PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of aemPermissionsJournals.ASM.orgJournalAEM ArticleJournal InfoAuthorsReviewers
 
Appl Environ Microbiol. 2010 April; 76(8): 2681–2683.
Published online 2010 March 5. doi:  10.1128/AEM.02841-09
PMCID: PMC2849223

Heterologous Expression of the Oxytetracycline Biosynthetic Pathway in Myxococcus xanthus[down-pointing small open triangle]

Abstract

New natural products for drug discovery may be accessed by heterologous expression of bacterial biosynthetic pathways in metagenomic DNA libraries. However, a “universal” host is needed for this experiment. Herein, we show that Myxococcus xanthus is a potential “universal” host for heterologous expression of polyketide biosynthetic gene clusters.

Bacterial natural products are excellent lead compounds for drug discovery and have played major roles in the development of pharmaceutical agents in nearly all therapeutic areas (1, 7, 9). Unfortunately, the rate of discovery of new bacterial natural products has decreased, due in part to frequent rediscovery of known compounds (7). An enormous and currently inaccessible reservoir of new natural products is located in the biosynthetic pathways found in the genomes of uncultivated bacteria (18). Heterologous expression of these biosynthetic gene clusters represents a powerful tool for discovering new natural products (20, 21). Herein, we demonstrate that the deltaproteobacterium Myxococcus xanthus is an effective host for heterologous expression of aromatic polyketide biosynthetic pathways. This work expands the scope of polyketide biosynthetic pathways which can be heterologously expressed in M. xanthus and suggests that M. xanthus may be a suitable general host for heterologous expression.

Molecular phylogenetic studies have shown that bacterial diversity is enormous, and the vast majority of the diversity is found in uncultivated bacterial species (18). Estimates suggest that 99% of bacteria from the environment are uncultivatable using standard techniques (2, 15, 16). Culture-independent analyses of metagenomic DNA libraries from soil and marine environments indicate that there is a wealth of natural product diversity in these uncultivated strains. For example, analysis of a soil metagenome for a highly conserved region of polyketide synthase genes showed that none of the sequences found were present in the known public databases (5). Polyketide synthases are key enzymes responsible for the production of the polyketide family of natural products in proteobacteria, actinobacteria, and “low-G+C Gram-positive bacteria” (4, 12, 19). Polyketide natural products have been developed into antibiotic, anticancer, and immunosuppressant clinical agents (1, 6, 8). Based on these observations, metagenomic DNA libraries are expected to possess a large number of new polyketide biosynthetic pathways, representing substantial new chemical diversity for drug discovery.

Heterologous expression of biosynthetic pathways can play a major role in interrogating metagenomic DNA libraries for new polyketide biosynthetic pathways. Heterologous production of polyketides in hosts such as Streptomyces coelicolor and Streptomyces lividans is an important tool in the identification and characterization of these pathways (6, 8, 17). Results from these studies have shown that Streptomyces strains are good hosts for heterologous production of many polyketides, particularly those from actinomycetes. However, Streptomyces strains have proved to be poor hosts for expression of deltaproteobacterial polyketide biosynthetic pathways, such as those in myxobacteria (10, 17). As polyketide biosynthetic pathways in metagenomic DNA libraries contain both actinomycete- and deltaproteobacterium-derived pathways, a heterologous expression host competent to express pathways of both origins is needed.

We examined the ability of the deltaproteobacterium M. xanthus to act as a general heterologous expression host. M. xanthus is a predatory bacterium that undergoes multicellular development in response to nutrient starvation. During development, M. xanthus is known to be an effective host for the heterologous expression of the deltaproteobacterium-derived epothilone D biosynthetic pathway and has been used for the production of epothilone D for clinical trials (17). M. xanthus has also been shown to be an excellent host for the heterologous expression of several other myxobacterial metabolites, including myxothiazol and myxochromide S (3, 11, 22). We demonstrate that M. xanthus can also heterologously express the Streptomyces rimosus oxytetracycline biosynthetic pathway, producing oxytetracycline. This is the first example of a polyketide from a nonmyxobacterial species heterologously expressed in a myxobacterium.

