(766h) Rational Design of Metabolic Engineering Strategies for Xylose Metabolism With Scheffersomyces Stipitis and Saccharomyces Cerevisiae

Rational design of metabolic engineering strategies for xylose metabolism with Scheffersomyces stipitis and Saccharomyces cerevisiae

Meng Liang¹, Q. Peter He², Jin Wang¹

1. Department of Chemical Engineering, Auburn University
2. Department of Chemical Engineering, Tuskegee University

Lignocellulosic ethanol production represents an attractive alternative source for long-term renewable energy supply due to the rising concerns over energy sustainability, global warming and feed stock availability¹.  However, many barriers exist for industrializing lignocellulosic ethanol processes and one of them is the effective conversion of xylose, the second abundant mono-saccharide and representative pentose in the hydrolysate of lignocellulosic biomass.

Saccharomyces cerevisiae is the most promising candidate for lignocellulosic ethanol production due to its excellent glucose fermentation capability, high ethanol tolerance, and resistance to inhibitors presented in lignocellulosic hydrolysate².  However, native S. cerevisiae strains cannot utilize xylose for either growth or ethanol production.  Despite of the inability to utilize xylose, S. cerevisiae has the metabolic pathway to convert xylulose, an isomerized product of xylose, into ethanol.  Thus, numerous studies have been carried out to link extracellular xylose with intracellular xylulose and to enhance the capacity of xylulose fermentation so as to further increase the efficiency of xylose fermentation³.

As the most promising native strain for xylose fermentation⁴, Scheffersomyces stipitis (previously named Pichia stipitis) has been chosen as one of the most common gene providers for recombinant S. cerevisiae.  Besides working as a gene provider, S. stipitis itself also shows good overall performance on lignocellulosic hydrolysate⁵.  Understanding its metabolism, especially the central carbon metabolism, is very important to improve the strain, or to provide hints for metabolic adjustment of other strains.  However, limited results have been published on the metabolic engineering of S. stipitis.

Redox couples NAD+/NADH and NADP+/NADPH have been reported to play an important role in energy metabolism and product formation of yeasts⁶.  For S. stipitis, it has been reported that its product distribution is very sensitive to oxygen transfer rate due to redox imbalance.  Meanwhile, it has been recognized that the heterologous expression of xylose reductase (XR) and xylitol dehydrogenase (XDH) in recombinant S. cerevisiae would cause perturbation to the intrinsic redox balance, which has been considered as one of the reasons of some recombinant S. cerevisiae’s subordinate performance on xylose fermentation⁷.  Therefore, understanding the mechanism of redox balance and tuning up the redox conditions by cofactor engineering or adjusting other NADH- or NADPH-related steps have significant impacts on ethanol production for xylose fermentation.

Besides the redox balance, there are other bottlenecks reported for recombinant S. cerevisiae ^{7, 8}, such as xylose transporter with low efficiency, insufficient capacity from xylulose to xylulose-5-P, limited flux of non-oxidative pentose phosphate pathway.  The metabolic changes caused by the heterologous expression of XR and XDH will propagate through the metabolic network and interact with other bottlenecks.  Therefore, the design of metabolic engineering strategy should be considered systematically.

In this work, we reconstructed the central carbon metabolic network model of S. stipitis and modified published genome-scale metabolic network model of S. cerevisiae to mimic real genetic modifications.  The two models were used to gain better understanding of xylose metabolism of the two strains, as well as to evaluate the various metabolic engineering strategies for xylose fermentation to ethanol and their influences to glucose fermentation. By analyzing the modeling results we propose the candidate strategies for optimizing ethanol production from xylose with the two strains.

Specifically, we first reconstructed the central carbon metabolic network model of S. stipitis by integrating genomic, biochemical and physiological information available for this microorganism and other related yeast.  Meanwhile a published genome-scale metabolic network model of S. cerevisiae was modified by adding additional reactions and constraints to simulate the recombinant S. cerevisiae with introduced XYL1 and XYL2 from S. stipitis.  Then we propose a system-identification based framework to characterize the xylose metabolism under various conditions, such as different oxygen conditions or varied cofactor preferences of the enzymes.  The reactions most influenced by introducing various perturbations, whose capacities would also affect the introduced perturbations most, as well as the desired regulations directions (e.g., depress or up-regulating reaction activity), have been identified.  The reactions chosen by other researchers as the targets of the metabolic engineering for xylose metabolism in recombinant S. cerevisiae have all been identified by the proposed approach.

With the reactions identified, the published metabolic engineering strategies were evaluated using the models through constraint-based FBA.  The modification, deletion, depression or overexpression of the genes have been simulated by applying hard constraints or flux ratio constraints.  The simulation predictions agreed well with the reported experimental results.  In addition, the influences of the strategies on glucose fermentation were evaluated as well.  Cofactor engineering by varying cofactor preferences of the enzymes through protein engineering has been characterized.  Based on these results, the optimal metabolic strategies for both strains have been proposed which predicted significantly increased ethanol yield compared to the previously published strategies.

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