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Inducible expression systems are pivotal for governing gene expression in strain engineering and synthetic biotechnological applications. Therefore, a critical need persists for the development of versatile and efficient inducible expression mechanisms. In this study, the xylose-responsive promoter xylA5p and its transcriptional regulator XylR were identified in Parageobacillus thermoglucosidasius DSM 2542. By combining promoter xylA5p with its regulator XylR, fine-tuning the expression strength of XylR, and reducing the glucose catabolite repression on xylose uptake, we successfully devised a xylose-inducible and glucose-insensitive expression system, denoted as IExyl*. This system exhibited diverse promoter strengths upon induction with xylose at varying concentrations and remained unhindered in the presence of glucose. Moreover, we showed the applicability of IExyl* in P. thermoglucosidasius by redirecting metabolic flux towards riboflavin biosynthesis, culminating in a 2.8-fold increase in riboflavin production compared to that of the starting strain. This glucose-insensitive and xylose-responsive expression system provides valuable tools for designing optimized biosynthetic pathways for high-value products and facilitates future synthetic biology investigations in Parageobacillus. Key points: • A xylose-inducible and glucose-insensitive expression system IExyl* was developed. • IExyl* was applied to enhance the riboflavin production in P. thermoglucosidasius • A tool for metabolic engineering and synthetic biology research in Parageobacillus strains. [ABSTRACT FROM AUTHOR]

Saccharomyces cerevisiae cannot assimilate xylose, second to glucose derived from lignocellulosic biomass. Here, the engineered S. cerevisiae strains INVSc-XI and INVSc-XI/XT were constructed using xylA and Xltr1p to co-utilize xylose and glucose, achieving economic viability and sustainable production of fuels. The xylose utilization rate of INVSc-XI/XT was 2.3-fold higher than that of INVSc-XI, indicating that overexpressing Xltr1p could further enhance xylose utilization. In mixed sugar media, a small amount of glucose enhanced the consumption of xylose by INVSc-XI/XT. Transcriptome analysis showed that glucose increased the upregulation of acetate of coenzyme A synthetase (ACS), alcohol dehydrogenase (ADH), and transketolase (TKL) gene expression in INVSc-XI/XT, further promoting xylose utilization and ethanol yield. The highest ethanol titer of 2.91 g/L with a yield of 0.29 g/g at 96 h by INVSc-XI/XT was 56.9% and 63.0% of the theoretical ethanol yield from glucose and xylose, respectively. These results showed overexpression of xylA and Xltr1p is a promising strategy for improving xylose and glucose conversion to ethanol. Although the ability of strain INVSc-XI/XT to produce ethanol was not very satisfactory, glucose was discovered to influence xylose utilization in strain INVSc-XI/XT. Altering the glucose concentration is a promising strategy to improve the xylose and glucose co-utilization. HIGHLIGHTS: INVSc-XI and INVSc-XI/XT strains were newly constructed to utilize xylose and glucose. XylA, in combination with xylose transporter Xltr1p, enhances xylose consumption. A small amount of glucose enhanced xylose utilization in INVSc-XI/XT strain. The expression of ACS, ADH, and TKL genes is upregulated in the media containing mixed sugars. The highest ethanol yield of 0.29 g/g was produced in a 2-L scale-up fermenter. [ABSTRACT FROM AUTHOR]

BACKGROUND: The need to mitigate and substitute the use of fossil fuels as the main energy matrix has led to the study and development of biofuels as an alternative. Second-generation (2G) ethanol arises as one biofuel with great potential, due to not only maintaining food security, but also as a product from economically interesting crops such as energy-cane. One of the main challenges of 2G ethanol is the inefficient uptake of pentose sugars by industrial yeast Saccharomyces cerevisiae, the main organism used for ethanol production. Understanding the main drivers for xylose assimilation and identify novel and efficient transporters is a key step to make the 2G process economically viable. RESULTS: By implementing a strategy of searching for present motifs that may be responsible for xylose transport and past adaptations of sugar transporters in xylose fermenting species, we obtained a classifying model which was successfully used to select four different candidate transporters for evaluation in the S. cerevisiae hxt-null strain, EBY.VW4000, harbouring the xylose consumption pathway. Yeast cells expressing the transporters SpX, SpH and SpG showed a superior uptake performance in xylose compared to traditional literature control Gxf1. CONCLUSIONS: Modelling xylose transport with the small data available for yeast and bacteria proved a challenge that was overcome through different statistical strategies. Through this strategy, we present four novel xylose transporters which expands the repertoire of candidates targeting yeast genetic engineering for industrial fermentation. The repeated use of the model for characterizing new transporters will be useful both into finding the best candidates for industrial utilization and to increase the models predictive capabilities.

