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Xiaoming Bao - One of the best experts on this subject based on the ideXlab platform.
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engineering of saccharomyces cerevisiae for the efficient co utilization of glucose and Xylose
Fems Yeast Research, 2017Co-Authors: Jin Hou, Chenxi Qiu, Yu Shen, Xiaoming BaoAbstract:The rapid co-fermentation of both glucose and Xylose is important for the efficient conversion of lignocellulose biomass into fuels and chemicals. Saccharomyces cerevisiae is considered to be a potential cell factory and has been used to produce various fuels and chemicals, but it cannot metabolize Xylose, which has greatly limited the utilization of lignocellulose materials. Therefore, numerous studies have attempted to develop Xylose fermenting strains in past decades. The simple introduction of the Xylose metabolic pathway does not enable yeast to rapidly utilize Xylose, and several limitations still need to be addressed, including glucose repression and slow Xylose transport, cofactor imbalance in the Xylose reductase/xylitol dehydrogenase pathway, functional expression of a heterologous Xylose isomerase, the low efficiency of downstream pathways and low ethanol production. In this review, we will discuss strategies to overcome these limitations and the recent progress in engineering Xylose fermenting S. cerevisiae strains.
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an efficient Xylose fermenting recombinant saccharomyces cerevisiae strain obtained through adaptive evolution and its global transcription profile
Applied Microbiology and Biotechnology, 2012Co-Authors: Yu Shen, Jin Hou, Bingyin Peng, Xiao Chen, Liyuan Chen, Xiaoming BaoAbstract:Factors related to ethanol production from Xylose in engineered Saccharomyces cerevisiae that contain an exogenous initial metabolic pathway are still to be elucidated. In the present study, a strain that expresses the Xylose isomerase gene of Piromyces sp. Pi-xylA and overexpresses XKS1, RPE1, RKI1, TAL1, and TKL1, with deleted GRE3 and COX4 genes was constructed. The Xylose utilization capacity of the respiratory deficiency strain was poor but improved via adaptive evolution in Xylose. The μ max of the evolved strain in 20 g l−1 Xylose is 0.11 ± 0.00 h−1, and the evolved strain consumed 17.83 g l−1 Xylose within 72 h, with an ethanol yield of 0.43 g g−1 total consumed sugars during glucose–Xylose cofermentation. Global transcriptional changes and effect of several specific genes were studied. The result revealed that the increased Xylose isomerase acivity, the upregulation of enzymes involved in glycolysis and glutamate synthesis, and the downregulation of trehalose and glycogen synthesis, may have contributed to the improved Xylose utilization of the strain. Furthermore, the deletion of PHO13 decreased the Xylose growth in the respiration deficiency strain although deleting PHO13 can improve the Xylose metabolism in other strains.
David Runquist - One of the best experts on this subject based on the ideXlab platform.
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the glucose Xylose facilitator gxf1 from candida intermedia expressed in a Xylose fermenting industrial strain of saccharomyces cerevisiae increases Xylose uptake in sscf of wheat straw
Enzyme and Microbial Technology, 2011Co-Authors: Cesar Fonseca, David Runquist, Kim Olofsson, Luis L Fonseca, Carla Ferreira, Barbel Hahnhagerdal, Gunnar LidenAbstract:Ethanolic fermentation of lignocellulose raw materials requires industrial Xylose-fermenting strains capable of complete and efficient D-Xylose consumption. A central question in Xylose fermentation by Saccharomyces cerevisiae engineered for Xylose fermentation is to improve the Xylose uptake. In the current study, the glucose/Xylose facilitator Gxf1 from Candida intermedia, was expressed in three different Xylose-fermenting S. cerevisiae strains of industrial origin. The in vivo effect on aerobic Xylose growth and the initial Xylose uptake rate were assessed. The expression of Gxf1 resulted in enhanced aerobic Xylose growth only for the TMB3400 based strain. It displayed more than a 2-fold higher affinity for D-Xylose than the parental strain and approximately 2-fold higher initial specific growth rate at 4 g/L D-Xylose. Enhanced Xylose consumption was furthermore observed when the GXF1-strain was assessed in simultaneous saccharification and co-fermentation (SSCF) of pretreated wheat straw. However, the ethanol yield remained unchanged due to increased by-product formation. Metabolic flux analysis suggested that the expression of the Gxf1 transporter had shifted the control of Xylose catabolism from transport to the NAD(+) dependent oxidation of xylitol to xylulose. (C) 2011 Elsevier Inc. All rights reserved. (Less)
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increased expression of the oxidative pentose phosphate pathway and gluconeogenesis in anaerobically growing Xylose utilizing saccharomyces cerevisiae
Microbial Cell Factories, 2009Co-Authors: David Runquist, Barbel Hahnhagerdal, Maurizio BettigaAbstract: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. 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. 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.
