基于基因组学的多粘类芽孢杆菌合成(2R,3R)-2,3-丁二醇研究

(2R,3R)-2,3-butanediol (R,R-BD) can be widely used in chemical, food, fuel, and medicine fields. Paenibacillus polymyxa is the optimal R, R-BD-producing strain to date, while the present shortage problems of high concentrations of byproducts and low optical purity of the product hindered its industrialization. Additionally, the metabolic background at genome level of P. polymyxa is not clear, thus it is an obstacle for rationally engineering strains to get potential strain with better fermentation performance. Our team has completed two R, R-BD-producing P. polymyxa strains' genome sequencing, annotation and metabolic network model construction. And some key enzymes in metabolic pathway were also functionally identified at biochemical level. This project intends to adopt systems metabolic engineering approach to change intracellular carbon source flux distribution, and therefore to improve yield and optical purity of the final product. Combination with cofactor engineering strategy, the expression levels of glucose dehydrogenase and rate-limiting enzyme R,R-BD dehydrogenase will also be optimized to reveal the effects of series metabolic engineering on the cell growth, fermentation performance and intracellular redox state. These general conclusions will provide fundamental information for strains improvement to get the higher titer and yield for R,R-BD and analogous polyols production.

(2R,3R)-2,3-丁二醇（R,R-BD）可广泛应用于化工、食品、能源、医药等领域。多粘类芽孢杆菌是目前最优的R,R-BD生产菌种，但存在副产物浓度高和产物光学纯度不足的问题。长期以来，多粘类芽孢杆菌基因组层面上的代谢背景不清晰，给菌株的理性改造带来了困难。项目组前期完成了两株高产R,R-BD的多粘类芽孢杆菌基因组测序、功能注释和代谢网络模型构建工作，并对代谢途径部分关键酶进行了鉴定。在此基础上，本项目拟采用系统代谢工程的多途径优化方法改变胞内碳架物质流量分布，提高产物的转化率和光学纯度；同时利用辅酶调控策略对葡萄糖脱氢酶和关键限速酶R,R-BD脱氢酶进行过量表达来优化胞内NADH/NAD+比率；研究系列改造对菌体生长、发酵性能和氧化还原状态的影响，揭示胞内氧化还原状态对代谢网络的调控机制，为R,R-BD高产菌株的理性改造奠定理论基础，同时为类似多元醇生产菌的改造提供有意义的指导。

本项目利用全基因组序列信息对Paenibacillus polymyxa的相关代谢网络模型进行修正和完善，并获得了主代谢途径、副产物代谢途径关键基因的DNA序列。对主代谢途径关键酶进行克隆表达以及酶学性质分析，阐明了P. polymyxa合成(R,R)-2,3-丁二醇、meso-2,3-丁二醇、(R)-乙偶因、(S)-乙偶因的分子生物学机理。建立了P. polymyxa的分子生物学技术平台，利用多途径方法对菌株进行遗传改造，显著提高(R,R)-2,3-丁二醇的浓度和光学纯度。利用合成生物学、代谢工程、辅酶工程方法对底盘细胞Escherichia coli进行遗传改造，构建了高效合成光学纯(R,R)-2,3-丁二醇的细胞工厂。开展了廉价生物质原料的生物炼制研究，对复合酶解工艺、同步糖化工艺和发酵罐补料发酵工艺进行优化，提高了产物的浓度、转化率和生产强度，(R,R)-2,3-丁二醇浓度最高可达118.8 g/L，光学纯度99%。