To generate an M. xanthus strain capable of heterologously expressing oxytetracycline, the Streptomyces rimosus oxytetracycline biosynthetic pathway (Fig. (Fig.1)1) was inserted via homologous recombination into the asgE locus of M. xanthus. The asgE locus of M. xanthus was amplified and inserted into the BglII site of pET28b (Novagen) to produce pMRH02. The oligonucleotides used for the amplification of the asgE locus were 5′-GACGAGATCTGTTGGAAGGTCGGCAACTGG-3′ and 5′-CTTAAGATCTTCCGTGAAGTACTGGCGCAC-3′. The asgE locus provides a chromosomal region for single-crossover homologous recombination into the M. xanthus chromosome. The 32-kb oxytetracycline pathway in S. rimosus was excised from pYT264 (24) and cloned into the EcoRI site of pMRH02 to produce pMRH08. M. xanthus DK1622 was electroporated under standard conditions (13) with pMRH08 to provide an M. xanthus ΔasgE Kanr mutant. Positive selection for the chromosomal insertion was maintained throughout all experiments by use of kanamycin supplementation (40 μg/ml). This large genomic insertion significantly increased the doubling time for the strain (doubling time, ≈10 h).

FIG. 1.
Oxytetracycline biosynthetic pathway. (A) Enzymatic pathway responsible for formation of oxytetracycline. (B) Oxytetracycline biosynthesis gene cluster from S. rimosus.

Oxytetracycline was heterologously produced in M. xanthus under standard rich medium culture conditions and detected in culture broth by liquid chromatography-mass spectrometry (LC-MS). A liquid culture of the mutant strain containing the oxytetracycline gene cluster was cultured for 10 days at 33°C in CTTYE (1.0% Casitone, 0.5% yeast extract, 10.0 mM Tris-HCl, 1.0 mM KH2PO4, and 8.0 mM MgSO4; 100 ml). Acetone (10%, vol/vol) was added to the culture and vigorously mixed. The resulting mixture was extracted with 3 volumes of ethyl acetate to remove the organic soluble materials, including oxytetracycline. The organic extracts were concentrated in vacuo and resuspended in methanol (100 μl). LC-MS analyses were carried out using an Altima Hypersil C18 column (3-μm particle size; 150 mm by 2.1 mm) with a linear gradient of water-acetonitrile (5 to 95%) with 0.05% formic acid over 90 min (0.20 ml/min), followed by positive-ion electrospray ionization (5,500 V) and analysis with a Shimadzu 2010A single quadrupole mass spectrometer. LC-MS analysis indicated that oxytetracycline was present in the fermentation broth (Fig. (Fig.2).2). The titer of oxytetracycline was determined to be approximately 10 mg per liter of fermentation broth. Quantification was performed in triplicate by LC-MS analysis using a standard curve generated from commercial oxytetracycline. Negative controls of M. xanthus DK1622 cultures processed under identical conditions did not contain detectable levels of oxytetracycline.

FIG. 2.
LC-MS ion extraction analysis of the molecular ion [M+H]+ of standard and culture extracts. (A) Oxytetracycline standard. (B) M. xanthus ΔasgE Kanr mutant containing the oxytetracycline biosynthetic pathway. (C) Wild-type M. xanthus ...

These data indicate that M. xanthus can heterologously express the oxytetracycline polyketide synthase biosynthetic pathway in S. rimosus. Several factors affect the successful heterologous production of polyketide synthase pathways, including codon usage, mRNA stability, functionality of regulatory elements, and the presence of all necessary starter and extender units (14). As codon usages between M. xanthus and the genus Streptomyces are very similar and myxobacteria are known to produce polyketide products requiring a wide diversity of starter and extender units, neither codon usage nor starter and extender unit availability was considered likely to affect the ability of M. xanthus to heterologously express streptomycete biosynthetic pathways. As Streptomyces strains do not appear to be effective at heterologous expression of myxobacterial biosynthetic pathways, we were concerned that Myxococcus and Streptomyces strains may possess substantially different regulatory elements. Our data indicate that the regulatory elements present in streptomycete-derived biosynthetic pathways are sufficient to enable expression of the biosynthetic genes in M. xanthus. Further work exploring the regulatory elements present in myxobacterial polyketide biosynthetic gene clusters is needed to evaluate this hypothesis.

