解淀粉芽孢杆菌2,3-丁二醇逆向转化3-羟基丁酮的代谢机制及调控研究

Nowadays, microbial production of 2,3-butanediol and 3-hydroxy-2-butanone is attracting more and more attentions due to that they have wide application in food and industrial biotechnology field. In our previous work, a GRAS strain Bacillus amyloliquefaciens B10-127, capable of producing 2,3-BD effectively, was isolated. When glucose was used as carbon source in fermentation, the fermentation characterizations of this strain were analyzed and a new phenomenon of reverse transforming 2,3-butanediol which was synthesized from glucose to acetoin was detected. Before 96 h, glucose which was mainly transformed to 2,3-butanediol and acetoin was totally consumed, while at the end of fermentation, the yield of acetoin was greatly improved but 2,3-butanediol was declined（World J Microbiol Biotechnol. 2011, 27:2785-2790）. To reveal the new phenomenon of reverse transforming 2,3-butanediol to acetoin at the end of fermentation, we will investigate substrate consumption, productions biosynthesis, key enzyme characteristics, and the strain's key physiological features such as level of energy charge and oxidation-reduction state at different fermentation stages. Based on these properties, we will try to regulate specific metabolites synthetize through altering the key physiological features of cells and modifying key genes. Consequently, the expected results the metabolic regulation of the new phenomenon of reverse transformation would provide techniques and references for other microbial metabolic engineering research.

发酵法生产2,3-丁二醇和3-羟基丁酮是目前国内外食品与工业生物技术领域研究热点之一。课题组前期筛选获得一株环境安全高产2,3-丁二醇解淀粉芽孢杆菌，发现该菌发酵前期主要积累2,3-丁二醇，后期转变为积累前体物质3-羟基丁酮（World J Microbiol Biotechnol. 2011, 27:2785-2790）。为揭示发酵产物逆向转化现象，课题拟从微生物生理和代谢水平对可逆代谢机制进行深入研究，探索发酵过程理性调控策略。课题拟通过发酵各阶段氧化还原状态、细胞能荷水平等重要生理生化分析，以及代谢流和代谢关键酶分析，建立细胞各生理状态、细胞代谢网络与产物逆向转化过程的相互关系。在此基础上，采用生理调控手段和分子修饰手段，进行针对性产物代谢调控，并反馈产物逆向转化的形成机制。课题通过探索解淀粉芽孢杆菌产物可逆代谢的形成机制及其调控策略，将为微生物代谢工程的研究进一步开拓思路。

项目针对芽孢杆菌在发酵过程中存在3-羟基丁酮和2,3-丁二醇相互转化的现象在芽孢杆菌遗传表达体系改造、胞内代谢途径优化、辅因子再生等方面做了大量研究。得到如下结论：. （1）克隆并分析了代谢途径中辅酶依赖型3-羟基丁酮还原酶/2,3-丁二醇脱氢酶（AR/BDH），发现该酶氧化方向和还原方向的最适反应pH存在明显差异，氧化方向最适pH为8.5，还原方向最适pH为6.5，并由此推测出发酵过程中3-羟基丁酮和2,3-丁二醇相互转化是受到胞内pH和辅酶水平变化调控的。. （2）通过添加不同氧化还原态底物和利用基因工程手段降低胞内NADH水平并且调节胞内NADH/NADPH水平进而重新分配3-羟基丁酮与2,3-丁二醇比例。结果表明不同的氧化还原态底物能够有效地操纵胞内辅酶水平。. （3）首次研究了来源于芽孢杆菌的NADH氧化酶YDOC和芽孢杆菌适度表达型启动子PbdhA。将YODC在芽孢杆菌中进行适度表达会有效降低2,3-丁二醇和NADH依赖型副产物的合成。. （4）通过共表达AR/BDH和YODC构建NAD+辅酶再生系统，可有效降低胞内NADH浓度和NADH/NAD+比例，同时获得高产3-羟基丁酮的全细胞转化体系。 . 本研究解析了芽孢杆菌主要发酵产物3-羟基丁酮和2,3-丁二醇在胞内的可逆转化现象，并在此基础上利用代谢工程改造和辅因子工程分别获得高产3-羟基丁酮和2,3-丁二醇的菌株。项目资助期间，相关研究成果发表在Metabolic Engineering、Biotechnology for Biofuels、Appl Microbiol Biotechnol、Microbial Cell Factories、Amino Acids等期刊（共发表SCI 论文14篇，其中IF>6的论文2篇，6>IF>3的论文6篇，单篇SCI最高IF为8.2，累计SCI的IF>50），获授权中国发明专利5项，为工业化利用芽孢杆菌合成代谢产物提供借鉴。