基因替换偶联NADH辅酶再生策略调控Paenibacillus polymyxa合成R,R-2,3-丁二醇的研究

R,R-2,3-butanediol, which is a kind of microbial production of four carbon chiral alcohol, has attracted increasing interest of researchers in the chemical and material engineering field because of its diverse industrial applications in the chiral synthesis in recent years. The R,R-2,3-butanediol at higher than 99.9% optical purity has practical uses in the synthesis of some chiral electronic material and chiral pharmaceuticals. In this research, the new method about the genetic manipulation of R,R-2,3-butanediol metabolic pathway in Paenibacillus polymyxa will be studied in order to reduce the small by-product meso-2,3-butanediol and achieve cefactor NADH regeneration. The gene of diacetyl reductase in P. polymyxa will be skillfully replaced with the gene of NAD-dependent formate dehydrogenase using the λ Red homologous recombination technique, and the synthesis of meso-2,3-butanediol will be interrupted as the same time the coenzyme NADH is regenerated. Then, the metabolic flux in the recombinant strain are efficiently regulated to optimize the coenzyme and carbon balance. Finally,the R,R-2,3-butanediol at higher than 99.9% optical purity and at higher yield will be achieved success. This work will promote the technological development of industrial R,R-2,3-butanediol production and its application in the chiral synthesis, and provide a kind of method, which will be utilized in the study of the chiral small molecule organic compounds, such as other chiral alcohol, chiral amino acid and so on. Through above-mentioned research, the huge economic and social benefit could been obtained.

R,R-2,3-丁二醇是一种新兴的微生物四碳手性醇，超高光学纯度（≥99.9%）的该产品是合成手性电子材料、医药中间体的重要原料，近年来，受到了化工、材料界的广泛关注。本课题针对Paenibacillus polymyxa发酵制备R,R-2,3-丁二醇过程中存在少量光学副产物meso-2,3-丁二醇以及NADH辅酶再生问题，巧妙地运用基因替换策略，构建新型λRed 同源重组系统，将该菌中丁二酮还原酶基因替换为NAD+依赖型甲酸脱氢酶基因，在阻断meso-2,3-丁二醇合成的同时偶联NADH辅酶再生，并借助最佳代谢流优化方案的设计，对辅酶、碳平衡进行有效调控，最终实现超高光学纯R,R-2,3-丁二醇的高效制备，促进其生产技术的发展以及在手性产品合成中的应用，并为类似手性醇、手性氨基酸等手性有机小分子的研究提供借鉴，具有重要的学术意义和实用价值。

R,R-2,3-丁二醇是近年来新兴的一种微生物四碳手性醇，是合成手性液晶材料、手性药物、手性试剂和手性配体的重要中间体，在手性天然产物的合成中也有潜在的重要应用。针对Paenibacillus polymyxa代谢还原合成R,R-2,3-丁二醇时，产物产量偏低、NADH过量消耗以及少量的meso-2,3-丁二醇副产物存在等关键问题，课题组对原始菌开展了发酵工艺优化、NADH辅助再生和meso-2,3-丁二醇代谢途径敲除等方面的研究，达到发酵提高R,R-2,3-丁二醇的产量和光学纯度，促进R,R-2,3-丁二醇工业化发酵制备，同时为P. polymyxa菌株分子遗传改造提供借鉴。.在7.5 L发酵罐上考察了P. polymyxa ZJ-9混合发酵菊粉和葡萄糖合成R,R-2,3-丁二醇的工艺条件。结果表明，初始菊粉75.0 g/L，当发酵到24 h，31 h时，分别添加15.0 g/L的葡萄糖，发酵效果最佳，发酵44 h时，产物产量达到最高值47.8 g/L，与分批发酵相比，糖转化率由原来的34.9%提高到45.5%，生产强度由原来的0.70 g/L/h提高到1.09 g/L/h，并且副产物乙偶姻，残糖浓度相对较低。.将来源于Candida boidinii中的NAD+依赖型甲酸脱氢酶基因（fdh）导入P. polymyxa ZJ-9中，考察该基因对P. polymyxa ZJ-9菌体生长，FDH酶活，胞内NADH/NAD+以及合成 R,R-2,3-2,3-BD 等代谢流的影响。结果表明，在7.5 L罐中，批式发酵以及补料分批发酵R,R-2,3-丁二醇最高产量分别达到36.8 g/L和51.3 g/L，与野生菌发酵结果相比，产量分别提高了10.2 %和8.0 %。该研究显示，通过NADH辅酶再生工程完全可以调控菌体内物质代谢，为依赖NADH的产物代谢合成提供一种行之有效手段。. 分别以P. polymyxa ZJ-9基因组DNA和质粒pSUP202为模板，PCR 扩增获得丁二酮还原酶基因（Dud A）片段和氯霉素基因（Cm）片段。构建敲除质粒pRN5101-Dud A-Cm并通过电转化成功导入野生菌P. polymyxa ZJ-9中。构建了多粘类芽胞杆菌的基因敲除体系，拓展了pRN5101的使用范围，为研究多粘类芽胞杆菌的基因功能提供了高效的遗传操作工具。