Promotion of spinosad biosynthesis by chromosomal integration of the Vitreoscilla hemoglobin gene in Saccharopolyspora spinosa  

作　　者：LUO YuShuang KOU XiaoXiao DING XueZhi HU ShengBiao TANG Ying LI WenPing HUANG Fan YANG Qi CHEN HanNa XIA LiQiu 

机构地区：[1]State Key Laboratory of Breeding Base of Microbial Molecular Biology,College of Life Science,Hunan Normal University,Changsha 410081,China

出　　处：《Science China(Life Sciences)》2012年第2期172-180,共9页中国科学（生命科学英文版）

基　　金：supported by the National Basic Research Program of China (Grant Nos. 2012CB722301 and 2011CB111605);the National High Technology Research and Development Project of China (Grant No. 2011AA10A203);the National Natural Science Foundation of China (Grant No. 31070006)

摘　　要：To promote spinosad biosynthesis by improving the limited oxygen supply during high-density fermentation of Saccharopolyspora spinosa, the open reading frame of the Vitreoscilla hemoglobin gene was placed under the control of the promoter for the erythromycin resistance gene by splicing using overlapping extension PCR. This was cloned into the integrating vector pSET152, yielding the Vitreoscilla hemoglobin gene expression plasmid pSET152EVHB. This was then introduced into S. spinosa SP06081 by conjugal transfer, and integrated into the chromosome by site-specific recombination at the integration site ФC31 on pSET152EVHB. The resultant conjugant, S. spinosa S078-1101, was genetically stable. The integration was further confirmed by PCR and Southern blotting analysis. A carbon monoxide differential spectrum assay showed that active Vitreoscilla hemoglobin was successfully expressed in S. spinosa S078-1101. Fermentation results revealed that expression of the Vitreoscilla hemoglobin gene significantly promoted spinosad biosynthesis under normal oxygen and moderately oxygen-limiting conditions （P〈0.01）. These findings demonstrate that integrating expression of the Vitreoscilla hemoglobin gene improves oxygen uptake and is an effective means for the genetic improvement of S. spinosa fermentation. Saccharopolyspora spinosa, spinosad, Vitreoscilla hemoglobin, integrating vector, homologous recombinationTo promote spinosad biosynthesis by improving the limited oxygen supply during high-density fermentation of Saccharopolyspora spinosa, the open reading frame of the Vitreoscilla hemoglobin gene was placed under the control of the promoter for the erythromycin resistance gene by splicing using overlapping extension PCR. This was cloned into the integrating vector pSET152, yielding the Vitreoscilla hemoglobin gene expression plasmid pSET152EVHB. This was then introduced into S. spinosa SP06081 by conjugal transfer, and integrated into the chromosome by site-specific recombination at the integration site ΦC31 on pSET152EVHB. The resultant conjugant, S. spinosa S078-1101, was genetically stable. The integration was further confirmed by PCR and Southern blotting analysis. A carbon monoxide differential spectrum assay showed that active Vitreoscilla hemoglobin was successfully expressed in S. spinosa S078-1101. Fermentation results revealed that expression of the Vitreoscilla hemoglobin gene significantly promoted spinosad biosynthesis under normal oxygen and moderately oxygen-limiting conditions (P<0.01). These findings demonstrate that integrating expression of the Vitreoscilla hemoglobin gene improves oxygen uptake and is an effective means for the genetic improvement of S. spinosa fermentation.

关 键 词：Saccharopolyspora spinosa SPINOSAD Vitreoscilla hemoglobin integrating vector homologous recombination 

分 类 号：Q78[生物学—分子生物学] S482.3[农业科学—农药学]