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bioRxiv · 10.1101/2023.07.16.549225

Biosensor-Assisted Laboratory Evolution of Malonyl-CoA production in Saccharomyces cerevisiae

Ferreira, R.·Hedin, K. A.·Nielsen, J.·David, F.
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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.

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Ferreira, R., Hedin, K. A., Nielsen, J., David, F.. 2023-07-17. Biosensor-Assisted Laboratory Evolution of Malonyl-CoA production in Saccharomyces cerevisiae. https://doi.org/10.1101/2023.07.16.549225

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