Genomic and transcriptional changes in response to pinene tolerance and overproduction in evolved Escherichia coli

Abstract

α-Pinene is an important monoterpene, which is widely used as a flavoring agent and in fragrances, pharmaceuticals and biofuels. Although an evolved strain Escherichia coli YZFP, which had higher tolerance to pinene and titer, has been successfully used to produce high levels of pinene, the pinene titer is much lower than that of hemiterpene (isoprene) and sesquiterpenes (farnesene) to date. Moreover, the overall cellular physiological and metabolic changes caused by higher tolerance to pinene and overproduction of pinene remains unclear. To reveal the mechanism of Escherichia coli YZFP with the higher tolerance to pinene and titer, a comparative genomics and transcriptional level analyses combining with CRISPR activation (CRISPRa) and interference (CRISPRi) were carried out. The results show that the tolerance to pinene and the overproduction of pinene in E. coli may be associated with: 1) the mutations of the DXP pathway genes, the rpoA and some membrane protein genes, and their upregulations of transcription levels; and 2) the mutations of some genes and their downregulation of transcriptional levels. These comparative omics analyses provided some genetic modification strategies to further improve pinene production. Overexpression of the mutated cbpA, tabA, pitA, rpoA, sufBCDS, mutS, ispH, oppF, dusB, dnaK, dxs, dxr and flgFGH genes further improved pinene production. This study also demonstrated that combining comparative omics analysis with CRISPRa and CRISPRi is an efficient technology to quickly find a new metabolic engineering strategy.

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].…”

“…Furthermore, a different terpene, α‐pinene, which is widely used as a flavoring agent as well as in fragrances, pharmaceuticals, and biofuels, has been produced by a combinatorial approach of CRISPRa and CRISPRi. Thereby, a higher tolerance and titer of α‐pinene was revealed [96].…”

Programmable gene regulation for metabolic engineering using decoy transcription factor binding sites

“…Several studies have demonstrated that E. coli can cope with pinene-induced stress using endogenous genes (37)(38)(39).…”

“…UlaR is a regulator of the ula operon that is responsible for L-ascorbate metabolism. UlaE has been reported to significantly enhance pinene tolerance (39), however, the specific mechanism by which this operon increases tolerance is not well understood. AcrR regulates expression of the multidrug efflux pump AcrAB-TolC, which is also known to increase pinene tolerance (37).…”

The Potential Production of the Bioactive Compound Pinene Using Whey Permeate

“…The catalytic process of mevalonic kinase and mevalonate diphosphate decarboxylase were identified as the bottlenecks of the MVA pathway in recombinant E. coli [36]. A strain of laboratory involved pinene-producing E. coli strain was whole genome sequenced, then aligned with the E. coli reference strain MG1655 genome sequence, then non-synonymous mutant genes were up and downregulated [37]. Using comparative genomics and transcriptional level analysis, it was shown that mutations and up-regulation of transcription levels of the MEP pathway genes (dxr, dxs, ispH, and ispU) and some protein membrane genes (gsiA, nlpA, sufBCDS, opgB, setC, oppF, ypjK, btuB, pitA and cusA) are related to increased pinene tolerance and production in E. coli [37].…”