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

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.

Integration of genome-scale metabolic model with biorefinery process model reveals market-competitive carbon-negative sustainable aviation fuel utilizing microbial cell mass lipids and biogenic CO2

“…The majority of reported titers, rates, and yields of terpenes from bench-scale studies (Table A1) are below 5 g/L, 0.15 g/L/h, and 35% of the theoretical yield, primarily utilizing glucose as the sole carbon source. Isoprenol exhibits a range of titer, rate, and yield, spanning from 8.5 to 12.4 g/L, 0.1 to 0.15 g/L/h, and 34 to 44 % of the maximum theoretical yield (Wang et al 2022a;Kim and Lee 2023;Kang et al 2019). However, even if the theoretical yield is achieved, approximately 60 to 70% of starting carbon material is not converted to the desired molecule.…”