Multiple gene expression by chromosomal integration and CRE-loxP mediated marker recycling in Saccharomyces cerevisiae

Multiple gene expression can be introduced in a yeast strain with using only two markers by means of the two new vectors described, the expression vector pB3 PGK and the CRE recombinase vector pCRE3. The pB3 PGK has a zeocin-selectable marker flanked by loxP sequences and an expression cassette consisting of the strong PGK1 promoter and the GCY1 terminator. The gene of interest (YFG1) is cloned between the promoter and terminator of pB3 PGK. The pB3 PGK-YFG1 is integrated into the genome by a single restriction cut within the YFG1 gene and integrated in the YFG1 locus. The strain is further transformed with the pCRE3 vector. The CRE recombinase expressed from this vector removes the zeocin marker and makes it possible to use the pB3 PGK vector over again in the same strain after curing of the pCRE3 vector. The 2 micro -based pCRE3 carries the aureobasidin A, zeocin and URA3 markers. pCRE3 is easily cured by growth in nonselective medium without active counterselection. The screening for loss of the chromosomal zeocin marker, as well as curing of the pCRE3 vector, is done in one step, by scoring zeocin sensitivity. This can be done because the zeocin marker is present in both the pB3 PGK and pCRE3. The S. cerevisiae pentose phosphate pathway genes RK11, RPE1, TAL1, and TKL1 were cloned in pB3 PGK and integrated in the locus of the respective gene, resulting in simultaneous overexpression of the genes in the xylose-fermenting S. cerevisiae strain TMB3001.(undefined

Nutrient requirements of lactococci in defined growth media

Many attempts have been made for the last six decades to design defined media for species of the lactococcus group. The general outcome of the studies suggests that this group is heterogeneous with respect to specific requirements for nutrients. Lactococcal species are limited in various metabolic pathways. Early attempts to trace the required nutrients were not always successful because of the poor quality of analysis and the presence of impurities in the medium components

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

<p>Abstract</p> <p>Background</p> <p>Fermentation of xylose to ethanol has been achieved in <it>S. cerevisiae </it>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 (<it>XYL1 </it>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.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>Co-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.</p

Comparison of heterologous xylose transporters in recombinant Saccharomyces cerevisiae

Abstract Background Baker's yeast (Saccharomyces cerevisiae) has been engineered for xylose utilization to enable production of fuel ethanol from lignocellulose raw material. One unresolved challenge is that S. cerevisiae lacks a dedicated transport system for pentose sugars, which means that xylose is transported by non-specific Hxt transporters with comparatively low transport rate and affinity for xylose. Results In this study, we compared three heterologous xylose transporters that have recently been shown to improve xylose uptake under different experimental conditions. The transporters Gxf1, Sut1 and At5g59250 from Candida intermedia, Pichia stipitis and Arabidopsis thaliana, respectively, were expressed in isogenic strains of S. cerevisiae and the transport kinetics and utilization of xylose was evaluated. Expression of the Gxf1 and Sut1 transporters led to significantly increased affinity and transport rates of xylose. In batch cultivation at 4 g/L xylose concentration, improved transport kinetics led to a corresponding increase in xylose utilization, whereas no correlation could be demonstrated at xylose concentrations greater than 15 g/L. The relative contribution of native sugar transporters to the overall xylose transport capacity was also estimated during growth on glucose and xylose. Conclusions Kinetic characterization and aerobic batch cultivation of strains expressing the Gxf1, Sut1 and At5g59250 transporters showed a direct relationship between transport kinetics and xylose growth. The Gxf1 transporter had the highest transport capacity and the highest xylose growth rate, followed by the Sut1 transporter. The range in which transport controlled the growth rate was determined to between 0 and 15 g/L xylose. The role of catabolite repression in regulation of native transporters was also confirmed by the observation that xylose transport by native S. cerevisiae transporters increased significantly during cultivation in xylose and at low glucose concentration.</p

Xylose reductase from Pichia stipitis with altered coenzyme preference improves ethanolic xylose fermentation by recombinant Saccharomyces cerevisiae

