期刊
METABOLIC ENGINEERING
卷 65, 期 -, 页码 30-41出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ymben.2021.03.001
关键词
CRISPR interference; Klebsiella pneumoniae; Glycerol; 3-Hydroxypropionic acid; Biomass
资金
- National Key Research and Development Program of China [2018YFA0901800]
- Hebei Project for the Development of Science and Technology Guided by Central Universities [206Z2902G]
- National Natural Science Foundation of China [21476011]
- National High Technology Research and Development Program (863 Program) [2015AA021003]
A newly discovered CRISPR interference system can switch glycerol metabolism pathways in Klebsiella pneumoniae, improving the production of 3-HP. Coexpressing CRISPR components and 3-HP biosynthetic enzyme significantly increased 3-HP yield, while implementing circulation feeding further improved production efficiency.
One grand challenge for bioproduction of desired metabolites is how to coordinate cell growth and product synthesis. Here we report that a tryptophan operon-assisted CRISPR interference (CRISPRi) system can switch glycerol oxidation and reduction pathways in Klebsiella pneumoniae, whereby the oxidation pathway provides energy to sustain growth, and the reduction pathway generates 1,3-propanediol and 3-hydroxypropionic acid (3HP), two economically important chemicals. Reverse transcription and quantitative PCR (RT-qPCR) showed that this CRISPRi-dependent switch affected the expression of glycerol metabolism-related genes and in turn improved 3-HP production. In shake-flask cultivation, the strain coexpressing dCas9-sgRNA and PuuC (an aldehyde dehydrogenase native to K. pneumoniae for 3-HP biosynthesis) produced 3.6 g/L 3-HP, which was 1.62 times that of the strain only overexpressing PuuC. In a 5 L bioreactor, this CRISPRi strain produced 58.9 g/L 3HP. When circulation feeding was implemented to alleviate metabolic stress, biomass was substantially improved and 88.8 g/L 3-HP was produced. These results indicated that this CRISPRi-dependent switch can efficiently reconcile biomass formation and 3-HP biosynthesis. Furthermore, this is the first report of coupling CRISPRi system with trp operon, and this architecture holds huge potential in regulating gene expression and allocating metabolic flux.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据