中国农业科学Issue(18)：3577-3587,11.DOI:10.3864/j.issn.0578-1752.2014.18.006
环腺苷酸受体蛋白基因的过表达对刺糖多孢菌生长和多杀菌素合成的影响
Impact on Strain Growth and Spinosad Biosynthesis by Overexpression of Cyclic AMP Receptor Protein Gene in Saccharopolyspora spinosa
摘要
Abstract
[Objective]The objective of this study is to promote spinosad biosynthesis in Saccharopolyspora spinosa and to study the effects on aspects of strain growth and morphological development through overexpression of Streptomyces global regulatory factor cyclic AMP receptor protein gene (crp). [Method] crp was amplified by PCR, and an intermediate vector pOJ260-cm-PermE-crp was constructed by restriction enzymes digestion and ligation, in which crp was placed under the control of erythromycin enhanced promoter PermE. The PermE-crp frangment was amplified from pOJ260-cm-PermE-crp and subcloned into Escherichia coli-Streptomyces shuttle vector pUC-spn stored in authors’ lab by Red/ET homologous recombination technology, generating Crp expression vector pUC-spn-PermE-crp. Then, The vector pUC-spn-PermE-crp was introduced into S. spinosa by conjugal transfer, and integrated into the chromosome via single-cross homologous recombination, and the strain whose chromosome was integrated by original plasmid pUC-spn was used as control strain in this study. The apramycin resistant gene and the target gene were amplified by PCR to confirme positive transconjugants. The morphological comparison of the engineering strain S. spinosa-Crp and the control strain S. spinosa-1 on different media were observed. Growth curves were compared between S. spinosa-Crp and S. spinosa-1 in liquid medium. The mycelial morphologies between S. spinosa-Crp and S. spinosa-1 were observed by scanning electron microscopy and the spinosad production of S. spinosa was detected by high performance liquid chromatography.[Result]A vector pUC-spn-PermE-crp expressing Crp was constructed successfully by molecular biology method, and was transferred into S. spinosa by conjugation. PCR detection results exhibited that the 2 kb long target band cm-PermE-crp could be amplified in engineering strain S. spinosa-Crp, suggesting that crp was integrated into chromosome of S. spinosa successfully. The recombinant engineering strain S. spinosa-Crp abtained was genetically stable. On BHI and ISP-2 media, the spore germination and formation rate of engineering strain S. spinosa-Crp delayed compared to the control strain S. spinosa-1, however, the spore germination and formation rate didn’t show significant difference between S. spinosa-Crp and S. spinosa-1 in R6 medium. Compared to the control strain of S. spinosa-1, the secondary growth phenomenon of engineering strains S. spinosa-Crp disappeared cultured in liquid medium, and its biomass became higher. Under the scanning electron microscopy it was showed that the engneering strain had a higher degree of mycelial fragments and less branches, which can improve the level of dissolved oxygen in S. spinosa-Crp in the process of fermentation. Shaking flask fermentation results revealed that spinosad yield of the engineering strain increased by 128%than that of the control strain.[Conclusion]Overexpression of crp had a certain effect on the mycelial morphology and growth of S. spinosa, and effectively promoted the biosynthesis of spinosad, which lay an important foundation for improvming spinosad production by overexpression of other positive regulatory genes.关键词
刺糖多孢菌/环腺苷酸受体蛋白/过量表达/多杀菌素/Red/ET重组Key words
Saccharopolyspora spinosa/cyclic AMP receptor protein/overexpression/spinosad/Red/ET recombination引用本文复制引用
徐妙,邬洋,杨燕,夏立秋..环腺苷酸受体蛋白基因的过表达对刺糖多孢菌生长和多杀菌素合成的影响[J].中国农业科学,2014,(18):3577-3587,11.基金项目
国家高技术研究发展计划（2011AA10A203）、国家自然科学基金 （）