The raw material cost of lactic acid fermentation accounts for the main part of the production cost, and this necessitates the exploration of the efficient use of cheap raw materials in lactic acid production. We compared the outcomes of the homologous expressions of xylose transporters (xylFGH, xylE, araE, and xylT), 6-phosphofructokinase (pfkA), fructose-bisphosphate aldolase (fbaA), and their co-expression in Lactococcus lactis IO-1 on lactic acid production using xylose as the raw material. We found that the production rate of lactic acid on xylose fermentation by L. lactis IO-1 overexpressing fbaA was the highest (14.42%). Among the xylose transporters investigated, XylT had the strongest xylose transport capacity in L. lactis IO-1, with an increase in the lactic acid production rate by 10.38%. The genes near the overexpression of fbaA or xylT in the metabolic pathway were more upregulated than the distant genes. The co-expression of fbaA and xylT increased the production rate of lactic acid by 27.84% on xylose fermentation by L. lactis IO-1. This work presents a novel strategy for the simultaneous enhancement of the expression of important genes at the beginning and midway of the xylose metabolic pathway of L. lactis IO-1, which could greatly improve the target production. [ABSTRACT FROM AUTHOR]

Abstract Glucose and xylose are two major components of lignocellulose. Simultaneous consumption of glucose and xylose is critical for engineered microorganisms to produce fuels and chemicals from lignocellulosic biomass. Although many production limitations have been resolved, glucose‐induced inhibition of xylose transport remains a challenge. In this study, a cell growth‐based screening strategy was designed to identify xylose transporters uninhibited by glucose. The glucose pathway was genetically blocked in Escherichia coli so that glucose functions only as an inhibitor and cells need xylose as the carbon source for survival. Through adaptive evolution, omics analysis and reverse metabolic engineering, a new phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS) galactitol transporter (GalABC, encoded by EcolC_1640, EcolC_1641, and EcolC_1642 genes) that is not inhibited by glucose was identified. Inactivation of adenylate cyclase led to increased expression of the EcolC_1642 gene, and a point mutation in gene EcolC_1642 (N13S) further enhanced xylose transport. During the second round of gene mining, AraE and a new ABC transporter (AraFGH) of xylose were identified. A point mutation in the transcription regulator araC (L156I) caused increased expression of araE and araFGH genes without arabinose induction, and a point mutation in araE (D223Y) further enhanced xylose transport. These newly identified xylose transporters can support the simultaneous consumption of glucose and xylose and have potential use in producing chemicals from lignocellulose.

Abstract Background The need to mitigate and substitute the use of fossil fuels as the main energy matrix has led to the study and development of biofuels as an alternative. Second-generation (2G) ethanol arises as one biofuel with great potential, due to not only maintaining food security, but also as a product from economically interesting crops such as energy-cane. One of the main challenges of 2G ethanol is the inefficient uptake of pentose sugars by industrial yeast Saccharomyces cerevisiae, the main organism used for ethanol production. Understanding the main drivers for xylose assimilation and identify novel and efficient transporters is a key step to make the 2G process economically viable. Results By implementing a strategy of searching for present motifs that may be responsible for xylose transport and past adaptations of sugar transporters in xylose fermenting species, we obtained a classifying model which was successfully used to select four different candidate transporters for evaluation in the S. cerevisiae hxt-null strain, EBY.VW4000, harbouring the xylose consumption pathway. Yeast cells expressing the transporters SpX, SpH and SpG showed a superior uptake performance in xylose compared to traditional literature control Gxf1. Conclusions Modelling xylose transport with the small data available for yeast and bacteria proved a challenge that was overcome through different statistical strategies. Through this strategy, we present four novel xylose transporters which expands the repertoire of candidates targeting yeast genetic engineering for industrial fermentation. The repeated use of the model for characterizing new transporters will be useful both into finding the best candidates for industrial utilization and to increase the model’s predictive capabilities. Graphical Abstract

