In vivo characterization of the dTDP-d-desosamine pathway of the megalomicin gene cluster from Micromonospora megalomicea

In vivo reconstitution of the dTDP-d-desosamine pathway of the megalomicin gene cluster from Micromonospora megalomicea was achieved by expression of the genes in Escherichia coli. LC/MS/MS analysis of the dTDP-sugar intermediates produced by operons containing different sets of genes showed that production of dTDP-d-desosamine from dtdp-4-keto-6-deoxy-d-glucose requires only four biosynthetic steps, catalysed by MegCIV, MegCV, MegDII and MegDIII, and that MegCII is not involved. Instead, bioconversion studies demonstrated that MegCII is needed together with MegCIII to catalyse transfer of d-desosamine to 3-α-mycarosylerythronolide B.

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Genetic localization and in vivo characterization of a Monascus azaphilone pigment biosynthetic gene cluster

Applied Microbiology and Biotechnology ◽

10.1007/s00253-013-4745-9 ◽

2013 ◽

Vol 97 (14) ◽

pp. 6337-6345 ◽

Cited By ~ 79

Author(s):

Bijinu Balakrishnan ◽

Suman Karki ◽

Shih-Hau Chiu ◽

Hyun-Ju Kim ◽

Jae-Won Suh ◽

...

Keyword(s):

Gene Cluster ◽

Biosynthetic Gene Cluster ◽

Biosynthetic Gene ◽

Genetic Localization ◽

In Vivo Characterization ◽

Azaphilone Pigment

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In vivo cloning and characterization of the gabCTDP gene cluster of Escherichia coli K-12.

Journal of Bacteriology ◽

10.1128/jb.172.6.3250-3256.1990 ◽

1990 ◽

Vol 172 (6) ◽

pp. 3250-3256 ◽

Cited By ~ 22

Author(s):

E Metzer ◽

Y S Halpern

Keyword(s):

Escherichia Coli ◽

Gene Cluster ◽

K 12

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In vivo characterization of the activities of novel cyclodipeptide oxidases: new tools for increasing chemical diversity of bioproduced 2,5-diketopiperazines in Escherichia coli

Microbial Cell Factories ◽

10.1186/s12934-020-01432-y ◽

2020 ◽

Vol 19 (1) ◽

Author(s):

Fabien Le Chevalier ◽

Isabelle Correia ◽

Lucrèce Matheron ◽

Morgan Babin ◽

Mireille Moutiez ◽

...

Keyword(s):

Escherichia Coli ◽

Biological Activities ◽

Gene Clusters ◽

Chemical Diversity ◽

E Coli ◽

Chemical Structures ◽

In Vivo Characterization ◽

Culture Supernatants

Abstract Background Cyclodipeptide oxidases (CDOs) are enzymes involved in the biosynthesis of 2,5-diketopiperazines, a class of naturally occurring compounds with a large range of pharmaceutical activities. CDOs belong to cyclodipeptide synthase (CDPS)-dependent pathways, in which they play an early role in the chemical diversification of cyclodipeptides by introducing Cα-Cβ dehydrogenations. Although the activities of more than 100 CDPSs have been determined, the activities of only a few CDOs have been characterized. Furthermore, the assessment of the CDO activities on chemically-synthesized cyclodipeptides has shown these enzymes to be relatively promiscuous, making them interesting tools for cyclodipeptide chemical diversification. The purpose of this study is to provide the first completely microbial toolkit for the efficient bioproduction of a variety of dehydrogenated 2,5-diketopiperazines. Results We mined genomes for CDOs encoded in biosynthetic gene clusters of CDPS-dependent pathways and selected several for characterization. We co-expressed each with their associated CDPS in the pathway using Escherichia coli as a chassis and showed that the cyclodipeptides and the dehydrogenated derivatives were produced in the culture supernatants. We determined the biological activities of the six novel CDOs by solving the chemical structures of the biologically produced dehydrogenated cyclodipeptides. Then, we assessed the six novel CDOs plus two previously characterized CDOs in combinatorial engineering experiments in E. coli. We co-expressed each of the eight CDOs with each of 18 CDPSs selected for the diversity of cyclodipeptides they synthesize. We detected more than 50 dehydrogenated cyclodipeptides and determined the best CDPS/CDO combinations to optimize the production of 23. Conclusions Our study establishes the usefulness of CDPS and CDO for the bioproduction of dehydrogenated cyclodipeptides. It constitutes the first step toward the bioproduction of more complex and diverse 2,5-diketopiperazines.

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