U.S. patent application number 09/870012 was filed with the patent office on 2001-09-27 for method to produce novel polyketides.
Invention is credited to Crane, David E., Kao, Camilla, Khosla, Chaitan, Luo, Guanglin, Pieper, Rembert.
Application Number | 20010024811 09/870012 |
Document ID | / |
Family ID | 25406015 |
Filed Date | 2001-09-27 |
United States Patent
Application |
20010024811 |
Kind Code |
A1 |
Khosla, Chaitan ; et
al. |
September 27, 2001 |
Method to produce novel polyketides
Abstract
Modified PKS gene clusters which produce novel polyketides in an
efficient system in a host cell or in a cell free extract are
described.
Inventors: |
Khosla, Chaitan; (Stanford,
CA) ; Pieper, Rembert; (Washington, DC) ; Luo,
Guanglin; (Madison, CT) ; Crane, David E.;
(Providence, RI) ; Kao, Camilla; (Palo Alto,
CA) |
Correspondence
Address: |
Kate H. Murashige
Morrison & Foerster LLP
Suite 500
3811 Valley Centre Drive
San Diego
CA
92130-2332
US
|
Family ID: |
25406015 |
Appl. No.: |
09/870012 |
Filed: |
May 29, 2001 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09870012 |
May 29, 2001 |
|
|
|
09434289 |
Nov 5, 1999 |
|
|
|
6261816 |
|
|
|
|
09434289 |
Nov 5, 1999 |
|
|
|
08896323 |
Jul 17, 1997 |
|
|
|
6066721 |
|
|
|
|
08896323 |
Jul 17, 1997 |
|
|
|
08675817 |
Jul 5, 1996 |
|
|
|
6080555 |
|
|
|
|
60003338 |
Jul 6, 1995 |
|
|
|
Current U.S.
Class: |
435/91.4 ;
435/118; 435/124; 435/91.1 |
Current CPC
Class: |
C12N 15/52 20130101;
C12P 17/06 20130101; C12P 17/08 20130101; C07H 17/08 20130101 |
Class at
Publication: |
435/91.4 ;
435/91.1; 435/118; 435/124 |
International
Class: |
C12P 019/34; C12N
015/64; C12P 017/16; C12P 017/10; C12P 017/08 |
Goverment Interests
[0002] This invention was made with U.S. government support from
the National Institutes of Health (GM22172 and CA66736-01). The
government has certain rights in this invention.
Claims
1. A modified modular polyketide synthase (PKS) comprising at least
two modules, wherein said PKS has been modified to prevent its
utilization of the native starter unit for said modular PKS.
2. The modified PKS of claim 1 wherein the ketosynthase (KS)
catalytic domain of module 1 has been inactivated.
3. The modified PKS of claim 1 wherein said modules are modules of
the DEBS PKS.
4. The modified of PKS of claim 2 wherein said modules are modules
of the DEBS PKS.
5. The modified PKS of claim 1 which is a complete PKS.
6. A PKS gene cluster which encodes a modified PKS wherein said
modified PKS has been modified to prevent its utilization of the
native starter unit for said modular PKS.
7. The gene cluster of claim 6 wherein the ketosynthase (KS)
catalytic domain of module 1 has been inactivated.
8. The gene cluster of claim 6 wherein said modules are modules of
the DEBS PKS.
9. The gene cluster of claim 7 wherein said modules are modules of
the DEBS PKS.
10. The gene cluster of claim 6 which encodes a complete PKS.
11. A recombinant host cell modified to contain the gene cluster of
claim 6.
12. The host cell of claim 10 which is a Streptomyces.
13. The host cell of claim 10 which is free of any endogenous PKS
activity.
14. A method to prepare a polyketide, which method comprises
providing a thioester diketide substrate for the modified PKS of
claim 1.
15. The method of claim 14 which is conducted in a host cell.
16. The method of claim 14 which is conducted in a cell free
system.
17. A novel polyketide which has the structure shown as formula 6,
7 or 8 in FIG. 2.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. Ser. No.
08/675,817 filed Jul. 5, 1996 which claims priority under 35 USC
119(e)(1) from provisional application Ser. No. 60/003,338 filed
Jul. 6, 1995. The contents of these applications are incorporated
herein by reference.
TECHNICAL FIELD
[0003] The invention relates to methods to synthesize polyketides
which are novel using modified modular polyketides synthases (PKS)
which cannot utilize a natural first module starter unit.
