows acyl-CoA analysis in S. cerevisiae. The level
of .sup.3H detected in fractions collected from HPLC of cell-free
extracts from [.sup.3H] .beta.-alanine-fed S. cerevisiae after
growth for 24 hours (solid trace), 48 hours (dashed trace) and 66
hours (dotted trace) is shown. The yeast strain InvScl (Kealey, J.
T., et al., Production of a polyketide natural product in
nonpolyketide-producing prokaryotic and eukaryotic hosts. Proc Natl
Acad Sci USA, 1998. 95(2): p. 505-9; incorporated herein by
reference), grown in synthetic YNB media lacking pantothenate and
.beta.-alanine, was used for acyl-CoA analysis. Yeast cultures
starved of .beta.-alanine were fed [.sup.3H] .beta.-alanine and the
cultures were grown for 24, 48 and 66 hours at 30.degree. C. Cells
were disrupted with glass beads in the presence of 10% cold TCA and
acyl-CoAs were separated by HPLC and quantified by scintillation
counting. The yeast CoA pools were labeled with [.sup.3H], but the
extent of isotope dilution remains unclear. One can measure the
specific activity of total CoA in these strains to ascertain the
extent of isotope dilution.
[0221] For PKS genes and initial studies of metabolic pathway
genes, one can employ the analogous sets of bluescript cloning
vectors and yeast 2 micron replicating shuttle vectors used in
6-MSA production (Kealey, J. T., et al., Production of a polyketide
natural product in nonpolyketide-producing prokaryotic and
eukaryotic hosts. Proc Natl Acad Sci USA, 1998. 95(2): p. 505-9;
incorporated herein by reference). With these vectors, yeast
expression is driven by the alcohol dehydrogenase 2 (ADH2)
promoter, which is tightly repressed by glucose and is highly
active following glucose depletion that occurs after the culture
reaches high density. Both vector sets have a "common cloning
cassette" that contains, from 5' to 3', a polylinker (L1), the ADH2
(or other) promoter, a Nde I restriction site, a polylinker (L2),
an ADH2 (or other) terminator, and a polylinker (L3). Due to excess
restriction sites in the yeast shuttle vectors, genes of interest
are first introduced into intermediate bluescript cloning vectors
via the Nde I site, to generate the ATG start codon, and a
downstream restriction site in the L2 polylinker that is common to
the bluescript and yeast shuttle vectors (shown in FIG. 8). The
promoter-gene cassette is then excised as an L1-L2 fragment and
transferred to the yeast expression vector containing the
transcriptional terminator.
[0222] Host strains for model systems include commonly available
yeast strains with nutritional deficiencies (Ura, Trp, His, Leu)
that can harbor at least three replicating vectors (see below). If
it is necessary to express more than three PKS genes
simultaneously, one can clone multiple promoter-PKS gene-terminator
cassettes into the same vector or use a fourth replicating vector
with a different nutritional marker (i.e. Leu) or an antibiotic
marker (i.e. G418). One can also construct an analogous set of
bluescript cloning and yeast expression/shuttle vectors containing
a galactose-inducible promoter. The galactose promoter-Gal4
activator system is more tightly regulated than the ADH2 promoter,
and may be beneficial or necessary for expression of proteins that
are toxic to yeast (Mylin, L. M., et al., Regulated GAL4 expression
cassette providing controllable and high-level outputfrom high-copy
galactose promoters in yeast. Methods Enzymol, 1990. 185: p.
297-308; incorporated herein by reference).
[0223] Genes involved in the production of substrates (eg.
methylmalonyl-CoA and/or propionyl-CoA), and the sfp gene can
preferably be stably integrated into the yeast chromosome in
appropriate copy number to produce adequate levels of desired
acyl-CoAs and post translational PKS modifications. Genes can first
be introduced into the intermediate bluescript cloning vector as
described. Then, the fragment containing the
promoter-gene-terminator cassette can be transferred as a L1-L3
fragment to a yeast "delta integration" vector (Lee, F. W. and N.
