Dataset Information

Reconstitution and Minimization of a Micrococcin Biosynthetic Pathway in Bacillus subtilis.

ABSTRACT: UNLABELLED:Thiopeptides represent one of several families of highly modified peptide antibiotics that hold great promise for natural product engineering. These macrocyclic peptides are produced by a combination of ribosomal synthesis and extensive posttranslational modification by dedicated processing enzymes. We previously identified a compact, plasmid-borne gene cluster for the biosynthesis of micrococcin P1 (MP1), an archetypal thiopeptide antibiotic. In an effort to genetically dissect this pathway, we have reconstituted it in Bacillus subtilis Successful MP1 production required promoter engineering and the reassembly of essential biosynthetic genes in a modular plasmid. The resulting system allows for rapid pathway manipulation, including protein tagging and gene deletion. We find that 8 processing proteins are sufficient for the production of MP1 and that the tailoring enzyme TclS catalyzes a C-terminal reduction step that distinguishes MP1 from its sister compound micrococcin P2. IMPORTANCE:The emergence of antibiotic resistance is one of the most urgent human health concerns of our day. A crucial component in an integrated strategy for countering antibiotic resistance is the ability to engineer pathways for the biosynthesis of natural and derivatized antimicrobial compounds. In this study, the model organism B. subtilis was employed to reconstitute and genetically modularize a 9-gene system for the biosynthesis of micrococcin, the founding member of a growing family of thiopeptide antibiotics.

SUBMITTER: Bennallack PR

PROVIDER: S-EPMC4999933 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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Reconstitution and Minimization of a Micrococcin Biosynthetic Pathway in Bacillus subtilis.

Bennallack Philip R PR Bewley Kathryn D KD Burlingame Mark A MA Robison Richard A RA Miller Susan M SM Griffitts Joel S JS

Journal of bacteriology 20160825 18

<h4>Unlabelled</h4>Thiopeptides represent one of several families of highly modified peptide antibiotics that hold great promise for natural product engineering. These macrocyclic peptides are produced by a combination of ribosomal synthesis and extensive posttranslational modification by dedicated processing enzymes. We previously identified a compact, plasmid-borne gene cluster for the biosynthesis of micrococcin P1 (MP1), an archetypal thiopeptide antibiotic. In an effort to genetically disse ...[more]

PMID: 27381911

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Project description:The pentose phosphate (PP) pathway is one of the major sources of cellular NADPH. A B. subtiliszwf mutant that lacks glucose-6-phosphate dehydrogenase (the enzyme that catalyzes the first step of the PP pathway) showed inoculum-dose-dependent growth. This growth defect was suppressed by glcP disruption, which causes the upregulation of an autoinducer neotrehalosadiamine (NTD)/kanosamine biosynthetic pathway. A metabolome analysis showed that the stimulation of NTD/kanosamine biosynthesis caused significant accumulation of TCA cycle intermediates and NADPH. Because the major malic enzyme YtsJ concomitantly generates NADPH through malate-to-pyruvate conversion, de novo NTD/kanosamine biosynthesis can result in an increase in the intracellular NADPH pool via the accumulation of malate. In fact, a zwf mutant grew in malate-supplemented medium. Artificial induction of glcP in the zwf mutant caused a reduction in the intracellular NADPH pool. Moreover, the correlation between the expression level of the NTD/kanosamine biosynthesis operon ntdABC and the intracellular NADPH pool was confirmed. Our results suggest that NTD/kanosamine has the potential to modulate the carbon-energy metabolism through an autoinduction mechanism.ImportanceAutoinducers enable bacteria to sense cell density and to coordinate collective behavior. NTD/kanosamine is an autoinducer produced by B. subtilis and several close relatives, although its physiological function remains unknown. The most important finding of this study was the significance of de novo NTD/kanosamine biosynthesis in the modulation of the central carbon metabolism in B. subtilis We showed that NTD/kanosamine biosynthesis caused an increase in the NADPH pool via the accumulation of TCA cycle intermediates. These results suggest a possible role for NTD/kanosamine in the carbon-energy metabolism. As Bacillus species are widely used for the industrial production of various useful enzymes and compounds, the NTD/kanosamine biosynthetic pathway might be utilized to control metabolic pathways in these industrial strains.

Dataset Information

Reconstitution and Minimization of a Micrococcin Biosynthetic Pathway in Bacillus subtilis.

Publications

Reconstitution and Minimization of a Micrococcin Biosynthetic Pathway in Bacillus subtilis.

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