Abstract Background The need to restructure the world’s energy matrix based on fossil fuels and mitigate greenhouse gas emissions stimulated the development of new biobased technologies for renewable energy. One promising and cleaner alternative is the use of second-generation (2G) fuels, produced from lignocellulosic biomass sugars. A major challenge on 2G technologies establishment is the inefficient assimilation of the five-carbon sugar xylose by engineered Saccharomyces cerevisiae strains, increasing fermentation time. The uptake of xylose across the plasma membrane is a critical limiting step and the budding yeast S. cerevisiae is not designed with a broad transport system and regulatory mechanisms to assimilate xylose in a wide range of concentrations present in 2G processes. Results Assessing diverse microbiomes such as the digestive tract of plague insects and several decayed lignocellulosic biomasses, we isolated several yeast species capable of using xylose. Comparative fermentations selected the yeast Candida sojae as a potential source of high-affinity transporters. Comparative genomic analysis elects four potential xylose transporters whose properties were evaluated in the transporter null EBY.VW4000 strain carrying the xylose-utilizing pathway integrated into the genome. While the traditional xylose transporter Gxf1 allows an improved growth at lower concentrations (10 g/L), strains containing Cs3894 and Cs4130 show opposite responses with superior xylose uptake at higher concentrations (up to 50 g/L). Docking and normal mode analysis of Cs4130 and Gxf1 variants pointed out important residues related to xylose transport, identifying key differences regarding substrate translocation comparing both transporters. Conclusions Considering that xylose concentrations in second-generation hydrolysates can reach high values in several designed processes, Cs4130 is a promising novel candidate for xylose uptake. Here, we demonstrate a novel eukaryotic molecular transporter protein that improves growth at high xylose concentrations and can be used as a promising target towards engineering efficient pentose utilization in yeast.

Abstract Xylose is a major component of lignocellulose and the second most abundant sugar present in nature. Efficient utilization of xylose is required for the development of economically viable processes to produce biofuels and chemicals from biomass. However, there are still some bottlenecks in the bioconversion of xylose, including the fact that some microorganisms cannot assimilate xylose naturally and that the uptake and metabolism of xylose are inhibited by glucose, which is usually present with xylose in lignocellulose hydrolysate. To overcome these issues, numerous efforts have been made to discover, characterize, and engineer the transporters and enzymes involved in xylose utilization to relieve glucose inhibition and to develop recombinant microorganisms to produce fuels and chemicals from xylose. Here we describe a recent advancement focusing on xylose-utilizing pathways, biosynthesis of chemicals from xylose, and engineering strategies used to improve the conversion efficiency of xylose.

Background: The need to restructure the world's energy matrix based on fossil fuels and mitigate greenhouse gas emissions stimulated the development of new biobased technologies for renewable energy. One promising and cleaner alternative is the use of second-generation (2G) fuels, produced from lignocellulosic biomass sugars. A major challenge on 2G technologies establishment is the inefficient assimilation of the five-carbon sugar xylose by engineered Saccharomyces cerevisiae strains, increasing fermentation time. The uptake of xylose across the plasma membrane is a critical limiting step and the budding yeast S. cerevisiae is not designed with a broad transport system and regulatory mechanisms to assimilate xylose in a wide range of concentrations present in 2G processes. Results: Assessing diverse microbiomes such as the digestive tract of plague insects and several decayed lignocellulosic biomasses, we isolated several yeast species capable of using xylose. Comparative fermentations selected the yeast Candida sojae as a potential source of high-affinity transporters. Comparative genomic analysis elects four potential xylose transporters whose properties were evaluated in the transporter null EBY.VW4000 strain carrying the xylose-utilizing pathway integrated into the genome. While the traditional xylose transporter Gxf1 allows an improved growth at lower concentrations (10 g/L), strains containing Cs3894 and Cs4130 show opposite responses with superior xylose uptake at higher concentrations (up to 50 g/L). Docking and normal mode analysis of Cs4130 and Gxf1 variants pointed out important residues related to xylose transport, identifying key differences regarding substrate translocation comparing both transporters. Conclusions: Considering that xylose concentrations in second-generation hydrolysates can reach high values in several designed processes, Cs4130 is a promising novel candidate for xylose uptake. Here, we demonstrate a novel eukaryotic molecular transporter protein that improves growth at high xylose concentrations and can be used as a promising target towards engineering efficient pentose utilization in yeast. [ABSTRACT FROM AUTHOR]

