Enhancing isoprenol production by systematically tuning metabolic pathways using CRISPR interference in E. coli

Jinho Kim; Taek Soon Lee

doi:10.3389/fbioe.2023.1296132

Enhancing isoprenol production by systematically tuning metabolic pathways using CRISPR interference in E. coli

Front Bioeng Biotechnol. 2023 Nov 6:11:1296132. doi: 10.3389/fbioe.2023.1296132. eCollection 2023.

Authors

Jinho Kim^{1

2}, Taek Soon Lee^{1

2}

Affiliations

¹ Joint BioEnergy Institute, Emeryville, CA, United States.
² Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA, United States.

Abstract

Regulation of metabolic gene expression is crucial for maximizing bioproduction titers. Recent engineering tools including CRISPR/Cas9, CRISPR interference (CRISPRi), and CRISPR activation (CRISPRa) have enabled effective knock-out, knock-down, and overexpression of endogenous pathway genes, respectively, for advanced strain engineering. CRISPRi in particular has emerged as a powerful tool for gene repression through the use of a deactivated Cas9 (dCas9) protein and target guide RNA (gRNA). By constructing gRNA arrays, CRISPRi has the capacity for multiplexed gene downregulation across multiple orthogonal pathways for enhanced bioproduction titers. In this study, we harnessed CRISPRi to downregulate 32 essential and non-essential genes in E. coli strains heterologously expressing either the original mevalonate pathway or isopentenyl diphosphate (IPP) bypass pathway for isoprenol biosynthesis. Isoprenol remains a candidate bioproduct both as a drop-in blend additive and as a precursor for the high-performance sustainable aviation fuel, 1,4-dimethylcyclooctane (DMCO). Of the 32 gRNAs targeting genes associated with isoprenol biosynthesis, a subset was found to vastly improve product titers. Construction of a multiplexed gRNA library based on single guide RNA (sgRNA) performance enabled simultaneous gene repression, yielding a 3 to 4.5-fold increase in isoprenol titer (1.82 ± 0.19 g/L) on M9-MOPS minimal medium. We then scaled the best performing CRISPRi strain to 2-L fed-batch cultivation and demonstrated translatable titer improvements, ultimately obtaining 12.4 ± 1.3 g/L isoprenol. Our strategy further establishes CRISPRi as a powerful tool for tuning metabolic flux in production hosts and that titer improvements are readily scalable with potential for applications in industrial bioproduction.

Keywords: CRISPR interference; IPP-bypass pathway; fed-batch cultivation; isoprenol; mevalonate pathway; multiplexed CRISPRi arrays.

This work is authored by Jinho Kim and Taek Soon Lee on behalf of the U.S. Government and as regards Dr. Kim, Dr. Lee and the U.S. Government, is not subject to copyright protection in the United States. Foreign and other copyrights may apply.

Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was supported by Small Business Innovation Research (SBIR) Program of the US Department of Energy, Office of Science through Cooperative Research and Development Agreement (CRADA) FP00003594 between the Regents of the University of California/Lawrence Berkeley National Laboratory and Technology Holding LLC. This work was part of the DOE Joint BioEnergy Institute (http://www.jbei.org) supported by the US Department of Energy, Office of Science, Office of Biological and Environmental Research, through Contract DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the US Department of Energy. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article, or the decision to submit it for publication.