<p>Abstract</p> <p>Background</p> <p>Xylose reductase (XR) and xylitol dehydrogenase (XDH) from <it>Pichia stipitis </it>are the two enzymes most commonly used in recombinant <it>Saccharomyces cerevisiae </it>strains engineered for xylose utilization. The availability of NAD⁺for XDH is limited during anaerobic xylose fermentation because of the preference of XR for NADPH. This in turn results in xylitol formation and reduced ethanol yield. The coenzyme preference of <it>P. stipitis </it>XR was changed by site-directed mutagenesis with the aim to engineer it towards NADH-preference.</p> <p>Results</p> <p>XR variants were evaluated in <it>S. cerevisiae </it>strains with the following genetic modifications: overexpressed native <it>P. stipitis </it>XDH, overexpressed xylulokinase, overexpressed non-oxidative pentose phosphate pathway and deleted GRE3 gene encoding an NADPH dependent aldose reductase. All overexpressed genes were chromosomally integrated to ensure stable expression. Crude extracts of four different strains overexpressing genes encoding native <it>P. stipitis </it>XR, K270M and K270R mutants, as well as <it>Candida parapsilosis </it>XR, were enzymatically characterized. The physiological effects of the mutations were investigated in anaerobic xylose fermentation. The strain overexpressing <it>P. stipitis </it>XR with the K270R mutation gave an ethanol yield of 0.39 g (g consumed sugars)^-1, a xylitol yield of 0.05 g (g consumed xylose)^-1and a xylose consumption rate of 0.28 g (g biomass)^-1h^-1in continuous fermentation at a dilution rate of 0.12 h^-1, with 10 g l^-1glucose and 10 g l^-1xylose as carbon sources.</p> <p>Conclusion</p> <p>The cofactor preference of <it>P. stipitis </it>XR was altered by site-directed mutagenesis. When the K270R XR was combined with a metabolic engineering strategy that ensures high xylose utilization capabilities, a recombinant <it>S. cerevisiae </it>strain was created that provides a unique combination of high xylose consumption rate, high ethanol yield and low xylitol yield during ethanolic xylose fermentation.</p

A mutated xylose reductase increases bioethanol production more than a glucose/xylose facilitator in simultaneous fermentation and co-fermentation of wheat straw

Genetically engineered Saccharomyces cerevisiae strains are able to ferment xylose present in lignocellulosic biomass. However, better xylose fermenting strains are required to reach complete xylose uptake in simultaneous saccharification and co-fermentation (SSCF) of lignocellulosic hydrolyzates. In the current study, haploid Saccharomyces cerevisiae strains expressing a heterologous xylose pathway including either the native xylose reductase (XR) from P. stipitis, a mutated variant of XR (mXR) with altered co-factor preference, a glucose/xylose facilitator (Gxf1) from Candida intermedia or both mXR and Gxf1 were assessed in SSCF of acid-pretreated non-detoxified wheat straw. The xylose conversion in SSCF was doubled with the S. cerevisiae strain expressing mXR compared to the isogenic strain expressing the native XR, converting 76% and 38%, respectively. The xylitol yield was less than half using mXR in comparison with the native variant. As a result of this, the ethanol yield increased from 0.33 to 0.39 g g-1 when the native XR was replaced by mXR. In contrast, the expression of Gxf1 only slightly increased the xylose uptake, and did not increase the ethanol production. The results suggest that ethanolic xylose fermentation under SSCF conditions is controlled primarily by the XR activity and to a much lesser extent by xylose transport

Comparing the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways in arabinose and xylose fermenting Saccharomyces cerevisiae strains

