Transcription Analysis of Recombinant Trichoderma reesei HJ-48 to Compare the Molecular Basis for Fermentation of Glucose and Xylose
National Engineering Research Center for Non-Food Biorefinery, State Key Laboratory of Non-Food Biomass and Enzyme Technology, Guangxi Key Laboratory of Biorefinery, Guangxi Biomass Engineering Technology Research Center, Guangxi Academy of Sciences, Nanning 530007, P.R. ChinaCorrespondence to:
J. Microbiol. Biotechnol. 2020; 30(10): 1467-1479
Published October 28, 2020
Copyright © The Korean Society for Microbiology and Biotechnology.
Lignocellulosic biomass, such as corn stover, sugarcane bagasse, and straw, are among the most attractive feedstocks for bioethanol production. Sugar derived from plant biomass is a mixture of hexoses and pentoses. The yeast species
Filamentous fungi are the primary source of cellulase and possess a remarkable capacity for extracellular protein production. With regard to commercial production of cellulases,
Genome-wide studies involving expressed sequence tags, cDNA microarrays, and transcriptomics have been carried out to study the various genes related to glucose metabolism [10, 11]. We previously performed a comprehensive metabolome analysis using transcriptomics between the recombinant strain HJ48 and the parent strain CICC40360 during fermentation with glucose, and demonstrated that a series of glycolysis enzymes are upregulated in the recombinant strain HJ48 under fermentation conditions . Although these studies provide a metabolic analysis for the low ethanol productivity of CICC40360 compared to HJ48, little is known about transcriptional differences between aerobic glucose and xylose fermentation by the recombinant strain HJ48. In this research we investigated the genes involved in the high ethanol productivity when grown using either xylose or glucose as a sole carbon source medium in the HJ48 strain. This study provides a genome-wide analysis of the transcriptional landscape of
Materials and Methods
Strains and Fermentation
Total RNA from samples was isolated using TRIzol (Invitrogen Life Technologies, USA) according to the manufacturer’s instructions, and treated with RNase-free DNase I (Tiangen, China) to remove any DNA contamination. Qubit RNA Assay Kit (Life Technologies, USA) and Nano 6000 Assay Kit (Agilent Technologies, USA) were used to check the RNA concentration and integrity. Three micrograms of RNA per sample was used for the RNA sample preparations. The sequencing libraries were created by NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, USA). mRNA was purified from total RNA using Poly-T oligo-attached magnetic beads. RNA fragmentation and cDNA synthesis were performed according to a protocol from a previous study . Library preparations were sequenced on an Illumina HiSeq 2500 platform, and 100 bp paired-end reads were generated. Clean data obtained in the FASTQ format were performed through in-house Perl scripts. Clean data were then analyzed by removing adapter, low-quality reads, and reads containing ploy-N from the raw data. The Q20, Q30, and GC content of the clean data were calculated. The
EdgeR was used for each sequenced library via one scaling normalized factor to adjust the read counts. DEGSeq R package (1.12.0) was used to analysis differentially expressed genes of two conditions, a q-value (corrected
Real-Time Quantitative PCR (qPCR)
qPCR was used to check the expression of thirteen genes involved in different metabolism pathways. Reverse transcription was done using the PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa Japan) and qPCR was done using the TB Green Premix Ex Taq II Kit (TaKaRa) in Agilent Technologies AriaMx Real-Time PCR, each with three repeats. The relative expression levels of each gene were implemented using the 2-ΔΔCT method, with the expression of glucose fermentation as the control and the expression of the tubulin gene as the internal standard. Primers for qPCR are listed in Table 1.
To study the effects of different carbon sources, specifically glucose and xylose, on ethanol fermentation, fermentation by the recombinant
Time course of glucose consumption, xylose consumption (A), and ethanol production (B) by HJ48 under anaerobic conditions in GM medium containing 50 g/l glucose and XM medium containing 50 g/l xylose.The values are means of triplicate experiments. Error bars indicate standard deviations.