This study demonstrates that M. xanthus can heterologously express streptomycete-derived polyketide biosynthetic pathways in addition to myxobacterial polyketide biosynthetic pathways. The observed titer of 10 mg/liter of culture broth is comparable to titers reported for the heterologous expression of myxobacterial polyketide biosynthetic pathways in myxobacteria (11) and streptomycete-derived polyketide biosynthetic pathways in Streptomyces (14, 23) and is sufficient for characterization of the polyketide product. Pseudomonas putida, which has a more favorable growth profile, has been shown to be a good host for heterologous expression of myxobacterial polyketide biosynthetic pathways, with product titers in the range of 0.6 to 40 mg/liter of culture broth (14, 21, 23). The observed breadth of polyketide pathways accessible and the titers of the polyketide products produced make M. xanthus an attractive potential candidate for a “universal” host for facilitating heterologous expression of polyketide biosynthetic pathways derived from environmental samples of metagenomic DNA.

Acknowledgments

We gratefully acknowledge Yi Tang (UCLA) for providing the oxytetracycline gene cluster, Anthony Garza for providing DK1622, and Roy Welch and Anthony Garza for helpful discussion.

This work was supported by Syracuse University, Shimadzu Scientific, and Syracuse University Department of Biology (K.A.M.).

D.C.S. and M.R.H contributed equally to this work.

Footnotes

[down-pointing small open triangle]Published ahead of print on 5 March 2010.

