Phylogenomic analysis of natural products biosynthetic gene clusters allows discovery of arseno-organic metabolites in model streptomycetes

We are indebted with Marnix Medema, Paul Straight and Sean Rovito, for useful discussions and critical reading of the manuscript, as well as with Alicia Chagolla and Yolanda Rodriguez of the MS Service of Unidad Irapuato, Cinvestav, and Araceli Fernandez for technical support in high-performance computing. This work was funded by Conacyt Mexico (grants No. 179290 and 177568) and FINNOVA Mexico (grant No. 214716) to FBG. PCM was funded by Conacyt scholarship (No. 28830) and a Cinvestav posdoctoral fellowship. JF and JFK acknowledge funding from the College of Physical Sciences, University of Aberdeen, UK.Peer reviewedPublisher PD

ActDES- a Curated Actinobacterial Database for Evolutionary Studies

Actinobacteria is a large and diverse phylum of bacteria that contains medically and ecologically relevant organisms. Many members are valuable sources of bioactive natural products and chemical precursors that are exploited in the clinic and made using the enzyme pathways encoded in their complex genomes. Whilst the number of sequenced genomes has increased rapidly in the last 20 years, the large size, complexity and high G+C content of many actinobacterial genomes means that the sequences remain incomplete and consist of large numbers of contigs with poor annotation, which hinders large-scale comparative genomic and evolutionary studies. To enable greater understanding and exploitation of actinobacterial genomes, specialized genomic databases must be linked to high-quality genome sequences. Here, we provide a curated database of 612 high-quality actinobacterial genomes from 80 genera, chosen to represent a broad phylogenetic group with equivalent genome re-annotation. Utilizing this database will provide researchers with a framework for evolutionary and metabolic studies, to enable a foundation for genome and metabolic engineering, to facilitate discovery of novel bioactive therapeutics and studies on gene family evolution. This article contains data hosted by Microreact

Evolution of substrate specificity in a recipient's enzyme following horizontal gene transfer

Despite the prominent role of horizontal gene transfer (HGT) in shaping bacterial metabolism, little is known about the impact of HGT on the evolution of enzyme function. Specifically, what is the influence of a recently acquired gene on the function of an existing gene? For example, certain members of the genus Corynebacterium have horizontally acquired a whole L-tryptophan biosynthetic operon, whereas in certain closely related actinobacteria, for example, Mycobacterium, the trpF gene is missing. In Mycobacterium, the function of the trpF gene is performed by a dual-substrate (βα)8 phosphoribosyl isomerase (priA gene) also involved in L-histidine (hisA gene) biosynthesis. We investigated the effect of a HGT-acquired TrpF enzyme upon PriA’s substrate specificity in Corynebacterium through comparative genomics and phylogenetic reconstructions. After comprehensive in vivo and enzyme kinetic analyses of selected PriA homologs, a novel (βα)8 isomerase subfamily with a specialized function in L-histidine biosynthesis, termed subHisA, was confirmed. X-ray crystallography was used to reveal active-site mutations in subHisA important for narrowing of substrate specificity, which when mutated to the naturally occurring amino acid in PriA led to gain of function. Moreover, in silico molecular dynamic analyses demonstrated that the narrowing of substrate specificity of subHisA is concomitant with loss of ancestral protein conformational states. Our results show the importance of HGT in shaping enzyme evolution and metabolism

Minimum Information about a Biosynthetic Gene cluster

A wide variety of enzymatic pathways that produce specialized metabolites in bacteria, fungi and plants are known to be encoded in biosynthetic gene clusters. Information about these clusters, pathways and metabolites is currently dispersed throughout the literature, making it difficult to exploit. To facilitate consistent and systematic deposition and retrieval of data on biosynthetic gene clusters, we propose the Minimum Information about a Biosynthetic Gene cluster (MIBiG) data standard.Chemistry and Chemical Biolog

Multicopy proC in Streptomyces coelicolor A3(2) elicits a transient production of prodiginines, while proC deletion does not yield a proline auxotroph

The last step of proline biosynthesis is typically catalysed by the enzyme Delta(1)-pyrroline-5-carboxylate reductase, encoded by the proC gene. Complete genome sequencing of Streptomyces coelicolor, a soil-dwelling Gram-positive bacterium that uses proline as a precursor for synthesis of prodiginine, revealed a single copy of this gene. Unexpectedly, disruption of this proC homologue (Sco3337) in S. coelicolor M145 yielded a prototrophic strain, yet the reductase activity of Sco3337 was confirmed by complementation of an Escherichia coli proC mutant. Multicopy proC within different genetic contexts elicited a transient production of prodiginines, which showed differential production kinetics of the two most common forms of this natural product produced by S. coelicolor, i.e. streptorubin B (cyclic) and undecylprodigiosin (linear). The metabolic and evolutionary implications of these observations are discussed. Copyright (C) 2010 S. Karger AG, Base

Erratum : a new family of ATP-dependent oligomerization-macrocyclization biocatalysts