When xylose was provided as a sole carbon source, half of the xylose was consumed by HJ48 after 72 h of cultivation, demonstrating that the recombinant strain HJ48 consumes xylose at a slower rate than it does glucose after 72 h of cultivation. Additionally, an ethanol concentration of 1.1 g/l was detected. The maximum ethanol production of the recombinant strain HJ48 achieved in fermentation with XM was lower than that observed with GM.
Transcriptome Analysis of
T. reesei Strain HJ48 Growth on Xylose Versus Glucose
The transcriptome of
Figure 2. Overview of the differentially expressed genes between xylose and glucose.
We next performed gene ontology (GO) term analysis to classify the functions of the differentially expressed genes. Surprisingly, we did not identify any significant enrichment in GO terms (
Histogram of gene ontology classification.Gene ontology (GO) enrichment analysis of differentially expressed genes was implemented by the goseq R package. The results are summarized in three main categories: biological process, cellular component, and molecular function.
The relationship of upregulated GO terms involved in cellular components.Gene enrichment is represented by color, and the darker colors signify increased enrichment.
In order to categorize the genes found to be differentially expressed into functional pathways between xylose and glucose utilized by the recombinant strain HJ48, the genes were classified based on KEGG enrichment using FDR ≤ 0.05. The upregulated expression genes between xylose and glucose utilized by the recombinant strain HJ48 were predominantly functionally categorized into 20 pathways, including ribosome [tre03010], proteasome [tre03050], peroxisome [tre04146], alanine, aspartate and glutamate metabolism [tre00250], metabolic pathways [tre01100], aminoacyl-tRNA biosynthesis [tre00970], valine, leucine and isoleucine degradation [tre00280], and amino sugar and nucleotide sugar metabolism [tre00520]. Statistical analysis showed that the differentially expressed genes were enriched for those involved in ribosome [tre03010] (FDR ≤ 0.05). It was interesting to note that the upregulated expression genes were enriched for sugar metabolism involved in citrate cycle (TCA cycle)[tre00020], glycolysis/gluconeogenesis [tre00010], pentose phosphate pathway [tre00030], carbon metabolism [tre01200], starch and sucrose metabolism [tre00500], and fructose and mannose metabolism [tre00051], although this enrichment was not statistically significant (FDR ≤ 0.05).
In order to assess the accuracy of the RNA-Seq results, qPCR was implemented. Thirteen genes involved in different metabolism pathways were randomly selected (S1). The values shown are the mean values of three parallel experiments. As expected, the results of qPCR were consistent with those of RNA-Seq, indicating that the RNA-Seq results were credible (Fig. 5).
qPCR analysis of the selected genes.G: glucose. X: xylose. (XR: xylose reductase; XDH: xylitol dehydrogenase; G6PDH: glucose-6-phosphate dehydrogenase; TAL: transaldolase; TKL: transketolase; PYK: pyruvate kinase; PCK: phosphoenolpyruvate carboxykinase; PDC: pyruvate decarboxylase; RPE1: ribulose-5-phosphate 3-epimerase1; PC: pyruvate carboxylase; FH: fumarate hydratase; ADH: alcohol dehydrogenase)
Analysis of Genes Involved in Central Carbon Pathways
The fermentation of xylose to ethanol is achieved in
Expression profiles of genes involved in central carbon metabolism (including glycolysis, PPP, and TCA cycle) in HJ48 during xylose and glucose fermentation.The red boxes indicate transcriptional upregulation; the green boxes indicate transcriptional downregulation; the white boxes indicate no significant change in transcription; the yellow boxes indicate differences in the expression of isoenzymes.
