Abstract
BackgroundFermentation of xylose to ethanol has been achieved in S. cerevisiae by genetic engineering. Xylose utilization is however slow compared to glucose, and during anaerobic conditions addition of glucose has been necessary for cellular growth. In the current study, the xylose-utilizing strain TMB 3415 was employed to investigate differences between anaerobic utilization of glucose and xylose. This strain carried a xylose reductase (XYL1 K270R) engineered for increased NADH utilization and was capable of sustained anaerobic growth on xylose as sole carbon source. Metabolic and transcriptional characterization could thus for the first time be performed without addition of a co-substrate or oxygen.ResultsAnalysis of metabolic fluxes showed that although the specific ethanol productivity was an order of magnitude lower on xylose than on glucose, product yields were similar for the two substrates. In addition, transcription analysis identified clear regulatory differences between glucose and xylose. Respiro-fermentative metabolism on glucose during aerobic conditions caused repression of cellular respiration, while metabolism on xylose under the same conditions was fully respiratory. During anaerobic conditions, xylose repressed respiratory pathways, although notably more weakly than glucose. It was also observed that anaerobic xylose growth caused up-regulation of the oxidative pentose phosphate pathway and gluconeogenesis, which may be driven by an increased demand for NADPH during anaerobic xylose catabolism.ConclusionCo-factor imbalance in the initial twp steps of xylose utilization may reduce ethanol productivity by increasing the need for NADP+ reduction and consequently increase reverse flux in glycolysis.
Highlights
Production of fuel ethanol has increased several fold during the last decade due to increasing oil prices and environmental concerns [1]
Aerobic and anaerobic batch cultivation TMB 3415 was cultivated in synthetic medium containing either glucose or xylose as the sole carbon source, under aerobic and anaerobic conditions
Balanced exponential cell growth was seen for glucose and xylose under both aerobic and anaerobic conditions, allowing for the assumption of pseudo steadystate conditions [24]
Summary
Production of fuel ethanol has increased several fold during the last decade due to increasing oil prices and environmental concerns [1]. Poor xylose utilization has been ascribed to potentially rate-controlling metabolic steps including: low substrate affinity of the recombinant enzymes [8]; cofactor imbalance in the XR-XDH reactions [7,14]; low xylose transport capacity [15,16]; and failure to recognize xylose as a fermentable carbon source [17,18]. Transcriptional characterization of anaerobic xylose metabolism has remained elusive, regardless of the importance of this particular condition in a production setting. The xylose-utilizing strain TMB 3415 was employed to investigate differences between anaerobic utilization of glucose and xylose. This strain carried a xylose reductase (XYL1 K270R) engineered for increased NADH utilization and was capable of sustained anaerobic growth on xylose as sole carbon source. Metabolic and transcriptional characterization could for the first time be performed without addition of a co-substrate or oxygen
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