Ethanol production improvement driven by genome-scale metabolic modeling and sensitivity analysis in Scheffersomyces stipitis

Abstract

The yeast Scheffersomyces stipitis naturally produces ethanol from xylose, however reaching high ethanol yields is strongly dependent on aeration conditions. It has been reported that changes in the availability of NAD(H/+) cofactors can improve fermentation in some microorganisms. In this work genome-scale metabolic modeling and phenotypic phase plane analysis were used to characterize metabolic response on a range of uptake rates. Sensitivity analysis was used to assess the effect of ARC on ethanol production indicating that modifying ARC by inhibiting the respiratory chain ethanol production can be improved. It was shown experimentally in batch culture using Rotenone as an inhibitor of the mitochondrial NADH dehydrogenase complex I (CINADH), increasing ethanol yield by 18%. Furthermore, trajectories for uptakes rates, specific productivity and specific growth rate were determined by modeling the batch culture, to calculate ARC associated to the addition of CINADH inhibitor. Results showed that the increment in ethanol production via respiratory inhibition is due to excess in ARC, which generates an increase in ethanol production. Thus ethanol production improvement could be predicted by a change in ARC.

Elucidating redox balance shift in Scheffersomyces stipitis’ fermentative metabolism using a modified genome-scale metabolic model

“…The reduced fluxes through both NADH shuttles were confirmed by the transcriptomic data as genes associated with both NADH shuttles show reduced expression levels. It is worth noting that it has been reported inhibiting NADHDH shuttle can increase ethanol production as suggested by the SID-based analysis; however, the inhibition only resulted in 18% of increase in ethanol production, and such less than expected increase can be explained by the operation of the GLYC3P shuttle, which was not considered in [ 29 ].…”

Metabolic Engineering of Bacterial Respiration: High vs. Low P/O and the Case of Zymomonas mobilis

“…Decreasing of respiration may be achieved by respiratory inhibitors, keeping in mind that inhibitor effects may serve as a proof of principle, yet are hardly applicable to industrial bioprocesses. Thus, Acevedo et al (2017), using rotenone, the inhibitor of the mitochondrial NADH dehydrogenase complex I, demonstrated an increase of aerobic ethanol yield from xylose in the yeast Scheffersomyces stipitis . Cyanide at submillimolar concentrations was shown to improve growth and ethanol yield in aerobic culture of ethanologenic bacterium Zymomonas mobilis (Kalnenieks et al, 2000).…”

“…It may complicate, for example, long-term directed evolution experiments, like those aimed at engineering of the E. coli fermentative metabolism (Portnoy et al, 2008), where complete absence of oxygen is essential. On the other hand, many yeast species performing ethanol fermentation require limited presence of oxygen (Visser et al, 1990), often within a certain narrow concentration range for optimum ethanol yield (Acevedo et al, 2017). From the technical point of view, keeping low, steady, micro-aerobic oxygen levels in bioreactors (especially for larger volumes) may be difficult, due to the sensitivity of oxygen mass transfer coefficient to the agitation rate, insufficient responsiveness of the dissolved oxygen probe and spatial in homogeneities (Wu et al, 2015a).…”

Reconstruction and analysis of genome-scale metabolic model of weak Crabtree positive yeast Lachancea kluyveri

“…GEMs have been used to analyze disease phenotypes and improvise the production of chemicals from cell factories. Fungal GEMs have been widely used for metabolic engineering efforts to overproduce target metabolites like fuels and chemicals [20,21,14,13]. As an obvious target, S. cerevisiae's GEM was constructed iteratively due to its immense industrial and academic importance.…”