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.
Highlights
Reprogramming complex cellular metabolism requires simultaneous regulation of multigene expression
Base Editor-Targeted and Template-free Expression Regulation (BETTER) was first used to diversify the expression of a green fluorescence protein (GFP) reporter in C. glutamicum, a major workhorse in industrial biotechnology used for the production of approximately 70 natural and nonnatural compounds[20]
The sequencing data revealed an editing bias toward the five Gs distal to protospacer-adjacent motif (PAM) (Fig. 1b), which is consistent with the reported five-base editing window of cytidine base editors[13]
Summary
Reprogramming complex cellular metabolism requires simultaneous regulation of multigene expression. In order to obtain optimal yield and productivity, the expression of pathway genes must be appropriately balanced to avoid metabolic bottlenecks or burdens[2,3] Cloning methods such as Gibson assembly[4] allow ex situ construction of combinatorial libraries for multiple genes controlled by diverse promoters or RBSs, the reported maximum number of target genes is limited to five with a maximum pool size of 3,125 (55, five genes × five promoters)[5]. Synthesis of complex DNA template pools, expression of multiple guide-RNAs (gRNAs), and cellular toxicity of DSBs limit the number of targetable genes These limitations make the existing methods only viable for a limited number of microorganisms with high DNA transformation and incorporation efficiencies, namely Escherichia coli[2,6,8,9] and Saccharomyces cerevisiae[3,5,7]. Expression of up to ten genes is simultaneously tuned for enhanced substrate uptake or natural product biosynthesis
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