Abstract The production of bio-based chemicals and fuels through microbial engineering offers a promising and sustainable alternative to petroleum-based fuels and chemicals, with the potential for scalability. However, engineering microbes and continuously evolving them to enhance the production of industrially relevant products is a complex and challenging task, requiring precise selection of genetic traits to achieve desired outcomes. In this study, we report the development of a novel counter-selectable growth-sensitive malonyl-CoA platform strain by coupling the malonyl-CoA repressor FapR from Bacillus subtilis to essential gene promoters involved in glucose growth and the plasma membrane arginine permease. This platform strain was then coupled with a CRISPR-dCas9 guide-RNA (gRNA) library, which after multiple rounds of dilutions and library sequencing, resulted in the enrichment for gRNAs that increased fluxes towards malonyl-CoA. The enriched gRNAs were validated for their effects on growth enhancement, gene regulation, and the production of an industrially relevant malonyl-CoA product, namely 3-hydroxypropionic acid. This study highlights an innovative approach to microbial engineering and opens up avenues for further exploration in the field of laboratory continuous evolution.

Biosensor-Assisted Laboratory Evolution of Malonyl-CoA production inSaccharomyces cerevisiae