<p>Abstract</p> <p>Background</p> <p>Ethanolic fermentation of lignocellulosic biomass is a sustainable option for the production of bioethanol. This process would greatly benefit from recombinant <it>Saccharomyces cerevisiae </it>strains also able to ferment, besides the hexose sugar fraction, the pentose sugars, arabinose and xylose. Different pathways can be introduced in <it>S. cerevisiae </it>to provide arabinose and xylose utilisation. In this study, the bacterial arabinose isomerase pathway was combined with two different xylose utilisation pathways: the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways, respectively, in genetically identical strains. The strains were compared with respect to aerobic growth in arabinose and xylose batch culture and in anaerobic batch fermentation of a mixture of glucose, arabinose and xylose.</p> <p>Results</p> <p>The specific aerobic arabinose growth rate was identical, 0.03 h^-1, for the xylose reductase/xylitol dehydrogenase and xylose isomerase strain. The xylose reductase/xylitol dehydrogenase strain displayed higher aerobic growth rate on xylose, 0.14 h^-1, and higher specific xylose consumption rate in anaerobic batch fermentation, 0.09 g (g cells)^-1h^-1than the xylose isomerase strain, which only reached 0.03 h^-1and 0.02 g (g cells)^-1h^-1, respectively. Whereas the xylose reductase/xylitol dehydrogenase strain produced higher ethanol yield on total sugars, 0.23 g g^-1compared with 0.18 g g^-1for the xylose isomerase strain, the xylose isomerase strain achieved higher ethanol yield on consumed sugars, 0.41 g g^-1compared with 0.32 g g^-1for the xylose reductase/xylitol dehydrogenase strain. Anaerobic fermentation of a mixture of glucose, arabinose and xylose resulted in higher final ethanol concentration, 14.7 g l^-1for the xylose reductase/xylitol dehydrogenase strain compared with 11.8 g l^-1for the xylose isomerase strain, and in higher specific ethanol productivity, 0.024 g (g cells)^-1h^-1compared with 0.01 g (g cells)^-1h^-1for the xylose reductase/xylitol dehydrogenase strain and the xylose isomerase strain, respectively.</p> <p>Conclusion</p> <p>The combination of the xylose reductase/xylitol dehydrogenase pathway and the bacterial arabinose isomerase pathway resulted in both higher pentose sugar uptake and higher overall ethanol production than the combination of the xylose isomerase pathway and the bacterial arabinose isomerase pathway. Moreover, the flux through the bacterial arabinose pathway did not increase when combined with the xylose isomerase pathway. This suggests that the low activity of the bacterial arabinose pathway cannot be ascribed to arabitol formation via the xylose reductase enzyme.</p

PGM2 overexpression improves anaerobic galactose fermentation in Saccharomyces cerevisiae

<p>Abstract</p> <p>Background</p> <p>In <it>Saccharomyces cerevisiae </it>galactose is initially metabolized through the Leloir pathway after which glucose 6-phosphate enters glycolysis. Galactose is controlled both by glucose repression and by galactose induction. The gene <it>PGM2 </it>encodes the last enzyme of the Leloir pathway, phosphoglucomutase 2 (Pgm2p), which catalyses the reversible conversion of glucose 1-phosphate to glucose 6-phosphate. Overexpression of <it>PGM2 </it>has previously been shown to enhance aerobic growth of <it>S. cerevisiae </it>in galactose medium.</p> <p>Results</p> <p>In the present study we show that overexpression of <it>PGM2 </it>under control of the <it>HXT7'</it>promoter from an integrative plasmid increased the PGM activity 5 to 6 times, which significantly reduced the lag phase of glucose-pregrown cells in an anaerobic galactose culture. <it>PGM2 </it>overexpression also increased the anaerobic specific growth rate whereas ethanol production was less influenced. When <it>PGM2 </it>was overexpressed from a multicopy plasmid instead, the PGM activity increased almost 32 times. However, this increase of PGM activity did not further improve aerobic galactose fermentation as compared to the strain carrying <it>PGM2 </it>on the integrative plasmid.</p> <p>Conclusion</p> <p><it>PGM2 </it>overexpression in <it>S. cerevisiae </it>from an integrative plasmid is sufficient to reduce the lag phase and to enhance the growth rate in anaerobic galactose fermentation, which results in an overall decrease in fermentation duration. This observation is of particular importance for the future development of stable industrial strains with enhanced PGM activity.</p

The NILE Project — Advances in the Conversion of Lignocellulosic Materials into Ethanol

