U.S. patent application number 10/607809 was filed with the patent office on 2003-12-25 for production of polyketides.
Invention is credited to Katz, Leonard, Revill, Peter.
Application Number | 20030235892 10/607809 |
Document ID | / |
Family ID | 22581090 |
Filed Date | 2003-12-25 |
United States Patent
Application |
20030235892 |
Kind Code |
A1 |
Katz, Leonard ; et
al. |
December 25, 2003 |
Production of polyketides
Abstract
Recombinant host cells that comprise recombinant DNA expression
vectors that drive expression of a product and a precursor for
biosynthesis of that product can be used to produce useful products
such as polyketides in host cells that do not naturally produce the
product or produce the product at low levels due to the absence of
the precursor or the presence of the precursor in rate limiting
amounts.
Inventors: |
Katz, Leonard; (Oakland,
CA) ; Revill, Peter; (Oakland, CA) |
Correspondence
Address: |
MORRISON & FOERSTER LLP
3811 VALLEY CENTRE DRIVE
SUITE 500
SAN DIEGO
CA
92130-2332
US
|
Family ID: |
22581090 |
Appl. No.: |
10/607809 |
Filed: |
June 27, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10607809 |
Jun 27, 2003 |
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09697022 |
Oct 25, 2000 |
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6627427 |
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60161414 |
Oct 25, 1999 |
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Current U.S.
Class: |
435/76 ; 435/193;
435/252.3; 435/320.1; 435/69.1; 536/23.2 |
Current CPC
Class: |
C12P 19/62 20130101;
C12P 11/00 20130101; C12N 15/52 20130101 |
Class at
Publication: |
435/76 ;
435/69.1; 435/193; 435/252.3; 435/320.1; 536/23.2 |
International
Class: |
C12P 019/62; C07H
021/04; C12P 021/02; C12N 005/06; C12N 001/21; C12N 015/74; C12N
009/10 |
Claims
1. A recombinant polyketide synthase gene that encodes a loading
module comprising a KS.sup.Q domain, an AT specific for
ethylmalonyl CoA, and an ACP domain.
2. A host cell that comprises the recombinant polyketide synthase
gene of claim 1, wherein said host cell further comprises a
recombinant gene selected from the group consisting of recombinant
ccr and icm genes.
3. A method for the production of 14,15-propenylerythromycin and/or
the corresponding 14,15-propenyl-6-deoxyerythronolide B, which
comprises culturing a recombinant host cell that expresses
isobutyryl CoA mutase, valine dehydrogenase, butyryl CoA
dehydrogenase, and 6-deoxyerythronolide polyketide synthase.
4. The method of claim 3, wherein said host cell is a
Saccharopolyspora erythraea host cell.
5. The method of claim 4, wherein said host cell does not express a
functional eryM gene product.
6. The method of claim 3, wherein said butyryl CoA dehydrogenase is
expressed from a gene isolated from Clostridium acetobutylicum or
Mycobacterium tuberculosis (fadE25).
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. patent application
Serial No. 60/161,414, filed Oct. 25, 1999, and is related to U.S.
patent application Serial No. 60/161,703, filed Oct. 27, 1999, and
No. 60/206,082, filed May 18, 2000, each of which is incorporated
herein by reference.
FIELD OF THE INVENTION
[0002] 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
[0003] 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,
megalomycin, 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; and
5,712,146; 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.
[0004] 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 "fungal" PKS enzymes.
[0005] Modular PKSs are responsible for producing a large number of
12-, 14-, and 16-membered macrolide antibiotics including
erythromycin, megalomycin, 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).
[0006] 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 No. WO
98/49315, incorporated herein by reference).
[0007] The ability to control aspects of polyketide biosynthesis,
such as monomer selection and degree of 3-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.
[0008] One example of this technology involves the use of a PKS in
which the first extender module is inactivated by mutation and
synthetic molecules, called diketides. These diketides are provided
to the altered PKS and bind to the second extender module. The
diketides are then processed by the PKS in the normal fashion to
yield a polyketide. If the diketide provided differs in structure
from the corresponding diketide that is the product of the first
extender module, then the polyketide will correspondingly differ
from the natural polyketide produced by the intact PKS. See PCT
patent publication Nos. 97/02358 and 99/03986, each of which is
incorporated herein by reference. One important compound produced
by this technology resulted from feeding a propyl diketide to DEBS
to produce 15-methyl-6-dEB. This molecule is referred to herein as
propyl-6-dEB, because it has a C-13 propyl group where 6-dEB has a
C-13 ethyl group.
[0009] While the diketide feeding technology provides useful
amounts of compound, the cost of producing polyketides by that
technology is increased by the need to prepare the synthetic
diketide. Moreover, certain polyketide producing cells degrade some
of the diketide before it can be incorporated into a polyketide by
the PKS, thus increasing the cost of production. Thus, there
remains a need for methods to produce polyketides by other means.
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
[0010] The present invention provides recombinant host cells and
expression vectors for making propyl-6-dEB and compounds derived
therefrom. The present invention also provides methods for
increasing the amounts of propyl-6-dEB produced in a host cell by
providing recombinant biosynthetic pathways for production of a
precursor utilized in the biosynthesis of the compound and
optionally altering other biosynthetic pathways in the cell.
[0011] In one embodiment, the host cell does not produce
propyl-6-dEB, and the host cell is modified by introduction of a
recombinant expression vector so that it can produce the compound.
In another embodiment, propyl-6-dEB 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 compound
in larger amounts. In a preferred embodiment, the host cell is
altered to produce the precursor butyryl CoA by transferring the
genes that encode the enzymes that produce butyryl CoA from a first
cell to the host cell. The transfer is accomplished using an
expression vector of the invention. The expression vector drives
expression of the genes and production of butyryl CoA in the second
cell.
[0012] In another embodiment, the product is a polyketide other
than 6-dEB that is made by a PKS that utilizes propionyl CoA. The
polyketide produced by the host cell of the invention containing
the PKS differs from the usual product produced by the PKS in that
butyryl CoA instead of propionyl CoA is utilized by the PKS in
producing the polyketide. The polyketide is a polyketide
synthesized by either a modular, iterative, or fungal PKS. In one
preferred embodiment, the polyketide is synthesized by a modular
PKS.
[0013] In one embodiment, the host cell is either a procaryotic or
eukaryotic host cell. In one embodiment, the host cell is a
Saccharopolyspora host cell, including but not limited to S.
erythraea. In another embodiment, the host cell is a Streptomyces
host cell, including but not limited to S. coelicolor, S. lividans,
and S. venezuelae. In another 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 a plant host
cell.
[0014] 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 butyryl CoA. 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.
[0015] 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.
[0016] 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
[0017] FIG. 1 shows the modules and domains of DEBS and the
biosynthesis of 6-dEB from propionyl CoA and methylmalonyl CoA.
[0018] FIG. 2 shows biosynthetic pathways for the production of
butyryl CoA and ethylmalonyl CoA.
DETAILED DESCRIPTION OF THE INVENTION
[0019] 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. 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.
[0020] 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 a 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.
[0021] 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.
[0022] Thus, in a preferred embodiment, the product produced by the
cell is a polyketide.
[0023] 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, butyryl CoA, propionyl CoA,
methylmalonyl CoA, ethylmalonyl CoA, and hydroxymalonyl CoA. In an
especially preferred embodiment the polyketide utilizes butyryl or
ethylmalonyl CoA in its biosynthesis. In another preferred
embodiment, the polyketide is synthesized by a modular PKS.
[0024] 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.
[0025] Macrolides such as erythromycin and megalomycin 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).
[0026] 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/DH/ER, 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.
[0027] More particularly, the loading module of DEBS consists of
two domains, an acyl-transferase (AT) domain and an acyl carrier
protein (ACP) domain. In some other PKS enzymes, the loading module
is not composed of an AT and an ACP but instead utilizes an
inactivated KS, an AT, and an ACP. This 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 activity. The AT domain of
the loading module recognizes a particular acyl CoA (propionyl for
DEBS, which can also accept acetyl or butyryl) 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.
[0028] 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 polyketone, 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.
[0029] 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 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.
[0030] DEBS is produced naturally in Saccharopolyspora erythraea
and has been transferred via recombinant DNA methodology 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 (using the propionyl
CoA starter unit) and 8,8a-deoxyoleandolide (using the acetyl CoA
starter unit). These two compounds differ from one another with
regard to the substituent at the C-13 position: 6-dEB has an ethyl
and 8,8a-deoxyoleandolide a methyl at the C-13 position. Using a
recombinant version of the DEBSI protein, researchers reported that
DEBS could also accept a butyryl Co A starter unit (see Pieper et
al., 1996, Biochemistry 35: 2054-2060, incorporated herein by
reference). If the complete DEBS enzyme utilized butyryl CoA as a
starter unit, then the product of the PKS would be
propyl-6-dEB.
[0031] Propyl-6-dEB has also been prepared using a recombinant DEBS
enzyme in which the KS domain of extender module 1 was inactivated
by a mutation changing the active site cysteine residue to an
alanine residue. When the recombinant PKS was provided an activated
form of a synthetic diketide having the structure of a diketide
produced by the DEBS-mediated condensation of a butyryl CoA starter
unit and a methylmalonyl CoA extender unit, propyl-6-dEB was
produced. Moreover, when propyl-6-dEB was provided to
Saccharopolyspora erythraea cells, the cells could take up the
aglycone and convert it to the corresponding propyl analogs of
erythromycin A, B, C, and D. See PCT patent publication Nos.
99/03986, 97/02358, and 98/49315, incorporated herein by reference.
Propyl erythromycin A is a particularly useful compound in that it
has potent antibiotic activity and can be readily converted to even
more active compounds, such as the ketolides (see PCT patent
applications US00/09914 and US00/09915, both of which are
incorporated herein by reference) by chemical methodology.
[0032] Although the above results demonstrate that DEBS can accept
butyryl CoA as a starter unit, complete the synthesis of
propyl-6-dEB, and then modify the aglycone to yield the propyl
erythromycins, the propyl erythromycins have not been reported in
fermentations of erythromycin producing Saccharopolyspora erythraea
strains. In one embodiment, the present invention provides
recombinant S. erythraea strains that produce propyl-6-dEB and the
propyl erythromycins.
[0033] In a first aspect, this embodiment of the invention is
exemplified by recombinant Saccharopolyspora erythraea strains in
which the eryM gene has been inactivated by mutation. The eryM gene
encodes a malonyl decarboxylase that converts methylmalonyl CoA to
propionyl CoA. When the eryM gene is inactivated by mutation, the
production of erythromycin in S. erythraea in minimal media is
dependent on the feeding of exogenous propionic acid. See, Hsieh
& Kolattukudy, 1994, J. Bacteriol. 176: 714-724, incorporated
herein by reference. In accordance with a method of the invention,
an eryM mutant strain is provided with exogenous butyric acid and
fermented to produce propyl-6-dEB and the propyl erythromycins.
[0034] While the above method can result in the production of the
desired compounds, the requirement of adding exogenous butyric acid
to the fermentation media is not only inconvenient but also limits
production to the amount of butyric acid that can be taken up by
the cells. This can be addressed by the addition of transporter and
CoA ligase genes of the appropriate specificity (see, e.g., U.S.
patent application Serial No. 60/206,082, filed May 18, 2000,
incorporated herein by reference). As another means of overcoming
this potential limitation, the present invention provides
recombinant host cells that make butyryl CoA. Because these host
cells can make butyryl CoA, providing exogenous butyric acid to the
media is not required, although it may in some instances increase
the amount of propyl-6-dEB and propyl erythromycins produced.