Xylose must first be converted into xylulose, which is then phosphorylated by xylulokinase. Xylose reductase (XR), the first enzyme in the oxidoredutive pathway, is fundamental for xylose utilization . Interestingly, the expression of xylose reductase and xylitol dehydrogenase (XDH) was found to be upregulated in this study. The xylulose kinase (XK) however, was not differentially expressed in
The pentose phosphate pathway is subdivided into two biochemical branches known as the oxidative and non-oxidative pentose phosphate pathway . In the oxidative component of the pentose phosphate pathway, the enzymatic reactions are considered unidirectional. In order to help replenish NADPH-reducing equivalents, the enzymes glucose-6-phosphate dehydrogenase (G6PDH) and NADPH-producing 6-phosphogluconate dehydrogenase (6PGDH) are significantly upregulated with xylose in
The expression levels of several genes in the tricarboxylic acid cycles were upregulated with xylose compared to glucose. ATP-citrate synthase subunit 1 (ACL1) and ATP-citrate synthase subunit 2 (ACL2), which catalyze the condensation of acetyl coenzyme A and oxaloacetate to form citrate, were upregulated on xylose. Aconitate hydratase (ACO), which is repressed by ethanol, was expressed at a relatively low level during the xylose fermentation. The expression level of isocitrate dehydrogenase remained unchanged, whereas the expression of isocitrate dehydrogenase subunit 1 (IDH1), a mitochondrial NADP+-dependent enzyme, decreased with xylose. As for the KGD genes that encode 2-oxoglutarate dehydrogenase, the expression level of KGD1 and KGD2 encoding 2-oxoglutarate dehydrogenase E1 and 2-oxoglutarate dehydrogenase E2 components, respectively, were unchanged. Succinyl-CoA synthetase, various subunits of succinate dehydrogenase, as well as fumarate hydratase were all significantly upregulated with xylose. The expression level of cytoplasmic malate dehydrogenase (MDH), which is involved in the gluconeogenesis and glyoxylate cycles, was found to be unchanged. We also observed that genes encoding enzymes of the tricarboxylic acid cycle and respiration were upregulated during xylose metabolism. Moreover, Hap2, a subunit of the transcriptional activator complex Hap2/3/5, was upregulated with xylose under anaerobic conditions. In contrast, Hap3 was found to be upregulated during glucose fermentation.
We next analyzed the expression level of genes involved in glycolysis and alcohol fermentation during xylose and glucose fermentation. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of hexokinases, which catalyze the conversion of glucose to glucose-6-phosphate, revealed that three genes were differentially expressed (TRIREDRAFT_73665, TRIREDRAFT_80231, TRIREDRAFT_79677). TRIREDRAFT_73665 and TRIREDRAFT_80231 were more highly expressed during xylose fermentation than during glucose fermentation. In contrast, the gene TRIREDRAFT_79677 was expressed more highly with glucose than with xylose. In
The expression levels of several enzymes in the lower half of the glycolytic pathway were unchanged, with the exception of enolase (ENO) and fructose-bisphosphate aldolase (FBA), which were downregulated, and pyruvate kinase and phosphoenolpyruvate carboxykinase (PCK), which were upregulated, with xylose compared to that with glucose. In the ethanol fermentation pathway, several genes showed significantly different expression. The isozymes of the pyruvate decarboxylase (PDC) gene TRIREDRAFT_121534 were upregulated with xylose, whereas expression of TRIREDRAFT_59267 was not significantly different. Transcripts of ADH, which encodes alcohol dehydrogenase, decreased with xylose. The result is consistent with our previous finding that only one alcohol dehydrogenase gene (TRIREDRAFT_22633), which catalyzes the reduction of acetaldehyde to ethanol, was upregulated in
Analysis of Genes Involved in Sugar Transport
In the present study, several of the transporter genes in
We demonstrated from the genomic level the enormous advantages of using the filamentous fungus,
Xylose is converted to xylulose in filamentous fungi via the oxidoreductive pathway, which includes two reactions. First, xylose is reduced to xylitol by a NAD(P)H-dependent xylose reductase. Then, xylitol is oxidized to xylulose by a NADP+-dependent xylitol dehydrogenase [29, 30].