Xylose is a major component of lignocellulose and the second most abundant sugar present in nature. Efficient utilization of xylose is required for the development of economically viable processes to produce biofuels and chemicals from biomass. However, there are still some bottlenecks in the bioconversion of xylose, including the fact that some microorganisms cannot assimilate xylose naturally and that the uptake and metabolism of xylose are inhibited by glucose, which is usually present with xylose in lignocellulose hydrolysate. To overcome these issues, numerous efforts have been made to discover, characterize, and engineer the transporters and enzymes involved in xylose utilization to relieve glucose inhibition and to develop recombinant microorganisms to produce fuels and chemicals from xylose. Here we describe a recent advancement focusing on xylose-utilizing pathways, biosynthesis of chemicals from xylose, and engineering strategies used to improve the conversion efficiency of xylose. [ABSTRACT FROM AUTHOR]

Lignocellulosic ethanol has been considered as an alternative transportation fuel. Utilization of hemicellulosic fraction in lignocelluloses is crucial in economical production of lignocellulosic ethanol. However, this fraction has not efficiently been utilized by traditional yeast Saccharomyces cerevisiae . Genetically modified S. cerevisiae , which can utilize xylose, has several limitations including low ethanol yield, redox imbalance, and undesired metabolite formation similar to native xylose utilizing yeasts. Besides, xylose uptake is a major issue, where sugar transport system plays an important role. These genetically modified and wild-type yeast strains have further been engineered for improved xylose uptake. Various techniques have been employed to facilitate the xylose transportation in these strains. The present review is focused on the sugar transport machineries, mechanisms of xylose transport, limitations and how to deal with xylose transport for xylose assimilation in yeast cells. The recent advances in different techniques to facilitate the xylose transportation have also been discussed. [ABSTRACT FROM AUTHOR]

Pentoses, including xylose and arabinose, are the second most prevalent sugars in lignocellulosic biomass that can be harnessed for biological conversion. Although Yarrowia lipolytica has emerged as a promising industrial microorganism for production of high-value chemicals and biofuels, its native pentose metabolism is poorly understood. Our previous study demonstrated that Y. lipolytica (ATCC MYA-2613) has endogenous enzymes for D-xylose assimilation, but inefficient xylitol dehydrogenase causes Y. lipolytica to assimilate xylose poorly. In this study, we investigated the functional roles of native sugar-specific transporters for activating the dormant pentose metabolism in Y. lipolytica. By screening a comprehensive set of 16 putative pentose-specific transporters, we identified two candidates, YALI0C04730p and YALI0B00396p, that enhanced xylose assimilation. The engineered mutants YlSR207 and YlSR223, overexpressing YALI0C04730p and YALI0B00396p, respectively, improved xylose assimilation approximately 23% and 50% in comparison to YlSR102, a parental engineered strain overexpressing solely the native xylitol dehydrogenase gene. Further, we activated and elucidated a widely unknown native L-arabinose assimilation pathway in Y. lipolytica through transcriptomic and metabolic analyses. We discovered that Y. lipolytica can coconsume xylose and arabinose, where arabinose utilization shares transporters and metabolic enzymes of some intermediate steps of the xylose assimilation pathway. Arabinose assimilation is synergistically enhanced in the presence of xylose, while xylose assimilation is competitively inhibited by arabinose. L-Arabitol dehydrogenase is the rate-limiting step responsible for poor arabinose utilization in Y. lipolytica. Overall, this study sheds light on the cryptic pentose metabolism of Y. lipolytica and, further, helps guide strain engineering of Y. lipolytica for enhanced assimilation of pentose sugars. [ABSTRACT FROM AUTHOR]

