title: Molecular cloning of a novel bioH gene from an environmental metagenome encoding a carboxylesterase with exceptional tolerance to organic solvents.
abstract: BACKGROUND
  BioH 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.
  
  
  RESULTS
  Screening 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.
  
  
  CONCLUSIONS
  This 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.
authors: Key Laboratory of Quality and Safety Risk Assessment for Aquatic Products on Storage and Preservation (Shanghai), China Ministry of Agriculture, Engineering Centre for Quality Control and Risk Assessment of Aquatic Products, College of Food Science and Technology, Shanghai Ocean University, 999 Hu Cheng Huan Road, 201306, Shanghai, PR China.
authors: Yuping
authors: Y
authors: Shi
authors: Yingjie
authors: Y
authors: Pan
authors: Bailin
authors: B
authors: Li
authors: Wei
authors: W
authors: He
authors: Qunxin
authors: Q
authors: She
authors: Lanming
authors: L
authors: Chen
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D043182:Carboxylesterase / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D015723:Gene Library; D016680:Genome, Bacterial; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D005810:Multigene Family; D010802:Phylogeny; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D016415:Sequence Alignment; D012997:Solvents / Q000737:chemistry*; D013379:Substrate Specificity; D013696:Temperature; D014280:Triglycerides / Q000633:toxicity
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D012997:Solvents; D014280:Triglycerides; D043182:Carboxylesterase; C005830:tributyrin