On the contrary, the genome of
The efficiency of the pentose phosphate pathway has been considered a barrier to efficient xylose utilization by
Many genes coding for enzymes of the tricarboxylic acid cycle were not repressed in glucose-rich medium. However, those genes were still expressed at a relatively high level in xylose-rich medium compared to glucose in our study (Figs. 5 and 6). Meanwhile, the genes encoding respiratory enzymes were also upregulated during xylose metabolism compared to glucose. The Hap complex consists of four subunits:
The transport of xylose into the cell is the initial rate-limiting step of xylose utilization [48, 49]. The predominant sugar transporters in
Furthermore, transporter genes can be engineered to improve xylose uptake activity by introducing directed evolution. Using directed evolution, four novel xylose transporters that remained inhibited by glucose were created. Young
Industrial microorganisms that can produce alcohol utilizing both hexose and pentose sugars simultaneously are essential for reducing the cost of lignocellulose conversion to bioethanol.
Supplementary data for this paper are available on-line only at http://jmb.or.kr.
This study was funded by the General Program of the Natural Science Foundation of Guangxi (2018GXNSFAA 294118,2018GXNSFAA138111,2018GXNSFAA294035,2020GXNSFBA159021,2020GXNSFAA259021), the central government directs special funds for local science and technology development projects (ZY1949015), Guangxi major science and technology innovation base construction project (2018-15-Z03-1208/2018-15-Z03-1209).
Conflict of Interest
The authors have no financial conflicts of interest to declare.
- Zheng JN, Negi A, Khomlaem C, Kim BS. 2019. Comparison of bioethanol production by Candida molischiana and Saccharomyces cerevisiae from glucose, cellobiose, and cellulose. J. Microbiol. Biotechnol. 29: 905-912.
- Kumar V, Binod P, Sindhu R, Gnansounou E, Ahluwalia V. 2018. Bioconversion of pentose sugars to value added chemicals and fuels:Recent trends, challenges and possibilities. Bioresour. Technol. 269: 443-451.
- Tang HT, Hou J, Shen Y, Xu LL, Yang H, Fang X, et al. 2013. High beta-glucosidase secretion in Saccharomyces cerevisiae improves the efficiency of cellulase hydrolysis and ethanol production in simultaneous saccharification and fermentation. J. Microbiol. Biotechnol. 23: 1577-1585.
- Nijland JG, Vos E, Shin HY, de Waal PP, Klaassen P, Driessen AJ. 2016. Improving pentose fermentation by preventing ubiquitination of hexose transporters in Saccharomyces cerevisiae. Biotechnol. Biofuels 9: 158.
- Liu H, Sun J, Chang JS, Shukla P. 2018. Engineering microbes for direct fermentation of cellulose to bioethanol. Crit. Rev. Biotechnol. 38: 1089-1105.
- Zou ZS, Zhao YY, Zhang TZ, Xu JX, He AY, Deng Y. 2018. Efficient isolation and characterization of a cellulase hyperproducing mutant strain of Trichoderma reesei. J. Microbiol. Biotechnol. 28: 1473-1481.
- Xu Q, Singh A, Himmel ME. 2009. Perspectives and new directions for the production of bioethanol using consolidated bioprocessing of lignocellulose. Curr. Opin. Biotechnol. 20: 364-371.
- Li YH, Zhang XY, Zhang F, Peng LC, Zhang DB, Kondo A, et al. 2018. Optimization of cellulolytic enzyme components through engineering Trichoderma reesei and on-site fermentation using the soluble inducer for cellulosic ethanol production from corn stover. Biotechnol. Biofuels 11: 49.
- Huang J, Chen D, Wei Y, Wang Q, Li Z, Chen Y, et al. 2014. Direct ethanol production from lignocellulosic sugars and sugarcane bagasse by a recombinant Trichoderma reesei strain HJ48. ScientificWorldJournal. 2014: 798683.