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Expression of the Gxf1 transporter from Candida intermedia improves fermentation performance in recombinant Xylose-utilizing Saccharomyces cerevisiae
Applied Microbiology and Biotechnology, 2009Co-Authors: David Runquist, Isabel Spencer-martins, C Fonseca, Peter Rådström, Bärbel Hahn-hägerdalAbstract:The glucose/Xylose facilitator Gxf1 from Candida intermedia was expressed in the recombinant Xylose-fermenting Saccharomyces cerevisiae strain TMB 3057. The new strain, TMB 3411, displayed approximately two times lower K _m for Xylose transport compared to a control strain not expressing Gxf1. In aerobic batch cultivation, the specific growth rate was significantly higher at low Xylose concentration, 4 g/L, when Gxf1 was expressed, whereas it remained unchanged at high Xylose concentration, 40 g/L. Similarly, in aerobic-Xylose-limited chemostat culture, the Gxf1-expressing strain consumed more Xylose than the control strain at low dilution rates (low Xylose concentration), whereas the situation was reversed at higher dilution rates (high Xylose concentration). Also, under anaerobic conditions, the Gxf1-expressing strain showed faster Xylose uptake and ethanol formation at low substrate concentrations. The results are discussed in relation to previous observations, which suggested that transport controlled Xylose utilization in recombinant Xylose-utilizing S. cerevisiae only at low Xylose concentrations.
Paes, Bárbara Gomes - One of the best experts on this subject based on the ideXlab platform.
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Engenharia metabólica de Saccharomyces cerevisiae para aproveitamento de xilose na produção de etanol lignocelulósico
2015Co-Authors: Paes, Bárbara GomesAbstract:O aumento na demanda por energias sustentáveis impulsiona o desenvolvimento de estratégias biotecnológicas para a produção de biocombustíveis. Neste contexto, o aproveitamento eficiente da biomassa lignocelulósica como matéria prima é fundamental para a produção de etanol de segunda geração. A levedura Saccharomyces cerevisiae, organismo mais utilizado na produção industrial de bioetanol, é incapaz de utilizar pentoses, como a xilose, que é o segundo açúcar mais abundante em algumas biomassas. Neste trabalho, plasmídeos epissomais foram construídos para expressão de genes codificadores para xilose isomerase (XI) de Piromyces sp. e xiluloquinase (XK) de S. cerevisiae. As linhagens laboratoriais de S. cerevisiae CEN.PK 113.14A Δtrp1-289 (L2) e CEN.PK 113.3C Δtrp1-289, Δura-52 (L7) foram transformadas com os plasmídeos gerados. Desta forma, foram construídas linhagens recombinantes de S. cerevisiae expressando XI isoladamente (L2XI) ou em conjunto com XK (L7XIXK). Uma terceira linhagem, L7XIΦ, expressando XI e com o segundo plasmídeo vazio foi construída como controle. A linhagem L7XIXK apresentou melhores taxas fermentativas que as demais linhagens, confirmando o efeito positivo da expressão de XK. As linhagens L2XI, L7XIΦ e L7XIXK obtidas foram submetidas a um processo de condicionamento em meio seletivo contendo xilose como única fonte de carbono. Ao final do condicionamento, quando comparadas com as originais, apresentaram menor fase lag de crescimento e maior taxa de crescimento, maior consumo de xilose (entre 1,8 e 18,5 vezes) e rendimento de etanol (47% para L7XIXK), concomitante à diminuição do rendimento de xilitol (entre 87,6% e 91,8%). A linhagem L2XI, investigada anaerobicamente, também apresentou aumento no consumo específico de xilose (49,8%), rendimento de etanol (19%) e redução no rendimento de xilitol (75%). As linhagens condicionadas foram submetidas então a um processo de cura para remoção dos plasmídeos. Uma das linhagens obtidas, LC7, derivada de L7XIXK perdeu a capacidade de crescer em meio mínimo, indicando a perda dos plasmídeos. Entretanto o gene para XI ainda foi identificado na levedura. A retransformação desta levedura curada com os plasmídeos originais demonstrou que as melhorias da levedura condicionada podem estar associadas a mutações fora do plasmídeo. Esta linhagem curada tem grande potencial para desenvolvimento de uma linhagem de seleção para novas enzimas da via do catabolismo de xilose. ____________________________________________________________________________________ ABSTRACTThe increasing demand for sustainable energy drives the development of biotechnological strategies for the production of biofuels. In this context, efficient utilization of lignocellulosic biomass as feedstock is essential for the production of second generation biofuels. The yeast S. cerevisiae, main organism utilized in the industrial production of bioethanol, is unable to use pentoses, such as Xylose, which is the second most abundant sugar in some biomasses. In this work, multi-copy plasmids were constructed for the expression of genes coding Xylose isomerase (XI) from Piromyces sp. and xylulokinase (XK) from S. cerevisiae. The laboratory strains of S. cerevisiae CEN.PK 113.14A Δtrp1-289 (L2) and CEN.PK 113.3C Δtrp1-289, Δura-52 (L7) were transformed with the generated plasmids. Thus, recombinant strains of S. cerevisiae expressing solely XI (L2XI), or combined with XK (L7XIXK) were obtained. A third strain, L7XIΦ, expressing XI and with an empty second plasmid, was constructed as control. The L7XIXK strain presented better fermentative rates than the other strains, confirming the positive effect of XK expression. The obtained strains L2XI, L7XIΦ and L7XIXK underwent a conditioning process in selective medium with Xylose as sole carbon source. At the end of the process, when compared to the original strains, conditioned strains presented shorter lag growth phase, and increased growth rate, increased Xylose consumption (1,8 to 18,5 fold) and ethanol yield (47% for L7XIXK), along with reduction in xylitol production (between 87,6 and 91,8%). The strain L2XI was investigated under anaerobic conditions and also has presented improved Xylose specific consumption (49,8%), ethanol yield (19%) and xylitol reduction (75%). The conditioned strains underwent a curing process, in order to remove the plasmids. One of the obtained strains, LC7, which derived from L7XIXK, has lost its ability to grow on minimal medium, a result consistent with the loss of plasmids. However the XI gene was still identified in the yeast. The retransformation of this curated yeast with the originally constructed plasmids showed that the improvements observed in conditioned strains may be associated with mutations outside of the plasmids. The curated strain has a great potential for the development of a screening strain for new enzymes of the Xylose catabolic pathway
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Engenharia metabólica de Saccharomyces cerevisiae para aproveitamento de xilose na produção de etanol lignocelulósico
'Biblioteca Central da UNB', 2015Co-Authors: Paes, Bárbara GomesAbstract:Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, Programa de Pós-Graduação em Biologia Molecular, 2015.O aumento na demanda por energias sustentáveis impulsiona o desenvolvimento de estratégias biotecnológicas para a produção de biocombustíveis. Neste contexto, o aproveitamento eficiente da biomassa lignocelulósica como matéria prima é fundamental para a produção de etanol de segunda geração. A levedura Saccharomyces cerevisiae, organismo mais utilizado na produção industrial de bioetanol, é incapaz de utilizar pentoses, como a xilose, que é o segundo açúcar mais abundante em algumas biomassas. Neste trabalho, plasmídeos epissomais foram construídos para expressão de genes codificadores para xilose isomerase (XI) de Piromyces sp. e xiluloquinase (XK) de S. cerevisiae. As linhagens laboratoriais de S. cerevisiae CEN.PK 113.14A Δtrp1-289 (L2) e CEN.PK 113.3C Δtrp1-289, Δura-52 (L7) foram transformadas com os plasmídeos gerados. Desta forma, foram construídas linhagens recombinantes de S. cerevisiae expressando XI isoladamente (L2XI) ou em conjunto com XK (L7XIXK). Uma terceira linhagem, L7XIΦ, expressando XI e com o segundo plasmídeo vazio foi construída como controle. A linhagem L7XIXK apresentou melhores taxas fermentativas que as demais linhagens, confirmando o efeito positivo da expressão de XK. As linhagens L2XI, L7XIΦ e L7XIXK obtidas foram submetidas a um processo de condicionamento em meio seletivo contendo xilose como única fonte de carbono. Ao final do condicionamento, quando comparadas com as originais, apresentaram menor fase lag de crescimento e maior taxa de crescimento, maior consumo de xilose (entre 1,8 e 18,5 vezes) e rendimento de etanol (47% para L7XIXK), concomitante à diminuição do rendimento de xilitol (entre 87,6% e 91,8%). A linhagem L2XI, investigada anaerobicamente, também apresentou aumento no consumo específico de xilose (49,8%), rendimento de etanol (19%) e redução no rendimento de xilitol (75%). As linhagens condicionadas foram submetidas então a um processo de cura para remoção dos plasmídeos. Uma das linhagens obtidas, LC7, derivada de L7XIXK perdeu a capacidade de crescer em meio mínimo, indicando a perda dos plasmídeos. Entretanto o gene para XI ainda foi identificado na levedura. A retransformação desta levedura curada com os plasmídeos originais demonstrou que as melhorias da levedura condicionada podem estar associadas a mutações fora do plasmídeo. Esta linhagem curada tem grande potencial para desenvolvimento de uma linhagem de seleção para novas enzimas da via do catabolismo de xilose.The increasing demand for sustainable energy drives the development of biotechnological strategies for the production of biofuels. In this context, efficient utilization of lignocellulosic biomass as feedstock is essential for the production of second generation biofuels. The yeast S. cerevisiae, main organism utilized in the industrial production of bioethanol, is unable