[0035] The present invention provides a wide variety of such host
cells. In general, these host cells comprise one or more
biosynthetic pathways for producing butyryl CoA. FIG. 2 shows two
illustrative pathways. On the left side of the Figure, the pathway
starting with valine is shown. On the right side of the Figure, the
pathway starting with acetyl CoA is shown. The host cells of the
invention can comprise either or both of these pathways. In one
embodiment, the recombinant host cell is derived from a host cell
that lacks either pathway, and the genes for one or both pathways
are added to provide the host cell of the invention. In another
embodiment, the recombinant host cell is derived from a host cell
that has one pathway but lacks the other, and the genes for the
other pathway are added to provide the host cell of the
invention.
[0036] As shown on the left side of FIG. 2, butyryl CoA can be
synthesized from valine by a pathway in which valine is first
converted to isobutyryl CoA by the enzyme valine dehydrogenase.
This enzyme is the product of the vdh gene, which can be isolated
from Streptomyces coelicolor, S. fradiae, S. cinnamonensis, and
other host cells. Isobutyryl CoA is then converted to butyryl CoA
by isobutyryl CoA mutase. This enzyme is present in S.
cinnamonensis in a two subunit form; the subunits are the products
of the icmA and icmB genes. Genes encoding this enzyme can also be
isolated from other host cells. As shown on the right side of FIG.
2, butyryl CoA can be synthesized from acetyl CoA by a pathway in
which acetyl CoA is first converted to acetoacetyl CoA by the
enzyme acetoacetyl CoA synthetase. This enzyme is the product of
the acsA gene, which can be isolated from Streptomyces coelicolor
and other host cells. Acetoacetyl CoA is then converted to
beta-hydroxybutyryl CoA by the enzyme beta-hydroxybutyryl CoA
dehydrogenase. The gene (bdh) encoding this enzyme can be isolated
from Streptomyces coelicolor and other host cells.
Beta-hydroxybutyryl CoA is then converted to crotonyl CoA by the
enzyme enoyl CoA hydratase. The gene (ech) encoding this enzyme can
be isolated from Streptomyces coelicolor and other host cells.
Crotonyl CoA is then converted to butyryl CoA by the enzyme
crotonyl CoA reductase. The gene. (ccr) encoding this enzyme can be
isolated from Streptomyces coelicolor and other host cells.
[0037] Those of skill in the art will recognize that, depending on
the host cell selected, not all of the genes in a pathway may be
required. This is because some host cells will express one or more
genes in a pathway naturally, requiring the addition of only a few
of the genes to complete the pathway. This aspect of the invention
is illustrated with Saccharopolyspora erythraea, which is modified
in accordance with the invention to make propyl-6-dEB and the
propyl erythromycins instead of 6-dEB and the erythromycins. Thus,
as discussed above, one can modify S. erythraea cells to knock out
or otherwise inactivate the eryM gene. The thus modified host cells
can be cultured in fermentation media comprising butyric acid to
provide the desired propyl-6-dEB and propyl erythromycins.
[0038] To improve production of the desired compounds, the cells
can be transformed with a recombinant DNA expression vector that
comprises the ccr gene. The cells may contain enough endogenous
product of acsA, bdh, and ech genes that merely the addition of the
ccr gene will increase the production of propyl-6-dEB and the
propyl erythromycins. Optionally, however, the cells are
transformed with a recombinant DNA expression vector that comprises
the ccr, acsA, bdh, and ech genes to provide the propyl-6-dEB and
propyl erythromycins in high yield.
[0039] Alternatively, the cells can be transformed with a
recombinant DNA expression vector that comprises the icmA and icmB
genes (hereinafter referred to as the icm gene). The cells may
contain enough endogenous product of the vdh gene that merely the
addition of the icm gene will increase the production of
propyl-6-dEB and the propyl erythromycins. Optionally, however, the
cells are transformed with a recombinant DNA expression vector that
comprises the icm and vdh genes to provide the propyl-6-dEB and
propyl erythromycins in high yield.
[0040] The invention also provides recombinant Saccharopolyspora
erythraea host cells in which various combinations of recombinant
genes are expressed. These host cells include but are not limited
to host cells that comprise the following sets of genes:
[0041] (i) ccr and icm;
[0042] (ii) ccr, acsA, bdh, ech, and icm;
[0043] (iii) ccr, acsA, bdh, ech, icm, and vdh; and
[0044] (iv) icm, vdh, and ccr.
[0045] Thus, the invention provides a diverse collection of
recombinant S. erythraea host cells that produce propyl-6-dEB and
the propyl erythromycins in high yield.
[0046] In another embodiment, the invention provides a method for
the production of 14,15-propenylerythromycin and/or the
corresponding 14,15-propenyl-6-deoxyerythronolide B using a
recombinant host cell that comprises isobutyryl CoA mutase, valine
dehydrogenase, and butyryl CoA dehydrogenase genes. In a preferred
embodiment, the host cell is a Saccharopolyspora erythraea host
cell that optionally does not express a functional eryM gene
product. In one embodiment, the butyryl CoA dehydrogenase (bcd)
gene is obtained from Clostridium acetobutylicum or Mycobacterium
tuberculosis (fadE25).
[0047] The host cells of the invention can also be employed for the
expression of recombinant PKS genes for a variety of purposes. As
but one non-limiting example, any PKS gene that contains at least
one AT domain that binds ethylmalonyl CoA can be expressed in
recombinant host cells of the invention that express a carboxylase
that converts butyryl CoA to ethylmalonyl CoA. A number of PKS
enzymes contain AT domains that bind ethylmalonyl CoA, including
but not limited to, the niddamycin PKS and the FK-520 PKS.
[0048] The present invention also provides a recombinant PKS gene
that encodes a PKS that has an ethylmalonyl CoA specific AT domain.
These recombinant PKS genes of the invention are useful in the
production of propyl-6-dEB and are characterized by having a
loading module comprised of a KS.sup.Q domain, an ethylmalonyl CoA
specific AT domain, and an ACP domain, followed by 6 extender
modules specific for methylmalonyl CoA. PKS genes that are
especially suitable for modification to yield the hybrid PKS of the
invention include the DEBS eryA genes, the oleA genes (for
oleandomycin), and the meg genes (for megalomycin).
[0049] In another embodiment, the present invention provides a
hybrid PKS in which the loading module is comprised of a KS.sup.Q
domain, an ethylmalonyl CoA specific AT domain, and an ACP domain,
and which also contains two or more, preferably 6, but optionally
7, extender modules, wherein at least one extender module has an AT
domain specific for malonyl CoA. For example, the loading module
can be linked to the 5 extender modules of methymycin or the 6
extender modules of narbomycin or picromycin.
[0050] The host cells of the invention are particularly suited for
expressing such hybrid PKS genes, because the host cells make
butyryl CoA, which is converted to ethylmalonyl CoA. The loading
module AT domain binds the ethylmalonyl CoA produced, and the
KS.sup.Q domain decarboxylates the ethylmalonyl CoA during its
incorporation into the polyketide. Thus, the polyketide produced is
propyl-6-dEB if the extender modules are those, encoded by the
eryA, oleA, or megA genes, and propyl-narbonolide if the extender
modules are those encoded by the picA genes.
[0051] 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. 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
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.
[0052] 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 butyryl 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
butyryl CoA and may also include additional elements for the
expression and/or regulation of expression of these genes, e.g.
promoter sequences.
[0053] 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 described 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.
[0054] 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.
[0055] In one important embodiment, the invention provides methods
for the heterologous expression of one or more of the biosynthetic
genes involved in butyryl 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.
[0056] 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,
p1, and pBR.
[0057] 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.
[0058] 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.
[0059] 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.
[0060] 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 erythraea 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. Erythromcyin 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, propyl-6-dEB are converted in S. erythraea to the
corresponding derivatives of erythromycins A, B, C, and D. 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). Another mutant strain useful in this
regard is the KS 1 null mutant strain that is typically employed in
diketide feeding, as this strain is unable to produce erythromycins
in the absence of added diketide. 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.
[0061] 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.
[0062] 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.
[0063] 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 confers 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).
[0064] 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.
[0065] 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, 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.
[0066] A hybrid PKS for purposes of the present invention can
result not only:
[0067] (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,
[0068] but also:
[0069] (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,
[0070] (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
[0071] (iv) from combinations of the foregoing. Various hybrid PKSs
of the invention illustrating these various alternatives are
described herein.
[0072] 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).
[0073] 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).
[0074] 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 Serial
No. 60/129,731, filed Apr. 16, 1999, and PCT Pub. No. 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 utlizing 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.
[0075] 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.
[0076] Avermectin
[0077] U.S. Pat. No. 5,252,474 to Merck.
[0078] 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.
[0079] MacNeil et al., 1992, Gene 115: 119-125, Complex
Organization of the Streptomyces avermitilis genes encoding the
avermectin polyketide synthase.
[0080] Candicidin (FR008)
[0081] Hu et al., 1994, Mol. Microbiol. 14: 163-172.
[0082] Epothilone
[0083] PCT patent publication No. WO US99/43653 to Kosan.
[0084] Erythromycin
[0085] PCT Pub. No. 93/13663 to Abbott.
[0086] U.S. Pat. No. 5,824,513 to Abbott.
[0087] Donadio et al., 1991, Science 252:675-9.
[0088] Cortes et al., Nov. 8, 1990, Nature 348:176-8, An unusually
large multifunctional polypeptide in the erythromycin producing
polyketide synthase of Saccharopolyspora erythraea.
[0089] Glycosylation Enzymes
[0090] PCT Pat. App. Pub. No. 97/23630 to Abbott.
[0091] FK-506
[0092] Motamedi et al., 1998, The biosynthetic gene cluster for the
macrolactone ring of the immunosuppressant FK506, Eur. J. biochem.
256: 528-534.
[0093] 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.
[0094] Methyltransferase
[0095] U.S. Pat. No. 5,264,355, issued Nov. 23, 1993, Methylating
enzyme from Streptomyces MA6858. 31-O-desmethyl-FK506
methyltransferase.
[0096] 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.
[0097] FK-520
[0098] PCT patent publication No. WO US00/020601 to Kosan.
[0099] See also Nielsen et al., 1991, Biochem. 30:5789-96
(enzymology of pipecolate incorporation).
[0100] Lovastatin
[0101] U.S. Pat. No. 5,744,350 to Merck.
[0102] Narbomycin (and Picromycin)
[0103] PCT patent publication No. WO US99/61599 to Kosan.
[0104] Nemadectin
[0105] MacNeil et al., 1993, supra.
[0106] Niddamycin
[0107] Kakavas et al., 1997, Identification and characterization of
the niddamycin polyketide synthase genes from Streptomyces
caelestis, J. Bacteriol. 179: 7515-7522.
[0108] Oleandomycin
[0109] 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.
[0110] PCT patent publication No. WO US00/026349 to Kosan.
[0111] 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.
[0112] Platenolide
[0113] EP Pat. App. Pub. No. 791,656 to Lilly.
[0114] Rapamycin
[0115] Schwecke et al., August 1995, The biosynthetic gene cluster
for the polyketide rapamycin, Proc. Natl. Acad. Sci. USA
92:7839-7843.
[0116] 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.
[0117] Rifamycin
[0118] August et al., Feb. 13, 1998, Biosynthesis of the ansamycin
antibiotic rifamycin: deductions from the molecular analysis of the
rifbiosynthetic gene cluster of Amycolatopsis mediterranei S669,
Chemistry & Biology, 5(2): 69-79.
[0119] Soraphen
[0120] U.S. Pat. No. 5,716,849 to Novartis.
[0121] 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.
[0122] Spiramycin
[0123] U.S. Pat. No. 5,098,837 to Lilly.
[0124] Activator Gene
[0125] U.S. Pat. No. 5,514,544 to Lilly.
[0126] Tylosin
[0127] EP Pub. No. 791,655 to Lilly.