- Walker ME, Nguyen TD, Liccioli T, Schmid F, Kalatzis N, Sundstrom JF, et al. 2014. Genome-wide identification of the Fermentome;genes required for successful and timely completion of wine-like fermentation by Saccharomyces cerevisiae. BMC Genomics 15: 552.
- Matsushika A, Goshima T, Hoshino T. 2014. Transcription analysis of recombinant industrial and laboratory Saccharomyces cerevisiae strains reveals the molecular basis for fermentation of glucose and xylose. Microb. Cell Fact. 13: 16.
- Huang J, Wu R, Chen D, Wang Q, Huang R. 2016. Transcriptional profiling of the Trichoderma reesei recombinant strain HJ48 by RNA-Seq. J. Microbiol. Biotechnol. 26: 1242-1251.13.
- Yang M, Xu L, Liu Y, Yang P. 2015. RNA-Seq uncovers SNPs and alternative splicing events in asian lotus (Nelumbo nucifera). PLoS One. 10: e0125702.
- Martinez D, Berka RM, Henrissat B, Saloheimo M, Arvas M, Baker SE, et al. 2008. Genome sequencing and analysis of the biomassdegrading fungus Trichoderma reesei (syn. Hypocrea jecorina). Nat. Biotechnol. 26: 553-560.
- Kim D, Pertea G, Trapnell C, Pimentel H, Kelley R, Salzberg SL. 2013. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 14: R36.
- Anders S, Pyl PT, Huber W. 2015. HTSeq-a Python framework to work with high-throughput sequencing data. Bioinformatics 31:166-169.
- Mortazavi A, Williams BA, Mccue K, Schaeffer L, Wold B. 2008. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat. Methods 5: 621-628.
- Wang L, Feng Z, Wang X, Wang X, Zhang X. 2010. DEGseq: an R package for identifying differentially expressed genes from RNAseq data. Bioinformatics 26: 136-138.
- Benjamini Y, Hochberg Y. 1995. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. R. Statist. Soc. B 1: 289-300.
- Anders S, Huber W. 2010. Differential expression analysis for sequence count data. Genome Biol. 11: R106.
- Mao XZ, Cai T, Olyarchuk JG, Wei LP. 2005. Automated genome annotation and pathway identification using the KEGG Orthology (KO) as a controlled vocabulary. Bioinformatics 21: 3787-3793.
- Li J, Lin L, Li H, Tian C, Ma Y. 2014. Transcriptional comparison of the filamentous fungus Neurospora crassa growing on three major monosaccharides D-glucose, D-xylose and L-arabinose. Biotechnol. Biofuels 7: 31.
- Stincone A, Prigione A, Cramer T, Wamelink MM, Campbell K, Cheung E, et al. 2015. The return of metabolism: biochemistry and physiology of the pentose phosphate pathway. Biol. Rev. Camb. Philos. Soc. 90: 927-963.
- Matsushika A, Goshima T, Hoshino T. 2014. Transcription analysis of recombinant industrial and laboratory Saccharomyces cerevisiae strains reveals the molecular basis for fermentation of glucose and xylose. Microb. Cell Fact. 13: 16.
- Runquist D, Hahn-Hagerdal B, Bettiga M. 2009. Increased expression of the oxidative pentose phosphate pathway and gluconeogenesis in anaerobically growing xylose-utilizing Saccharomyces cerevisiae. Microb. Cell Fact. 8: 49.
- Rodriguez A, de la Cera T, Herrero P, Moreno F. 2001. The hexokinase 2 protein regulates the expression of the GLK1, HXK1 and HXK2 genes of Saccharomyces cerevisiae. Biochem. J. 355: 625-631.