Wood sugars such as xylose can be used as an inexpensive carbon source for biotechnological applications. The model cyanobacterium Synechocystis sp. PCC 6803 lacks the ability to catabolize wood sugars as an energy source. Here, we generated four Synechocystis strains that heterologously expressed XylAB enzymes, which mediate xylose catabolism, either in combination with or without one of three xylose transporters, namely XylE, GalP, or Glf. Except for glf, which is derived from the bacterium Zymomonas mobilis ZM4, the heterologous genes were sourced from Escherichia coli K-12. All of the recombinant strains were able to utilize xylose in the absence of catabolite repression. When xylose was the lone source of organic carbon, strains possessing the XylE and Glf transporters were most efficient in terms of dry biomass production and xylose consumption and the strain lacking a heterologous transporter was the least efficient. However, in the presence of a xylose-glucose mixed sugar source, the strains exhibited similar levels of growth and xylose consumption. This study demonstrates that various bacterial xylose transporters can boost xylose catabolism in transgenic Synechocystis strains, and paves the way for the sustainable production of bio-compounds and green fuels from lignocellulosic biomass.

Wood sugars such as xylose can be used as an inexpensive carbon source for biotechnological applications. The model cyanobacterium Synechocystis sp. PCC 6803 lacks the ability to catabolize wood sugars as an energy source. Here, we generated four Synechocystis strains that heterologously expressed XylAB enzymes, which mediate xylose catabolism, either in combination with or without one of three xylose transporters, namely XylE, GalP, or Glf. Except for glf, which is derived from the bacterium Zymomonas mobilis ZM4, the heterologous genes were sourced from Escherichia coli K-12. All of the recombinant strains were able to utilize xylose in the absence of catabolite repression. When xylose was the lone source of organic carbon, strains possessing the XylE and Glf transporters were most efficient in terms of dry biomass production and xylose consumption and the strain lacking a heterologous transporter was the least efficient. However, in the presence of a xylose-glucose mixed sugar source, the strains exhibited similar levels of growth and xylose consumption. This study demonstrates that various bacterial xylose transporters can boost xylose catabolism in transgenic Synechocystis strains, and paves the way for the sustainable production of bio-compounds and green fuels from lignocellulosic biomass. [ABSTRACT FROM AUTHOR]

Abstract: Genetically engineered Saccharomyces cerevisiae strains for renewable biofuels conversion often show limited xylose utilization and no satisfactory strain is currently available for sustainable cellulosic ethanol production. Using a genetically engineered industrial yeast strain NRRL Y-50049-YXI with a chromosomally integrated unique codon-optimized xylose isomerase as a host, we developed six new genotypes using heterologous xylose transporter genes from Scheffersomyces stipitis namely Y-50049-YXI-XUT4, -XUT5, -XUT6, -XUT7, -RGT2, and -SUT4, respectively. While a functional YXI is necessary to enable xylose utilization for Y-50049, introduction of the heterologous xylose transporter genes further improved cell growth and fermentation for all newly developed genotypes. Under aerobic conditions, all new genotypes displayed significant improvement of cell growth and xylose consumption. Among which, Genotypes Y-50049-YXI-RGT2, -XUT7, and -XUT6 showed a volumetric consumption rate ranging from 0.399 to 0.535g/L/h that is comparable with the native xylose utilizing yeast Scheffersomyces stipitis. Under oxygen-limited fermentation conditions, genotypes Y-50049-YXI-XUT7, -RGT2, and -SUT4 displayed approximately 50% increase of ethanol productivity and ethanol yield compared with their parental strain. We found RGT2 is closely related to GXS1, a glucose-xylose symporter from Candida intermedia, and functioned well as a xylose transporter gene. No obvious inhibition of xylose utilization by glucose was observed in the mixed sugars of glucose and xylose for all genotypes examined. Enhanced expression of YXI by most characterized xylose transporters suggested Y-50049-YXI as a promising host to improve xylose utilization for the next generation biocatalyst development. [Copyright &y& Elsevier]