BACKGROUND ART
[0004] Modular polyketide syntheses are typified by the
organization of deoxyerythronolide B synthase (DEBS) which produces
.beta.-deoxyerythronolide B (6-dEB) the parent macrolactone of the
broad spectrum antibiotic erythromycin. DEBS consists of three
large polypeptides each containing about 10 distinctive active
sites. FIG. 1 shows, diagramatically, the nature of the three DEBS
modules encoded by the three genes eryAI, eryAII and eryAIII.
[0005] Various strategies have been suggested for genetic
manipulation of PKS to produce novel polyketides. New polyketides
have been generated through module deletion (Kao, C. M. et al., J.
Am. Chem. Soc. (1995) 117:9105-9106; Kao, C. M. et al, J. Am. Chem.
Soc. (1996) 118:9184-9185). Also reported to provide novel
polyketides are loss of function mutagenesis within reductive
domains (Donadio, S. et al., Science (1991) 252:675-679; Donadio,
S. et al, Proc. Natl. Acad. Sci. USA (1993) 90:7119-7123; Bedford,
D. et al., Chem. Biol. (1996) 3:827-831) and replacement of acyl
transferase domains to alter starter or extender unit specificity
(Oliynyk, M et al., Chem. Biol.-(1996) 3:833-839; Kuhstoss, S. et
al., Gene (1996) 183:231-236), as well as gain of function
mutagenesis to introduce new catalytic activities within existing
modules (McDaniel, R. et al., J. Am. Chem. Soc. (1997) in press).
In some of these reports, downstream enzymes in the polyketide
pathway have been shown to process non-natural intermediates.
However, these methods for providing novel polyketides suffer from
the disadvantages of requiring investment in cloning and DNA
sequencing, the systems used being limited to producer organisms
for which gene replacement techniques have been developed, primer
and extender units that can only be derived from metabolically
accessible CoA thioesters, and the fact that only limited auxiliary
catalytic functions can be employed.
[0006] The DEBS system in particular has been shown to accept
non-natural primer units such as acetyl and butyryl-CoA (Wiesmann,
KEH et al, Chem. Biol. (1995)2:583-589; Pieper, R. et al, J. Am.
Chem. Soc. (1995) 117:11373-11374) as well as N-acetylcysteamine
(NAC) thioesters of their corresponding ketides (Pieper, R. et al.,
Nature (1995) 378:263-266). However, it has become clear that even
though such unnatural substrates can be utilized, competition from
the natural starter unit has drastically lowered yield. Even if
starter units are not supplied artificially, they can be inherently
generated from decarboxylation of the methylmalonyl extender units
employed by the DEBS system (Pieper, R. et al., Biochemistry (1996)
35:2054-2060; Pieper, R. et al., Biochemistry
(1997)36:1846-1851).
[0007] Accordingly, it would be advantageous to provide a mutant
form of the modular polyketide synthesis system which cannot employ
the natural starter unit. Such systems can be induced to make novel
polyketides by supplying, instead, a suitable diketide as an NAC
thioester or other suitable thioester. Mutations have been made in
the past to eliminate the competition from natural materials (Daum,
S. J. et al., Ann. Rev. Microbiol. (1979) 33:241-265). Novel
avermectin derivatives have been synthesized using a randomly
generated mutant strain of the overmectin producing organism
(Dutton, C. J. et al., Tetrahedron Letters (1994) 35:327-330;
Dutton, C. J. et al., J. Antibiot. (1991) 44:357-365). This
strategy is, however, not generally applicable due to
inefficiencies in both mutagenesis and incorporation of the
substrates.
[0008] Thus, there is a need for a more efficient system to prepare
novel polyketides by inhibiting competitive production of the
natural product.
DISCLOSURE OF THE INVENTION
[0009] The invention is directed to methods to prepare novel
polyketides using modified modular polyketide synthase systems
wherein directed modification incapacitates the system from using
its natural starting material. Novel polyketides can then be
synthesized by overriding the starter module and supplying a
variety of suitable diketide substrates.
[0010] Thus, in one aspect, the invention is directed to a method
to prepare a novel polyketide which method comprises providing a
thioester diketide substrate to a modular PKS comprising at least
two modules under conditions wherein said substrate is converted by
said modular PKS to a product polyketide, wherein said PKS has been
modified to prevent its utilization of the native starter unit. In
other aspects, the invention is directed to the modified modular
PKS which is disarmed with respect to utilization of the native
starter substrate supplying the initial two carbon unit, and to
suitable cells modified to contain this disarmed PKS. The invention
is further directed to recombinant materials for production of the
modified PKS and to the novel polyketides produced by this
system.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 shows a schematic representation of the DEBS modular
PKS.