REFERENCES

1. Berdy, J. 2005. Bioactive microbial metabolites. J. Antibiot. (Tokyo) 58:1-26. [PubMed]
2. Davis, K. E., S. J. Joseph, and P. H. Janssen. 2005. Effects of growth medium, inoculum size, and incubation time on culturability and isolation of soil bacteria. Appl. Environ. Microbiol. 71:826-834. [PMC free article] [PubMed]
3. Fu, J., S. C. Wenzel, O. Perlova, J. Wang, F. Gross, Z. Tang, Y. Yin, A. F. Stewart, R. Muller, and Y. Zhang. 2008. Efficient transfer of two large secondary metabolite pathway gene clusters into heterologous hosts by transposition. Nucleic Acids Res. 36:e113. [PMC free article] [PubMed]
4. Gerth, K., S. Pradella, O. Perlova, S. Beyer, and R. Müller. 2003. Myxobacteria: proficient producers of novel natural products with various biological activities—past and future biotechnological aspects with the focus on the genus Sorangium. J. Biotechnol. 106:233-253. [PubMed]
5. Ginolhac, A., C. Jarrin, B. Gillet, P. Robe, P. Pujic, K. Tuphile, H. Bertrand, T. M. Vogel, G. Perriere, P. Simonet, and R. Nalin. 2004. Phylogenetic analysis of polyketide synthase I domains from soil metagenomic libraries allows selection of promising clones. Appl. Environ. Microbiol. 70:5522-5527. [PMC free article] [PubMed]
6. Kao, C. M., L. Katz, and C. Khosla. 1994. Engineered biosynthesis of a complete macrolactone in a heterologous host. Science 265:509-512. [PubMed]
7. Lam, K. S. 2007. New aspects of natural products in drug discovery. Trends Microbiol. 15:279-289. [PubMed]
8. Liu, H., H. Jiang, B. Haltli, K. Kulowski, E. Muszynska, X. Feng, M. Summers, M. Young, E. Graziani, F. Koehn, G. T. Carter, and M. He. 2009. Rapid cloning and heterologous expression of the meridamycin biosynthetic gene cluster using a versatile Escherichia coli-Streptomyces artificial chromosome vector, pSBAC (perpendicular). J. Nat. Prod. 72:389-395. [PubMed]
9. Newman, D. J., and G. M. Cragg. 2007. Natural products as sources of new drugs over the last 25 years. J. Nat. Prod. 70:461-477. [PubMed]
10. Park, S. R., J. W. Park, W. S. Jung, A. R. Han, Y. H. Ban, E. J. Kim, J. K. Sohng, S. J. Sim, and Y. J. Yoon. 2008. Heterologous production of epothilones B and D in Streptomyces venezuelae. Appl. Microbiol. Biotechnol. 81:109-117. [PubMed]
11. Perlova, O., J. Fu, S. Kuhlmann, D. Krug, A. F. Stewart, Y. Zhang, and R. Muller. 2006. Reconstitution of the myxothiazol biosynthetic gene cluster by Red/ET recombination and heterologous expression in Myxococcus xanthus. Appl. Environ. Microbiol. 72:7485-7494. [PMC free article] [PubMed]
12. Petković, H., J. Cullum, D. Hranueli, I. S. Hunter, N. Perić-Concha, J. Pigac, A. Thamchaipenet, D. Vujaklija, and P. F. Long. 2006. Genetics of Streptomyces rimosus, the oxytetracycline producer. Microbiol. Mol. Biol. Rev. 70:704-728. [PMC free article] [PubMed]
13. Plamann, L., J. M. Davis, B. Cantwell, and J. Mayor. 1994. Evidence that asgB encodes a DNA-binding protein essential for growth and development of Myxococcus xanthus. J. Bacteriol. 176:2013-2020. [PMC free article] [PubMed]
14. Rodriguez, E., H. G. Menzella, and H. Gramajo. 2009. Heterologous production of polyketides in bacteria. Methods Enzymol. 459:339-365. [PubMed]
15. Sait, M., P. Hugenholtz, and P. H. Janssen. 2002. Cultivation of globally distributed soil bacteria from phylogenetic lineages previously only detected in cultivation-independent surveys. Environ. Microbiol. 4:654-666. [PubMed]
16. Staley, J. T., and A. Konopka. 1985. Measurement of in situ activities of nonphotosynthetic microorganisms in aquatic and terrestrial habitats. Annu. Rev. Microbiol. 39:321-346. [PubMed]
17. Tang, L., S. Shah, L. Chung, J. Carney, L. Katz, C. Khosla, and B. Julien. 2000. Cloning and heterologous expression of the epothilone gene cluster. Science 287:640-642. [PubMed]
18. Van Lanen, S. G., and B. Shen. 2006. Microbial genomics for the improvement of natural product discovery. Curr. Opin. Microbiol. 9:252-260. [PubMed]
19. Walsh, C. T. 2004. Polyketide and nonribosomal peptide antibiotics: modularity and versatility. Science 303:1805-1810. [PubMed]
20. Watanabe, K., and H. Oikawa. 2007. Robust platform for de novo production of heterologous polyketides and nonribosomal peptides in Escherichia coli. Org. Biomol. Chem. 5:593-602. [PubMed]
21. Wenzel, S. C., F. Gross, Y. Zhang, J. Fu, A. F. Stewart, and R. Muller. 2005. Heterologous expression of a myxobacterial natural products assembly line in pseudomonads via red/ET recombineering. Chem. Biol. 12:349-356. [PubMed]
22. Wenzel, S. C., and R. Muller. 2009. Myxobacteria—′microbial factories' for the production of bioactive secondary metabolites. Mol. Biosyst. 5:567-574. [PubMed]
23. Zhang, H., Y. Wang, and B. A. Pfeifer. 2008. Bacterial hosts for natural product production. Mol. Pharm. 5:212-225. [PubMed]
24. Zhang, W., B. D. Ames, S. C. Tsai, and Y. Tang. 2006. Engineered biosynthesis of a novel amidated polyketide, using the malonamyl-specific initiation module from the oxytetracycline polyketide synthase. Appl. Environ. Microbiol. 72:2573-2580. [PMC free article] [PubMed]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)