Heterologous Production of Polyketides in Streptomyces coelicolor and Escherichia coli

James T. Kealey

doi:10.1128/9781555816827.ch26

Chapter 26 : Heterologous Production of Polyketides in Streptomyces coelicolor and Escherichia coli

Author: James T. Kealey

Content Type: Reference

Publication Year: 2010

Category: Applied and Industrial Microbiology

Book DOI: 10.1128/9781555816827

Chapter DOI: 10.1128/9781555816827.ch26

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Abstract:

This chapter reviews the development of heterologous polyketide production systems in Streptomyces coelicolor and Escherichia coli, providing specific examples of polyketides produced in these hosts to illustrate the utility of the approach. The first section covers S. coelicolor, an actinomycete that produces a number of polyketides, most notably the blue pigment actinorhodin. The success with S. coelicolor provided motivation to develop heterologous production systems in E. coli and Saccharomyces cerevisiae, user-friendly organisms for which advanced molecular biology tools and fermentation systems are available. In the second section, the development of an E. coli production system is described. Standard Streptomyces transformation methods are employed to introduce expression plasmids into S. coelicolor CH999, a strain that contains a chromosomal deletion of the actinorhodin gene cluster encoding the activities required for the synthesis of the aromatic polyketide actinorhodin. The epothilones are mixed polyketides/nonribosomal peptides that have microtubule-stabilizing activities similar to those of the taxanes but are active against Taxol-resistant tumors. Many polyketides, including epothilone and certain analogs of 6-deoxyerythronolide B (6Deb), are produced at low levels in the S. coelicolor heterologous production system. The availability of heterologous E. coli production systems, together with recent advances in synthetic biology, has accelerated studies of polyketide synthase (PKS) modularity and specificity. With further refinement of the connectivity rules, it is anticipated that virtually any polyketide can be produced in E. coli by coexpression of a suitable set of synthetic PKS modules.

Citation: Kealey J. 2010. Heterologous Production of Polyketides in Streptomyces coelicolor and Escherichia coli, p 380-390. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch26

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Heterologous Production of Polyketides in Streptomyces coelicolor and Escherichia coli

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FIGURE 1

Module and domain organization of PKSs expressed in S. coelicolor and/or E. coli: 6-MSAS (A); DEBS (B); DEBS with AT6 replaced with AT2 (malonyl-CoA specific) from RAPS (C); DEBS with KS1 inactivated by a point mutation (D); DEBS with KR2 replaced with dehydratase (DH)/enoylreductase (ER)/KR1 from RAPS, KR5 inactivated by insertion of a stuffer fragment, and AT6 replaced with AT2 (malonyl-CoA specific) from RAPS (E); and epothilone PKS (F). KSy, inactive ketosynthase; TE, thioesterase; NRPS, nonribosomal peptide synthase; MT, methyltransferase; C, condensation; A, adenylation; PCP, peptidyl carrier protein; Ox, oxidase; linker, interpeptide linker that facilitates interaction between the DEBS proteins.

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FIGURE 1

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FIGURE 2

Polyketides heterologously produced in S. coelicolor and/or E. coli. For analogs, arrows indicate the site(s) of functional group modifications with respect to the 6dEB core structure (see the text for details).

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FIGURE 2

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FIGURE 3

Heterologous biosynthetic pathways for (2S)-methylmalonyl-CoA introduced into E. coli.

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FIGURE 3

Heterologous biosynthetic pathways for (2S)-methylmalonyl-CoA introduced into E. coli.

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Tables

Heterologous Production of Polyketides in Streptomyces coelicolor and Escherichia coli

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TABLE 1

Streptomyces shuttle vectors and strains

TABLE 1

Streptomyces shuttle vectors and strains

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TABLE 2

E. coli vectors and strains

TABLE 2

E. coli vectors and strains