聚羟基丁酸路径在克雷伯氏菌中的构建

Construction of polyhydroxybutyrate pathway in Klebsiella pneumoniae

以生物柴油的副产物甘油生产高附加值的1,3-丙二醇,现已成为提升生物柴油产业链经济性的重要途径,而中间代谢产物3-羟基丙醛积累造成细胞死亡,发酵异常终止是生物法生产1,3-丙二醇过程中的关键问题。不同于传统的降低3-羟基丙醛积累的思路,本文从增强克雷伯氏菌对3-羟基丙醛的抗逆性出发,改善克雷伯氏菌1,3-丙二醇的生产性能,首次将聚羟基丁酸路径引入克雷伯氏菌中,构建了新型基因工程菌,并对其1,3-丙二醇发酵性能及聚羟基丁酸代谢进行了初步的研究。经IPTG诱导,工程菌中检测到聚羟基丁酸,其含量随IPTG浓度增加而增大。优化的IPTG浓度为0.5 mmol/L。初始甘油50 g/L时,野生菌可正常发酵生产1,3-丙二醇,1,3-丙二醇浓度达到22.1 g/L,其质量得率为46.4%。当初始甘油达到70 g/L时,由于高浓度3-HPA积累,野生菌发酵终止,而工程菌可正常发酵生产1,3-丙二醇,PDO产量可达31.3 g/L,其质量得率为43.9%。同时检测到聚羟基丁酸积累。研究结果有助于加深对克雷伯氏菌1,3-丙二醇代谢机理的认识,为克雷伯氏菌的进一步优化提供了新的思路。

Production of 1,3-propanediol, a byproduct of biodiesel production, is important to increase the economic benefit of biodiesel industry. Accumulation of 3-hydroxypropionaldehyde is one of the key problems in the 1,3-propanediol fermentation process, leading to the cell death and the fermentation abnormal ceasing. Different from the traditional way of reducing the accumulation of the 3-hydroxypropionaldehyde, we introduced the polyhydroxybutyrate pathway into the Klebsiella pneumoniae for the first time to enhance the tolerance of K. pneumoniae to 3-hydroxypropionaldehyde, at the same time, to improve the 1,3-propanediol production. Plasmid pDK containing phbC, phbA, phbB gene was constructed and transformed into K. pneumoniae successfully. PHB was detected in the engineered K. pneumoniae after IPTG induction and its content was enhanced with the IPTG concentration increasing. The optimized IPTG concentration was 0.5 mmol/L. The constructed K. pneumoniae could produce 1,3-propanediol normally, at the same time accumulate polyhydroxybutyrate. With the constructed strain, the fermentation proceeds normally; 1,3-propanediol concentration and yield reached 31.3 g/L and 43.9% at 72 h. Our work is helpful for the deep understanding of 1,3-propanediol metabolic mechanism of Klebsiella pneumoniae, and also provides a new way for strain optimization of Klebsiella pneumoniae.