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

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.