基于多组学技术的淀粉酶产色链霉菌突变株高产ε-聚赖氨酸作用机制研究

ε-Polylysine has a wide range of antimicrobial activity and has been used as a bio-preservative. ε-Polylysine and ε-Polylysine•HCl have been approved used as new kinds of food additives by NHFPC in1994. But its high production costs restrict its wide application in china. Breeding of high yield strain through physical and chemical mutagenesis is a feasible method to reduce the ε-polylysine production costs. Analysis of mutant mechanism has become a scientific problem to be solved urgently at present. The high-yield mutant ε-polylysine producing strain Streptomyces diastatochromogenes 6#-7 was obtained through physical and chemical mutation methods, the yield increased by 41.07% compared with the starting strain Streptomyces diastatochromogenes TUST, which provides a good study material for the study of high yield ε-Polylysine synthesis mechanism of mutant strain. This project intends to determine the influence and regulation of ε-Polylysine synthesis key targets by analysis of genes, transcription, protein and metabolite levels differences between wild type ε-Polylysine producing strain and high yield mutant through use genomics, transcriptomics, proteomics and metabolomics technology，and systematic explain the mutant high yield ε-Polylysine mechanism, and provide a theoretical basis for the further improve the ε-Polylysine fermentation level. The results obtained in this study also can be used as a reference to reveal the mechanism of high yield mutants by physical and chemical mutagenesis.

ε-聚赖氨酸是一种广谱生物防腐剂，2014 年我国批准ε-聚赖氨酸及其盐酸盐作为新型食品防腐剂使用。由于生产成本高，限制了其在国内的广泛应用。通过理化诱变选育高产菌株是降低ε-聚赖氨酸生产成本的可行方法，解析高产突变株作用机制就成为目前亟待解决的科学问题。前期以ε-聚赖氨酸产生菌淀粉酶产色链霉菌TUST作为出发菌通过理化诱变手段得到高产突变株6#-7，产量较出发菌株提高41.07%，为研究突变株高产ε-聚赖氨酸合成机制提供了良好研究材料。项目拟采用基因组学、转录组学、蛋白质组学和代谢组学技术，对ε-聚赖氨酸产生菌原始菌株和高产突变株从基因、转录、蛋白质和代谢水平进行比较分析，确定影响ε-聚赖氨酸合成和调控的关键靶点，系统阐述突变株高效合成ε-聚赖氨酸的作用机制，为进一步提高ε-聚赖氨酸发酵水平提供理论依据。项目对于揭示其他通过理化诱变手段提高微生物产品发酵水平的高产机制也具有借鉴意义。

ε-聚赖氨酸是一种具有良好的热稳定性、安全性和广谱抑菌性的天然食品防腐剂。由于生产成本高，限制了其广泛应用。本研究在获得聚赖氨酸高产菌株的基础上，基于酶学研究、代谢组学、转录组学、蛋白组学、基因组学等方法对淀粉酶产色链霉菌高产聚赖氨酸的生物合成机制进行探讨。（1）通过对不同淀粉酶产色链霉菌各个途径关键酶活的测定，发现在高产ε-PL菌株中，己糖激酶的增强提供了较多的磷酸烯醇式丙酮酸，丙酮酸激酶增强，使发酵中后期有足够的乙酰辅酶A；柠檬酸合成酶活性的减低，一定程度上减少草酰乙酸的消耗；值得注意的是，聚赖氨酸合成酶酶活的高低并不是限制ε-PL积累的关键因素。（2）通过代谢组学分析结果，分析发现诱变菌株的糖酵解途径和三羧酸循环、二氨基庚二酸途径显著增强，脂肪酸代谢途径受到抑制。另外，海藻糖、谷氨酸、脯氨酸等保护性代谢物质的增加，使得菌株自身抵御聚赖氨酸的能力加强。（3）对诱变前后菌株转录组学数据进行分析，发现菌体生长期，诱变菌株较原始菌株显著上调基因为487个，显著下调基因为1237个；产酸期，诱变菌株较原始菌株显著上调基因为510个，显著下调基因为1867个。糖酵解通路6-磷酸果糖激酶表达上调、丙酮酸激酶表达上调；磷酸戊糖途径相关酶表达显著减弱，另外，二氨基庚二酸途径大部分酶表达上调，为聚赖氨酸生物合成提供充足的前体物赖氨酸；脂肪酸代谢途径的减弱，也在一定程度上减少了乙酰辅酶A的消耗，使得代谢流更多的流向TCA循环，也影响了细胞膜的流动性。（4）在相同条件下对S. diastatochromogenes 6#-7和S. diastatochromogenes TUST进行基于iTRAQ技术的蛋白组学研究。共鉴定到3387种蛋白，其中菌体生长期上调蛋白个数为193个，下调蛋白个数为269个；产酸期上调蛋白数244个，下调蛋白数247个。（5）对原始菌株S. diastatochromogenes TUST和高产突变株6#-7测序结果表明突变株存在近500个单核苷酸位点变异(single nucleotide variants, SNV)，近200个插入缺失位点（InDel，Insertion/Deletion）。项目系统阐述突变株高效合成ε-聚赖氨酸的作用机制，为进一步提高ε-聚赖氨酸发酵水平提供理论依据。