to use pentoses, such as Xylose, which is the second most abundant sugar in some biomasses. In this work, multi-copy plasmids were constructed for the expression of genes coding Xylose isomerase (XI) from Piromyces sp. and xylulokinase (XK) from S. cerevisiae. The laboratory strains of S. cerevisiae CEN.PK 113.14A Δtrp1-289 (L2) and CEN.PK 113.3C Δtrp1-289, Δura-52 (L7) were transformed with the generated plasmids. Thus, recombinant strains of S. cerevisiae expressing solely XI (L2XI), or combined with XK (L7XIXK) were obtained. A third strain, L7XIΦ, expressing XI and with an empty second plasmid, was constructed as control. The L7XIXK strain presented better fermentative rates than the other strains, confirming the positive effect of XK expression. The obtained strains L2XI, L7XIΦ and L7XIXK underwent a conditioning process in selective medium with Xylose as sole carbon source. At the end of the process, when compared to the original strains, conditioned strains presented shorter lag growth phase, and increased growth rate, increased Xylose consumption (1,8 to 18,5 fold) and ethanol yield (47% for L7XIXK), along with reduction in xylitol production (between 87,6 and 91,8%). The strain L2XI was investigated under anaerobic conditions and also has presented improved Xylose specific consumption (49,8%), ethanol yield (19%) and xylitol reduction (75%). The conditioned strains underwent a curing process, in order to remove the plasmids. One of the obtained strains, LC7, which derived from L7XIXK, has lost its ability to grow on minimal medium, a result consistent with the loss of plasmids. However the XI gene was still identified in the yeast. The retransformation of this curated yeast with the originally constructed plasmids showed that the improvements observed in conditioned strains may be associated with mutations outside of the plasmids. The curated strain has a great potential for the development of a screening strain for new enzymes of the Xylose catabolic pathway
Yu Shen - One of the best experts on this subject based on the ideXlab platform.
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engineering of saccharomyces cerevisiae for the efficient co utilization of glucose and Xylose
Fems Yeast Research, 2017Co-Authors: Jin Hou, Chenxi Qiu, Yu Shen, Xiaoming BaoAbstract:The rapid co-fermentation of both glucose and Xylose is important for the efficient conversion of lignocellulose biomass into fuels and chemicals. Saccharomyces cerevisiae is considered to be a potential cell factory and has been used to produce various fuels and chemicals, but it cannot metabolize Xylose, which has greatly limited the utilization of lignocellulose materials. Therefore, numerous studies have attempted to develop Xylose fermenting strains in past decades. The simple introduction of the Xylose metabolic pathway does not enable yeast to rapidly utilize Xylose, and several limitations still need to be addressed, including glucose repression and slow Xylose transport, cofactor imbalance in the Xylose reductase/xylitol dehydrogenase pathway, functional expression of a heterologous Xylose isomerase, the low efficiency of downstream pathways and low ethanol production. In this review, we will discuss strategies to overcome these limitations and the recent progress in engineering Xylose fermenting S. cerevisiae strains.
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an efficient Xylose fermenting recombinant saccharomyces cerevisiae strain obtained through adaptive evolution and its global transcription profile
Applied Microbiology and Biotechnology, 2012Co-Authors: Yu Shen, Jin Hou, Bingyin Peng, Xiao Chen, Liyuan Chen, Xiaoming BaoAbstract:Factors related to ethanol production from Xylose in engineered Saccharomyces cerevisiae that contain an exogenous initial metabolic pathway are still to be elucidated. In the present study, a strain that expresses the Xylose isomerase gene of Piromyces sp. Pi-xylA and overexpresses XKS1, RPE1, RKI1, TAL1, and TKL1, with deleted GRE3 and COX4 genes was constructed. The Xylose utilization capacity of the respiratory deficiency strain was poor but improved via adaptive evolution in Xylose. The μ max of the evolved strain in 20 g l−1 Xylose is 0.11 ± 0.00 h−1, and the evolved strain consumed 17.83 g l−1 Xylose within 72 h, with an ethanol yield of 0.43 g g−1 total consumed sugars during glucose–Xylose cofermentation. Global transcriptional changes and effect of several specific genes were studied. The result revealed that the increased Xylose isomerase acivity, the upregulation of enzymes involved in glycolysis and glutamate synthesis, and the downregulation of trehalose and glycogen synthesis, may have contributed to the improved Xylose utilization of the strain. Furthermore, the deletion of PHO13 decreased the Xylose growth in the respiration deficiency strain although deleting PHO13 can improve the Xylose metabolism in other strains.
