Redirecting Metabolic Flux via Combinatorial Multiplex CRISPRi-Mediated Repression for Isopentenol Production in Escherichia coli

Tian, Tian; Kang, Jing Wei; Kang, Aram; Lee, Taek Soon

doi:10.1021/acssynbio.8b00429

Title: Redirecting Metabolic Flux via Combinatorial Multiplex CRISPRi-Mediated Repression for Isopentenol Production in Escherichia coli

Journal Article · Fri Jan 04 00:00:00 EST 2019 · ACS Synthetic Biology

DOI:https://doi.org/10.1021/acssynbio.8b00429· OSTI ID:1580375

Tian, Tian ^[1]; Kang, Jing Wei ^[2]; Kang, Aram ^[1];

^[1]

Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Univ. of California, Berkeley, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)

CRISPR interference (CRISPRi) via target guide RNA (gRNA) arrays and a deactivated Cas9 (dCas9) protein has been shown to simultaneously repress expression of multiple genomic DNA loci. By knocking down endogenous genes in competing pathways, CRISPRi technology can be utilized to redirect metabolic flux toward target metabolite. In this study, we constructed a CRISPRi-mediated multiplex repression system to silence transcription of several endogenous genes to increase precursor availability in a heterologous isopentenol biosynthesis pathway. To identify genomic knockdown targets in competing pathways, we first designed a single-gRNA library with 15 individual targets, where 3 gRNA cassettes targeting gene asnA, prpE, and gldA increased isopentenol titer by 18-24%. We then combined the 3 single-gRNA cassettes into a two- or three-gRNA array and observed up to 98% enhancement in production by fine-tuning the repression level through titrating dCas9 expression. Our strategy shows that multiplex combinatorial knockdown of competing genes using CRISPRi can increase production of the target metabolite, while the repression level needs to be adjusted to balance the metabolic network and achieve the maximum titer improvement.

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Research Organization:: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES)

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 1580375

Journal Information:: ACS Synthetic Biology, Vol. 8, Issue 2; ISSN 2161-5063

Publisher:: American Chemical Society (ACS)Copyright Statement

Country of Publication:: United States

Language:: English

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