Switching metabolic flux by engineering tryptophan operon-assisted CRISPR interference system in Klebsiella pneumoniae

Metab Eng. 2021 May:65:30-41. doi: 10.1016/j.ymben.2021.03.001. Epub 2021 Mar 5.

Abstract

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 (3-HP), 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 3-HP. 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.

Keywords: 3-Hydroxypropionic acid; Biomass; CRISPR interference; Glycerol; Klebsiella pneumoniae.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Clustered Regularly Interspaced Short Palindromic Repeats*
  • Glycerol
  • Klebsiella pneumoniae* / genetics
  • Metabolic Engineering
  • Operon / genetics
  • Tryptophan / genetics

Substances

  • Tryptophan
  • Glycerol