须糖多孢菌Saccharopolyspora pogona的核糖体工程改造对丁烯基多杀菌素合成的影响

Effect of ribosome engineering on butenyl-spinosyns synthesis of Saccharopolyspora pogona

摘要:

核糖体工程是以微生物的各类抗生素抗性突变为筛选标记，高效获得次生代谢产物合成能力提高的突变株的一种育种新方法。通过核糖体工程技术，使用链霉素对须糖多孢菌Saccharopolyspora pogona进行抗性选育，以获得高产丁烯基多杀菌素突变菌株。对原始菌株和所获得的突变菌株代谢产物的研究发现，相对于原始菌株，其中突变株S13的丁烯基多杀菌素产量提高幅度最大，相比原始菌株提高了1.79倍。经质谱测定表明，其代谢物中比原始菌株多了一种丁烯基多杀菌素组分Spinosyn α1。对抗性突变株S13的DNA序列进行分析，发现在编码核糖体S12蛋白的rpsL基因保守区域中出现点突变，第314位和第320位的胞嘧啶(C)分别突变为腺嘌呤(A)和胸腺嘧啶(T)，对应的氨基酸残基分别由脯氨酸突变为谷氨酰胺，丙氨酸突变为缬氨酸。研究显示，突变株S13遗传稳定性良好。

Abstract:

Through introducing mutations into ribosomes by obtaining spontaneous drug resistance of microorganisms, ribosome engineering technology is an effective approach to develop mutant strains that overproduce secondary metabolites. In this study, ribosome engineering was used to improve the yield of butenyl-spinosyns produced by Saccharopolyspora pogona by screening streptomycin resistant mutants. The yields of butenyl-spinosyns were then analyzed and compared with the parent strain. Among the mutants, S13 displayed the greatest increase in the yield of butenyl-spinosyns, which was 1.79 fold higher than that in the parent strain. Further analysis of the metabolite profile of S13 by mass spectrometry lead to the discovery of Spinosyn α1, which was absent from the parent strain. DNA sequencing showed that there existed two point mutations in the conserved regions of rpsL gene which encodes ribosomal protein S12 in S13. The mutations occurred a C to A and a C to T transversion mutations occurred at nucleotide pair 314 and 320 respectively, which resulted in the mutations of Proline (105) to Glutamine and Alanine (107) to Valine. It also demonstrated that S13 exhibited genetic stability even after five passages.