Akinori Matsushika - One of the best experts on this subject based on the ideXlab platform.
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Xylose and Xylose glucose co fermentation by recombinant saccharomyces cerevisiae strains expressing individual hexose transporters
Enzyme and Microbial Technology, 2014Co-Authors: Davi Goncalves, Tetsuya Goshima, Akinori Matsushika, Belisa B De Sales, Elba P S Bon, Boris U StambukAbstract:Since the uptake of Xylose is believed to be one of the rate-limiting steps for Xylose ethanol fermentation by recombinant Saccharomyces cerevisiae strains, we transformed a hxt-null strain lacking the major hexose transporters (hxt1Δ-hxt7Δ and gal2Δ) with an integrative plasmid to overexpress the genes for Xylose reductase (XYL1), xylitol dehydrogenase (XYL2) and xylulokinase (XKS1), and analyzed the impact that overexpression of the HXT1, HXT2, HXT5 or HXT7 permeases have in anaerobic batch fermentations using Xylose, glucose, or Xylose plus glucose as carbon sources. Our results revealed that the low-affinity HXT1 permease allowed the maximal consumption of sugars and ethanol production rates during Xylose/glucose co-fermentations, but was incapable to allow Xylose uptake when this sugar was the only carbon source. The moderately high-affinity HXT5 permease was a poor glucose transporter, and it also did not allow significant Xylose uptake by the cells. The moderately high-affinity HXT2 permease allowed Xylose uptake with the same rates as those observed during glucose consumption, even under co-fermentation conditions, but had the drawback of producing incomplete fermentations. Finally, the high-affinity HXT7 permease allowed efficient Xylose fermentation, but during Xylose/glucose co-fermentations this permease showed a clear preference for glucose. Thus, our results indicate that approaches to engineer S. cerevisiae HXT transporters to improve second generation bioethanol production need to consider the composition of the biomass sugar syrup, whereby the HXT1 transporter seems more suitable for hydrolysates containing Xylose/glucose blends, whereas the HXT7 permease would be a better choice for Xylose-enriched sugar streams.
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characterization of non oxidative transaldolase and transketolase enzymes in the pentose phosphate pathway with regard to Xylose utilization by recombinant saccharomyces cerevisiae
Enzyme and Microbial Technology, 2012Co-Authors: Akinori Matsushika, Tetsuya Goshima, Shigeki Sawayama, Tatsuya Fujii, Hiroyuki Inoue, Shinichi YanoAbstract:Abstract The activity of transaldolase and transketolase, key enzymes in the non-oxidative pentose phosphate pathway, is rate-limiting for Xylose utilization in recombinant Saccharomyces cerevisiae . Overexpression of TAL1 and TKL1 , the major transaldolase and transketolase genes, increases the flux from the pentose phosphate pathway into the glycolytic pathway. However, the functional roles of NQM1 and TKL2 , the secondary transaldolase and transketolase genes, especially in Xylose utilization, remain unclear. This study focused on characterization of NQM1 and TKL2 , together with TAL1 and TKL1 , regarding their roles in Xylose utilization and fermentation. Knockout or overexpression of these four genes on the phenotype of Xylose-utilizing S . cerevisiae strains was also examined. Transcriptional analysis indicated that the expression of TAL1 , NQM1 , and TKL1 was up-regulated in the presence of Xylose. A significant decrease in both growth on Xylose and Xylose-fermenting ability in tal1Δ and tkl1Δ mutants confirmed that TAL1 and TKL1 are essential for Xylose assimilation and fermentation. Gene disruption analysis using a tkl1Δ mutant revealed that TKL1 is also required for utilization of glucose. Growth on Xylose and Xylose-fermenting ability were slightly influenced by deletion of NQM1 or TKL2 when Xylose was used as the sole carbon source. Moreover, the rate of Xylose consumption and ethanol production was slightly impaired in TKL1 - and TKL2 -overexpressing strains. NQM1 and TKL2 may thus play a physiological role via an effect on the non-oxidative pentose phosphate pathway in the Xylose metabolic pathway, although their roles in Xylose utilization and fermentation are less important than those of TAL1 and TKL1 .
