title: Promotion of spinosad biosynthesis by chromosomal integration of the Vitreoscilla hemoglobin gene in Saccharopolyspora spinosa.
abstract: 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 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.
authors.affiliation: State Key Laboratory of Breeding Base of Microbial Molecular Biology, College of Life Science, Hunan Normal University, Changsha 410081, China.
authors.forename: Yushuang
authors.initials: Y
authors.lastname: Luo
authors.forename: Xiaoxiao
authors.initials: X
authors.lastname: Kou
authors.forename: Xuezhi
authors.initials: X
authors.lastname: Ding
authors.forename: Shengbiao
authors.initials: S
authors.lastname: Hu
authors.forename: Ying
authors.initials: Y
authors.lastname: Tang
authors.forename: Wenping
authors.initials: W
authors.lastname: Li
authors.forename: Fan
authors.initials: F
authors.lastname: Huang
authors.forename: Qi
authors.initials: Q
authors.lastname: Yang
authors.forename: Hanna
authors.initials: H
authors.lastname: Chen
authors.forename: Liqiu
authors.initials: L
authors.lastname: Xia
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D015139:Blotting, Southern; D002876:Chromosomes, Bacterial / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004338:Drug Combinations; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D018014:Gene Transfer Techniques; D005818:Genetic Engineering / Q000379:methods; D007218:Industrial Microbiology / Q000379:methods; D018942:Macrolides / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D016930:Saccharopolyspora / Q000235:genetics* / Q000378:metabolism*; D054793:Truncated Hemoglobins / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004338:Drug Combinations; D018942:Macrolides; D054793:Truncated Hemoglobins; C082613:hemoglobin protein, Vitreoscilla; D010100:Oxygen; C415329:spinosad