[0128] Kuhstoss et al., 1996,: Gene 183:231-6, Production of a
novel polyketide through the construction of a hybrid polyketide
synthase.
[0129] U.S. Pat. No. 5,876,991 to Lilly.,
[0130] Tailoring Enzymes
[0131] 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.
[0132] 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.
[0133] 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., Apr. 16,
1999, Dissecting and Exploiting Intermodular Communication in
Polyketide Synthases", Science 284: 482-485, incorporated herein by
reference.
[0134] 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.
[0135] 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 (KS 1) 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 KS 1
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.
[0136] 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.
[0137] 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.
[0138] 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.
[0139] 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.
[0140] 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.
[0141] 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.
[0142] 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.
[0143] 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.
[0144] 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.
[0145] 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.
[0146] 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.
[0147] 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.
[0148] 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.
[0149] 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 erythraea or Streptomyces venezuelae or
other host cell to make the conversion, preferably using mutants
unable to synthesize macrolides, as discussed above.
[0150] 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.
[0151] 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
oreryF (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.
[0152] 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. 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.
[0153] 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.
[0154] 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 Peeters, 1999, Motilide
Web Site, http://www.med.kuleuven.ac.be/med/gih/m- otilid.htm, and
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.
[0155] 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.
[0156] 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.
[0157] 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.
[0158] 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.
[0159] 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.
[0160] 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.
[0161] 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.
[0162] It will be understood, however, that the specific dose level
for any particlular 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.
[0163] A detailed description of the invention having been provided
above, the following examples are given for the purpose of
illustrating the present invention and shall not be construed as
being a limitation on the scope of the invention or claims.
EXAMPLE 1
Construction of eryM Knockout Strains and Production of
15-Methyl-Erythromycins
[0164] This example describes the construction of two recombinant
DNA vectors designed to disrupt the eryM gene in Saccharopolyspora
erythraea by single crossover. These vectors can be used to
generate a strain of S. erythraea that produces higher titers of
15-methyl erythromycin A or C than does wild-type S. erythraea
under the same conditions without the need for the addition of an
exogenous diketide. The desired strain differs from the wild-type
strain in that intracellular pools of propanoyl-CoA are greatly
reduced, pools of butanoyl-CoA are greatly elevated, and pools of
methylmalonyl-CoA remain high. It has been shown in vitro that
6-dEB synthase accepts butanoyl-CoA as the starter equally
efficiently as propanoyl-CoA (Pieper et al., 1996, Biochemistry
35:2054). It has been reported that disruption of the eryM gene,
which encodes methylmalonyl decarboxylase, causes loss of
erythromycin production that can be restored by feeding propionate,
methylpropionate, or propanol in a wild-type strain of S. erythraea
(Hsieh and Kolattukudy, 1994, J. Bact. 176:714). This results
suggests that the EryM decarboxylase catalyzes the primary flux
from methylmalonyl CoA to propionyl CoA. This example shows how to
disrupt eryM by single-crossover for illustrative purposes.
Preferred strains of the invention are modified by deletion of the
eryM gene by double crossover, leaving no marker in the
chromosome.
[0165] The Saccharopolyspora erythraea eryM gene was isolated by
PCR of the coding region. The cloning vectors pWHM3 and pOJ260 are
well-known Streptomyces vectors. An internal fragment of the eryM
gene was isolated by PCR and cloned into the XbaI and HindIII sites
of the vectors pWHM3 (confers thiostrepton resistance) and pOJ260
(confers apramycin resistance) for gene disruption. The resulting
vectors were propagated in E. coli ET 12567 to obtain unmethylated
DNA. The eryM gene sequence showing the engineered XbaI and native.
HindIII sites used to clone the internal fragment into the vectors
is shown below. The XbaI site introduces a stop codon into the
reading frame, ensuring that insertion by homologous recombination
will disrupt the gene.
1 GATCTGGATGTCGAAGCCGGGACGGAGCGGGATGACGGCGTCAGCGG CGTCTTCCATGTG 60
GAACTCCTTATCCGGACGACTCGACCTGGTTGGCTAAGCG- GAGATTAG GTCTGCGCGCGC 120
GAAACCGCCCAGCGGAGCGCCGAGATCCTCACCTGATCAGGTAAGGA TCTTCATTCGATG 180
TCATGTAGCCAGATTTCGGCTGAACTGGTCCACGATCCC- GATTCGTGA CCATGCGTGTCC 240
ACTTTGGAGCGGGTGCGTTCGTTCGGCCTAGTGGCGTGCTCCGCGGTG ATCAAGTGTTAG 300
GTTAGCCTCAGCTCAGCGGGGTCGACGGATGGAGTGAACG- GCGTGGC GGGCGACGTGGAA 360
V A G D V E XbaI CTCGCGGACAGGGCTCGACGACGCG- CGTGCCGGCTGCTCAGGCGTTGG
CTGGCCGAGACG 420 L A D R A R R R A C R L L R R W L A E T
CACACTCCGGTGGAGCCCGGCCCGCTGTCCCTGCGGATCGGCCCGGTG CGGGTGTCGGCC 480 H
T P V E P G P L S L R L G P V R V S A
GAGGTCGCTTACCGCTCGCCGACGGGCGCCCACGGGTTCGGCCCGATC CGCGTCCTCGAT 540 E
V A Y R S P T G A H G F G P I R V L D
GCCGAGGGTGTGCCGGTGGCGCTCGCCGATCCGGTGCTGCTGGCGGCC GCCTGCTCGGCG 600 A
E G V P V A L A D P V L L A A A C S A
GACTCGCGGAGCCGCTCGCTGCCGAGCGCGCCGATCAACGCCCCG- GA CGCCGGTACCGCT 660
D S R S R S L P S A P I N A P D A G T A
GTCGACTGGGTGCTCTCGTCGCTCGCCGACGACGAGGAC- GACGAGGTG CCCGCCGGCATG 720
V D W V L S S L A D D E D D E V P A G M
ACCGCGGAGGAGGCGGTGCGCCTGCTGTCGCGG- CAGGTCGACGACCT GCCGCGGTCGCCG 780
T A E E A V R L L S R Q V D D L P R S P
GGCGCCGACCCGTGGTCGCTGGTCGCC- GGCCCGCTGGCGGCCATCGGG CGGTTCGGGCGG 840
G A D P W S L V A G P L A A I G R F G R
GCCGGGATCGCCGACGAGTGCTGGTTGCTGGAGGTGCTCGCCGGGCG GCTCCGCGCGGTC 900 A
G L A D E C W L L E V L A G R L R A V
GACGACGACCTGTCCCGCTCGTGGCTGAGCAGTCCGACGCTCGCCGAC CGCGCTGTGCTC 960 D
D D L S R S W L S S P T L A D R A V L
GTGGGTGAGGGGTTGCGCTACCGGCCGGATGTGCGGCCGGTGCCGTTC GACGTGCCGAAC 1020
V G E G L R Y R P D V R P V P F D V P N
CCGCTGCACGAGGGCAAGTCCGACGTCCCGCCGCCGCCCGTGC- CCGTG CTGGGCGGGCCG
1080 P L H E G K S D V P P P P V P V L G G P
TGGTCGCTGCGTCCGGTCGAGGTCGCGGTCCACGG- GGATGGCGGGCCT GACGTCGCACTG
1140 WSLRPVEVAVHGDGGPDVAL GTGCACCGCTGGATGAACACCCCGCACGTCGCGCAC-
CACTGGAACCA GGCGTGGCCGCTG 1200 V H R W M N T P H V A I I H W N Q A
W P L GAGCGCTGGCGGGAGGAACTCGCCCAC- CAGCTCGGCGGTGAGCACTC
CCTGCCCTGCGTG 1260 E R W R E E L A H Q L G G E H S L P C V
GTCGGACACGAGGGACGCGAGGTCGCGTATCTGGAGCTCTACCGGGT GACCCGCGACAAG 1320
VGHEGREVAYLELYRVTRDK HindIII
CTTGCGGGCTGCTACCCGTACGGGCCGCACGACCTCGGGGTCCACATC GCGATCGGCGAG 1380
L A G C Y P Y G P H I D L G V H J A I G E
CGGGAGGTGCTCGGGCGCGGTTTCGGGTCGTCGCTGCTGCGCGCGGT- C GCGGGTGCGCTG
1440 R E V L G R G F G S S L L R A V A G A L
CTGGACGCCGATCCGCGGTGCGCGCGGGTGGTCGCCGAG- CCGAATGT GCACAACGAGGCT
1500 L D A D P R C A R V V A E P N V H N E A
TCGGTGCGCGCCTTCGCCAAGGCCGGGTTCG- TCCGGGAGAGGGAGATC GGCCTGCCCGCC
1560 S V R A F A K A G F V R E R E I G L P A
AAGAACTCGGCTCTGATGGTCTT- CTCCCGGGTCTGACGACCGGTCATG CCCCTGTGTGAA
1620 K N S A L M V F S R V *> CGCGTGAGTAAGCGCACCGTGACGTGAT-
CCCCCGCTTGAACCAAGGTT AGCCTTACTTTT 1680
ATTGGTGGAGAACGATGCCGGAGCGCTCCGCCGTGTCGTTGCCGCTGA CCACAGCGCAGT 1740
AGGGCATCTGGTTCGCCCAGCAACTCGACCGGACGAACC- CGATCTACA ACACCGGCGAGT
1800 GCGTCGAGATCAGCGGCCCGGTGGAGCCGGTGGTGTTCGAGCAGGCC CTGCGGTGGGGCG
1860 TGGCGGAGGCCGAGGCGCTGCGAGCCCGCGTGGTCGTC- GACGGCGAC
GAGCCGCGCCAGG 1920 TCGTGGAGCCGGAGGTGGACTTCCCGCTGCCGTGCTCGACGTCAGCGC
CGAGGCGGACCC 1980
[0166] The above constructs were then introduced into a
high-producing Saccharopolyspora erythraea strain for gene
disruption by homologous recombination. Protoplast transformation
of this strain was very difficult, transformants were only obtained
only using alkali-denatured, non-methylated DNA of only the
pOJ260-derived construct. The transformant strains were grown in
TSB for DNA isolation and in a standard two-stage shake flask
fermentation procedure to evaluate production (two days growth in
vegetative medium, 10% crossing volume into fermentation medium and
daily feeds of 0.6% soy oil and 40 mM propanol over nine days).
Metabolites were quantitated by ion counting in a mass spectrometer
relative to a roxithromycin internal standard.
[0167] Putative eryM knockout transformants were shown to be
correct by Southern blot hybridization. The mutant displayed the
same morphology as the parent strain, both in liquid medium and on
agar plates (i.e., gray colonies with brown pigment in agar).
[0168] The parent strain and two isolates of the eryM mutant were
grown using the shake flask procedure. In addition to the oil plus
propanol feed, culture flasks were fed equivalent levels of oil
alone, oil plus butanol, oil plus propionate, and oil plus
butyrate. The cultures were killed by the propionate and butyrate
feeds, and these flasks were discarded. Samples were taken from the
other flasks each day and the set was analyzed by ion counting. The
results are shown graphically below. The first graph shows a
time-course of production of erythromycins A and B for the
wild-type and mutant strains with the different feeding regimes.
The second graph shows the same for 15-methyl-erythromycins A and
B. The ion count at 748.6 amu is not all due to
15-methyl-erythromycin A. LC-MS analysis of ethyl acetate extracts
of day 7 samples fed oil alone or oil and butanol suggested that
only about 10% of the 748.6 peak was 15-methyl-erythromycin A. The
exact amount of PrEryA produced by the strains remains to be
determined.
