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首页> 外文期刊>Applied Microbiology and Biotechnology >Characterization of the chromosomal integration of Saccharopolyspora plasmid pCM32 and its application to improve production of spinosyn in Saccharopolyspora spinosa
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Characterization of the chromosomal integration of Saccharopolyspora plasmid pCM32 and its application to improve production of spinosyn in Saccharopolyspora spinosa

机译：糖多孢菌质粒pCM32的染色体整合特性及其在提高多刺糖多孢菌中多杀菌素生产中的应用

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摘要

Saccharopolyspora spinosa produces tetra-cyclic macrolide spinosyns, a group of highly efficient pesticidal agents. However, this species lacks efficient vectors for genetic manipulation. In this study, the circular plasmid pCM32 was newly isolated from Saccharopolyspora endophytica YIM 61095. The complete nucleotide sequence of pCM32 consists of 14,611 bp and is predicted to encode 17 open reading frames (ORFs). Interestingly, a putative int gene in pCM32 was predicted by homologous alignment to encode an integrase belonging to the tyrosine family of integrases/recombinases. Plasmid pCM238 containing this int locus derived from pCM32 could be transferred by conjugation from Escherichia coli into Sa. spinosa at a high frequency. Integration of pCM238 in the host chromosome was demonstrated as site-specific recombination (at the tRNA (Ser) gene) via a 56-bp core sequence within the attP/attB sites. Plasmid pCM265, a shuttle vector containing the int and attP sequences of pCM32, was constructed to introduce foreign genes into Sa. spinosa. The production of spinosad approximately doubled in Sa. spinosa NRRL18395 after introducing pCM265-derived plasmids carrying the genes for phosphofructokinase (PFK) or anthranilate synthase. These results indicate that plasmid pCM32 is an actinomycete integrative and conjugative element (AICE) and that its derived integrative vectors are useful for efficiently introducing foreign DNA into Sa. spinosa.
机译：棘孢糖多孢菌产生四环大环内酯多杀菌素，一组高效杀虫剂。但是，该物种缺乏用于基因操作的有效载体。在这项研究中，环状质粒pCM32是从内生糖多孢菌YIM 61095中新分离的。pCM32的完整核苷酸序列由14,611 bp组成，预计可编码17个开放阅读框（ORF）。有趣的是，通过同源比对预测pCM32中的推定的int基因编码属于整合/重组酶的酪氨酸家族的整合酶。含有pCM32衍生的int位点的质粒pCM238可以通过结合从大肠杆菌转移到Sa中。棘突的频率很高。 pCM238在宿主染色体中的整合通过atP / attB位点的56 bp核心序列以位点特异性重组（在tRNA（Ser）基因处）证实。构建了质粒pCM265，它是一种包含pCM32的int和attP序列的穿梭载体，可将外源基因导入Sa。棘。在Sa中多杀菌素的产量大约增加了一倍。引入带有磷酸果糖激酶（PFK）或邻氨基苯甲酸合酶基因的pCM265衍生质粒后，Spinosa NRRL18395。这些结果表明质粒pCM32是放线菌的整合和结合元件（AICE），并且其衍生的整合载体可用于有效地将外源DNA引入Sa。棘。

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