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Chapter 29 : Genetic Engineering of Myxobacterial Natural Product Biosynthetic Genes

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Genetic Engineering of Myxobacterial Natural Product Biosynthetic Genes, Page 1 of 2

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Abstract:

The majority of myxobacterial natural products have been isolated from strains of (48%), (20%), and (10%). This chapter summarizes progress made with genetic engineering novel analogs of epothilone and ambruticin, an antifungal compound isolated from more than 30 years ago. The authors describe the cloning of the gene clusters from , the development of as a heterologous expression host, and the development of tools and techniques to genetically manipulate the natural producer for generating new analogs. The epothilone biosynthetic gene cluster was built in the genome of in a two-step process due to its large size. The first step required introduction of fragments of the epothilone gene cluster for use as recombination sites. Subsequent steps introduced large fragments of the gene cluster from cosmids into the chromosome. Although this work involved time-consuming techniques, it proved the viability of using the myxobacterium as a heterologous host for production of myxobacterial natural products, and led the way for combinatorial biosynthesis of novel epothilone analogs. Thus, the heterologous expression of the epothilone gene cluster in provided the opportunity for making novel analogs and understanding novel enzyme mechanisms. The junctions between the ketide synthase (KS), acyltransferase (AT) AT, and acyl carrier protein (ACP) were based on the engineering of the AT in the epothilone biosynthetic genes.

Citation: Julien B, Rodriguez E. 2010. Genetic Engineering of Myxobacterial Natural Product Biosynthetic Genes, p 426-437. 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.ch29
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Image of FIGURE 1
FIGURE 1

The PKS/NRPS assembly line for epothilone biosynthesis. The six polypeptides of the megasynthase are shown at the top (EpoA to EpoF) as arrows (N to C terminus). The domain composition is shown schematically. The numbered domains on the polypeptides were targets for genetic engineering as described in section 29.5.1 . The gene encodes a cytochrome P450 protein which adds an epoxide to epothilones C and D to produce epothilone A and B, respectively.

Citation: Julien B, Rodriguez E. 2010. Genetic Engineering of Myxobacterial Natural Product Biosynthetic Genes, p 426-437. 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.ch29
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Image of FIGURE 2
FIGURE 2

(A) Schematic representation of cloning and identification of the PKS/NRPS gene cluster from a natural producer. (B) Schematic steps for using the transposon derivative for identification of PKS/NRPS in myxobacteria.

Citation: Julien B, Rodriguez E. 2010. Genetic Engineering of Myxobacterial Natural Product Biosynthetic Genes, p 426-437. 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.ch29
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Image of FIGURE 3
FIGURE 3

Novel epothilone analogs generated by manipulation of the PKS. Most of the successful changes were produced through inactivation of domains involved in the reductive cycle. (A) KR. (B) KR. (C) DH. (D) ER.

Citation: Julien B, Rodriguez E. 2010. Genetic Engineering of Myxobacterial Natural Product Biosynthetic Genes, p 426-437. 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.ch29
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Image of FIGURE 4
FIGURE 4

Scheme for engineering an AT replacement in the chromosome of carrying the epothilone gene cluster. (A) Schematic representation of the epothilone gene cluster. (B) Step 1: replacement of AT with the genes through double recombination using a linear delivery vector. (C) Step 2: a cassette, consisting of a heterologous AT flanked by upstream and downstream regions, is introduced into the strain constructed in step 1. The first crossover can occur with either of the flanking regions. (D) Step 3: the second crossover is selected using plates containing galactose, resulting in the recombination event to remove duplicated regions and segments with the kanamycin resistance and genes. Fermentation of the Gal Kan Phle strain allows selection of the correct construct.

Citation: Julien B, Rodriguez E. 2010. Genetic Engineering of Myxobacterial Natural Product Biosynthetic Genes, p 426-437. 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.ch29
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Image of FIGURE 5
FIGURE 5

Pathway proposed for the biosynthesis of ambruticin. Inactivation of genes from the ambruticin gene cluster allows the isolation of intermediates in the biosynthesis of ambruticins.

Citation: Julien B, Rodriguez E. 2010. Genetic Engineering of Myxobacterial Natural Product Biosynthetic Genes, p 426-437. 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.ch29
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Image of FIGURE 6
FIGURE 6

PKS engineering in using a single crossover event. The first PKS gene, , encodes the loading module consisting of KS′, a methylmalonyl-CoA specific AT (AT), and an ACP. A plasmid is constructed carrying an in-frame deletion at the 59 end of the KS (KS), an AT domain replaced with one specific for malonyl-CoA (AT), and an ACP under the expression of a heterologous promoter, P. Isolation of a transformant in which crossover has occurred at the KS’ sequence allows the expression of the engineered loading module gene carrying the malonyl-CoA-specific AT domain under the P promoter, while the expression of remaining downstream PKS genes is under the heterologous promoter . The expression of the 5′ truncated loading module gene carrying the AT domain is maintained to avoid a polar effect over the downstream PKS genes.

Citation: Julien B, Rodriguez E. 2010. Genetic Engineering of Myxobacterial Natural Product Biosynthetic Genes, p 426-437. 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.ch29
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