文献类型：期刊文献

英文题名：Engineering CRISPR interference system in Klebsiella pneumoniae for attenuating lactic acid synthesis

作者：Wang, Jingxuan[1];Zhao, Peng[1];Li, Ying[2];Xu, Lida[1];Tian, Pingfang[1]

第一作者：Wang, Jingxuan

通讯作者：Tian, PF[1]

机构：[1]Beijing Univ Chem Technol, Coll Life Sci & Technol, Beijing Key Lab Bioproc, Beijing 100029, Peoples R China;[2]Beijing Union Univ, Coll Biochem Engn, Beijing 100023, Peoples R China

第一机构：Beijing Univ Chem Technol, Coll Life Sci & Technol, Beijing Key Lab Bioproc, Beijing 100029, Peoples R China

通讯机构：[1]corresponding author), Beijing Univ Chem Technol, Coll Life Sci & Technol, Beijing Key Lab Bioproc, Beijing 100029, Peoples R China.

年份：2018

卷号：17

期号：1

外文期刊名：MICROBIAL CELL FACTORIES

收录：;WOS:【SCI-EXPANDED(收录号:WOS:000429282800002)】；

基金：This work was supported by grants from National High Technology Research and Development Program (863 Program) (No. 2015AA021003), National Natural Science Foundation of China (Nos. 21276014, 21476011), National Basic Research Program of China (973 Program) (No. 2012CB725200), and Fundamental Research Funds for the Central Universities (YS1407).

语种：英文

外文关键词：CRISPR interference; Klebsiella pneumoniae; 3-Hydroxypropionic acid; Lactic acid; Lactate dehydrogenase

摘要：Background: Klebsiella pneumoniae is a promising industrial species for bioproduction of bulk chemicals such as 1,3-propanediol,2,3-butanediol and 3-hydroxypropionic acid (3-HP). However, lactic acid is a troublesome by-product when optimizing for 3-HP production. Therefore, it is highly desirable to minimize lactic acid. Results: Here, we show that lactic acid synthesis can be largely blocked by an engineered CRISPR interference (CRISPRi) system in K. pneumoniae. EGFP was recruited as a reporter of this CRISPRi system. Fluorescence assay of this CRISPRi system showed that enhanced green fluorescent protein (EGFP) expression level was repressed by 85-90%. To further test this CRISPRi system, guide RNAs were designed to individually or simultaneously target four lactate-producing enzyme genes. Results showed that all lactate-producing enzyme genes were significantly repressed. Notably, D-lactate dehydrogenase (ldhA) was shown to be the most influential enzyme for lactic acid formation in micro-aerobic conditions, as inhibiting ldhA alone led to lactic acid level similar to simultaneously repressing four genes. In shake flask cultivation, the strain coexpressing puuC (an aldehyde dehydrogenase catalyzing 3-hydroxypropionaldehyde to 3-HP) and dCas9-sgRNA inhibiting ldhA produced 1.37-fold 3-HP relative to the reference strain. Furthermore, in bioreactor cultivation, this CRISPRi strain inhibiting ldhA produced 36.7 g/L 3-HP, but only generated 1 g/L lactic acid. Clearly, this engineered CRISPRi system largely simplified downstream separation of 3-HP from its isomer lactic acid, an extreme challenge for 3-HP bioprocess. Conclusions: This study offers a deep understanding of lactic acid metabolism in diverse species, and we believe that this CRISPRi system will facilitate biomanufacturing and functional genome studies of K. pneumoniae or beyond.