Generating lower ethanol yields in fermentations by Saccharomyces cerevisiae via diversion of carbon flux towards the production of fructo-oligosaccharides

[摘要] ENGLISH ABSTRACT: There is a growing international consumer demand for the production of lower ethanol wines.This can be attributed to various qualitative, social, economic and health concerns that areassociated with high ethanol wines (Kutyna et al., 2010; Varela et al., 2012). There iscontinuous development and research into methods and technologies to lower the ethanolconcentration in wine. However, in addition to the added cost and complexity these technologiesall have various shortcomings. The development of yeast strains with lower ethanol productivity,yet desirable organoleptic and fermentation capacity, therefore remains a highly sought afterresearch and development target in the wine industry.Biologically based approaches aim to generate yeast strains with the capacity to divertcarbon from ethanol production towards targeted metabolic endpoints (Kutyna et al., 2010). Thisshould ultimately be achieved without the production of unwanted metabolites that cannegatively affect wine characteristics. In the context of these challenges, this study aimed toinvestigate the use of fructans as carbon sinks during fermentation to divert fructose fromglycolysis and ethanol production toward intracellular fructan production by generating levanproducing strains. In addition, the impact of fructan production on metabolic carbon flux duringfermentation by these strains was analyzed. This was the first attempt to analyze intracellularfructan production in Saccharomyces cerevisiae under fermentative conditions with fructansacting as carbon sinks.Fructans are fructose polymers that act as storage molecules in certain plants andfunction as part of the extracellular matrix in microbial biofilms, and are intensively studied dueto their economic interest. Here we undertook the heterologous expression of a levansucrase(LS) M1FT from Leuconostoc mesenteroides, an enzyme producing β(2-6) levan-type fructans,in the S. cerevisiae BY4742Δsuc2 strains without invertase activity (encoded by SUC2).Levansucrases indeed utilize sucrose as both fructose donor and initial polymerizationsubstrate, and the sucrose concentration is of import to maintain transfructosylation activity ofenzyme. High intracellular sucrose accumulation was achieved by the heterologous expressionof either a sucrose synthase (Susy; cloned from potato) or by growing strains expressing thespinach sucrose transporter (SUT) in sucrose containing media. Endogenous sucrose synthesiswas of specific interest to the overall goal of the project, which was to reroute carbon flux awayfrom glycolysis in grape must containing only hexoses as carbon source. In addition, thisapproach of combining intracellular sucrose production with intracellular levan production couldbe used in various applications to limit the need for sucrose in media as both carbon source and LS substrate. The extracellular LS M1FT was introduced into Susy and SUT strains as either the completegene (M1FT) or 50bp truncation (M1FTΔsp) without the predicted signal peptide. The data showthat intracellular levan accumulation occurred in aerobic, but not anaerobic conditions. The dataalso suggest that the production of levan did not impact negatively on general yeast physiologyor metabolism in these conditions. However, no significant reduction in ethanol yields wereobserved, suggesting that further optimisation of the expression system is required. This is thefirst report of levan synthesis by S. cerevisiae, and contributes towards expanding thepossibilities for further industrial applications of these compounds.

[发布机构] Stellenbosch University