Abstract
BackgroundBioH is one of the key enzymes to produce the precursor pimeloyl-ACP to initiate biotin biosynthesis de novo in bacteria. To date, very few bioH genes have been characterized. In this study, we cloned and identified a novel bioH gene, bioHx, from an environmental metagenome by a functional metagenomic approach. The bioHx gene, encoding an enzyme that is capable of hydrolysis of p-nitrophenyl esters of fatty acids, was expressed in Escherichia coli BL21 using the pET expression system. The biochemical property of the purified BioHx protein was also investigated.ResultsScreening of an unamplified metagenomic library with a tributyrin-containing medium led to the isolation of a clone exhibiting lipolytic activity. This clone carried a 4,570-bp DNA fragment encoding for six genes, designated bioF, bioHx, fabG, bioC, orf5 and sdh, four of which were implicated in the de novo biotin biosynthesis. The bioHx gene encodes a protein of 259 aa with a calculated molecular mass of 28.60 kDa, displaying 24-39% amino acid sequence identity to a few characterized bacterial BioH enzymes. It contains a pentapeptide motif (Gly76-Trp77-Ser78-Met79-Gly80) and a catalytic triad (Ser78-His230-Asp202), both of which are characteristic for lipolytic enzymes. BioHx was expressed as a recombinant protein and characterized. The purified BioHx protein displayed carboxylesterase activity, and it was most active on p-nitrophenyl esters of fatty acids substrate with a short acyl chain (C4). Comparing BioHx with other known BioH proteins revealed interesting diversity in their sensitivity to ionic and nonionic detergents and organic solvents, and BioHx exhibited exceptional resistance to organic solvents, being the most tolerant one amongst all known BioH enzymes. This ascribed BioHx as a novel carboxylesterase with a strong potential in industrial applications.ConclusionsThis study constituted the first investigation of a novel bioHx gene in a biotin biosynthetic gene cluster cloned from an environmental metagenome. The bioHx gene was successfully cloned, expressed and characterized. The results demonstrated that BioHx is a novel carboxylesterase, displaying distinct biochemical properties with strong application potential in industry. Our results also provided the evidence for the effectiveness of functional metagenomic approach for identifying novel bioH genes from complex ecosystem.
Highlights
BioH is one of the key enzymes to produce the precursor pimeloyl-ACP to initiate biotin biosynthesis de novo in bacteria
In the BioC-BioH pathway, BioC catalyzes S-adenosylmethionine-dependent methylation of malonyl-CoA to malonyl-CoA methyl ester that enters fatty acid synthetic pathway, where an intermediate pimeloyl-ACP methyl ester was cleaved by BioH to produce pimeloyl-ACP that serves as the substrate for BioF to initiate biotin synthesis [2,5]
Our results indicate that BioHx is a novel carboxylesterase involved in the biotin biosynthesis, exhibiting good application potential in industry and biotechnological research
Summary
BioH is one of the key enzymes to produce the precursor pimeloyl-ACP to initiate biotin biosynthesis de novo in bacteria. Biotin ( known as vitamin H, or B7) is an essential enzyme cofactor involved in multiple important metabolic pathways including fatty acids, amino acids, and carbohydrates in all three domains of life [1]. This coenzyme is synthesized de novo in microorganisms [2] where enzymes catalyzing the last four steps in the biotin biosynthesis are well characterized. These include 7-keto-8-aminopelargonic acid (KAPA) synthase, 7, 8-diaminopelargonic acid synthase, dethiobiotin synthetase, and biotin synthase. BioH genes have been cloned from E. coli, Serratia marcescens, Serratia sp. and Kurthia sp. strains, and the encoded proteins exhibit carboxylesterase activity [6,7,8,9]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