[0169] Another fermentation experiment was performed using a
lighter medium with the oil omitted to evaluate a wider range of
feeding regimes. The results did not show a clear trend, except
that the oil feed is beneficial to good production for both
strains. Esters of propionate or butyrate, instead of the
corresponding alcohols, could also be fed but produced no
substantial improvement.
[0170] Because overexpression of crotonyl CoA reductase can
increase butyryl CoA levels, the Streptomyces coelicolor ccr gene
could be used to replace the eryM gene, i.e., with an expression
cassette for ccr overexpression. Because overexpression of
propionyl CoA carboxylase (pcc) can reduce propionyl CoA levels, a
similar construct for overexpression of pcc could also be used
advantageously with the methods and recombinant strains of the
present invention. In addition, in those strains that contain a
methylmalonyl CoA transcarboxylase that decarboxylates
methylmalonyl CoA to propionyl CoA (putative homologues of both
subunits of this enzyme are in the S. coelicolor genome), one could
disrupt the corresponding gene to improve production.
[0171] Production of erythromycin A and B by the eryM.sup.- mutant
is similar to that of the corresponding wild-type strain when fed
oil alone or oil and propanol in rich medium. For both strains,
production of erythromycin A and B was depressed with an oil and
butanol feed. While knockout of eryM does not reduce production of
erythromycin A and B in rich medium in the dramatic way reported by
Hsieh & Kolattukudy, the rich medium used (which is necessary
for high level production) probably masks the effect of the eryM
knockout.
[0172] The high-producing wild-type strain appeared to produce low
levels of 15-methyl-erythromycins when butanol is fed instead of
propanol. Thus, in one aspect the present invention provides a
method for producing 15-methyl-erythromycins by feeding a culture
of erythromycin-producing cells by culturing said cells in a medium
containing butanol. If the ion count at 748.6 amu for the culture
fed oil and propanol is subtracted as background, the oil and
butanol feed causes 15-methyl-erythromycin A and B production at up
to 5% of the total erythromycin production, although the actual
amount of 15-methyl-erythromycin A and B is probably much lower.
The eryM.sup.- mutant produced a higher maximum percentage
15-methyl-erythromycin A and B (.about.15%) with an oil and butanol
feed compared to the wild-type strain, demonstrating that propionyl
CoA levels are reduced in the eryM.sup.- strain and confirming that
the methods of the present invention can be used to increase
production of 15-methyl-erythromycins.
EXAMPLE 2
Construction of a Loading Domain for a DEBS PKS
[0173] While Example 1 illustrates the aspect of the invention in
which butyryl CoA is loaded by the loading domain of DEBS to
produce 15-methyl-6-deoxyerythronolide B, this and the following
Example illustrate an alternative aspect of the invention, in which
a recombinant PKS that comprises an altered loading domain is used
to produce the compound. This altered loading domain can be
employed with any number of extender modules from any one or more
PKS. In a preferred embodiment, the loading domain is used in
conjunction with the six extender modules of DEBS, or the
oleandolide PKS (see PCT patent publication No. WO 00/026349,
incorporated herein by reference), or the megalomicin PKS (see U.S.
patent application Serial No. 60/190,024, filed Mar. 17, 2000, and
the application Serial No. ______, attorney Morrison and Foerster
docket no. 30062-20047.20, filed Oct. 4, 2000, naming the same
inventors and claiming priority to the former applicaton, each of
which is incorporated herein by reference), to produce
15-methyl-erythromycins in Saccharopolyspora erythraea host
cells.
[0174] An illustrative hybrid PKS of the invention is made by
replacing the AT domain of the loading module of the oleandomycin
PKS with the ethylmalonyl-CoA specifying AT domain of the fourth
extender module of the FK520 PKS. The resulting hybrid PKS contains
the KS.sup.Q domain and downstream interdomain region of OleA1 (aa
1-562) fused to the FKAT4 domain (aa 562-896) fused to the OleA1.
AT-ACP interdomain region, adjoining OleA1 ACP of the loading
domain and the remainder of the OlePKS (897-end). The amino acid
sequence of the hybrid portion of this PKS is shown below.
2 MHVPGEENGHSIAIVGIACRLPGSATPQEFWRLLADSADALDEPPAGRFPTGSLSS PPAP-60
RGGFLDSIDTFDADFFNISPREAGVLDPQQRLALELGWEALEDAGIVPRHLRGTRT SVFM-120
GAMWDDYAHLAHARGEAALTRHSLTGTHRGMIANRLSYALGLQGPSLT- VDTGQ SSSLAAV-180
HMACESLARGESDLALVGGVNLVLDPAGTTGVERFGALSP- DGRCYTFDSRANG YARGEGG-240
VVVVLKPTHRALADGDTVYCEILGSALNNDGA- TEGLTVPSARAQADVLRQAWE RARVAPT-300
DVQYVELHGTGTPAGDPVEAEGLG- TALGTARPAEAPLLVGSVKTNIGHLEGAAG IAGLLK-360
TVLSIKNRHLPASLNFTSPNPRIDLDALRLRVHTAYGPWPSPDRPLVAGVSSFGMG GTNC-420
HVVLSELRNAGGDGAGKGPYTGTEDRLGATEAEKRPDPATGNGPDPAQDTHRY PPLILSA-480
RSDAALRAQAERLRHHLEHSPGQRLRDTAYSLATRRQVFERHAVVTGHDREDLL NGLRDL-540
ENGLPAPQVLLGRTPTPEPGGLVFVFPGQGPQWRGMGVELMAASPVFA- ARMRQ CADALIP-600
HTGWDPIAMLDDPEVTRRVDVVHPVCWAVMVSLAAVWEAA- GVRPDAVIGHSQ GEIAAACV-660
AGALTLEDGARLVALRSVLLLLRELAGRGAMG- SVALPAADVEADAARIDGVWV AGRNGAT-720
TTTVAGRPDAVETLIADYEARGVW- VRRIAVDCPTHTPFVDPLYDELQRIVADTTS RTPEI-780
PWFSTADERWIDAPLDDEYWFRNMRHPVGFATAVTAAREPGDTVFVEVSAHPV LLPAIDG-840
ATVATLRRGGGVHRLLTALAEAHTTGVPVDWAAVVPATATAHDLPTYAFHHER YWISHWL-900
PSGEAI-IPRPADDTESGTGRTEASPPRPHD-929
[0175] Another illustrative hybrid PKS of the invention is made by
fusing the following in order specified: the first 9 aa of OleAI
(1-9 in sequence below); 846 aa of the FK520 PKS encompassing the
KS and AT domains of module 4 as well as the KS-AT interdomain
region (10-855); the ATL-ACPL interdomain region of OleAI, followed
by the ACPL domain and the rest of the OLE PKS (856-end). The amino
acid sequence of the hybrid portion of this PKS is shown below.
3 MHVPGEENGEPLAIVGMACRLPGGVASPEDLWRLLESGGDGITAFPTDRGWDVD GLYDPD-60
PDHPGTSTVRHGGFLAGVADFDAAFFGISPREALAMDPQQRLVLETSWEALEHA GILPES-120
LRGSDTGVFMGAFSDGYGLGTDLGGFGATGTQTSVLSGRLSYFYGLEG- PAVTVD TACSSS-180
LVALHQAGQSLRSGECSLALVGGVTVMASPSGFVEFSQQR- GLAPDARCKAFADA ADGTGF-240
AEGSGVLIVERLSDAERNGFIRVLAVVRGSAV- NQDGASNGLSAPNGPSQERVIRQ
ALANAG-300 LTPADVDAVEAHGTGTRLGDPIE- AQAVLATYGQGRDTPVLLGSLKSNIGHTQAA
AGVAGV-360 IKMVLAMRHGTLPRTLHVDTPSSHVDWTAGAVELLTDARPWPETDRPRRAGVSS
FGVSGT-420 NAHVLLEAHPAGEPPAEEPSASKPGEPLIATPLTPLPVSARTATALDGQVRRLREH
LAAR-480 PGHDPRAIAAGLLARRTTFPHRAVLLDDDVVTGTALTEPRTVFVFPGQGPQWRG
MGVELM-540 AASPVFAARMRQCADALIPIITGWDPIAMLDDPEVTRRVDVVHPVC- WAVMVSLA
AVWEAAG-600 VRPDAVIGHSQGEIAAACVAGALTLEDGARLVALRSV-
LLLLRELAGRGAMGSVA LPAADV-660 EADAARIDGVWVAGRNGATTTTVAGRPDA-
VETLIADYEARGVWVRRIAVDCPT HTPFVDP-720
LYDELQRIVADTTSRTPEIPWFSTADERWIDAPLDDEYWFRNMRHPVGFATAVT AAREPG-780
DTVFVEVSAHPVLLPAIDGATVATLRRGGGVHRLLTALAEAHTTGVPVDWAAV VPATATA-840
HDLPTYAFHHERYWISHWLPSGEAHPRPADDTESGTGRTEASPPRPHD-889
[0176] The hybrid PKS above is then changed in the DNA sequence
corresponding to aa 177 so that the C is replaced by a Q residue in
the final hybrid PKS, yielding the following amino acid
sequence.
4 MHVPGEENGEPLAIVGMACRLPGGVASPEDLWRLLESGGDGITAFPTDRGWDVD GLYDPD-60
PDHPGTSTVRHGGFLAGVADFDAAFFGISPREALAMDPQQRLVLETSWEALEHA GILPES-120
LRGSDTGVFMGAFSDGYGLGTDLGGFGATGTQTSVLSGRLSYFYGLEG- PAVTVD TAQSSS-180
LVALHQAGQSLRSGECSLALVGGVTVMASPSGFVEFSQQR- GLAPDARCKAFADA ADGTGF-240
AEGSGVLIVERLSDAERNGIJRVLAVVRGSAV- NQDGASNGLSAPNGPSQERVIRQ
ALANAG-300 LTPADVDAVEAHGTGTRLGDPIE- AQAVLATYGQGRDTPVLLGSLKSNIGHTQAA
AGVAGV-360 IKMVLAMRHGTLPRTLHVDTPSSHVDWTAGAVELLTDARPWPETDRPRRAGVSS
FGVSGT-420
NAHVLLEALIPAGEPPAEEPSASKPGEPLIATPLTPLPVSARTATALDGQVRRLREH LAAR-480
PGHDPRAIAAGLLARRTTFPFLRAVLLDDDVVTGTALTEPRTVFVFPGQGPQW- RG
MGYELM-540 AASPVFAARMRQCADALIPHTGWDPIAMLDDPEVTRRVDVVHPV- CWAVMVSLA
AVWEAAG-600 VRPDAVIGILSQGEIAAACVAGALTLEDGARLVALR-
SVLLLLRELAGRGAMGSVA LPAADV-660 EADAARIDGVWVAGRNGATTTTVAGRP-
DAVETLIADYEARGVWVRRIAVDCPT HTPFVDP-720
LYDELQRIVADTTSRTPEIPWFSTADERWIDAPLDDEYWFRNMRHPVGFATAVT AAREPG-780
DTVFVEVSAHPVLLPAIDGATVATLRRGGGVHRLLTALAEAHTTGVPVDWAAV VPATATA-840
HDLPTYAFHHERYWISHWLPSGEAHPRPADDTESGTGRTEASPPRPHD-889
[0177] These hybrid PKS loading domains can be employed in the
methods of the invention as described and illustrated in Example
3.