Xylose utilization is inhibited by glucose uptake in xylose-assimilating yeasts, including Candida tropicalis, resulting in limitation of xylose uptake during the fermentation of glucose/xylose mixtures. In this study, a heterologous xylose transporter gene ( At5g17010) from Arabidopsis thaliana was selected because of its high affinity for xylose and was codon-optimized for functional expression in C. tropicalis. The codon-optimized gene was placed under the control of the GAPDH promoter and was integrated into the genome of C. tropicalis strain LXU1 which is xyl2-disrupted and NXRG (codon-optimized Neurospora crassa xylose reductase) introduced. The xylose uptake rate was increased by 37-73 % in the transporter expression-enhanced strains depending on the glucose/xylose mixture ratio. The recombinant strain LXT2 in 500-mL flask culture using glucose/xylose mixtures showed a xylose uptake rate that was 29 % higher and a xylitol volumetric productivity (1.14 g/L/h) that was 25 % higher than the corresponding rates for control strain LXU1. Membrane protein extraction and Western blot analysis confirmed the successful heterologous expression and membrane localization of the xylose transporter in C. tropicalis. [ABSTRACT FROM AUTHOR]

Abstract: It has been known that wild type Bacillus subtilis cannot grow rapidly in a minimal medium containing xylose as a sole carbon source because it does not have a xylose-specific transporter. In this study, the arabinose:H+ symporter, AraE protein from B. subtilis was expressed in B. subtilis 168 in order to transport xylose efficiently. The AraE expression cassette was constructed to contain the xylose-inducible xylA promoter, araE gene and fba terminator, and integrated into the chromosomal amyE gene in B. subtilis 168. Batch cultures in a defined medium with xylose only or a mixture of xylose and glucose showed that expression of AraE led to fast and complete consumption of initially added xylose and hence a considerable increase in cell growth of the recombinant B. subtilis JY123 expressing AraE. Considering the systematic analysis of cell growth, sugar consumption, respiratory quotient and xylulokinase activity, it was certain that AraE protein could transport xylose into B. subtilis efficiently. [Copyright &y& Elsevier]

Abstract: Efficient use of xylose is necessary for economic production of biochemicals and biofuels from lignocellulosic materials. Current studies on xylose uptake for various microorganisms have been hampered by the lack of a facile assay for xylose transport. In this work, a rapid in vivo, label-free method for measuring xylose transport in Escherichia coli was developed by taking advantage of the Bacillus pumilus xylosidase (XynB), which cleaved a commercially available xylose analog, p-nitrophenyl-β-d-xylopyranoside (pNPX), to release a chromogenic group, p-nitrophenol (pNP). XynB was expressed alone or in conjunction with a Zymomonas mobilis glucose facilitator protein (Glf) capable of transporting xylose. This XynB-mediated transport assay was demonstrated in test tubes and 96-well plates with submicromolar concentrations of pNPX. Kinetic inhibition experiments validated that pNPX and xylose were competitive substrates for the transport process, and the addition of glucose (20g/L) in the culture medium clearly diminished the transmembrane transport of pNPX and, thus, mimicked its inhibitory action on xylose uptake. This method should be useful for engineering of the xylose transport process in E. coli, and similar assay schemes can be extended to other microorganisms. [Copyright &y& Elsevier]

Glucose and xylose are two major components of lignocellulose. Simultaneous consumption of glucose and xylose is critical for engineered microorganisms to produce fuels and chemicals from lignocellulosic biomass. Although many production limitations have been resolved, glucose-induced inhibition of xylose transport remains a challenge. In this study, a cell growth-based screening strategy was designed to identify xylose transporters uninhibited by glucose. The glucose pathway was genetically blocked in Escherichia coli so that glucose functions only as an inhibitor and cells need xylose as the carbon source for survival. Through adaptive evolution, omics analysis and reverse metabolic engineering, a new phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS) galactitol transporter (GalABC, encoded by EcolC_1640 , EcolC_1641 , and EcolC_1642 genes) that is not inhibited by glucose was identified. Inactivation of adenylate cyclase led to increased expression of the EcolC_1642 gene, and a point mutation in gene EcolC_1642 (N13S) further enhanced xylose transport. During the second round of gene mining, AraE and a new ABC transporter (AraFGH) of xylose were identified. A point mutation in the transcription regulator araC (L156I) caused increased expression of araE and araFGH genes without arabinose induction, and a point mutation in araE (D223Y) further enhanced xylose transport. These newly identified xylose transporters can support the simultaneous consumption of glucose and xylose and have potential use in producing chemicals from lignocellulose., Competing Interests: This study has been included in patent applications by the Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences., (© 2022 The Authors. mLife published by John Wiley & Sons Australia, Ltd. on behalf of Institute of Microbiology, Chinese Academy of Sciences.)

