Optimization, reconstruction and heterologous expression of the gene cluster encoding toluene/o-xylene monooxygenase from Pseudomonas stutzeri in Escherichia coli and its successive hydroxylation of toluene and benzene

ABSTRACT In vivo reconstitution of the TDP-l-megosamine pathway from the megalomicin gene cluster of Micromonospora megalomicea was accomplished by the heterologous expression of its biosynthetic genes in Escherichia coli. Mass spectrometric analysis of the TDP-sugar intermediates produced from operons containing different sets of genes showed that the production of TDP-l-megosamine from TDP-4-keto-6-deoxy-d-glucose requires only five biosynthetic steps, catalyzed by MegBVI, MegDII, MegDIII, MegDIV, and MegDV. Bioconversion studies demonstrated that the sugar transferase MegDI, along with the helper protein MegDVI, catalyzes the transfer of l-megosamine to either erythromycin C or erythromycin D, suggesting two possible routes for the production of megalomicin A. Analysis in vivo of the hydroxylation step by MegK indicated that erythromycin C is the intermediate of megalomicin A biosynthesis.

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Heterologous Production of Cyanobacterial Mycosporine-Like Amino Acids Mycosporine-Ornithine and Mycosporine-Lysine in Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.01632-16 ◽

2016 ◽

Vol 82 (20) ◽

pp. 6167-6173 ◽

Cited By ~ 24

Author(s):

Meenu Katoch ◽

Rabia Mazmouz ◽

Rocky Chau ◽

Leanne A. Pearson ◽

Russell Pickford ◽

...

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Heterologous Expression ◽

Gene Cluster ◽

Uv Radiation ◽

Active Ingredient ◽

Stress Factors ◽

Heterologous Production ◽

Content Type ◽

E Coli

ABSTRACTMycosporine-like amino acids (MAAs) are an important class of secondary metabolites known for their protection against UV radiation and other stress factors. Cyanobacteria produce a variety of MAAs, including shinorine, the active ingredient in many sunscreen creams. Bioinformatic analysis of the genome of the soil-dwelling cyanobacteriumCylindrospermum stagnalePCC 7417 revealed a new gene cluster with homology to MAA synthase fromNostoc punctiforme. This newly identified gene cluster is unusual because it has five biosynthesis genes (mylAtomylE), compared to the four found in other MAA gene clusters. Heterologous expression ofmylAtomylEinEscherichia coliresulted in the production of mycosporine-lysine and the novel compound mycosporine-ornithine. To our knowledge, this is the first time these compounds have been heterologously produced inE. coliand structurally characterized via direct spectral guidance. This study offers insight into the diversity, biosynthesis, and structure of cyanobacterial MAAs and highlights their amenability to heterologous production methods.IMPORTANCEMycosporine-like amino acids (MAAs) are significant from an environmental microbiological perspective as they offer microbes protection against a variety of stress factors, including UV radiation. The heterologous expression of MAAs inE. coliis also significant from a biotechnological perspective as MAAs are the active ingredient in next-generation sunscreens.

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Biochemical characterization and substrate specificity of the gene cluster for biosyntheses of K-252a and its analogs by in vitro heterologous expression system of Escherichia coli

Molecular BioSystems ◽

10.1039/b912395b ◽

2009 ◽

Vol 5 (10) ◽

pp. 1192 ◽

Cited By ~ 10

Author(s):

Hsien-Tai Chiu ◽

Yu-Chin Lin ◽

Meng-Na Lee ◽

Yi-Lin Chen ◽

Mei-Sin Wang ◽

...

Keyword(s):

Escherichia Coli ◽

Substrate Specificity ◽

Heterologous Expression ◽

Gene Cluster ◽

Biochemical Characterization ◽

Expression System ◽

Heterologous Expression System

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Targeted Capture and Heterologous Expression of the Pseudoalteromonas Alterochromide Gene Cluster in Escherichia coli Represents a Promising Natural Product Exploratory Platform

ACS Synthetic Biology ◽

10.1021/sb500280q ◽

2014 ◽

Vol 4 (4) ◽

pp. 414-420 ◽

Cited By ~ 62

Author(s):

Avena C. Ross ◽

Lauren E. S. Gulland ◽

Pieter C. Dorrestein ◽

Bradley S. Moore

Keyword(s):

Escherichia Coli ◽

Heterologous Expression ◽

Natural Product ◽

Gene Cluster ◽

Targeted Capture

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Methionine-Containing Rhabdopeptide/Xenortide-like Peptides from Heterologous Expression of the Biosynthetic Gene Cluster kj12ABC in Escherichia coli

Journal of Natural Products ◽

10.1021/acs.jnatprod.8b00425 ◽

2018 ◽

Vol 81 (10) ◽

pp. 2292-2295 ◽

Cited By ~ 1

Author(s):

Lei Zhao ◽

Xiaofeng Cai ◽

Marcel Kaiser ◽

Helge B. Bode

Keyword(s):

Escherichia Coli ◽

Heterologous Expression ◽

Gene Cluster ◽

Biosynthetic Gene Cluster ◽

Biosynthetic Gene

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Analysis of the Gene Cluster Encoding Toluene/o-Xylene Monooxygenase from Pseudomonas stutzeri OX1

Applied and Environmental Microbiology ◽

10.1128/aem.64.10.3626-3632.1998 ◽

1998 ◽

Vol 64 (10) ◽

pp. 3626-3632 ◽

Cited By ~ 91

Author(s):

Giovanni Bertoni ◽

Manuela Martino ◽

Enrica Galli ◽

Paola Barbieri

Keyword(s):

Escherichia Coli ◽

Gene Cluster ◽

Dimethyl Sulfide ◽

Pseudomonas Stutzeri ◽

Open Reading Frames ◽

Enzymatic System ◽

Wild Type Allele ◽

Wild Type ◽

Bacterial Strains ◽

Reading Frames

ABSTRACT The toluene/o-xylene monooxygenase cloned fromPseudomonas stutzeri OX1 displays a very broad range of substrates and a very peculiar regioselectivity, because it is able to hydroxylate more than one position on the aromatic ring of several hydrocarbons and phenols. The nucleotide sequence of the gene cluster coding for this enzymatic system has been determined. The sequence analysis revealed the presence of six open reading frames (ORFs) homologous to other genes clustered in operons coding for multicomponent monooxygenases found in benzene- and toluene-degradative pathways cloned from Pseudomonas strains. Significant similarities were also found with multicomponent monooxygenase systems for phenol, methane, alkene, and dimethyl sulfide cloned from different bacterial strains. The knockout of each ORF and complementation with the wild-type allele indicated that all six ORFs are essential for the full activity of the toluene/o-xylene monooxygenase inEscherichia coli. This analysis also shows that despite its activity on both hydrocarbons and phenols, toluene/ o-xylene monooxygenase belongs to a toluene multicomponent monooxygenase subfamily rather than to the monooxygenases active on phenols.

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