Using high-throughput data and dynamic flux balance modeling techniques to identify points of constraint in xylose utilization in Saccharomyces cerevisiae

Abstract

Background: Several enzymes and cofactors have been identified as contributing to the slow utilization of xylose by xylose-fermenting strains of Saccharomyces cerevisiae. However, there has been no consensus on which of these possible bottlenecks are the most important to address. A previous strain characterization study from our lab suggested that insufficient NAD+ limits fermentation and may be the most important bottleneck affecting utilization of xylose for the production of ethanol. The development and validation of a genome scale dynamic flux balance model would help to verify the existence and extent of this and other metabolic bottlenecks and suggest solutions to guide future strain development thereby minimizing bottleneck impact on process economics. Results: A dynamic flux balance model was developed to identify bottlenecks in several strains of S. cerevisiae, both with wild-type pentose phosphate pathway expression and with the pathway over expressed. ZWF1 was found to be limiting in the oxidative portion of the pentose phosphate pathway under oxygen replete conditions. This pathway is used to regenerate NADPH. Under oxygen limiting conditions, respiration of xylose was limited by the lack of oxygen as a terminal electron acceptor. Ethanol production was also limited under these conditions due to the inability to balance NAD+/NADH. The model suggests the use of the anaplerotic glyoxylate pathway to improve NAD+/NADH balance, increasing ethanol production by 50% while producing succinate as a coproduct at upwards of 20 g/l. Conclusion: In the production of high value chemicals from biomass, the use of the respiratory metabolism is a waste of feedstock carbon. Bottlenecks previously identified in the oxidative pentose phosphate pathway are currently only relevant under oxygen-replete conditions and cannot impact the partitioning of carbon between the respiratory and fermentative pathways. Focusing future efforts on the non-respiratory balancing of NAD+/NADH, perhaps through the glyoxylate pathway, would improve the economics of ethanol production both directly and through coproduct formation. Graphic abstract: [Figure not available: see fulltext.].