纳豆支链短链脂肪酸形成的关键基因挖掘及功能解析

Natto is prepared by fermenting with Bacillus subtilis natto, while its high concentration of branched short-chain fatty acids (BCFAs) leads to the problems of undesirable flavors and food safety. Explaining the genetic molecular mechanism of BCFAs formation can provide target genes for breeding the odor-free natto producing strain. The genes of phosphotransbutyrylase and aldehyde dehydrogenases are involved in the BCFAs formation in some bacteria, while similar six genes in B. subtilis have not been identified for BCFAs formation, hindering us to explain BCFAs formation mechanism and solve natto odor problems. Therefore, the key genes responsible for natto BCFAs formation will be screened from phosphotransbutyrylase and aldehyde dehydrogenases genes through knockout mutation, and the genetic function will be verified by genetic complementation and enhanced expression in vivo. Meanwhile, inducible expression of the key genes in E. coli will be performed to analyze the enzymatic function in vitro. This study will further explain the molecular mechanism of natto BCFAs formation. Finally, the relation of the key genes and the natto comprehensive quality will be investigated, which will provide the theoretical foundation to select the mutagenic targets for breeding the odor-free natto producing strain.

纳豆由枯草芽胞杆菌纳豆亚种(Bacillus subtilis natto)发酵而成，其高浓度的异味支链短链脂肪酸(BCFAs)导致不良风味和食品安全问题。从基因水平阐明BCFAs形成的分子机制，可为无异味纳豆生产菌的诱变育种提供靶基因。磷酸丁酰转移酶和醛脱氢酶基因在不同细菌中被证实参与合成BCFAs，但B. subtilis此类基因（共6个）在其合成BCFAs中的功能尚未解析，阻碍对纳豆BCFAs形成分子机制的阐释和异味问题的解决。因此，本研究拟缺失突变B. subtilis natto的磷酸丁酰转移酶和醛脱氢酶基因，从中挖掘参与纳豆BCFAs形成的关键基因，通过基因回补和强化表达验证关键基因在纳豆BCFAs形成中的体内功能，并通过大肠杆菌诱导表达解析其体外酶学功能，进一步阐释纳豆BCFAs形成的分子机制；并阐明关键基因和纳豆综合品质的相关性，为无异味纳豆生产菌的诱变靶点的选择提供依据。

为解决纳豆异味问题，本项目阐释了异味物质支链短链脂肪酸形成的分子机制。首先通过诱导表达解析了磷酸丁酰转移酶基因（ptb）和醛脱氢酶基因（gabD）在支链短链脂肪酸形成中的作用，并通过基因敲除及回补表达证实了亮氨酸脱氢酶基因（bcd）、α-酮酸脱氢酶复合体基因（bkdAB）和磷酸丁酰转移酶基因（ptb）在支链短链脂肪酸形成中的功能，进一步研究了缺失磷酸丁酰转移酶基因（ptb）和亮氨酸脱氢酶基因（bcd）与纳豆关键品质的相关性，最后以异味物质生物胺为拓展，探究了调控豆豉异味生物胺的关键微生物。本项目在一定程度上阐释了发酵豆制品异味物质的形成机制，为发酵豆制品异味问题的解决提供了理论依据。