NILE ("New Improvements for Lignocellulosic Ethanol") was an integrated European project (2005-2010) devoted to the conversion of lignocellulosic raw materials to ethanol. The main objectives were to design novel enzymes suitable for the hydrolysis of cellulose to glucose and new yeast strains able to efficiently converting all the sugars present in lignocellulose into ethanol. The project also included testing these new developments in an integrated pilot plant and evaluating the environmental and socio-economic impacts of implementing lignocellulosic ethanol on a large scale. Two model raw materials – spruce and wheat straw – both preconditioned with similar pretreatments, were used. Several approaches were explored to improve the saccharification of these pretreated raw materials such as searching for new efficient enzymes and enzyme engineering. Various genetic engineering methods were applied to obtain stable xylose- and arabinose-fermenting Saccharomyces cerevisiae strains that tolerate the toxic compounds present in lignocellulosic hydrolysates. The pilot plant was able to treat 2 tons of dry matter per day, and hydrolysis and fermentation could be run successively or simultaneously. A global model integrating the supply chain was used to assess the performance of lignocellulosic ethanol from an economical and environmental perspective. It was found that directed evolution of a specific enzyme of the cellulolytic cocktail produced by the industrial fungus, Trichoderma reesei, and modification of the composition of this cocktail led to improvements of the enzymatic hydrolysis of pretreated raw material. These results, however, were difficult to reproduce at a large scale. A substantial increase in the ethanol conversion yield and in specific ethanol productivity was obtained through a combination of metabolic engineering of yeast strains and fermentation process development. Pilot trials confirmed the good behaviour of the yeast strains in industrial conditions as well as the suitability of lignin residues as fuels. The ethanol cost and the greenhouse gas emissions were highly dependent on the supply chain but the best performing supply chains showed environmental and economic benefits. From a global standpoint, the results showed the necessity for an optimal integration of the process to co-develop all the steps of the process and to test the improvements in a flexible pilot plant, thus allowing the comparison of various configurations and their economic and environmental impacts to be determined. <br> Le projet NILE, acronyme de "New Improvements for Lignocellulosic Ethanol", était un projet européen (2005-2010) consacré à la conversion des matières premières lignocellulosiques en éthanol. Ses principaux objectifs étaient de concevoir de nouvelles enzymes adaptées à l’hydrolyse de la cellulose en glucose et de nouvelles souches de levure capables de convertir efficacement tous les sucres présents dans la lignocellulose en éthanol. Une autre partie du projet consistait à tester ces nouveaux systèmes dans une installation pilote et à évaluer les impacts environnementaux et socio-économiques de la production et utilisation à grande échelle d’éthanol lignocellulosique. Deux matières premières modèles (l’épicéa et la paille de blé) prétraitées de façon semblable, ont été étudiées. Différentes approches ont été tentées pour améliorer la saccharification de ces matières premières, par exemple, la recherche de nouvelles enzymes efficaces ou l’ingénierie d’enzymes. Plusieurs stratégies d’ingénierie génétique ont été utilisées pour obtenir des souches stables de Saccharomyces cerevisiae capables de fermenter le xylose et l’arabinose, et de tolérer les composés toxiques présents dans les hydrolysats lignocellulosiques. L’installation pilote pouvait traiter 2 tonnes de matières sèches par jour, et l’hydrolyse et la fermentation pouvaient être menées successivement ou simultanément. Un modèle global intégrant la chaîne d’approvisionnement en matière première a servi à évaluer les performances économiques et environnementales de la production d’éthanol lignocellulosique. L’évolution dirigée d’une enzyme du cocktail cellulolytique produit par le champignon Trichoderma reesei, et la modification de la composition de ce cocktail améliorent l’hydrolyse enzymatique des matières premières prétraitées. Cependant, ces résultats n’ont pu être reproduits à grande échelle. Le rendement de conversion et la productivité spécifique en éthanol ont été sensiblement augmentés grâce à l’ingénierie métabolique des souches de levure et au développement d’un procédé optimal de fermentation. Les essais en pilote ont confirmé le bon comportement de ces souches de levure en conditions industrielles ainsi que la possibilité d’utiliser les résidus riches en lignine comme combustible. Le coût de production de l’éthanol et le bilan des émissions de gaz à effet de serre étaient très dépendants des sources d’énergie utilisées. D’un point de vue plus global, les résultats ont montré que l’optimisation du procédé nécessite de codévelopper toutes les étapes de façon intégrée et de valider les améliorations dans une installation pilote, afin notamment de pouvoir comparer différentes configurations et d’en déterminer les effets sur l’économie du procédé et ses impacts environnementaux