CRISPRi-Based Dynamic Control of Carbon Flow for Efficient N-Acetyl Glucosamine Production and Its Metabolomic Effects in Escherichia coli

Abstract

Carbon competition between cell growth and product synthesis is the bottleneck in efficient N-acetyl glucosamine (GlcNAc) production in microbial cell factories. In this study, a xylose-induced T7 RNA polymerase−P T7 promoter system was introduced in Escherichia coli W3110 to control the GlcNAc synthesis. Meanwhile, an arabinose-induced CRISPR interference (CRISPRi) system was applied to adjust cell growth by attenuating the transcription of key growth-related genes. By designing proper sgRNAs, followed by elaborate adjustment of the addition time and concentration of the two inducers, the carbon flux between cell growth and GlcNAc synthesis was precisely redistributed. Comparative metabolomics analysis results confirmed that the repression of pf kA and zwf significantly attenuated the TCA cycle and the synthesis of related amino acids, saving more carbon for the GlcNAc synthesis. Finally, the simultaneous repression of pf kA and zwf in strain GLA-14 increased the GlcNAc titer by 47.6% compared with that in E. coli without the CRISPRi system in a shake flask. GLA-14 could produce 90.9 g/L GlcNAc within 40 h in a 5 L bioreactor, with a high productivity of 2.27 g/L/h. This dynamic strategy for rebalancing cell growth and product synthesis could be applied in the fermentative production of other chemicals derived from precursors synthesized via central carbon metabolism.

dCas9 regulator to neutralize competition in CRISPRi circuits

“…Although we did not investigate this aspect in this paper, the impact of resource competition on the dynamic behavior of a genetic circuit can also result in dramatic outcomes 30 . Further investigations are required to determine the benefit of the regulated dCas9 generator when dynamic behavior is of interest 31,32 .…”

Programming Cells by Multicopy Chromosomal Integration Using CRISPR-Associated Transposases

“…2A). 23,24 To achieve the insertional inac-tivation of the targeted genes and the transcriptional disruption of the downstream genes simultaneously, we used the sequence containing a bidirectional terminator tonB 25 and two monodirectional terminators fdTer 26 as cargo. The CRISPR RNAs (crRNAs) were…”

Engineering a CRISPRi Circuit for Autonomous Control of Metabolic Flux in Escherichia coli

“…With advantages including simple manipulation, multiplexed regulation, and high dynamic range, the CRISPRi system was widely implemented in building efficient microbial cell factories. 28,29 For example, by developing a tryptophan operon-assisted CRISPRi system in K. pneumoniae, cell growth and 3-hydroxypropionic acid synthesis were successfully decoupled. 30 By integrating a blue lightsensitive protein to control the expression of dCpf1, an optogenetic CRISPRi platform was constructed for improving muconic acid production.…”

“…The introduction of SPPs to trigger the transcription of sgRNA or dCas9 provided a valuable alternative in realizing autonomous dynamic control. With advantages including simple manipulation, multiplexed regulation, and high dynamic range, the CRISPRi system was widely implemented in building efficient microbial cell factories. , For example, by developing a tryptophan operon-assisted CRISPRi system in K. pneumoniae, cell growth and 3-hydroxypropionic acid synthesis were successfully decoupled .…”