A. Da Silva, Improved efficiency and stability of multiple cloned
gene insertions at the delta sequences of Saccharomyces cerevisiae.
Appl Microbiol Biotechnol, 1997. 48(3): p. 339-45; Lee, F. W. and
N. A. Da Silva, Sequential delta-integration for the regulated
insertion of cloned genes in Saccharomyces cerevisiae. Biotechnol
Prog, 1997. 13(4): p. 368-73; both incorporated herein by
reference) that allows chromosomal integration of the cassettes
into one or more of the ca. 425 delta sequences dispersed
throughout the yeast chromosome (see FIG. 9). These vectors have
cloning sites compatible with those in the L1-L3 linkers to permit
direct transfer of promoter-gene-terminator cassettes as L1-L3
fragments. They also contain the excisable Ura3 selection marker
flanked by two bacterial hisG repeats ("URA Blaster"), enabling
insertion of multiple identical or different genes into the yeast
chromosome by repetitive integrations. After selection for gene
integration on media lacking uracil, the Ura3 gene fragment is
removed by selecting for marker loss via excisional recombination
by positive selection with 5-fluoroorotic acid (FOA), which renders
the Ura3 gene toxic to yeast. This enables the introduction of
stable pathways needed for acyl-CoA precursors and Sfp into yeast,
while conserving the Ura marker to allow its subsequent use in
plasmids containing other genes.
[0224] The single-gene mutase, Sbm (Sleeping beauty mutase), from
E. coli (Haller, T., et al., Discovering new enzymes and metabolic
pathways: conversion of succinate to propionate by Escherichia
coli. Biochemistry, 2000. 39(16): p. 4622-9; incorporated herein by
reference), can be cloned as follows. Primers designed based on the
DNA sequence were used to PCR amplify the sbm gene from E. coli
genomic DNA as a NdeI-L2 fragment. The general strategy for cloning
the genes into yeast expression vectors follows that of Kealey et
al. (Kealey, J. T., et al., Production of a polyketide natural
product in nonpolyketide-producing prokaryotic and eukaryotic
hosts. Proc Natl Acad Sci USA, 1998. 95(2): p. 505-9; incorporated
herein by reference) (see FIG. 9). One can first clone the genes as
NdeI-L2 fragments into the intermediate bluescript cloning vector.
The promoter-gene-terminator cassette can then be excised as an
L1-L3 fragment, transferred to the yeast integrating vector,
restricted with L1/L3, and introduced into the yeast chromosome as
described above. As an alternative to Sbm, one can use the two-gene
mutase from P. shermanii; the translationally coupled genes have
each been amplified by PCR as NdeI-L2 fragments and can be
integrated into yeast as described above.
[0225] The genes encoding matABC have been cloned into a bluescript
vector (An, J. H. and Y. S. Kim, A gene cluster encoding
malonyl-CoA decarboxylase (MatA), malonyl-CoA synthetase (MatB) and
a putative dicarboxylate carrier protein (MatC) in Rhizobium
trifolii--cloning, sequencing, and expression of the enzymes in
Escherichia coli. Eur J Biochem, 1998. 257(2): p. 395-402;
incorporated herein by reference). One can isolate the matB
(methylmalonyl-CoA ligase) and matC (dicarboxylic acid transporter)
genes by PCR, each as a NdeI-L2 fragment, and integrate them into
the yeast chromosome as described above and shown in the schematic
below. Yeast transformed with matBC will be treated with
methylmalonic acid, and cells extracts can be analyzed for
methylmalonyl-CoA.
[0226] The pccB and accA1 genes involved in the propionyl-CoA
carboxylation pathway in S. coelicolor can be amplified by PCR from
genomic DNA. As shown in FIG. 9, the genes can be cloned into the
intermediate bluescript vector between Nde I and L2, then
transferred to the yeast integrating vector via L1/L3. One can
express the S. coelicolor genes shown to be effective in E. coli;
should codon usage be suboptimal, one can employ the B. subtilis
orthologs (discussed above).
