Metabolic pathway analysis of Scheffersomyces (Pichia) stipitis: effect of oxygen availability on ethanol synthesis and flux distributions

Abstract

Elementary mode analysis (EMA) identifies all possible metabolic states of the cell metabolic network. Investigation of these states can provide a detailed insight into the underlying metabolism in the cell. In this study, the flux states of Scheffersomyces (Pichia) stipitis metabolism were examined. It was shown that increasing oxygen levels led to a decrease of ethanol synthesis. This trend was confirmed by experimental evaluation of S. stipitis in glucose-xylose fermentation. The oxygen transfer rate for an optimal ethanol production was 1.8 mmol/l/h, which gave the ethanol yield of 0.40 g/g and the ethanol productivity of 0.25 g/l/h. For a better understanding of the cell's regulatory mechanism in response to oxygenation levels, EMA was used to examine metabolic flux patterns under different oxygen levels. Up- and downregulation of enzymes in the network during the change of culturing condition from oxygen limitation to oxygen sufficiency were identified. The results indicated the flexibility of S. stipitis metabolism to cope with oxygen availability. In addition, relevant genetic targets towards improved ethanol-producing strains under all oxygenation levels were identified. These targeted genes limited the metabolic functionality of the cell to function according to the most efficient ethanol synthesis pathways. The presented approach is promising and can contribute to the development of culture optimization and strain engineers for improved lignocellulosic ethanol production by S. stipitis.

A constraint-based model of Scheffersomyces stipitis for improved ethanol production

“…It was found that (i) the glycolytic flux wasn’t obviously affected by the oxygen levels by either glucose or xylose, but the flux channelled into tricarboxylic acid cycle (TCA cycle) increased about twofold when the cells were shifted from semiaerobic to aerobic, which were consistent with the reported results that genes involved in TCA cycle were downregulated as oxygen availability decreased [33]; (ii) at the node of pyruvate, the flux to acetaldehyde (leading to fermentation) increased about tenfold with the decreased oxygen level, which was in accordance with the experiment that the expression of key fermentative genes ( PDC , ALD and ADH ) for ethanol production were greatly improved under the oxygen-limited condition [34]. Meanwhile, the effects of different carbon sources (xylose and glucose) on the flux distribution of central metabolism were also analysed.…”

Reconstruction and analysis of a genome-scale metabolic network of Corynebacterium glutamicum S9114

“…We observed that: (1) Flux through the glycolysis pathway was almost unchanged with a decreased oxygen level but flux into the TCA cycle decreased nearly threefold. This result was in accordance with the finding that genes involved in the TCA cycle are downregulated as oxygen level decreases (Unrean and Nguyen, 2012). (2) The intracellular PYR pool was enhanced, resulting in increased M A N U S C R I P T…”

Effects of aeration on growth, ethanol and polyol accumulation by Spathaspora passalidarum NRRL Y‐27907 and Scheffersomyces stipitis NRRL Y‐7124

“…(Silva et al, ). Unrean and Nguyen () used an elementary mode analysis to predict that an OTR of 1.8 mmol/L h was optimal for S. stipitis in a 1‐L fermentor containing 15 g/L of glucose and 5 g/L of xylose. Slininger et al () suggested that S. stipitis ethanol production is growth associated and that ethanol production can occur even when the growth rate is as high as 0.5 h −1 ; the requisite dissolved oxygen necessary to support that rate of growth would be at least 1–3 mg O 2 /L (or 31–93 µmole O 2 /L) at 25°C.…”