In-situ generation of large numbers of genetic combinations for metabolic reprogramming via CRISPR-guided base editing

Abstract

Reprogramming complex cellular metabolism requires simultaneous regulation of multigene expression. Ex-situ cloning-based methods are commonly used, but the target gene number and combinatorial library size are severely limited by cloning and transformation efficiencies. In-situ methods such as multiplex automated genome engineering (MAGE) depends on high-efficiency transformation and incorporation of heterologous DNA donors, which are limited to few microorganisms. Here, we describe a Base Editor-Targeted and Template-free Expression Regulation (BETTER) method for simultaneously diversifying multigene expression. BETTER repurposes CRISPR-guided base editors and in-situ generates large numbers of genetic combinations of diverse ribosome binding sites, 5’ untranslated regions, or promoters, without library construction, transformation, and incorporation of DNA donors. We apply BETTER to simultaneously regulate expression of up to ten genes in industrial and model microorganisms Corynebacterium glutamicum and Bacillus subtilis. Variants with improved xylose catabolism, glycerol catabolism, or lycopene biosynthesis are respectively obtained. This technology will be useful for large-scale fine-tuning of multigene expression in both genetically tractable and intractable microorganisms.

Application of Corynebacterium glutamicum engineering display system in three generations of biorefinery

“…C. glutamicum still lacks an in-depth understanding of the basic regulatory principles for the synthesis of central metabolic enzymes under different environmental conditions and their effects on cell growth [ 117 ]. In recent years, research on the catalysis and regulation mechanism [ 118 – 121 ] and resistance of aromatic compounds [ 122 ], genome simplification, optimization of gene editing tools [ 123 , 124 ], and research on ribosomal switches [ 125 ] and biosensors [ 126 ] have gradually increased. With the developments in metabolic engineering, systems biology, and synthetic biology, C. glutamicum will hopefully become a promising and advantageous biorefinery factory.…”

Advances in promoter engineering: Novel applications and predefined transcriptional control

“…Recently, base editors were implemented for promoter engineering in the model industrial bacterial hosts, Corynebacterium glutamicum and Bacillus subtilis . [ 74 ] In this study, a short, weak RBS was placed within the promoters for genes/enzymes that catalyze sugar utilization or lycopene production metabolic pathways. Because of the initial RBS sequence was the same, the dCas9‐base editor targeted up to 10 RBSs simultaneously, creating stronger RBS mutants that tuned promoters in‐situ, maximizing the growth rate and utilization of certain molecules, such as glycerol.…”

Halomonasas a chassis

“…Most importantly, optimization of genetic parts, promoter, RBS [49] and tools, promoter library construction [92], inducible systems [93], effective CRISPR-based gene editing approach [94] and alternative expression vectors, have been demonstrated to strongly improve the programmability of Halomonas. Screening on chromosomal locus with low expression variance can provide applicable genomic sites for efficient integrations of multiple expression modules [95].…”