Engineering and Evolution of Methanol Assimilation in Saccharomyces cerevisiae

Microbial 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 yeast Saccharomyces 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 in S. cerevisiae, which was optimized using Adaptive Laboratory Evolution. Three independent lineages selected in liquid methanol-yeast extract medium evolved premature stop codons in YGR067C, which encodes an uncharacterised protein that has a predicted DNA-binding domain with homology to the ADR1 transcriptional regulator. Adr1p regulates genes involved in ethanol metabolism and peroxisomal proliferation, suggesting YGR067C has a related function. When one of the evolved YGR067C mutations was reverse engineered into the parental CEN.PK113-5D strain, there were up to 5-fold increases in 13C-labelling of intracellular metabolites from 13C-labelled methanol when 0.1 % yeast extract was a co-substrate, and a 44 % increase in final biomass. Transcriptomics and proteomics revealed that the reconstructed YGR067C mutation 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 in S. cerevisiae and pave the way for further development of native and synthetic one-carbon assimilation pathways in this model eukaryote.

Anaerobic biodegradation of 2,4,6-trichlorophenol by methanogenic granular sludge: role of co-substrates and methanogenic inhibition

The influence of several co-substrates in the anaerobic biodegradation of 2,4,6-trichlorophenol (246TCP) by methanogenic granular sludge as well as in methanogenesis inhibition by 246TCP has been studied. 4 g-COD·L−1 of lactate, sucrose, volatile fatty acids (VFA) acetate:propionate:butyrate 1:1:1, ethanol, methanol, yeast extract (YE), and 2 g-COD·L−1 of formate and methylamine were tested. Two concentrations of 246TCP: 80 mg·L−1 and 113 mg·L−1 (this last corresponding to the EC50 for acetotrophic methanogenesis) were tested. Three consecutive co-substrate and nutrient feedings were accomplished. 246TCP was added in the second feed, and the 246TCP removal rate increased considerably after the third feed. Accumulated metabolites after ortho-dechlorination, either 4-chlorophenol (4CP) (when methanol, ethanol or VFA were used as co-substrates) or 2,4-dichlorophenol (24DCP) (with lactate) avoided the complete dechlorination of 246TCP. With methylamine and formate this compound was degraded only partially. Monochlorophenols biodegradation was partially achieved with YE, but both 24DCP and 2,6-dichlorophenol (26DCP) were accumulated. In the presence of sucrose para-dechlorination was observed. 246TCP was better tolerated by methanogens when ethanol and methanol were added because of the highest specific methanogenic activity achieved with these co-substrates. Methanol and ethanol were the best co-substrates in the anaerobic biodegradation of 246TCP.