克雷伯氏肺炎杆菌2,3-丁二醇立体异构体合成机理研究

Many carbon sources that come from biomass can be used by Klebsiella pneumoniae for 2,3-butanediol synthesis. This process is efficient and has the potential to be used in industrial applications. The molecule of 2,3-butanediol contains two chiral carbons, resulting in three stereoisomers. Reports have shown that Klebsiella spp. produces meso-2,3-butanediol together with a small amount of 2S,3S-butanediol. However, the stereoisomers of the 2,3-butanediol produced by K. pneumoniae were analyzed by the applicant, and it was found to contain all three stereoisomers. Up to data, the mechanisms of the three 2,3-butanediol isomers formation and inter conversion were not full understanding. In this application, the enzymes that responsible for catalyzed the conversion of acetoin and 2,3-butanediol will be identified, and the enantioselectivity characteristics of these enzymes will be studied. The results of this part study will be got by the physical characteristic of special gene mutants and in vitro enzyme reactions. Another part of this application is to study the role of diacetyl in the 2,3-butanediol isomers formation. The genes involved in the 2,3-butanediol will be knockout and construction several mutants. The physical characteristics of the mutants will be investigated. The results of this application will present the mechanism of 2,3-butanediol stereoisomers formation and the regulations of there inter conversion. And answer the question that if K. pneumoniae can be used for single 2,3-butanediol stereoisomers production, and how to got.

克雷伯氏肺炎杆菌可以利用多种碳源合成2,3-丁二醇，并具有较高的底物转化率，是一种具有工业化应用前景的生产用菌。2,3-丁二醇具有三种立体异构体。文献报道克雷伯氏肺炎杆菌主要合成内消旋2,3-丁二醇和少量2S,3S-丁二醇。而项目申请人发现，克雷伯氏肺炎杆菌合成的2,3-丁二醇是三种异构体的混合物。目前三种立体异构体的合成机理和相互转化规律并不完全清楚。本项目拟通过基因突变和体外酶活实验鉴定催化乙偶姻到2,3-丁二醇的相关酶，及催化反应的立体专一性；通过构建2,3-丁二醇合成途径特定基因突变株，并考察突变株代谢特性变化等实验确定双乙酰的来源和在2,3-丁二醇异构体合成中的地位。通过这些工作确定克雷伯氏肺炎杆菌三种2,3-丁二醇异构体合成的机理和相互转化规律，确定是否能利用克雷伯氏肺炎杆菌生产单一构型2,3丁二醇，以及实现的方法。

克雷伯氏肺炎杆菌是一种重要的工业微生物，2,3-丁二醇是该菌一种重要的代谢产物。本研究对2,3-丁二醇合成的代谢机理进行了详细研究，主要取得以下研究成果。1. 2,3-丁二醇合成途径中丙酮酸合成乙酰乳酸由多个同工酶，包括budB编码的乙酰乳酸合成酶，ilvBN，ilvGM和ilvIH编码的乙酰羟酸合成酶。为了确定这些同工酶在2,3-丁二醇合成中的地位，分别构建了这些基因的突变株，考察2,3-丁二醇的变化。研究结果表明budB编码的乙酰乳酸合成酶是主要的功能酶，该酶的缺失将造成2,3-丁二醇合成的阻断。2. 乙偶姻还原形成2,3-丁二醇由budC编码的丁二醇合成酶催化，除了budC外，dhaD编码的甘油脱氢酶也具有丁二醇脱氢酶活性，并且特异性的将乙偶姻的酮基转化成R构型的羟基。另外gldA编码的甘油脱氢酶和acyI编码的短链酰基脱氢酶也被报道具有丁二醇脱氢酶活性。为此实验构建了budC、gldA、acyl单突变株，和kp-ΔbudC-ΔdhaD-ΔgldA-Δacyl四基因突变株。相比kp-ΔbudC，kp-ΔbudC-ΔdhaD-ΔgldA-Δacyl突变株2,3-丁二醇产量微有下降，但还是有2,3-丁二醇的生成，表明dhaD，gldA和acyl编码的酶蛋白对于2,3-丁二醇合成贡献微弱，同时细胞中可能还存在其他具有丁二醇脱氢酶功能的酶蛋白。3. budA突变株阻断了2,3-丁二醇合成途径，该菌株在酸性条件下培养积累大量2-酮基葡萄糖酸。2-酮基葡萄糖酸是葡萄糖氧化途径产物，位于周质空间。将葡萄糖酸脱氢酶失活后菌株积累葡萄糖酸。4. budA突变株在中性条件下培养积累2-酮基异戊酸，2-酮基异戊酸是支链氨基酸合成途径中缬氨酸的前体。