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Chapter 28 : Genetic Engineering To Regulate Production of Secondary Metabolites in

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Abstract:

Many early studies on penicillin and cephamycin biosynthesis were carried out in , but because of the commercial importance of clavulanic acid, most recent studies have focused on that compound. In particular, genetic engineering to generate improved clavulanic acid producer strains has been a main interest. grows and produces secondary metabolites optimally at 28 to 30°C, but it is unusually sensitive to elevated temperature and will not grow above 31 to 32°C. Liquid media employed by research groups studying the production of the various types of β-lactam metabolites produced by vary widely. Numerous bioprocess-type studies examining growth medium optimization as a means of improving production of clavulanic acid have appeared in recent years, typically reinforcing the association of soy-based growth medium constituents with high productivity. In , biosynthesis begins with the amino acids L-lysine, L-cysteine, and L-valine and proceeds through the synthesis of penicillin and cephalosporin intermediates to give the final cephamycin product. While improvements in productivity can be achieved by increasing production of pathway enzymes, another approach to achieving this is to identify genes encoding critical regulatory proteins and increase their expression levels. More global approaches to improvement of antibiotic production could also be coupled with strategies aimed at the pathway-specific regulators. Effects of greater magnitude are seen when pathway-specific regulators are targeted for overproduction, and combination approaches targeting both global and specific regulators along with individual pathway enzymes for overproduction, together with elimination of competing pathways, will likely yield even greater results.

Citation: Jensen S. 2010. Genetic Engineering To Regulate Production of Secondary Metabolites in , p 411-425. 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.ch28

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Clavulanic acid
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beta-Lactam Antibiotics
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beta-Lactamase Inhibitors
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Image of FIGURE 1
FIGURE 1

Secondary metabolites produced by

Citation: Jensen S. 2010. Genetic Engineering To Regulate Production of Secondary Metabolites in , p 411-425. 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.ch28
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Image of FIGURE 2
FIGURE 2

The cephamycin C-clavulanic acid gene supercluster in . Arrows filled with dots indicate genes for cephamycin C biosynthesis; arrows filled with slanted lines indicate genes for clavulanic acid production. Genes marked with asterisks are essential or important for cephamycin C or clavulanic acid production.

Citation: Jensen S. 2010. Genetic Engineering To Regulate Production of Secondary Metabolites in , p 411-425. 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.ch28
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Image of FIGURE 3
FIGURE 3

The biosynthetic pathway to cephamycin C in . Enzyme names are followed by the corresponding gene designation shown in italics.

Citation: Jensen S. 2010. Genetic Engineering To Regulate Production of Secondary Metabolites in , p 411-425. 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.ch28
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Image of FIGURE 4
FIGURE 4

The biosynthetic pathway to clavulanic acid and the 5 clavams in . Enzyme names are followed by the corresponding gene designation shown in italics. Parts of the pathway shown with broken arrows represent pathway steps that are proposed but not yet demonstrated.

Citation: Jensen S. 2010. Genetic Engineering To Regulate Production of Secondary Metabolites in , p 411-425. 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.ch28
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Image of FIGURE 5
FIGURE 5

The clavam and paralogue gene clusters in . Genes marked with asterisks are essential or important for 5 clavam production.

Citation: Jensen S. 2010. Genetic Engineering To Regulate Production of Secondary Metabolites in , p 411-425. 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.ch28
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Image of FIGURE 6
FIGURE 6

Generation of an A mutant via PCR-mediated mutagenesis using the REDIRECT system. An cassette targeting the gene was prepared in vitro via PCR and then electroporated into BW25113/pIJ790 for recombination with cosmid 14E10. The resulting mutant 14E10-Δ:: cosmid was then transferred by conjugation to where it exchanged with the resident gene to give a mutant strain of Introduction of plasmid pUWLftp to the mutant resulted in loss of the cassette, leaving an unmarked A mutant.

Citation: Jensen S. 2010. Genetic Engineering To Regulate Production of Secondary Metabolites in , p 411-425. 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.ch28
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