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Model-Assisted Fine-Tuning of Central Carbon Metabolism in Yeast through dCas9-Based Regulation

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journal contribution
posted on 14.10.2019, 12:33 by Raphael Ferreira, Christos Skrekas, Alex Hedin, Benjamín J. Sánchez, Verena Siewers, Jens Nielsen, Florian David
Engineering Saccharomyces cerevisiae for industrial-scale production of valuable chemicals involves extensive modulation of its metabolism. Here, we identified novel gene expression fine-tuning set-ups to enhance endogenous metabolic fluxes toward increasing levels of acetyl-CoA and malonyl-CoA. dCas9-based transcriptional regulation was combined together with a malonyl-CoA responsive intracellular biosensor to select for beneficial set-ups. The candidate genes for screening were predicted using a genome-scale metabolic model, and a gRNA library targeting a total of 168 selected genes was designed. After multiple rounds of fluorescence-activated cell sorting and library sequencing, the gRNAs that were functional and increased flux toward malonyl-CoA were assessed for their efficiency to enhance 3-hydroxypropionic acid (3-HP) production. 3-HP production was significantly improved upon fine-tuning genes involved in providing malonyl-CoA precursors, cofactor supply, as well as chromatin remodeling.

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