Biosensor-Coupled In Vivo Mutagenesis and Omics Analysis Reveals Reduced Lysine and Arginine Synthesis To Improve Malonyl-Coenzyme A Flux in Saccharomyces cerevisiae

Abstract

Malonyl-CoA is a key precursor for the production a variety of value-added chemicals. Although rational engineering has been performed to improve the synthesis of malonyl-CoA in S. cerevisiae , due to the complexity of the metabolism there is a need for evolving strains and analyzing new mechanism to improve malonyl-CoA flux.

CRISPR-mediated protein-tagging signal amplification systems for efficient transcriptional activation and repression inSaccharomyces cerevisiae

“…In addition to ACC1 , FAS1 and FAS2 , we also performed simultaneous regulation ARG3 gene. Arg3 is associated with the biosynthesis of the arginine precursor citrulline, and our recent study has reported that the deletion of ARG3 increased the accumulation of 3-HP ( 46 ). The results showed that combing FAS1 , FAS2 and ARG3 downregulation and ACC1 upregulation, the production of 3-HP was further increased by 16%, demonstrated the advantage of our CRISPR-based system ( Supplementary Figure S7 ).…”

Dynamic-tuning yeast storage carbohydrate improves the production of acetyl-CoA-derived chemicals

“…This result is consistent with our previous work, in which we found that PGM3 deletion increases 3-HP production and decreases glycogen accumulation. 18 However, in contrast to overexpressing GDB1 , UGP1 overexpression increases glycogen and trehalose accumulation and improves the 3-HP production. The 3-HP titer is found to be even higher than that in the GDB1 -overexpressing strain.…”

“…On the contrary, in situ mutations of these genes ( UGP1 I484M , UGP1 P386S , LAT1 T47A , and GDB1 I947T ) in the genome did not significantly increase 3-HP production. 18 Taken together, it can be concluded that the expression level, not the mutation, of these genes affects the malonyl-CoA flux.…”

“…The commonly changed genes with single nucleotide polymorphisms (SNPs) and insertions-deletions (InDels) were identified by whole-genome sequencing. 18 Here, we overexpressed the commonly changed genes with SNPs in the control strain and then determined the production of a malonyl-CoA downstream chemical 3-HP by expression of malonyl-CoA reductase (MCR) in these strains. Interestingly, we found that overexpression of the mutant versions of UGP1 , LAT1 , and GDB1 increases the 3-HP production by 40%–50% ( Figure 1 B).…”

“…In our previous study, we isolated strains with improved malonyl-CoA synthesis using biosensor-coupled in vivo mutagenesis. 18 Through whole-genome sequencing and transcriptome analysis, we found that reducing lysine and arginine synthesis and carbohydrate storage synthesis can improve malonyl-CoA availability. This demonstrates that weakening the carbon flux to other competitive metabolic pathways (such as glycogen or lysine and arginine) can improve malonyl-CoA synthesis.…”