[0227] FIG. 9 shows a general method for cloning genes into yeast
expression vectors.
[0228] In one embodiment, the recombinant yeast host cells of the
invention co-express the B. subtilis P-pant transferase, Sfp, with
a PKS to convert the apo PKS to its holo form. The sfp gene is
available on Bluescript.TM. (Stratagene) cloning and yeast
shuttle/expression vectors and is functional in yeast (Kealey, J.
T., et al., Production of a polyketide natural product in
nonpolyketide-producing prokaryotic and eukaryotic hosts. Proc Natl
Acad Sci USA, 1998. 95(2): p. 505-9; incorporated herein by
reference), so one can simply construct stable strains expressing
this gene. One to several copies (as determined optimal) of the sfp
gene can be introduced into delta sequences in the yeast chromosome
as described above. One can test the activity of the integrated sfp
gene by co-expressing 6-MSAS on a replicating vector, by measuring
the Sfp-dependent 6-MSA production (Kealey, J. T., et al.,
Production of a polyketide natural product in
nonpolyketide-producing prokaryotic and eukaryotic hosts. Proc Natl
Acad Sci USA, 1998. 95(2): p. 505-9; incorporated herein by
reference), and by quantifying the incorporation of [.sup.3H]
.beta.-alanine into the ACP domain of the PKS (see below). This
allows one to determine the optimal number of copies of the sfp
gene needed for maximal polyketide production.
[0229] The gene for the modular PKS, DEBS1+TE, is available as a
NdeI-EcoRI fragment, which can be readily introduced into a yeast
shuttle/expression vector as indicated in FIG. 9. Yeast strains
expressing DEBS1+TE are analyzed for the
[.sup.3H]-phosphopantetheinylation of the PKS, and for production
of triketide lactone by liquid chromatography/mass
spectrometry.
[0230] .sup.3H labeling of intracellular Acyl-CoAs is carried out
as follows. Cells are treated with [.sup.3H] .beta.-alanine
(available at 50 Ci/mmol) in defined media lacking pantothenate,
enabling the radioactive precursor of pantothenate to enter the CoA
pool. Cells are then disrupted, CoA esters are separated by HPLC,
and the radioactivity quantified by liquid scintillation counting,
as described above.
[0231] Saccharomyces cerevisiae host cells are grown, and extracts
prepared as follows. Defined minimal YNB media (1 mL) lacking
pantothenate but containing 1 .mu.M .beta.-alanine are inoculated
with a single colony of S. cerevisiae (InvScl, or Fen2b deletion
strain) from a YPD plate. The culture is grown to stationary phase
and 10 .mu.l of the stationary culture are used to inoculate the
above media lacking .beta.-alanine and pantothenate. The culture is
incubated for 4 hours and 10 .mu.l of the "starved" culture is used
to inoculate media (1 mL) containing 10 .mu.Ci [.sup.3H]
.beta.-alanine (50 Ci/mmol; 0.2 mM final .beta.-alanine). After
culture growth for appropriate times, the cells from a 1 mL culture
are collected by centrifugation and washed with water. The cells
are suspended in 200 .mu.l of 10% cold trichloroacetic acid (TCA),
containing standard unlabeled acyl-CoAs as chromatography markers
(malonyl-, methylmalonyl-, succinyl-, acetyl-, propionyl-CoA, and
CoA). The cells are disrupted by vortexing with glass beads, and
the supernatent analyzed by HPLC.
