An evaluation of cellulose saccharification and fermentation with an engineered Saccharomyces cerevisiae capable of cellobiose and xylose utilization

Abstract

Commercial-scale cellulosic ethanol production has been hindered by high costs associated with cellulose-to-glucose conversion and hexose and pentose co-fermentation. Simultaneous saccharification and fermentation (SSF) with a yeast strain capable of xylose and cellobiose co-utilization has been proposed as a possible avenue to reduce these costs. The recently developed DA24-16 strain of Saccharomyces cerevisiae incorporates a xylose assimilation pathway and a cellodextrin transporter (CDT) that permit rapid growth on xylose and cellobiose. In the current work, a mechanistic kinetic model of cellulase-catalyzed hydrolysis of cellulose was combined with a multi-substrate model of microbial growth to investigate the ability of DA24-16 and improved cellobiose-consuming strains to obviate the need for exogenously added β-glucosidase and to assess the impact of cellobiose utilization on SSF and separate hydrolysis and fermentation (SHF). Results indicate that improved CDT-containing strains capable of growing on cellobiose as rapidly as on glucose produced ethanol nearly as rapidly as non-CDT-containing yeast supplemented with β-glucosidase. In producing 75 g/L ethanol, SSF with any strain did not result in shorter residence times than SHF with a 12 h saccharification step. Strains with improved cellobiose utilization are therefore unlikely to allow higher titers to be reached more quickly in SSF than in SHF.

Model‐guided strain improvement: Simultaneous hydrolysis and co‐fermentation of cellulosic sugars

“…Fox et al [4] simulated various SSF and separate hydrolysis and fermentation (SHF) conditions with a large number of empirical parameters. Using an extremely high Monod constant (K cellobiose = 350 mM, ~120 g cellobiose/L) for growth on cellobiose and a low starting inoculum of yeast cells (OD600 ~1.0), the model predicted that the co-fermenting strain of yeast produces ethanol more slowly from a mixture of cellulose and xylose than a control case where β-glucosidase was added extracellularly to a strain exhibiting the same glucose and xylose fermenting capability (Fig.…”

“…In order for co-fermenting strains that utilize intracellular β-glucosidase to be competitive, it will be necessary to reduce the Monod constant for growth on cellobiose. While the mechanistic model-based predictions of Fox et al [4] provide a quantitative means for understanding the dynamics of cellulose hydrolysis and fermentation in SSF scenarios and for improving yeast strains for commercial processes, there are always difficulties in extrapolating from the near ideal conditions used to derive the empirical parameters to the unknowns in a heterogeneous (solid/ liquid) and multi-component (cellulose, cellobiose, enzymes, yeast cells, and ethanol) system. For example, cellulases likely do not behave as modeled in high cellulose loadings (>10% weight/volume) due to unknown mechanisms that result in lower hydrolysis yields [5].…”

“…Additionally, the effect of ethanol inhibition on β-glucosidase activity needs to be evaluated. The model developed by Fox et al [4] will now allow for a "virtuous cycle" of comparing experimental results to a quantitative model and updating the parameters used. It will demonstrate its greatest benefit as the parameter values are further refined to reflect new empirical measurements of the performance of engineered strains and proposed processes for cellulosic ethanol.…”

“…Fox et al [4], in this issue of Biotechnology Journal, report a compreAs endo and exo-type cellulase enzymes are strongly inhibited by intermediate (cellodextin) and final products (glucose) of the hydrolysis reaction, efficient and timely removal of these inhibitory products via fermentation in simultaneous saccharification and fermentation (SSF) is a promising process for producing cellulosic ethanol. Historically, the crucial enzyme in cellulase cocktails that generate glucose from cellodextrins, β-glucosidase, must be supplemented due to relatively weak β-glucosidase activities of most fungal cellulase mixtures, and the fact that Saccharomyces cerevisiae, the workhorse yeast for ethanol production, cannot use cellodextrins such as cellobiose as a carbon source, but is superb at fermenting glucose.…”

Produção De Etanol Celulósico Por Sacarificação E Fermentação Simultâneas Da Biomassa De Palma Forrageira Pré-Tratada Em Meio Alcalino

“…Esta estratégia reduz o potencial de contaminação microbiológica, diminui os custos de equipamento e previne a ação enzimática de ser inibida pelos açúcares produzidos, aumentando assim o rendimento desta etapa (Fox et al, 2012).…”