[0012] FIG. 2 shows the products of a modified DEBS construct
wherein the ketosynthase in module 1 is disarmed.
[0013] FIG. 3 shows the processing of 6-dEB derivatives to
erythromycin-D derivatives.
MODES OF CARRYING OUT THE INVENTION
[0014] The invention provides modular PKS systems which are
disarmed with respect to loading the native starting material and
their corresponding genes. In a particularly preferred embodiment,
the ketosynthase (KS) of module 1 is inactivated so as to prevent
competition from the native starter unit. Other approaches to
similarly disarming the PKS involve inactivating the acyl
transferase (AT) or acyl carrier protein (ACP) functions of module
1.
[0015] The PKS of the invention must contain at least two modules
but may contain additional modules and, indeed may, represent
complete synthase systems. While the DEBS PKS system is used to
illustrate the invention, any modular PKS can be used, such as the
modular PKS resulting in the production of avermectin, rapamycin
and the like. Suitable mutations can be introduced by known site
specific mutagenesis techniques.
[0016] Other micro-organisms such as yeast and bacteria may also be
used. The novel polyketides may be synthesized in a suitable hosts,
such as a Streptomyces host, especially a Streptomyces host
modified so as to delete its own PKS. The polyketides may also be
synthesized using a cell-free system by producing the relevant PKS
proteins recombinantly and effecting their secretion or lysing the
cells containing them. A typical cell-free system would include the
appropriate PKS, NADPH and an appropriate buffer and substrates
required for the catalytic synthesis of polyketides. To produce the
novel polyketides thioesters of the extender units are employed
along with the thioester of a diketide.
[0017] The following examples are intended to illustrate but not to
limit the invention.
Preparation A
Starting Materials
[0018] Streptomyces coelicolor CH999, which has been engineered to
remove the native PKS gene cluster is constructed as described in
WO 95/08548. pRM5, a shuttle plasmid used for expressing PKS genes
in CH999 was also described in that application. Plasmid pCK7 which
contains the entire DEBS modular system was described in the
foregoing application as well.
EXAMPLE 1
Preparation of DEBS 1+2+TE
[0019] A modified DEBS PKS system containing only modules 1 and 2
and thioesterase (TE) activity, designated DEBS 1+2+TE, was
subjected to site directed mutagenesis to inactivate module 1 KS by
replacing the active site cysteine residue in the signature
sequence cys-ser-ser-ser-leu by alanine. The resulting expression
plasmid, designated pKAO179, encodes a 2-module PKS which is
inactive under the standard reaction conditions for synthesis of
the native product, i.e., propionyl-CoA, methylmalonyl-CoA, and
NADPH. The details of this construction are set forth in Kao, C. M.
et al, Biochemistry (1996) 35:12363-12368. When provided with the
diketide thioester (2S,
3R)-2-methyl-3,3-hydroxy-pentanoyl-N-acetylcystea- mine thioester,
and with methylmalonyl-CoA, and NADPH, the triketide product set
forth below is obtained.
[0020] The triketide product is produced under these conditions
when the PKS is incubated in a cell-free system or can be
duplicated in vivo by providing the appropriate diketide thioester
analogs to actively growing cultures of CH99 containing the
modified expression plasmid:
[0021] A culture of S. coelicolor CH999/pKAO179 is established by
inoculation of 200 mL of SMM medium (5% PEG-800, 0.06% MgSO.sub.4,
0.2% (NH.sub.4).sub.2SO.sub.4, 25 mM TES, pH 7.02, 25 mM
KH.sub.2PO.sub.4, 1.6% glucose, 0.5% casamino acids, trace
elements) with spores. The culture is incubated at 30.degree. C.
with shaking at 325 rpm. A solution of (2S,
3R)-2-methyl-3-hydroxypentanoyl N-acetlycysteamine thioester (100
mg) and 4-pentynoic (15 mg) in 1 mL of methylsulfoxide is added to
the culture in three parts: after 50 hours (400 mL); after 62 hours
(300 mL); and after 86 hours (300 mL). After a total of 144 hours,
the culture is centrifuged to remove mycelia. The fermentation
broth is saturated with NaCl and extracted with ethyl acetate
(5.times.100 mL). The combined organic extract is dried over
Na.sub.2SO.sub.4, filtered, and concentrated. Silica gel
chromatography yields (2R, 3S, 4S, 5R)-2,4-dimethyl-3,
5-dihydroxy-n-heptanoic acid .delta.-lactone.
