title: Cloning and expression of the first gene for biodegrading microcystin LR by Sphingopyxis sp. USTB-05.
abstract: Harmful cyanobacterial blooms are a growing environmental problem worldwide in natural waters, the biodegradation is found to be the most efficient method for removing microcystins (MCs) produced by harmful cyanobacteria. Based on the isolation of a promising bacterial strain of Sphingopyxis sp. USTB-05 for biodegrading MCs, we for the first time cloned and expressed a gene USTB-05-A (HM245411) that is responsible for the first step in the biodegradation of microcystin LR (MC-LR) in E. coli DH5alpha, with a cloning vector of pGEM-T easy and an expression vector of pGEX-4T-1, respectively. The cell-free extracts (CE) of recombinant E. coli DH5alpha containing USTB-05-A had high activity for biodegrading MC-LR. The initial MC-LR concentration of 40 mg/L was completely biodegraded within 1 hr in the presence of CE with a protein concentration of 0.35 mg/mL. Based on an analysis of the liquid chromatogram-mass spectrum (LC-MS), the enzyme encoded by gene USTB-OS-A was found to be active in cleaving the target peptide bond between 3-amino-9-methoxy-2,6, 8-trimethyl-10-phenyl-deca-4,6-dienoic acid (Adda) and arginine of MC-LR, and converting cyclic MC-LR to linear MC-LR as a first product that is much less toxic than parent MC-LR, which offered direct evidence for the first step on the pathway of MC-LR biodegradation by Sphingopyxis sp. USTB-05.
authors.affiliation: School of Chemical and Biological Engineering, University of Science and Technology Beijing, Beijing 100083, China. haiyan@ustb.edu.cn
authors.forename: Hai
authors.initials: H
authors.lastname: Yan
authors.forename: Huasheng
authors.initials: H
authors.lastname: Wang
authors.forename: Junfeng
authors.initials: J
authors.lastname: Wang
authors.forename: Chunhua
authors.initials: C
authors.lastname: Yin
authors.forename: Song
authors.initials: S
authors.lastname: Ma
authors.forename: Xiaolu
authors.initials: X
authors.lastname: Liu
authors.forename: Xueyao
authors.initials: X
authors.lastname: Yin
mesh_terms: D020561:Alphaproteobacteria / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D008387:Marine Toxins; D013058:Mass Spectrometry; D052998:Microcystins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D015394:Molecular Structure
chemical_list: D001426:Bacterial Proteins; D008387:Marine Toxins; D052998:Microcystins; C057862:cyanoginosin LR