Engineering a CRISPRi Circuit for Autonomous Control of Metabolic Flux in Escherichia coli

Abstract

Building autonomous switches is an effective approach for rewiring metabolic flux during microbial synthesis of chemicals. However, current autonomous switches largely rely on metabolite-responsive biosensors or quorum-sensing circuits. In this study, a stationary phase promoter (SPP) and a protein degradation tag (PDT) were combined with the CRISPR interference (CRISPRi) system to construct an autonomous repression system that could shut down multiple-gene expression depending on the cellular physiological state. With this autonomous CRISPRi system to regulate one target gene, a fermenter-scale titer of shikimic acid reached 21 g/L, which was the highest titer ever reported by Escherichia coli in a minimal medium without any chemical inducers. With three target genes repressed, 26 g/L glutaric acid could be achieved with decreased byproduct accumulation. These results highlight the applicability of the autonomous CRISPRi system for microbial production of value-added chemicals.

Enhancement of acyl‐CoA precursor supply for increased avermectin B1a production by engineering meilingmycin polyketide synthase and key primary metabolic pathway genes

“…Each of the key nodes we selected is involved in an essential cellular pathway, and inhibition of the above genes may therefore result in cell growth arrest. Gao et al (2021) constructed a CRISPRi circuit for autonomous control of metabolic flux in E. coli by designing sgRNA expressed by a stationary phase promoter. To avoid CRISPRi-mediated inhibition of selected key nodes affecting cell growth, we used S. avermitilis native temporal promoter pkn5p (active mainly in middle and late fermentation stages) (Hao et al, 2022) to regulate dcas9 expression.…”

Microbial cell factories based on filamentous bacteria, yeasts, and fungi

“…Furthermore, a modular synthetic biology toolkit can be built for filamentous fungi microorgnaisms, which can be more rapidly assembled in a standardized and modular manner [ 106 , 120 ]. Intelligent manipulations are mainly based on the new metabolic engineering tools for achieving the autonomous dynamic regulation for the synthesis of chemicals, such as N-acetylglucosamine [ 121 ], glucaric acid [ 122 ], shikimate [ 123 ], and other high-value compounds [ 124 ]. Sufficient understanding of microbial physiology, organelles, cell morphology, and artificial consortia can allow us to design artificial novel functions to regulate the pathway optimization and cell–cell communication [ 125 – 128 ].…”

“…Furthermore, a modular synthetic biology toolkit can be built for filamentous fungi microorgnaisms, which can be more rapidly assembled in a standardized and modular manner [ 106 , 120 ]. Intelligent manipulations are mainly based on the new metabolic engineering tools for achieving the autonomous dynamic regulation for the synthesis of chemicals, such as N-acetylglucosamine [ 121 ], glucaric acid [ 122 ], shikimate [ 123 ], and other high-value compounds [ 124 ].…”

Engineered Artificial Membraneless Organelles in Saccharomyces cerevisiae To Enhance Chemical Production

“…Currently, strategies to switch metabolic pathways have focused on optimizing key gene expression levels, introducing optogenetic circuits, developing biosensors, and controlling protein degradation. [28,33] In this study, the design of MLO complemented existing strategies. Next, to expand the bioconversion of acetyl-coA for improving the synthesis of high value added chemicals in yeast, the MORS-rigidity and GATHCYC pathway were introduced into n-butanol producing strain LH002-B4 [32] to generate strain ZP04-RB (Figure 7f).…”