EXAMPLE 3
Production of 15-Methyl-Erythromycins in Saccharopolyspora
erythraea and Streptomyces fradiae
[0178] This Example describes methods and recombinant host cells of
the invention for efficient and economical production of
15-methylerythromycin A and/or 15-methylerythromycin C, both of
which can be converted to ketolides with potent anti-bacterial
activity. The recombinant host cells of the invention directly
produce high levels of 15-methylerythromycin A or
15-methylerythromycin C without diketide feeding. Products from
these strains can be used in the production of potent ketolide
antibiotics. More specifically, this Example describes methods
to:
[0179] (1) introduce a series of genes to construct a pathway or
series of pathways in Sac. erythraea or an eryM.sup.- derivative to
produce butyryl-CoA or ethylmalonyl-CoA at levels sufficient to
permit high level synthesis of 15-methylerythromycins, and
concomitantly, determine whether the level of propionyl-CoA can be
reduced in a directed fashion without affecting the pools of other
required precursors, so that DEBS can make 15-methylerythromycins
exclusively;
[0180] (2) re-engineer the loading domain of DEBS to initiate the
synthesis of 15-methylerythromycin A with butyryl-CoA or
ethylmalonyl-CoA in a high-producing strain of Sac. erythraea;
and
[0181] (3) introduce a series of genes into a high-producing strain
of Streptomyces fradiae to enable production of
15-methylerythromycin C, including genes enabling the host to
produce TDP-desosamine, transfer desosamine and mycarose to the
erythromycin backbone, and hydroxylate 15-methylerythromycin at the
appropriate positions.
[0182] References cited in this example by reference number in
parentheses, and the numbered listing of references is located at
the end of the example. Compound numbers are bracketed.
[0183] The benefits provided by the present invention can be better
appreciated with some understanding of the need for improved
antibiotic compounds. Erythromycin A [1] and its semisynthetic
derivatives clarithromycin [2] and azithromycin [3] are widely used
antibiotics in human healthcare because of their broad spectrum of
activity and their minimal side-effects. They are used primarily
against respiratory tract pathogens (Streptococcus pneumoniae, St.
pyogenes, Hemophilus influenzae), some Gram-positive pathogens of
skin and soft-tissue (Staphylococcus aureus) and, to a lesser
extent, the opportunistic pathogens belonging to the Enterococcus
species. Clarithromycin is used in combination with a proton-pump
inhibitor in the treatment of the gastic ulcer-associated bacterium
Helicobacter pylori. World-wide sales of macrolides exceed US $3 B
annually.
[0184] Continued use of these macrolide antibiotics is threatened
by the rise of resistance to these agents, often accompanied by the
presence of other genetic determinants conferring resistance to
many antibiotics. Of importance is MRSA (methicillin-resistant
Staph. aureus), most strains of which also carry macrolide
resistance. Only vancomycin is currently available for treatment of
these agents. Macrolide-resistant Strep. pneumoniae is emerging in
the US, Europe, and Japan, particularly strains which have also
acquired penicillin resistance. There is a growing need, therefore,
to discover and develop novel antibiotics that can overcome current
resistance mechanisms. One approach is to discover agents that
attack essential bacterial targets not hit by existing drugs, and
thus not expected to exhibit pre-existing resistance patterns. This
approach is generally based upon screening either large
combinatorial chemical or natural product libraries. Another
approach is to modify existing agents, such as macrolides, so as to
overcome the resistance mechanisms. The ketolides fulfill this
second objective.
[0185] Macrolides block protein synthesis by binding to the 50S
ribosomal subunit and causing premature release of peptidyl tRNA
(1). The segment of nucleotides surrounding the A-2058 (Bacillus
subtilis numbering) residue in domain V of the RNA molecule
interacts with erythromycin and other macrolides (2) and is the
target of ERM methylases which confer resistance through
methylation of A-2058 and subsequent blocking of macrolide binding.
This resistance is also referred to as MLS
(macrolide-lincosamine-streptogramin B) resistance (3). Both
inducible and constitutive MLS resistance is found in pathogenic
bacteria. In inducible strains, methylase activity develops in the
presence of the antibiotic. In constitutive strains, the activity
is present even in the absence of the drug, although drug often
increases the level of resistance. Macrolides such as tylosin [4]
and spiramycin [5] do not appear to induce MLS resistance in
inducible strains, but constitutively MLS-resistant strains are
resistant to tylosin and other 16-membered macrolides. 1
[0186] A second mechanism of resistance in Gram positive pathogens,
efflux, uses export of the compound from the host to keep
intracellular concentrations of the drug low and can be either
inducible or constitutive. Several genes (mef) in Streptococcus sp.
have been found to confer efflux-based resistance (4). The
16-membered macrolides appear to bypass the efflux mechanism and
can be used against some of these strains.
[0187] A novel series of semisynthetic macrolides called ketolides
introduced by Hoechst-Marion-Roussel (HMR 3647 [6]) and Abbott
(ABT-773 [7]) show excellent activity against Gram positive
pathogens, including those carrying inducible MLS resistance and
many S. pneumoniae and S. pyogenes strains carrying constitutive
MLS resistance, (5,6). These compounds are also active against S.
pneumoniae strains carrying efflux resistance. It is believed that
the N11-aralkyl side chains of these molecules attach themselves to
the ribosome at a site distinct from domain V and thus enable
binding to methylated ribosomes. Compounds 6 and 7 are currently in
clinical development. 2
[0188] The biosynthesis of macrolide antibiotics is best understood
for erythromycin (in Saccharopolyspora erythraea) and tylosin (in
Streptomyces fradiae). For erythromycin (see the figure below), the
polyketide 6-deoxyerythronolide B [6-dEB; 8], is produced by the
successive condensation of one propionyl-CoA (p-CoA) and six
methylmalonyl-CoA (mm-CoA) molecules. The polyketide synthase (PKS)
that assembles 6-dEB, 6-deoxyerythronolide B synthase (DEBS), is
determined by the genes eryAI, eryAII and eryAIII. Polyketide
synthesis is followed by 6-hydroxylation (eryF) to yield
erythronolide B [9]. Addition of the sugar L-mycarose (via
TDP-mycarose) to yield 3-O-alpha-mycarosyl-erythron- olide B [10]
and the addition of desosamine (via TDP-desosamine) yields
erythromycin D [11]. The two sugars are produced by independent
pathways not shown here but controlled by the genes designated eryB
(mycarose) and eryC (desosamine). The final steps are hydroxylation
of 11 to yield erythromycin C. [12] by a second P450 enzyme (eryK)
and O-methylation of the mycarosyl residue (eryG) to yield the
cladinosyl moiety in erythromycin A [1]. A side product is
erythromycin B [13], which results from the methylation of 11 and
is only poorly converted to 1. 34
Pathway and genes for erythromycin biosynthesis in Sac.
erythraea
[0189] Tylosin [4] is produced in a similar fashion: formation of
the polyketide, oxidation of the C-6b methyl to the aldehyde,
addition of the three sugars in succession, and finally,
methylation of the sugar. In addition to the single additional
condensation required to construct the 16-membered macrolactone
ring, named protylonolide or tylactone, the ring itself is built
from condensations that employ the precursors in the following
order of use: p-CoA (or mm-CoA; see below), mm-CoA, mm-CoA,
malonyl-CoA (m-CoA), mm-CoA, ethylmalonyl-CoA (em-CoA), mm-CoA,
m-CoA.
[0190] Production of erythromycin depends upon the cells having a
supply of the precursors p-CoA and mm-CoA. Tylosin requires mm-CoA,
m-CoA and em-CoA, which is built from butyryl-CoA (b-CoA). Although
the wild type cultures of Sac. erythraea and S. fradiae produce
10-100 mg/L of erythromycin and tylosin, respectively, in
fermentation broths, strains of the two cultures exist that can
produce the compounds at higher levels. The biochemical basis for
high level production is not understood, but it is clear that the
supply of precursors does not limit the production of the drugs at
high levels in such hosts. Some work has been done on precursor
supply in macrolide producing organisms, but work on high producing
strains has not been reported. Schemes for the synthesis of the
components used for erythromycin synthesis, p-CoA and mm-CoA and
also required for tylosin synthesis, as well as m-CoA and em-CoA,
required for the latter are shown in FIG. 2 and below.
[0191] The degradation of valine through the route shown below has
been demonstrated in Streptomyces avermitilis, the producer of
avermectin, a complex polyketide that utilizes ib-CoA as the
starter, and employs mm-CoA in building the polyketide ring (7).
Valine utilization can produce the n-butyrate-derived units in
tylosin (8). In addition, the rate of valine degradation has been
shown to have major impact on tylosin and spiramycin production
(9-12). Addition of valine increases tylosin production and has
been shown to increase the level of ib-CoA (ib-CoA), most likely
resulting in increases in the em-CoA, p-CoA, and mm-CoA required
for tylosin synthesis. Conversion of ib-CoA to mm-CoA takes place
through methacrylyl-CoA (13) and is probably a major source of
mm-CoA from valine-fed fermentations (14,15). High utilization of
valine would require high flux through the pathway to mm-CoA, not
yet reported for high producing strains. It has been shown that
increasing the copy number of valine dehydrogenase in a low
tylosin-producing strain results in the increase in the titer of
tylosin produced (11,12). For Sac. erythraea, addition of
proteinaceous material to the fermentation also increases the
titers of erythromycin produced (16). It is possible that high
levels of mm-CoA required for the synthesis of erythromycin can be
achieved from the carboxylation of p-CoA by the enzyme propionyl
CoA carboxylase, although the disruption of p-CoA carboxylase
activity in a low erythromycin-producing Sac. erythraea strain did
not affect the antibiotic titer (17). On the other hand, the
finding that the disruption of the gene eryM, which encodes a
methylmalonyl CoA decarboxylase, results in the cell running out of
the supply of p-CoA and arresting erythromycin synthesis in a
low-producing strain of Sac. erythraea (18) suggests that p-CoA is
derived from mm-CoA and not that mm-CoA is derived from p-CoA. It
can be seen from the biosynthetic pathway shown below that p-CoA
can be produced from alternate sources such as the degradation of
leucine and the breakdown of odd-chain fatty acids, and it has been
shown that addition of oils can improve the titers of erythromycin
(19).
[0192] Several other routes to mm-CoA are shown below. One is the
conversion of succinyl-CoA to mm-CoA via the enzyme methylmalonyl
CoA mutase, which has been identified in S. cinnamonensis, producer
of the polyketide monensin (20). Succinyl-CoA arises from the
oxidation of acetyl-CoA via the Krebs cycle, but requires the input
of oxaloacetate, which would be derived from the breakdown of
glucose. Because the utilization of glucose is generally controlled
in high-titer fermentations, and because it serves as the principal
source for the synthesis of the deoxysugars present in macrolide
antibiotics, it is unlikely that it serves as a significant source
of precursors. Succinyl-CoA could also come from
alphaketoglutarate, produced from deamination of glutamate.
Fermentations fed with succinate or its purported precursors have
only shown marginal increases in titers of polyketides, indicating
that the succinyl-CoA pathway is not a major contributor to the
large precursor pools required for high level synthesis, but this
has not been ruled out in the high titer strains. The figure below
shows pathways for the synthesis of acyl-CoA precursors in
Streptomyces. Enzymes are shown in bold: VDH, valine dehydrogenase;
LDH, leucine dehydrogenase; MMCoA mutase, methylmalonyl CoA mutase.
56
[0193] A rather circuitous route to mm-CoA shown in the figure
above is the conversion of acetyl-CoA to b-CoA, conversion to
ib-CoA, and then degradation to mm-CoA. This pathway in its
entirety has been shown to exist in S. cinnamonensis (20), and the
enzyme that converts ib-CoA to methyacrylyl-CoA has been found in
S. coelicolor and S. avermitilis (13). This pathway is believed to
exist in S. fradiae. It allows acetyl-CoA, the most abundant
precursor in the cell, to be converted into mm-CoA. The pathway
depends on the enzyme crotonyl-CoA reductase (ccr), which reduces
crotonyl-CoA to b-CoA. CCR activity has not been demonstrated in
wild type Sac. erythraea, nor does this organism contain a DNA
sequence hybridizing with the Streptomyces collinus ccr gene
(21,22). Sac. erythraea thus probably cannot make mm-CoA from
acetyl-CoA, and probably relies on the degradation of valine, oils
and other amino acids to produce mm-CoA in sufficient quantities to
sustain high level synthesis of erythromycin. S. fradiae has a ccr
gene and is believed to contain a complete pathway to make mm-CoA
from acetyl-CoA (23). Thus, the mm-CoA precursor, at least for
tylosin, can be made via several sources.
