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journal1 ›› 2018, Vol. 34 ›› Issue (4): 625-638.DOI: 10.16409/j.cnki.2095-039x.2018.04.018

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The Effect of glnA Gene on Growth Development and Spinosad Biosynthesis in Saccharopolyspora spinosa

XIAO Jie, LIU Zhudong, PENG Shengnan, HE Haocheng, RANG Jie, LIU Xiong, DING Xuezhi, XIA Liqiu   

  1. Key Laboratory of Microbial Molecular Biology/State Key Laboratory of Development Biology of Freshwater Fish/College of Life Science, Hunan Normal University, Changsha 410081, China
  • Received:2018-01-30 Online:2018-08-08 Published:2018-07-30

Abstract: Glutamine synthetase (GS) encoded by glnA is the oldest enzyme and most widely exists in organisms. It participates in nitrogen and carbon metabolism in many organism and is one of the key enzymes for nitrogen metabolism, thus, it has played a crucial role in the life activities of microorganisms. This study explored the effect of glnA gene on the growth development and secondary metabolites of Saccharopolyspora spinosa. First, partial fragment of glnA gene was cloned from the genome of S. spinosa and ligated with shuttle vector pOJ260 to construct knockout vector pOJ260-glnA; Then, the recombinant vector was introduced into S. spinosa by conjugal transfer, and the knockout strain S. spinosa-△glnA was generated. Second, glnA gene was placed under the PermE strong promoter by overlap extension PCR, and the fusion fragment PermE-glnA was ligated with the shuttle vector pOJ260 to construct the overexpression vector pOJ260-PermE-glnA; Then, the recombinant vector was introduced into S. spoinosa by conjugal transfer and generated the over expression strain S. spinosa-glnA. Phenotypic analysis found that the knockout of glnA gene had a significant inhibition effect on the mycelial development and spore germination of S. spinosa. HPLC analysis demonstrated that spinosad A+D yield increased by 170% in S. spinosa-glnA strain compared to the parental strain. Thus, our study revealed that the glnA gene regulated the mycelia development and the spinosad synthesis of S. spinosa, which laid an important foundation on studying the function in the secondary metabolism of Streptomyces.

Key words: Saccharopolyspora spinosa, spinosad, glutamine synthetase, knockout, overexpression

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