Engineering and Evolution of Methanol Assimilation inSaccharomyces cerevisiae

Abstract

AbstractMicrobial fermentation for chemical production is becoming more broadly adopted as an alternative to petrochemical refining. Fermentation typically relies on sugar as a feedstock, however, one-carbon compounds like methanol are an attractive alternative as they can be derived from organic waste and natural gas. This study focused on engineering methanol assimilation in the yeastSaccharomyces cerevisiae.Three methanol assimilation pathways were engineered and tested: a synthetic xylulose monophosphate (XuMP), a ‘hybrid’ methanol dehydrogenase-XuMP, and a bacterial ribulose monophosphate (RuMP) pathway, with the latter identified as the most effective at assimilating methanol. Additionally,13C-methanol tracer analysis uncovered a native capacity for methanol assimilation inS. cerevisiae, which was optimized using Adaptive Laboratory Evolution. Three independent lineages selected in liquid methanol-yeast extract medium evolved premature stop codons inYGR067C, which encodes an uncharacterised protein that has a predicted DNA-binding domain with homology to theADR1transcriptional regulator. Adr1p regulates genes involved in ethanol metabolism and peroxisomal proliferation, suggestingYGR067Chas a related function. When one of the evolvedYGR067Cmutations was reverse engineered into the parental CEN.PK113-5D strain, there were up to 5-fold increases in13C-labelling of intracellular metabolites from13C-labelled methanol when 0.1 % yeast extract was a co-substrate, and a 44 % increase in final biomass. Transcriptomics and proteomics revealed that the reconstructedYGR067Cmutation results in down-regulation of genes in the TCA cycle, glyoxylate cycle, and gluconeogenesis, which would normally be up-regulated during growth on a non-fermentable carbon source. Combining the synthetic RuMP and XuMP pathways with the reconstructed Ygr067cp truncation led to further improvements in growth. These results identify a latent methylotrophic metabolism inS. cerevisiaeand pave the way for further development of native and synthetic one-carbon assimilation pathways in this model eukaryote.