[0194] Little is known about the supply of butyrate-derived
precursors in S. fradiae, although some work has been done in S.
collinus and S. cinnamonensis, both of which make b-CoA. The
pathway from acetyl-CoA to b-CoA shown above is best understood in
Clostridum acetobutylicum, which produces high levels of butanol.
Genes for the pathway have been cloned from various sources, but at
least one counterpart of each gene has been shown to be present in
the genome of Streptomyces coelicolor A3(2) (Sanger Web site).
Genes for the entire pathway from acetyl-CoA to b-CoA are thus
accessible. The S. collinus ccr gene supports incorporation of low
levels of em-CoA into 6-ethylerythromycin in a strain of Sac.
erythraea expressing a mutant DEBS (22), demonstrating that Sac.
erythraea has the capability of utilizing b-CoA as a precursor for
polyketide synthesis.
[0195] Wallace et al. (24) demonstrated that labeled valine was
incorporated into butyrate in Sac. erythraea, suggesting the
pathway valine->ib-CoA->b-CoA. Yet b-CoA, which can be
employed as a starter by DEBS, has not previously been demonstrated
to be incorporated into erythromycin in high titer fermentations.
The findings of Wallace et al. suggest the presence of an ib-CoA
mutase in Sac. erythraea, but this activity has not been examined.
The genes for ib-CoA mutase, icmA and icmB, have been cloned from
S. cinnamonensis (25,26). Thus, in accordance with the methods of
the invention, pathways to produce b-CoA in high levels in Sac.
erythraea are available and transferable from related polyketide
producers. Only a minimum number of additional steps are needed to
convert intermediates into b-CoA in Sac. erythraea.
[0196] DEBS has been extensively studied genetically and
biochemically, and the details will thus not be reviewed here. FIG.
1 shows the linear modular organization of the enzyme complex,
displaying the functional domains within the modules and the
structure of the growing acyl chain at the end of each cycle of
growth and reduction. DEBS consists of 6 extender modules and a
loading module. Precursor specificity is provided by the AT domains
(27). The DEBS loading module shows relaxed specificity, loading
various acyl-CoAs depending upon the environment (28-30). Tylosin
is produced similarly by a 7-module PKS. The loading module of the
tylosin PKS, fully sequenced (31) contains a KS.sup.Q domain that
has decarboxylase activity (32,33) and an AT domain having the
signature sequence for mm-CoA binding. It is likely, therefore,
that the tylPKS employs mm-CoA, malonyl-CoA and em-CoA to make
tylosin.
[0197] PKS genes for other complex polyketides such as rapamycin
(34), pikromycin (35), avermectin (36), FK506 (37) and rifamycin
(38) all show similar organizations as the erythromycin and tylosin
PKSs. AT, KR, DH and ER domains may be exchanged between different
PKSs, even in combinations to result in the creation of polyketides
with novel but predicted structures (39-42). The loading domain of
one PKS can be exchanged for a loading domain of another to produce
a hybrid polyketide (43,44). In accordance with the methods of the
present invention, a loading domain is exchanged for an extender
domain, and then converted back to a loading domain.
[0198] Heterologous expression of DEBS in S. coelicolor (45)
yielded a mixture of 6-dEB and 8,8a-deoxyoleandolide, at levels up
to 50 mg/L. DEBS can thus initiate polyketide synthesis in vivo
either from p-CoA or acetyl-CoA. Similarly, the DEBS1 protein
(containing the loading module and modules 1 and 2) was
re-engineered by placing the TE domain after the ACP domain of
module 2. In S. coelicolor, the. DEBS1-TE construct yielded the
predicted triketide products 2,4-dimethyl-3,5-dihydroxyheptanoic
acid delta-lactone [14] and 2,4,-dimethyl-3,5-dihydroxyhexanoic
acid-delta-lactone [15] shown below (46). It is thus not always
necessary to replace the DEBS loading domain to alter the starter
unit. FIG. 1 and the schematic below show the erythromycin PKS and
synthesis of 6-deoxyerythronolide B. 7
[0199] Inactivation of the module 1 KS domain of DEBS has been used
to bypass the loading domain specificity. This KS1 null (KS
1.degree.) mutation produces novel polyketides when supplied with
analogs of the normal product of module 1, a diketide thioester
(47,48). This technology has been used to convert (2S,
3R)-2-methyl-3-hydroxyhexanoate N-acetylcysteamine (SNAC) thioester
into 15-methyl-6-dEB (6-deoxy-15-methylerythronolide B) [16]. This
compound is converted to the antibiotic 15-methylerythromycin A
when fed to a KS1.degree. strain of Sac. erythraea. 8
[0200] The desosamine of erythromycin and the mycaminose of tylosin
differ only by a C4-hydroxyl, and are made by similar routes, shown
schematically below. Biosynthesis of TDP-mycaminose requires only
two steps past common intermediate 1', C-3 amination and
N-dimethylation, whereas TDP-desosamine synthesis requires
dehydration and reduction at C-4, then C-3 amination and
N-dimethylation. The genes for TDP-desosamine biosynthesis (eryC)
in Sac. erythraea cluster with the other ery biosynthesis genes. It
is believed that the dehydration and reduction functions are
encoded by the genes eryCIV and eryCV (49-51). The genes for
TDP-mycaminose synthesis have also been described (51a,b). Both
Sac. erythraea and S. fradiae make TDP-mycarose; thus, the gene
distinguishing the pathways in the two hosts is the one encoding
the mycarosyltransferase. In Sac. erythraea it is designated eryB
V. There is no expectation that the S. fradiae mycarosyltransferase
can use erythronolide B as a substrate should one desire to make an
erythromycin analog in this host.
[0201] The schematic below shows the pathways to TDP-desosamine and
TDP-mycaminose. 9
[0202] Novel ketolide compounds with improved antibacterial
activity over the currently marketed macrolides clarithromycin and
azithromycin have been produced. A number of diketide-SNAC
thioesters were synthesized and fed to S. coelicolor CH999/pJRJ2
(deleted for the actinorhodin PKS and carrying the [KS
1.degree.]-DEBS genes). The resulting macrolactones were purified
and fed to Sac. erythraea (KS1.degree. strain K39-14) to convert
the macrolactone to the erythromycin A analog. After preliminary
antibacterial testing, 15-methylerythromycin A [17] was of great
interest. Samples of 17 were chemically converted into the
3-descladinosyl-3-oxo-6-O-methyl-10,11-anhydro derivative [18] and
then to a number of ketolide derivatives, of general structure 19
with compound 20 as an example. Compounds related to 20 have been
subjected to extensive in vitro testing and in vivo testing.
1011
[0203] The initial transformation of 15-methylerythromycin A into
the 6-O-methyl derivative follows standard procedures (52) and
proceeds in good overall yield. The key intermediate in the
synthetic process is the 10,11-alkene [18], which is produced
similarly to reported ketolides (53). The 6-O-methyl analog is
subjected to acid-mediated removal of the cladinose, and the
resulting 3-hydroxy group is oxidized to the ketone after
protection of the desosamine 2'-hydroxyl as the acetate. The
11-hydroxyl group is converted to the mesylate, then eliminated by
treatment with diazabicycloundecene (DBU) to introduce a 10,
11-alkene functionality. This procedure results in an overall
10-15% yield of 18 starting from the initial 15-methylerythromycin
A. 12
[0204] Thus, it would be advantageous to produce
15-methylerythromycin A [15-MeEryA] or 15-methyl-erythromycin C
[15-MeEryC] in a recombinant strain of Sac. erythraea or S.
fradiae. This example describes three approaches, two using the
erythromycin producer Sac. erythraea and one using the tylosin
producer S. fradiae. In Sac. erythraea, the approaches employ
replacing p-CoA as the starter for 15-MeEryA synthesis with either
b-CoA or em-CoA. Both involve building a supply of b-CoA in the
host at sufficient levels so that it does not limit the synthesis
of the antibiotic. In S. fradiae, which is not limited for b-CoA
because it makes high levels of tylosin, genes concerned with the
production of the erythromycin polyketide and its C6 and C.sub.1-2
hydroxylation and glycosylation with desosamine are introduced.
[0205] The first method using Sac. erythraea aims to reduce the
level of p-CoA so that it does not compete with b-CoA for loading
of DEBS. As described in Example 1, disruption of the eryM gene,
which encodes a decarboxylase that converts mm-CoA to p-CoA (18),
did not result in a notable decrease in erythromycin production by
the strain of Sac. erythraea grown in a rich production medium that
supports high level antibiotic production. A minimal medium and
washed cells were used in the work that reported the lack of
erythromycin production by an eryM mutant (18). A considerable
decrease was anticipated if the supply of p-CoA made from mm-CoA
had been reduced markedly and caused a corresponding decrease in
the rate of initiation of 6dEB biosynthesis. The effect of the eryM
mutation on the p-CoA level has not been examined. One can reduce
the level of p-CoA through fermentation conditions (e.g.,
supplementation with butyric acid or butanol) and by overexpression
of p-CoA carboxylase, the enzyme that converts p-CoA to mm-CoA.
[0206] In the second method, the normal ATL domain of the loading
module of DEBS in Sac. erythraea is mutated to a form that
preferentially uses b-CoA over p-CoA or is replaced with an
extender AT.sub.E domain that is specific for em-CoA. In the latter
case, a KS.sup.Q domain is introduced immediately preceding the ATE
domain to decarboxylate the PKS-bound em-CoA and thus provide b-CoA
on the PKS to initiate 15-McEryA synthesis. Alternatively, the
entire loading module of DEBS1 is replaced with one that specifies
formation or utilization of a saturated C4 alkyl starter unit, such
as can be obtained from the rimocidin PKS genes that can be cloned
from the oxytetracycline producing Streptomyces rimosus strain
(53a).
[0207] These methods may require or benefit from the introduction
of a pathway to produce b-CoA or em-CoA derived from it in Sac.
erythraea in levels sufficient to support high level production of
15-MeEryA. The invention provides several different methods to this
end. First, intermediates from the breakdown of valine can be
directed into the b-CoA pathway by introducing and overexpressing
genes encoding the ib-CoA mutase. If the breakdown of valine is
limiting, one can enhance the pathway by introducing and
overexpressing the genes for branched chain alpha-keto acid
dehydrogenase (bkd). If this approach fails to provide the required
levels of b-CoA or in the alternative, one can introduce and
overexpress up to the four genes that encode enzymes required to
convert the abundant acetyl-CoA to b-CoA.
[0208] In an alternative method of the invention, the desired
compound can be produced in the tylosin producer S. fradiae.
Because the synthesis of tylosin employs em-CoA, synthesis of
15-MeEryA in this strain should not be limited by the supply of the
b-CoA precursor. However, the pathway to the erythromycins must be
assembled in S. fradiae using a modified DEBS that employs b-CoA or
em-CoA as the starter. This can be achieved by the method of Hu et
al. (53b) that enables the directed transfer of large DNA segments
between streptomycetes. In one embodiment, the entire erythromycin
gene cluster is transferred into an S. fradiae strain so as to
replace the tylosin PKS genes. As described below, the recombinant
DEBS genes employed contain a coding sequence for a hybrid loading
module, six extender modules, and a thioesterase. The coding
sequences for the six extender modules and thioesterase can
originate from, for example, the ery genes (involved in the
synthesis of erythromycin), the ole genes (involved in the
synthesis of oleandomycin), or the meg genes (involved in the
synthesis of megalomicin).
