Combining CRISPR and CRISPRi Systems for Metabolic Engineering of E. coli and 1,4-BDO Biosynthesis

Abstract

Biosynthesis of 1,4-butanediol (1,4-BDO) in E. coli requires an artificial pathway that involves six genes and time-consuming, iterative genome engineering. CRISPR is an effective gene editing tool, while CRISPR interference (CRISPRi) is repurposed for programmable gene suppression. This study aimed to combine both CRISPR and CRISPRi for metabolic engineering of E. coli and 1,4-BDO production. We first exploited CRISPR to perform point mutation of gltA, replacement of native lpdA with heterologous lpdA, knockout of sad and knock-in of two large (6.0 and 6.3 kb in length) gene cassettes encoding the six genes (cat1, sucD, 4hbd, cat2, bld, bdh) in the 1,4-BDO biosynthesis pathway. The successive E. coli engineering enabled production of 1,4-BDO to a titer of 0.9 g/L in 48 h. By combining the CRISPRi system to simultaneously suppress competing genes that divert the flux from the 1,4-BDO biosynthesis pathway (gabD, ybgC and tesB) for >85%, we further enhanced the 1,4-BDO titer for 100% to 1.8 g/L while reducing the titers of byproducts gamma-butyrolactone and succinate for 55% and 83%, respectively. These data demonstrate the potential of combining CRISPR and CRISPRi for genome engineering and metabolic flux regulation in microorganisms such as E. coli and production of chemicals (e.g., 1,4-BDO).

Impact of CRISPR interference on strain development in biotechnology

“…Mostly, CRISPRi applications have been described for the model organism E. coli , for example, to produce various products, such as terpenes [93–96], polyhydroxybutyrate (PHB) [97–101], or alcohols such as n ‐butanol [102] or 1,4 butandiol [103].…”

Strategies for Developing CRISPR‐Based Gene Editing Methods in Bacteria

“…The author has used CRISPRi to inhibit the expression of a transcription factor MetJ which globally repressed the SAM synthesis pathway, leading to a significantly improved yield of the final product, peonidin 3‐O‐glucoside. Similar strategy has been verified in many genera for manufacturing many biochemicals and pharmaceutic precursors, such as poly‐3‐hydroxbutyrate (PHB), hyaluronic acid, N‐acetylglucosamine, l ‐lysine, l ‐glutamate, citrulline, flavonoid, malate, butanol, 1,4‐butanediol, 3‐hydroxypropionic acid, mevalonate, and epothilones…”

Advancing biotechnology with CRISPR/Cas9: recent applications and patent landscape

“…Repression of genes involved in cell proliferation in order to switch towards a production phase [99] CRISPRi Escherichia coli…”

“…Once bound to, or in the vicinity of the transcriptional start site (TSS), the gRNA:dCas9 complex can significantly alter the transcriptional expression by physically interfering with RNA polymerase binding [14,43,79]. Wu et al recently exploited this strategy in E. coli where they did a selective knockdown of gene expression of enzymes that could divert the carbon flux away from the production of 1,4-Butanediol (BDO) [99]. They divided their study into two phases, (1) a heavy strain engineering approach through multiple genome edits such as gene knockouts, knockins, and point-mutations, and (2) optimization through fine tuning of gene expression of three genes competing with the production of BDO.…”