EXAMPLE 2
Preparation of Polyketides from the DEBS Cluster
[0022] The active site mutated module 1 KS domain of the eryAI
(DEBS 1 gene) is provided on plasmid pCK7, which contains the
eryAI, eryAII (DEBS 2) and eryAIII (DEBS 3 genes) under control of
the actI promoter. Expression from this plasmid pJRJ2 results in a
suitably modified full length PKS system. (Kao, C. M et al.,
Science (1994) 265:409-512. pJRJ2 was transformed into CH999 and
grown on R2YE medium. No detectable 6 DEB-like products were
produced.
[0023] In more detail, lawns of CH999/pJRJ2 were grown at
30.degree. C. on R2YE agar plates containing 0.3 mg/ml sodium
propionate. After three days, each agar plate was overlayed with
1.5 mL of a 20 mM substrate solution in 9:1 water:DMSO. After an
additional 4 days, the agar media (300 mL) were homogenized and
extracted three times with ethyl acetate. The solvent was dried
over magnesium sulfate and concentrated. Concentrated extracts were
purified by silica gel chromatography (gradient of ethyl acetate in
hexanes) to afford products.
[0024] However, when substrate 2, prepared by the method of Cane et
al., J. Am. Chem. Soc. (1993) 115:522-526; Cane, D. E. et al., J.
Antibiot. (1995) 48:647-651, shown in FIG. 2 (the NAC thioester of
the native diketide) was added to the system, the normal product, 6
dEB was produced in large quantities. Administration of 100 mg
substrate 2 to small scale cultures (300 ml grown on petri plates
as described above, resulted in production of 30 mg 6 dEB, 18%
yield.
EXAMPLE 3
Production of Novel Polyketides
[0025] Diketides with the structures shown in FIG. 2 as formulas 3,
4, and 5 were then administered to growing cultures of CH999/pJRJ2
under the conditions of Example 2. Substrates 3 and 4 were prepared
as described for Substrate 2 but substituting valeraldehyde and
phenylacetaldehyde, respectively for propionaldehyde in the aldol
reactions. The preparation of Substrate 5 was described by Yue, S.
et al., J. Am. Chem. Soc. (1987) 109:1253-1255. Substrates 3 and 4
provided 55 mg/L of product 6 and 22 mg/L of product 7.
respectively. Substrate 5 resulted in the production of 25 mg/L of
the 16 member lactone 8, an unexpected product.
EXAMPLE 4
Processing of the Polyketide Products
[0026] The successful processing of unnatural intermediates by the
"downstream" modules of DEBS prompted an experiment to determine
whether the post-PKS enzymes in the erthromycin biosynthetic
pathway might also accept unnatural substrates. In the natural
producer organism, Saccharopolyspora erythrea, 6dEB undergoes
several enzyme-catalyzed transformations. Oxidation at C6 and
glycosylations at C3 and C5 afford erythromycin D (formula 9 in
FIG. 3) and subsequent transformations afford erythromycins A, B,
and C. S. erythrea mutant (A34) (Weber, J. M. et al., J. Bactiol.
(1985) 164:425-433) is unable to synthesize 6dEB. This strain
produces no erythromycin when grown on R2YE plates (as judged by
the ability of extracts to inhibit growth of the
erythromycin-sensitive bacterium Bacillus cereus). However, when
6dEB (which has no antibacterial activity) is added to the culture
medium, extracts exhibited potent antibacterial activity.
[0027] Samples of 6dEB derivatives 6 and 7 were assayed for
conversion by this strain. Partially purified extracts demonstrated
inhibition of B. cereus growth, and mass spectrometry was used to
identify the major components of the extracts as formula 10 in FIG.
3 (from 6) and formula 11 (from 7).
[0028] In more detail, purified 6 and 7 (5 mg dissolved in 7.5 mL
50% aqueous ethanol) were layered onto R2YE plates (200 mL
media/experiment) and allowed to dry. S. erythrea A34 was then
applied so as to give lawns. After 7 days of growth, the media were
homogenized and extracted three times with 98.5:1.5 ethyl
acetate:triethylamine. Pooled extracts from each experiment were
dried over magensium sulfate and concentrated. Extracts were
partially purified by silica gel chromatography (gradient of
methanol and triethylamine in chloroform). The partially purified
extracts were examined by TLC and mass spectrometry. For
antibacterial activity analysis, filter discs were soaked in 400
.mu.M ethanolic solutions of erythromycin D, 10 and 11, as well as
a concentrated extract from S. erythrea A34 which had been grown
without addition of any 6-dEB analogs. Disks were dried and laid
over freshly-plated lawns of Bacillus cereus. After incubation for
12 h at 37.degree. C., inhibition of bacterial growth was evident
for all compounds but not for the control extract.
* * * * *