[0232] HPLC is performed using a 150.times.4.6 mm 5.mu. ODS-3
INERTSIL HPLC column purchased from Metachem technology. HPLC
buffer A is 100 mM sodium phosphate monobasic, 75 mM sodium
acetate, pH 4.6 and buffer B is 70% buffer A, 30% methanol. The
HPLC column is equilibrated at 10% buffer B at a flow rate of 1
mL/min. Following injection, a linear gradient to 40% buffer B is
implemented over 35 minutes, followed by a linear gradient to 90%
buffer B over 20 minutes. The gradient affords base-line separation
of the standard acyl-CoAs. The eluant is monitored at 260 nm and
fractions are collected and counted in a scintillation counter.
[0233] Determination of the specific activity of the total CoA pool
is carried out as follows. S. cerevisiae cultures are labeled with
100 .mu.Ci of [.sup.3H] .beta.-alanine as described above. The
yeast cells are disrupted and the extract is treated with 100 .mu.M
hydroxylamine, pH 8.5, to convert all acyl-CoAs to CoA. The labeled
CoA is isolated by HPLC as described above and converted to
acetyl-CoA with E. coli acetyl-CoA synthase (Sigma), using
[.sup.14C]-acetate as a substrate. The [.sup.3H,
.sup.14C]-acetyl-CoA is separated by HPLC and the dual labels
quantified by scintillation counting. The mmol CoA is determined by
.sup.14C, and specific activity of CoA determined from the .sup.3H
dpm per mmol CoA. The isotope dilution, reflecting endogenous
production of .beta.-alanine, is calculated by the specific
activity of [.sup.3H] CoA/specific activity [.sup.3H]
.beta.-alanine used in the test.
[0234] Analysis of PKS expression levels is carried out as follows.
Each ACP domain of each module of an active PKS is
post-translationally modified with phosphopantetheine derived from
CoA. Using yeast cells treated with [.sup.3H] .beta.-alanine
(described above), one can label the PKS with high specific
activity tritium. The protein will be separated on SDS-PAGE, eluted
and radioactivity determined by liquid scintillation counting.
EXAMPLE 3
Conversion of Erythronolides to Ervthromycins
[0235] A sample of a polyketide (.about.50 to 100 mg) is dissolved
in 0.6 mL of ethanol and diluted to 3 mL with sterile water. This
solution is used to overlay a three day old culture of
Saccharopolyspora erythraea WHM34 (an eryA mutant) grown on a 100
mm R2YE agar plate at 30.degree. C. After drying, the plate is
incubated at 30.degree. C. for four days. The agar is chopped and
then extracted three times with 100 mL portions of 1% triethylamine
in ethyl acetate. The extracts are combined and evaporated. The
crude product is purified by preparative HPLC (C-18 reversed phase,
water-acetonitrile gradient containing 1% acetic acid). Fractions
are analyzed by mass spectrometry, and those containing pure
compound are pooled, neutralized with triethylamine, and evaporated
to a syrup. The syrup is dissolved in water and extracted three
times with equal volumes of ethyl acetate. The organic extracts are
combined, washed once with saturated aqueous NaHCO.sub.3, dried
over Na.sub.2SO.sub.4, filtered, and evaporated to yield
.about.0.15 mg of product. The product is a glycosylated and
hydroxylated compound corresponding to erythromycin A, B, C, and D
but differing therefrom as the compound provided differed from
6-dEB.
EXAMPLE 4
Measurement of Antibacterial Activity
[0236] Antibacterial activity is determined using either disk
diffusion assays with Bacillus cereus as the test organism or by
measurement of minimum inhibitory concentrations (MIC) in liquid
culture against sensitive and resistant strains of Staphylococcus
pneumoniae.
EXAMPLE 5
Evaluation of Antiparasitic Activity
[0237] Compounds can initially screened in vitro using cultures of
P. falciparum FCR-3 and
[0238] K1 strains, then in vivo using mice infected with P.
berghei. Mammalian cell toxicity can be determined in FM3A or KB
cells. Compounds can also be screened for activity against P.
berhei. Compounds are also tested in animal studies and clinical
trials to test the antiparasitic activity broadly (antimalarial,
trypanosomiasis and Leishmaniasis).
