Dataset Information

Increased expression of the oxidative pentose phosphate pathway and gluconeogenesis in anaerobically growing xylose-utilizing Saccharomyces cerevisiae.

ABSTRACT: BACKGROUND: Fermentation 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. RESULTS: Analysis 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. CONCLUSION: Co-factor imbalance in the initial two steps of xylose utilization may reduce ethanol productivity by increasing the need for NADP+ reduction and consequently increase reverse flux in glycolysis.

SUBMITTER: Runquist D

PROVIDER: S-EPMC2760498 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature

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Increased expression of the oxidative pentose phosphate pathway and gluconeogenesis in anaerobically growing xylose-utilizing Saccharomyces cerevisiae.

Runquist David D Hahn-Hägerdal Bärbel B Bettiga Maurizio M

Microbial cell factories 20090924

<h4>Background</h4>Fermentation 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 utiliz ...[more]

PMID: 19778438

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Project description:Background:Engineered strains of Saccharomyces cerevisiae have significantly improved the prospects of biorefinery by improving the bioconversion yields in lignocellulosic bioethanol production and expanding the product profiles to include advanced biofuels and chemicals. However, the lignocellulosic biorefinery concept has not been fully applied using engineered strains in which either xylose utilization or advanced biofuel/chemical production pathways have been upgraded separately. Specifically, high-performance xylose-fermenting strains have rarely been employed as advanced biofuel and chemical production platforms and require further engineering to expand their product profiles. Results:In this study, we refactored a high-performance xylose-fermenting S. cerevisiae that could potentially serve as a platform strain for advanced biofuels and biochemical production. Through combinatorial CRISPR-Cas9-mediated rational and evolutionary engineering, we obtained a newly refactored isomerase-based xylose-fermenting strain, XUSE, that demonstrated efficient conversion of xylose into ethanol with a high yield of 0.43 g/g. In addition, XUSE exhibited the simultaneous fermentation of glucose and xylose with negligible glucose inhibition, indicating the potential of this isomerase-based xylose-utilizing strain for lignocellulosic biorefinery. The genomic and transcriptomic analysis of XUSE revealed beneficial mutations and changes in gene expression that are responsible for the enhanced xylose fermentation performance of XUSE. Conclusions:In this study, we developed a high-performance xylose-fermenting S. cerevisiae strain, XUSE, with high ethanol yield and negligible glucose inhibition. Understanding the genomic and transcriptomic characteristics of XUSE revealed isomerase-based engineering strategies for improved xylose fermentation in S. cerevisiae. With high xylose fermentation performance and room for further engineering, XUSE could serve as a promising platform strain for lignocellulosic biorefinery.

Project description:BackgroundThe current shift from a fossil-resource based economy to a more sustainable, bio-based economy requires development of alternative production routes based on utilization of biomass for the many chemicals that are currently produced from petroleum. Muconic acid is an attractive platform chemical for the bio-based economy because it can be converted in chemicals with wide industrial applicability, such as adipic and terephthalic acid, and because its two double bonds offer great versatility for chemical modification.ResultsWe have constructed a yeast cell factory converting glucose and xylose into muconic acid without formation of ethanol. We consecutively eliminated feedback inhibition in the shikimate pathway, inserted the heterologous pathway for muconic acid biosynthesis from 3-dehydroshikimate (DHS) by co-expression of DHS dehydratase from P. anserina, protocatechuic acid (PCA) decarboxylase (PCAD) from K. pneumoniae and oxygen-consuming catechol 1,2-dioxygenase (CDO) from C. albicans, eliminated ethanol production by deletion of the three PDC genes and minimized PCA production by enhancing PCAD overexpression and production of its co-factor. The yeast pitching rate was increased to lower high biomass formation caused by the compulsory aerobic conditions. Maximal titers of 4 g/L, 4.5 g/L and 3.8 g/L muconic acid were reached with glucose, xylose, and a mixture, respectively. The use of an elevated initial sugar level, resulting in muconic acid titers above 2.5 g/L, caused stuck fermentations with incomplete utilization of the sugar. Application of polypropylene glycol 4000 (PPG) as solvent for in situ product removal during the fermentation shows that this is not due to toxicity by the muconic acid produced.ConclusionsThis work has developed an industrial yeast strain able to produce muconic acid from glucose and also with great efficiency from xylose, without any ethanol production, minimal production of PCA and reaching the highest titers in batch fermentation reported up to now. Utilization of higher sugar levels remained conspicuously incomplete. Since this was not due to product inhibition by muconic acid or to loss of viability, an unknown, possibly metabolic bottleneck apparently arises during muconic acid fermentation with high sugar levels and blocks further sugar utilization.