In the version of this article initially published, an extra methyl group was inadvertently added to the structure of desferrioxamine G1 and related compounds. The error has been corrected in the HTML and PDF versions of the article

A new family of ATP-dependent oligomerization-macrocyclization biocatalysts

Oligomerization and macrocyclization reactions are key steps in the biosynthesis of many bioactive natural products. Important macrocycles include the antibiotic daptomycin ( 1; ref. 1), the immunosuppressant FK- 506 ( 2; ref. 2), the anthelmintic avermectin B1a ( 3; ref. 3) and the insecticide spinosyn A ( 4; ref. 4); important oligomeric macrocycles include the siderophores enterobactin ( 5; ref. 5) and desferrioxamine E ( 6; ref. 6). Biosynthetic oligomerization and macrocyclization reactions typically involve covalently tethered intermediates and are catalyzed by thioesterase domains of polyketide synthase and nonribosomal peptide synthetase multienzymes(7). Here we report that the purified recombinant desferrioxamine siderophore synthetase DesD from Streptomyces coelicolor M145 catalyzes ATP- dependent trimerization- macrocyclization of a chemically synthesized 10- aminocarboxylic acid substrate via noncovalently bound intermediates. DesD is dissimilar to other known synthetase families but is similar to other enzymes known or proposed to be required for the biosynthesis of omega- aminocarboxylic acid - derived cyclodimeric siderophores(8,9). This suggests that DesD is the first biochemically characterized member of a new family of oligomerizing and macrocyclizing synthetases

Increasing pinosylvin production in Escherichia coli by reducing the expression level of the gene fabI-encoded enoyl-acyl carrier protein reductase

Background: The plant secondary metabolite pinosylvin is a polyphenol from the stilbene family, which have positive effects on human health. Biotechnological production is an attractive alternative for obtaining this stilbene. In Escherichia coli, malonyl-CoA is the precursor for both stilbene and fatty acid syntheses. In this study, with the aim of increasing pinosylvin production, we evaluated a novel approach that is based on reducing the expression of the gene fabI, which encodes the enzyme enoyl-acyl carrier protein reductase that is involved in fatty acid synthesis. Results: A recombineering method was employed to eliminate the chromosomal -35 promoter sequence and the upstream region of the gene fabI in E. coli strain W3110. Analysis, employing RT-qPCR, showed that such modification caused a 60% reduction in the fabI transcript level in the mutant strain W3110Δ-35fabI::Cm compared to the wild type W3110. Synthetic genes encoding a mutant version of 4-coumaroyl-CoA ligase from Streptomyces coelicolor A3 with improved catalytic activity employing cinnamic acid as substrate and a stilbene synthase from Vitis vinifera were cloned to generate the plasmid pTrc-Sc4CL(M)-VvSTS. The production performance of strains W3110Δ-35fabI::Cm/pTrc-Sc4CL(M)-VvSTS and W3110/pTrc-Sc4CL(M)-VvSTS was determined in shake flask cultures with Luria-Bertani medium supplemented with 10 g/L glycerol and 3 mM cinnamic acid. Under these conditions, the strain W3110Δ-35fabI::Cm/pTrc-Sc4CL(M)-VvSTS produced 52.67 mg/L pinosylvin, a level 1.5-fold higher than that observed with W3110/pTrc-Sc4CL(M)-VvSTS. Conclusion: A reduction in the transcript level of fabI caused by the elimination of the -35 and upstream promoter sequences is a successful strategy to improve pinosylvin production in E. coli.How to cite: Salas-Navarrete, C., Hernández-Chávez, G., Flores, N., et al. Increasing pinosylvin production in Escherichia coli by reducing expression level of gene fabI-encoded enoyl-acyl carrier protein reductase. Electron J Biotechnol 2018;33. https://doi.org/10.1016/j.ejbt.2018.03.001. Keywords: 4-Coumaroyl-CoA ligase, Enoyl-acyl carrier protein reductase, Escherichia coli, Fatty acid synthesis, Metabolic engineering, Natural products, Pinosylvin, Plant secondary metabolites, Recombineering, Stilbene synthase, Stilbene

The structure/function relationship of a dual-substrate (beta alpha)(8)-isomerase

Two structures of phosphoribosyl isomerase A (PriA) from Streptomyces coelicolor, involved in both histidine and tryptophan biosynthesis, were solved at 1.8 angstrom resolution. A closed conformer was obtained, which represents the first complete structure of PriA, revealing hitherto unnoticed molecular interactions and the occurrence of conformational changes. Inspection of these conformers, including ligand-docking simulations, allowed identification of residues involved in substrate recognition, chemical catalysis and conformational changes. These predictions were validated by mutagenesis and functional analysis. Arg(19) and Ser(81) were shown to play critical roles within the carboxyl and amino phosphate-binding sites, respectively; the catalytic residues Asp(11) and Asp(130) are responsible for both activities; and Thr(166) and Asp(171), which make an unusual contact, are likely to elicit the conformational changes needed for adopting the active site architectures. This represents the first report of the structure/function relationship of this (beta alpha)(8)-isomerase. (c) 2007 Elsevier Inc. All rights reserved