Pentoses, including xylose and arabinose, are the second most prevalent sugars in lignocellulosic biomass that can be harnessed for biological conversion. Although Yarrowia lipolytica has emerged as a promising industrial microorganism for production of high-value chemicals and biofuels, its native pentose metabolism is poorly understood. Our previous study demonstrated that Y. lipolytica (ATCC MYA-2613) has endogenous enzymes for d-xylose assimilation, but inefficient xylitol dehydrogenase causes Y. lipolytica to assimilate xylose poorly. In this study, we investigated the functional roles of native sugar-specific transporters for activating the dormant pentose metabolism in Y. lipolytica By screening a comprehensive set of 16 putative pentose-specific transporters, we identified two candidates, YALI0C04730p and YALI0B00396p, that enhanced xylose assimilation. The engineered mutants YlSR207 and YlSR223, overexpressing YALI0C04730p and YALI0B00396p, respectively, improved xylose assimilation approximately 23% and 50% in comparison to YlSR102, a parental engineered strain overexpressing solely the native xylitol dehydrogenase gene. Further, we activated and elucidated a widely unknown native l-arabinose assimilation pathway in Y. lipolytica through transcriptomic and metabolic analyses. We discovered that Y. lipolytica can coconsume xylose and arabinose, where arabinose utilization shares transporters and metabolic enzymes of some intermediate steps of the xylose assimilation pathway. Arabinose assimilation is synergistically enhanced in the presence of xylose, while xylose assimilation is competitively inhibited by arabinose. l-Arabitol dehydrogenase is the rate-limiting step responsible for poor arabinose utilization in Y. lipolytica Overall, this study sheds light on the cryptic pentose metabolism of Y. lipolytica and, further, helps guide strain engineering of Y. lipolytica for enhanced assimilation of pentose sugars. IMPORTANCE The oleaginous yeast Yarrowia lipolytica is a promising industrial-platform microorganism for production of high-value chemicals and fuels. For decades since its isolation, Y. lipolytica has been known to be incapable of assimilating pentose sugars, xylose and arabinose, that are dominantly present in lignocellulosic biomass. Through bioinformatic, transcriptomic, and enzymatic studies, we have uncovered the dormant pentose metabolism of Y. lipolytica Remarkably, unlike most yeast strains, which share the same transporters for importing hexose and pentose sugars, we discovered that Y. lipolytica possesses the native pentose-specific transporters. By overexpressing these transporters together with the rate-limiting d-xylitol and l-arabitol dehydrogenases, we activated the dormant pentose metabolism of Y. lipolytica Overall, this study provides a fundamental understanding of the dormant pentose metabolism of Y. lipolytica and guides future metabolic engineering of Y. lipolytica for enhanced conversion of pentose sugars to high-value chemicals and fuels., (Copyright © 2018 American Society for Microbiology.)

Background: Engineering of Saccharomyces cerevisiae for the simultaneous utilization of hexose and pentose sugars is vital for cost-efficient cellulosic bioethanol production. This yeast lacks specific pentose transporters and depends on endogenous hexose transporters for low affinity pentose uptake. Consequently, engineered xylose-fermenting yeast strains first utilize D-glucose before D-xylose can be transported and metabolized., Results: We have used an evolutionary engineering approach that depends on a quadruple hexokinase deletion xylose-fermenting S. cerevisiae strain to select for growth on D-xylose in the presence of high D-glucose concentrations. This resulted in D-glucose-tolerant growth of the yeast of D-xylose. This could be attributed to mutations at N367 in the endogenous chimeric Hxt36 transporter, causing a defect in D-glucose transport while still allowing specific uptake of D-xylose. The Hxt36-N367A variant transports D-xylose with a high rate and improved affinity, enabling the efficient co-consumption of D-glucose and D-xylose., Conclusions: Engineering of yeast endogenous hexose transporters provides an effective strategy to construct glucose-insensitive xylose transporters that are well integrated in the carbon metabolism regulatory network, and that can be used for efficient lignocellulosic bioethanol production.