[0239] The invention having now been described by way of written
description and example, those of skill in the art will recognize
that the invention can be practiced in a variety of embodiments and
that the foregoing description and examples are for purposes of
illustration and not limitation of the following claims.
Sequence CWU 1
1
21447DNAArtificial SequenceIsolated and recombinant form of the
full epimerase gene sequence 1atg agt aat gag gat ctt ttc atc tgt
atc gat cac gtg gca tat gcg 48Met Ser Asn Glu Asp Leu Phe Ile Cys
Ile Asp His Val Ala Tyr Ala1 5 10 15tgc ccc gac gcc gac gag gct tcc
aag tac tac cag gag acc ttc ggc 96Cys Pro Asp Ala Asp Glu Ala Ser
Lys Tyr Tyr Gln Glu Thr Phe Gly20 25 30tgg cat gag ctc cac cgc gag
gag aac ccg gag cag gga gtc gtc gag 144Trp His Glu Leu His Arg Glu
Glu Asn Pro Glu Gln Gly Val Val Glu35 40 45atc atg atg gcc ccg gct
gcg aag ctg acc gag cac atg acc cag gtt 192Ile Met Met Ala Pro Ala
Ala Lys Leu Thr Glu His Met Thr Gln Val50 55 60cag gtc atg gcc ccg
ctc aac gac gag tcg acc gtt gcc aag tgg ctt 240Gln Val Met Ala Pro
Leu Asn Asp Glu Ser Thr Val Ala Lys Trp Leu65 70 75 80gcc aag cac
aat ggt cgc gcc gga ctg cac cac atg gca tgg cgt gtc 288Ala Lys His
Asn Gly Arg Ala Gly Leu His His Met Ala Trp Arg Val85 90 95gat gac
atc gac gcc gtc agc gcc acc ctg cgc gag cgc ggc gtg cag 336Asp Asp
Ile Asp Ala Val Ser Ala Thr Leu Arg Glu Arg Gly Val Gln100 105
110ctg ctg tat gac gag ccc aag ctc ggc acc ggc ggc aac cgc atc aac
384Leu Leu Tyr Asp Glu Pro Lys Leu Gly Thr Gly Gly Asn Arg Ile
Asn115 120 125ttc atg cat ccc aag tcg ggc aag ggc gtg ctc atc gag
ctc acc cag 432Phe Met His Pro Lys Ser Gly Lys Gly Val Leu Ile Glu
Leu Thr Gln130 135 140tac ccg aag aac tga 447Tyr Pro Lys
Asn1452148PRTArtificial SequenceDeduced amino acid sequence of the
epimerase gene sequence 2Met Ser Asn Glu Asp Leu Phe Ile Cys Ile
Asp His Val Ala Tyr Ala1 5 10 15Cys Pro Asp Ala Asp Glu Ala Ser Lys
Tyr Tyr Gln Glu Thr Phe Gly20 25 30Trp His Glu Leu His Arg Glu Glu
Asn Pro Glu Gln Gly Val Val Glu35 40 45Ile Met Met Ala Pro Ala Ala
Lys Leu Thr Glu His Met Thr Gln Val50 55 60Gln Val Met Ala Pro Leu
Asn Asp Glu Ser Thr Val Ala Lys Trp Leu65 70 75 80Ala Lys His Asn
Gly Arg Ala Gly Leu His His Met Ala Trp Arg Val85 90 95Asp Asp Ile
Asp Ala Val Ser Ala Thr Leu Arg Glu Arg Gly Val Gln100 105 110Leu
Leu Tyr Asp Glu Pro Lys Leu Gly Thr Gly Gly Asn Arg Ile Asn115 120
125Phe Met His Pro Lys Ser Gly Lys Gly Val Leu Ile Glu Leu Thr
Gln130 135 140Tyr Pro Lys Asn145
* * * * *