[0209] Because the strain can be used in the production of
15-MeEryA, one may wish to avoid employing methods that introduce
genes on autonomous plasmids that require the addition of a drug to
select for their maintenance in the culture. Such addition of drugs
could add significant costs to the production process. In addition,
because the cultures could ultimately be used in large scale
manufacturing processes, one may again wish to avoid adding drug
resistance genes to the producing cultures (Sac. erythraea or S.
fradiae) even if one does not intend to employ the drugs.
Therefore, the invention enable one to employ plasmids to deliver
genes that integrate in the chromosome through site-specific
integration or double-site homologous recombination. For the
integration, one can choose sites in the genome that are neutral
with respect to erythromycin biosynthesis. One such site is the
native attB site used by vectors that contain the phiC31 attP gene
and another is an approx. 5 kb region adjacent to the eryK gene in
Sac. erythraea, that when disrupted has been shown to have no
effect on erythromycin synthesis (54). In S. fradiae as well, a
number of neutral sites in the chromosome are available for
homologous recombination (55).
[0210] In constructing the strains of the invention, one may desire
to determine accurately the levels of acyl-CoAs in cells grown
under various conditions. The methods of Hosokawa et al. (56) and
Kikuchi et al. (57) can be used for extraction of the CoA esters
from cells and HPLC analysis employing authentic standards. Because
there are no published reports of acyl-CoA determinations in
actinomycetes, one must be concerned primarily with a potentially
high background that can obscure accurate measurement. Hosokawa et
al. (56) found that passage of liver tissue extracts over a Sep-Pak
C.sub.18 cartridge greatly reduced background and allowed accurate
determinations. This approach can be followed, with other columns
that can partially purify and concentrate CoA esters also employed.
The identities of the CoA esters can be confirmed by LC/MS
analysis. Inclusion of an internal standard such as benzoyl-CoA
will allow for quantitation of the acyl-CoA levels. One may wish to
correlate p-CoA and mm-CoA levels in an erythromycin producing
strain with erythromycin production under fermentative conditions.
In S. fradiae, one can repeat this for p-CoA, mm-CoA, and b-CoA. To
set a baseline for precursor effects, one can compare different
strains of Sac. erythraea that produce different amounts of
erythromycin for acyl CoA levels. In addition one can compare
levels when a given strain is grown under different conditions.
[0211] As shown in Example 1, disruption of the eryM gene in a
high-producing Sac. erythraea strain does not reduce erythromycin
synthesis noticeably in rich medium. If one determines that the
eryM mutation does not significantly affect the p-CoA level (by
comparing the levels of this compound in the eryM.sup.+ and eryM
mutant backgrounds in cells grown in rich medium), then one can
reduce levels of p-CoA further through its increased conversion to
mm-CoA. The genes for a p-CoA carboxylase activity (pccA and pccB)
of S. coelicolor can be employed for this purpose. These genes,
under the control of the strong ermE* promoter, can be inserted
into a site in the chromosome of the eryM strain, as described
above, and the effect on production of erythromycin and various
acyl-CoA levels can be examined. The desired result is a diminution
of erythromycin synthesis, a drop in p-CoA levels, and an increase
or no change in the level of mm-CoA. If this is not observed, one
can reduce the intracellular p-CoA levels by altering the
fermentation medium, e.g. by removing lipids and amino acids that
break down to p-CoA. This approach may also limit the supply of
mm-CoA, and if this occurs, one can alter the erythromycin PKS so
that it no longer will use p-CoA, as described in Example 2 and
below.
[0212] A novel method for altering the DEBS loading domain (or any
loading domain that has an AT domain that binds propionyl CoA)
provided by the present invention involves site specific mutation
of the AT.sub.L domain. An Arg residue in an AT.sub.E domain that
normally utilizes em-CoA was mutated to an Ala or Trp, and each of
the mutated AT domains was substituted for the wild-type AT.sub.L
domain of DEBSI. Tests of both mutant proteins by single turnover
experiments in vitro revealed that each one prefers b-CoA over
p-CoA as substrate. Acetyl-CoA appears to compete for b-CoA,
however. This behavior is consistent with the effect of the same
mutation on the substrate specificity of the AT domain in animal
FAS (57a). A plasmid containing the mutant DEBS1 plus DEBS2 and
DEBS3 genes can be constructed in accordance with the methods of
the present invention to assess whether either mutation results in
preferential formation of 15-methyl-6dEB instead of 6-dEB in vivo
under conditions that provide an adequate level of b-CoA and
minimize the amounts of acetyl-CoA and p-CoA.
[0213] If preferential formation of 15-methyl-6dEB is not observed
in a strain of interest, one can employ the following alternative
method provided by the present invention. A segment of DNA is
assembled through directed or PCR-cloning that encodes KS.sup.Q,
AT.sub.E (specific for em-CoA) and ACP.sub.L in the DEBS1 (or
other) loading module, as illustrated in Example 2. In this case,
synthesis is initiated with em-CoA, which is subsequently
decarboxylated to yield butyryl-ACP (33) that can be extended by
DEBS in the usual way. The DEBS1 gene mutant is combined with the
DEBS2 and DEBS3 genes (or analogous genes from, for example the meg
or ole gene clusters) to assess the effect on production of
15-methyl6dEB vs. 6-dEB in vivo.
[0214] PKS domain/module switches can result in significant
reductions in the amount of polyketide produced (40,42) even though
the predicted structure is made. It may be necessary to employ
several different KS.sup.Q, AT.sub.E and ACP.sub.L domains and
employ them in various combinations to construct the PKS capable of
producing 15-methyl-6dEB at satisfactory levels. KS.sup.Q domains
from the oleandomycin and picromycin/methymicin PKSs have been
cloned and characterized at Kosan and can be used to construct the
corresponding DEBS1 segment. The AT.sub.E domain from module 4 of
the FK520 PKS has also been sequenced, as have those in module 5 of
the niddamycin and tylosin gene clusters (31,58). These AT.sub.E
sequences can be employed in constructing the novel loading domain
coding sequences of the invention. For the ACP in the loading
domain one can use the natural DEBS domain or replace it with ones
from the cognate modules that are used for the KS.sup.Q or AT.sub.E
domains. One should take care to ensure that the native interdomain
sequences are maintained as much as is possible and that the
spacing distances are kept constant. Because one can test rapidly
how efficient each construct is in producing 15-methyl-6dEB, one
can let the results guide the domains, interdomain regions, and
other factors to achieve the best construct.
[0215] Another method of the invention utilizing a novel loading
domain involves cloning the portion of the genes from Streptomyces
rimosus ATCC. 10970 that encodes the loading module and extender
module 1 of the rimocidin PKS. Rimocidin is a polyene macrolide
produced by this strain along with oxytetracycline, a widely used
antibacterial drug. The structure of rimocidin [23] shows a
polyketide backbone built from a saturated four carbon starter
unit. This could simply be b-CoA or could be formed from acetyl-CoA
and m-CoA by the first module of the rimocidin PKS wherein carbon
chain assembly involves complete reduction of the initial diketide
intermediate. Either case would provide a means for making
15-methylerythromycins. One can clone the desired genes from a
cosmid library of S. rimosus DNA using a specific probe for the
type I PKS genes involved in rimocidin production. Sequence
analysis of approx. 7 kb of DNA from the end of the cluster of PKS
genes that contains module 1 of the rimocidin PKS will identify the
desired genes and reveal which way the first four carbons of the
polyketide backbone are built. If b-CoA is the starter unit, one
replace the loading domain of DEBS1 (or the analogous domain of
MEG1) with one from the rimocidin PKS, following the guidelines
above and then determine whether the plasmid containing the full
set of DEBS genes directs preferential production of
15-methyl-6dEB. In contrast, if the first four carbons are built
from acetyl-CoA and m-CoA by extender module 1 of the rimocidin
PKS, one can replace the loading module of DEBS1 with that module,
assuming that the butyryl-ACP formed by it will be accepted by
module 1 of DEBS1 to allow elongation of the polyketide chain.
Formation of a functional DEBS1 PKS by addition of a complete
module to form a trimodular PKS is precedented (58a). This
construct can be evaluated as described above for the other PKS
genes to assess whether 15-methyl-6dEB is formed preferentially.
Success would avoid establishing fermentation conditions favorable
to a high b-CoA instead of p-CoA level in vivo.
[0216] One can use well established conditions for preparation of
recombinant cells, their growth in liquid fermentation media, and
the isolation and assay of the desired products in all of the above
methods. In particular, to make 15-methyl-6dEB and then
15-methylEryA, the normal DEBS loading module can be exchanged with
the engineered loading module/module 1 in a Sac. erythraea strain
through homologous recombination employing crossover sites upstream
and downstream of the altered sequence in the eryA region. This can
be done to result in 15-methyl-6dEB or 15-methylEryA
production.
[0217] One can build a de novo b-CoA pathway in Sac. erythraea with
the aim of producing 15-MeEryA (at high level) in the absence of
background EryA. This will take place when the level of p-CoA is
low enough and that of b-CoA high enough to allow the optimal form
of DEBS employed to use b-CoA (or the em-CoA derived from b-CoA)
exclusively as the starter unit. Although the methods described
above involving introduction of the p-CoA carboxylase genes or
manipulation of the fermentation conditions can allow one to
achieve the desired goal, alternative methods are provided by the
present invention. Because there is no precedent for employing a
genetic approach to diversion of metabolism to produce a novel
secondary metabolite, an empirical approach is described here to
achieve this goal.
[0218] As described above, there are several routes to the
synthesis of the various acyl-CoAs required for polyketide
biosynthesis (illustrated in the schematic above). Because the
valine utilzation pathway represents a major route to the required
precursors, one can shunt some of the pathway intermediate ib-CoA
to b-CoA. First, one can check the levels of b-CoA in fermentations
fed with high valine content proteinaceous substrates to establish
a baseline. Then, one can introduce the S. cinnamonensis genes icmA
and icmB for ib CoA mutase into the Sac. erythraea host under the
control of the ermE* promoter and examine the cells for the amount
of 15-MeEryA and EryA produced. One can also examine cells for the
levels of b-CoA and ib-CoA. The degree of success of this method
depends on the host either containing a large pool of ib-CoA or
there being sufficient flux through the valine degradation pathway
to provide enough ib-CoA to be converted efficiently to the
required b-CoA to promote its high level incorporation in
polyketide synthesis.
[0219] If one does not see the desired effect of the icmA and B
genes on 15-MeEryA or b-CoA levels in a cell and the cells contain
ib-CoA mutase activity, particularly in cultures fed with high
valine containing proteins, one can examine pathway flux by
measuring the level of BKD activity in erythromycin-producing Sac.
erythraea cultures (see the schematic above). If one finds
indistinguishable levels of activity in low and high
erythromycin-producing strains, one can in accordance with the
methods of the invention overexpress the S. coelicolor bkdF, G and
H genes, which have been identified in the S. coelicolor genome, in
the appropriate Sac. erythraea host containing icmA and icmB and
determine the effect on 15-MeEryA and b-CoA levels.
[0220] A second method provided by the invention is to build the
b-CoA pathway from acetyl-COA or intermediates to b-CoA, such as
crotonyl-CoA, produced by fatty acid utilization. As a first step,
one can introduce an overexpressed ccr gene from S. collinus or
which is cloned from the chromosome of S. coelicolor or S. fradiae
into Sac. erythraea. The impact on 15-MeEryA production and b-CoA
levels is measured when the host is grown under conditions that
permit fatty acid oxidation or with the addition of crotonic acid
(or an esterified derivative). One can measure the activity of
crotonyl-CoA reductase in the host containing this gene. If the
activity of the enzyme is high (relative to published reports) but
the level of b-CoA is low, one can clone the remaining three genes
acsA, bdh and ech from the chromosome of S. coelicolor and
introduce them into the chromosome of Sac. erythraea under control
of the ermE* promoter. These genes can be combined, if necessary,
with the icmA and icmB (and bkdF, G & H) genes within a given
host.
