Enhancing the capability of Klebsiella pneumoniae to produce 1, 3-propanediol by overexpression and regulation through CRISPR-dCas9.

Abstract

Summary Klebsiella pneumoniae is a common strain of bacterial fermentation to produce 1, 3‐propanediol (1, 3‐PDO). In general, the production of 1, 3‐PDO by wild‐type K. pneumoniae is relatively low. Therefore, a new gene manipulation of K. pneumoniae was developed to improve the production of 1, 3‐PDO by overexpressing in the reduction pathway and attenuating the by‐products in the oxidation pathway. Firstly, dhaB and/or dhaT were overexpressed in the reduction pathway. Considering the cost of IPTG, the constitutive promoter P32 was selected to express the key gene. By comparing K.P. pET28a‐P32‐dhaT with the original strain, the production of 1, 3‐PDO was increased by 19.7%, from 12.97 to 15.53 g l−1 (in a 250 ml shaker flask). Secondly, three lldD and budC regulatory sites were selected in the by‐product pathway, respectively, using the CRISPR‐dCas9 system, and the optimal regulatory sites were selected following the 1, 3‐PDO production. As a result, the 1, 3‐PDO production by K.P. L1‐pRH2521 and K.P. B3‐pRH2521 reached up to 19.16 and 18.74 g l−1, which was increased by 47.7% and 44.5% respectively. Overexpressing dhaT and inhibiting expression of lldD and budC were combined to further enhance the ability of K. pneumoniae to produce 1, 3‐PDO. The 1, 3‐PDO production by K.P. L1‐B3‐PRH2521‐P32‐dhaT reached 57.85 g l−1 in a 7.5 l fermentation tank (with Na+ neutralizer), which is higher than that of the original strain. This is the first time that the 1, 3‐PDO production was improved in K. pneumoniae by overexpressing the key gene and attenuating by‐product synthesis in the CRISPR‐dCas9 system. This study reports an efficient approach to regulate the expression of genes in K. pneumoniae to increase the 1, 3‐PDO production, and such a strategy may be useful to modify other strains to produce valuable chemicals.