Project description:BackgroundPretreatment of biomass for lignocellulosic ethanol production generates compounds that can inhibit microbial metabolism. The furan aldehydes hydroxymethylfurfural (HMF) and furfural have received increasing attention recently. In the present study, the effects of HMF and furfural on redox metabolism, energy metabolism and gene expression were investigated in anaerobic chemostats where the inhibitors were added to the feed-medium.ResultsBy cultivating the xylose-utilizing Saccharomyces cerevisiae strain VTT C-10883 in the presence of HMF and furfural, it was found that the intracellular concentrations of the redox co-factors and the catabolic and anabolic reduction charges were significantly lower in the presence of furan aldehydes than in cultivations without inhibitors. The catabolic reduction charge decreased from 0.13(±0.005) to 0.08(±0.002) and the anabolic reduction charge decreased from 0.46(±0.11) to 0.27(±0.02) when HMF and furfural were present. The intracellular ATP concentration was lower when inhibitors were added, but resulted only in a modest decrease in the energy charge from 0.87(±0.002) to 0.85(±0.004) compared to the control. Transcriptome profiling followed by MIPS functional enrichment analysis of up-regulated genes revealed that the functional group "Cell rescue, defense and virulence" was over-represented when inhibitors were present compared to control cultivations. Among these, the ATP-binding efflux pumps PDR5 and YOR1 were identified as important for inhibitor efflux and possibly a reason for the lower intracellular ATP concentration in stressed cells. It was also found that genes involved in pseudohyphal growth were among the most up-regulated when inhibitors were present in the feed-medium suggesting nitrogen starvation. Genes involved in amino acid metabolism, glyoxylate cycle, electron transport and amino acid transport were enriched in the down-regulated gene set in response to HMF and furfural. It was hypothesized that the HMF and furfural-induced NADPH drainage could influence ammonia assimilation and thereby give rise to the nitrogen starvation response in the form of pseudohyphal growth and down-regulation of amino acid synthesis.ConclusionsThe redox metabolism was severely affected by HMF and furfural while the effects on energy metabolism were less evident, suggesting that engineering of the redox system represents a possible strategy to develop more robust strains for bioethanol production.

Project description:BackgroundPretreatment of lignocellulosic biomass generates a number of undesired degradation products that can inhibit microbial metabolism. Two of these compounds, the furan aldehydes 5-hydroxymethylfurfural (HMF) and 2-furaldehyde (furfural), have been shown to be an impediment for viable ethanol production. In the present study, HMF and furfural were pulse-added during either the glucose or the xylose consumption phase in order to dissect the effects of these inhibitors on energy state, redox metabolism, and gene expression of xylose-consuming Saccharomyces cerevisiae.ResultsPulsed addition of 3.9 g L-1 HMF and 1.2 g L-1 furfural during either the glucose or the xylose consumption phase resulted in distinct physiological responses. Addition of furan aldehydes in the glucose consumption phase was followed by a decrease in the specific growth rate and the glycerol yield, whereas the acetate yield increased 7.3-fold, suggesting that NAD(P)H for furan aldehyde conversion was generated by acetate synthesis. No change in the intracellular levels of NAD(P)H was observed 1 hour after pulsing, whereas the intracellular concentration of ATP increased by 58%. An investigation of the response at transcriptional level revealed changes known to be correlated with perturbations in the specific growth rate, such as protein and nucleotide biosynthesis. Addition of furan aldehydes during the xylose consumption phase brought about an increase in the glycerol and acetate yields, whereas the xylitol yield was severely reduced. The intracellular concentrations of NADH and NADPH decreased by 58 and 85%, respectively, hence suggesting that HMF and furfural drained the cells of reducing power. The intracellular concentration of ATP was reduced by 42% 1 hour after pulsing of inhibitors, suggesting that energy-requiring repair or maintenance processes were activated. Transcriptome profiling showed that NADPH-requiring processes such as amino acid biosynthesis and sulfate and nitrogen assimilation were induced 1 hour after pulsing.ConclusionsThe redox and energy metabolism were found to be more severely affected after pulsing of furan aldehydes during the xylose consumption phase than during glucose consumption. Conceivably, this discrepancy resulted from the low xylose utilization rate, hence suggesting that xylose metabolism is a feasible target for metabolic engineering of more robust xylose-utilizing yeast strains.

Dataset Information

Increased expression of the oxidative pentose phosphate pathway and gluconeogenesis in anaerobically growing xylose-utilizing Saccharomyces cerevisiae.

Publications

Increased expression of the oxidative pentose phosphate pathway and gluconeogenesis in anaerobically growing xylose-utilizing Saccharomyces cerevisiae.

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