[0221] The present invention also provides host cells in addition
to Sac. erythraea for production of 15-methylEryA. An illustrative
and preferred embodiment is an S. fradiae strain that makes high
titers of tylosin. This degree of success with this method depends
on an adequate supply of b-CoA. One can employ a model system to
establish first the basic requirements for preferential utilization
of b-CoA as the starter unit in a strain of interest. This involves
formation of 2,4-dimethyl-3,5-dihydro- xyheptanoic acid
delta-lactone [14] and its 2,4-dimethyl-3,5-dihydroxyocta- noic
acid delta-lactone homolog [21] in the strain. Compounds 14 and 21
are made from p-CoA and b-CoA, respectively; therefore,
preferential production of 21 will be an indicator of success.
Conditions that achieve this should also be the ones that will
favor production of 15-methylEryA over EryA.
[0222] To achieve a high level of production of 21 in S. fradiae,
one can clone segments of the DEBS1-TE gene behind the tylG
promoter (tylGp) from this strain. An 864 nt sequence immediately
upstream of the first tylosin PKS gene has been sequenced. This
sequence can be used to isolate the tylG promoter (along with the
KS.sup.Q domain) from a genomic library of S. fradiae DNA prepared
in a cosmid vector in E. coli. One can sequence the tylG promoter
region and then clone it into the vector used to introduce the
constructs into S. fradiae. The tylG promoter from the a high level
tylosin promoter is preferred, but other systems, such as for
example, the actIp-actII-ORF4 expression system, can be employed.
The latter system has been used to produce .about.200 mg/L of 6dEB
in S. coelicolor but has not been examined for polyketide synthesis
in S. fradiae. The tylG promoter in S. fradiae can produce high
levels of tylosin, so tylGp is a good choice for 21 production in
this host. One can standardize delta-lactone production in the
various constructs extracted from the same fermentation sample
because the syntheses of both tylosin and 21 will be under the
control of tylGp (see below). Furthermore, because one ultimately
desires to express the altered (or unaltered) DEBS such that it
produces 15-MeEryA under control of the tylGp, one can subsequently
transfer the ultimate construct within the context of the full DEBS
PKS into S. fradiae.
[0223] Various plasmids that either replicate autonomously or
integrate site-specifically at the phage-phiC31 attachment site
have been used to introduce genes into S. fradiae by protoplast
transformation (59). Versions of both sets of plasmids carrying the
oriT locus are available for conjugal transfer from E. coli (60).
One can employ a set of plasmids that replicate autonomously or
integrate into the chromosome site-specifically that contain or
lack oriT. One can examine the frequency of transfer of these
plasmids into the S. fradiae host employing the published methods
to determine which plasmid and which transfer system is best to use
to make the necessary constructs that make 21 and introduce them
into the S. fradiae strain.
[0224] The S. fradiae/DEBS1-TE constructs are examined for
production of the corresponding deltalactone 21 and tylosin, using
growth conditions and metabolite isolation and assay methods for
tylosin from the literature in conjunction with suitable methods
for the delta-lactone. One will grow a number of independent
isolates (transformants or transconjugants) of each strain under
precise fermentation conditions and assay replicate samples.
Polyketide products will be extracted from whole broth samples
using C.sub.18 reversed-phase resin and analyzed by LC-mass
spectroscopy. Low-level products will be quantitated by comparison
of integrated ion currents with a standard curve generated using
pure samples. High-level products will be quantitated by
evaporative light scattering. If necessary, one can also examine
the samples for the pool sizes of various acyl-CoA thioesters to
ensure that variations in the levels of production of 21 from the
constructs are not the result of precursor supply fluctuations.
[0225] Although the normal DEBS1-TE gene under the control of tylGp
can be used as a control for delta-lactone production in S.
fradiae, it cannot be stated with certainty for any particular
strain whether 14 or 21 will be made. This will depend upon the
relative pool sizes of p-CoA and b-CoA and their relative rates of
incorporation into the corresponding growing polyketide chains. If
21 is produced exclusively, it will not be necessary to re-engineer
DEBS for 15-MeEryA production in S. fradiae.
[0226] After one determines which of the constructs yields the
highest production of 21, one can use the exact segment to rebuild
a PKS that can make 15-methyl-6dEB in S. fradiae. The most
straightforward way to exchange the normal DEBS loading module with
the altered loading module is to exchange a suitable restriction
fragment containing tylGp-DEBS loading or altered DEBS loading
domains--DEBS module 1 (to a convenient restriction site) with the
corresponding fragment in eryA1 that includes sequences to
eliminate upstream promoters. This will yield a segment containing
the three DEBS (or modified DEBS) genes behind the tylG promoter.
One can then insert these genes in a vector that can site
specifically integrate in the chromosome of S. fradiae and disrupt
the resident tylG genes. If this approach yields too little
15-methyl-6dEB, one can engineer the replacement of the tylG genes
with the DEBS genes under the control of the tylG promoter. To
accomplish this, one can clone the segment immediately downstream
of the beginning of the tylG genes taking care to maintain the same
PKS-downstream gene orientation as was present in the tyl cluster.
This entire segment is placed in a suicide vector for the two-step
recombination required for exchange of the tyl PKS with the ery (or
modified ery) PKS genes. Production of 15-methyl-6dEB instead of
6dEB itself by this recombinant strain yields the desired
result.
[0227] With this achieved, one can introduce the entire set of
erythromycin biosynthesis genes, containing the engineered DEBS1
gene, into S. fradiae. Ideally, one will transfer the ery gene
cluster from a suitable donor strain into a properly marked S.
fradiae recipient by the method of Hu et al. (53b). One can employ
a plasmid containing the entire ery gene cluster from Sac.
erythraea and marked with a second gene tsr encoding thiostrepton
resistance. The ery genes in this construct are flanked with
approx. 2 kb segments of the tylosin gene cluster flanking the
region into which the engineered ery genes are to be inserted. The
eryA1 PKS gene is replaced with the engineered version of DEBS1
described above. Finally, the two tra genes from pIJ101 that
mediate the conjugal transfer of the ery gene cluster from the
donor strain to S. fradiae are cloned onto the vector or provided
in trans in the donor strain. Once these constructs and strains are
made, conjugal mating of the donor strain (preferably a
streptomycin-sensitive strain of S. coelicolor) with a streptomycin
resistant, thiostrepton sensitive S. fradiae recipient strain
followed by selection for thiostrepton resistance and
counterselection of the donor strain by streptomycin results in
transfer of the ery genes into S. fradiae. Preferably, the incoming
genes replace most of the tylosin gene cluster, which should be
sufficient to allow erythromycin production even if some of the
other tylosin biosynthesis genes are still present. Because one can
select for the desired result via thiostrepton, one can obtain the
recombinant albeit at a very low transfer frequency. Alternatively,
the following method can be employed.
[0228] One can provide S. fradiae the ability to produce and
transfer desosamine and mycarose to 15-methylEB in accordance with
the methods of the present invention. Because TDP-desosamine is
required for the synthesis of 15-MeEryA, one can provide for its
synthesis in S. fradiae as followings. The addition of the two
desosaminyl-specific genes, eryCIV and eryCV, which are adjacent to
each other and probably co-transcribed in Sac. erythraea, should
divert the mycaminosyl pathway to result in the synthesis of the
5-O-desosaminyltylonolide [22] when cloned in S. fradiae. 13
[0229] One can also add the gene eryCIII, the
desosaminyltransferase, that is required to make 15-MeEryA in S.
fradiae. The synthesis of 22 will require that 4' (see the
schematic above) is produced by the addition of eryCIV and eryC,
can be converted to TDP-desosamine by the enzyme that aminates 2'
(see the schematic above) to yield TDP-mycaminose, and that the
TDP-mycaminosyltransferase can add TDP-desosamine to the C-5
hydroxyl of tylonolide. Because one is not disrupting any of the
tyl genes at this step, this all-or-none result would take place
within the background of the host that still produces tylosin.
[0230] In the normal pathway of tylosin biosynthesis, the cognate
intermediate (5-O-mycaminosyltylonolide) undergoes glycosylation at
C-4' of the mycaminosyl moiety with TDP-mycarose. The absence of an
OH group at C-4' would be expected to interrupt the pathway and
result in the production of 22 as a final product. One can examine
the fermentation broth for the presence of 22 by LC/MS and perform
rigorous analyses to confirm the structure. If the compound is not
produced, one can add 3-O-alphamycarosylerythronolide B, the
natural substrate for the eryCIII-catalyzed transfer of
TDP-desosamine, and look for the production of erythromycin D in
the fermentation broth. Finding this compound will validate that
the pathway to desosamine has been achieved in S. fradiae, even if
one does not see 22 produced. If one does not observe erythromycin
D, one can add the additional genes eryCI and eryCVI to S. fradiae
and again look for the synthesis of 22 in unfed cultures or
erythromycin D in cultures fed with
3-O-alpha-mycarosylerythronolide B. Once the desosamine pathway in
S. fradiae is produced, one can then introduce the gene eryBV, the
transferase which adds mycarose to the C-3 OH position of
erythronolide B. One can confirm the activity of EryBV by the
identification of the compound 3-O-alphamycarosylerythronolide B in
fermentations which are fed with erythronolide B. If the presence
of the TDP-mycaminose pathway interferes with the production of 22,
one can disrupt tylJ to eliminate the synthesis of this sugar
(61).
[0231] The gene eryF (or a gene with similar activity) is
introduced, under the control of the ermE* promoter into the S.
fradiae host that contains the eryC and B genes. Its activity will
be confirmed by demonstrating efficient conversion of 6-dEB to
erythromycin D when 6-dEB is fed to fermentation cultures.
Similarly, eryK (or a gene with similar activity) is introduced in
the host containing the required eryB, eryC and eryF genes and its
function confirmed by demonstrating efficient conversion 6-dEB to
erythromycin C when 6-dEB is fed to fermentation cultures.
[0232] Placement of DEBS in the S. fradiae chromosome behind the
tylG promoter can be done in three steps: (i) a delivery plasmid
containing the DEBS genes behind tylGp and at least a 1 kb sequence
downstream of tylG5 (tylG5ds) is built using PCR or restriction
fragments from the genome of S. fradiae, the loading module from
tylGp-DEBS1 (or modified tylGp-DEBS1-TE), and the full DEBS genes;
(ii) the tylG genes are removed from the chromosome of S. fradiae
in a two-step homologous replacement experiment employing similar
tylGp and downstream sequences; and (iii) the tylGp-DEBS-tylG5ds
segment is exchanged in a two-step homologous recombination
experiment with the tylG-free sequence in the chromosome. This will
leave the host with DEBS free of tylG PKS sequences. Furthermore,
the expression of the DEBS genes under tylGp will be regulated
similarly to those of the tylG genes in the unmodifed S. fradiae
host. The cloning described allows the S. fradiae host to produce
15-MeEryC from b-CoA and mm-CoA.
[0233] Conversion of 15-MeEryC to 15-MeEryA is dependent upon the
presence of the gene eryG. Because ketolide antibacterials have no
sugar at the 3-position, either 15-MeEryC or 15-MeEryA is a
suitable starting material. Therefore, if one obtain 15-MeEryC
through the cloning in S. fradiae described here, one can attempt
its conversion to 15-MeEryA through the introduction of eryG if
desired, but this is not required if the strain is used to make an
intermediate used in ketolide production.
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[0302] 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.
* * * * *
References