- Brown SR, Staff M, Lee R, Love J, Parker DA, Aves SJ, et al. 2018. Design of experiments methodology to build a multifactorial statistical model describing the metabolic interactions of alcohol dehydrogenase isozymes in the ethanol biosynthetic pathway of the yeast Saccharomyces cerevisiae. Acs Synth. Biol. 7: 1676-1684.
- Yao YX, Li M, Zhai H, You CX, Hao YJ. 2011. Isolation and characterization of an apple cytosolic malate dehydrogenase gene reveal its function in malate synthesis. J. Plant Physiol. 168: 474-480.
- Zeng WY, Tang YQ, Gou M, Xia ZY, Kida K. 2016. Transcriptomes of a xylose-utilizing industrial flocculating Saccharomyces cerevisiae strain cultured in media containing different sugar sources. AMB Express 6: 51.
- Hossain S, Švec D, Mrša V, Teparić R. 2018. Overview of catalytic properties of fungal xylose reductases and molecular engineering approaches for improved xylose utilisation in yeast. Appl. Food Biotechnol. 5: 47-58.
- Li HB, Schmitz O, Alper HS. 2016. Enabling glucose/xylose co-transport in yeast through the directed evolution of a sugar transporter. Appl. Microbiol. Biotechnol. 100: 10215-10223.
- Moyses DN, Reis VC, de Almeida JR, de Moraes LM, Torres FA. 2016. Xylose fermentation by Saccharomyces cerevisiae: challenges and prospects. Int. J. Mol. Sci. 17: 207.
- Yang BX , Xie CY , Xia ZY, Wu YJ. 2020. The effect of xylose reductase genes on xylitol production by industrial Saccharomyces cerevisiae in fermentation of glucose and xylose. Process Biochem. 95: 122-130.
- Hong Y, Dashtban M, Kepka G, Chen S, Qin W. 2014. Overexpression of D-xylose reductase (xyl1) gene and antisense inhibition of D-xylulokinase (xyiH) gene increase xylitol production in Trichoderma reesei. Biomed. Res. Int. 2014: 169705.
- Perl A, Hanczko R, Telarico T, Oaks Z, Landas S. 2011. Oxidative stress, inflammation and carcinogenesis are controlled through the pentose phosphate pathway by transaldolase. Trends Mol. Med. 17: 395-403.
- Souto-Maior AM, Runquist D, Hahn-Hagerdal B. 2009. Crabtree-negative characteristics of recombinant xylose-utilizing Saccharomyces cerevisiae. J. Biotechnol. 143: 119-123.
- Kobayashi Y, Sahara T, Suzuki T, Kamachi S, Matsushika A, Hoshino T, et al. 2017. Genetic improvement of xylose metabolism by enhancing the expression of pentose phosphate pathway genes in Saccharomyces cerevisiae IR-2 for high-temperature ethanol production. J. Ind. Microbiol. Biotechnol. 44: 879-891.
- Feng Q, Liu ZL, Weber SA, Li S. 2018. Signature pathway expression of xylose utilization in the genetically engineered industrial yeast Saccharomyces cerevisiae. PLoS One 13: e0195633.
- Chomvong K, Bauer S, Benjamin DI, Li X, Nomura DK, Cate JHD. 2016. Bypassing the pentose phosphate pathway: towards modular utilization of xylose. PLoS One 11: e0158111.
- Kurylenko OO, Ruchala J, Vasylyshyn RV, Stasyk OV, Dmytruk OV, Dmytruk KV, et al. 2018. Peroxisomes and peroxisomal transketolase and transaldolase enzymes are essential for xylose alcoholic fermentation by the methylotrophic thermotolerant yeast, Ogataea (Hansenula) polymorpha. Biotechnol. Biofuels 11: 197.
- Jeppsson M, Johansson B, Hahn-Hagerdal B, Gorwa-Grauslund MF. 2002. Reduced oxidative pentose phosphate pathway flux in recombinant xylose-utilizing Saccharomyces cerevisiae strains improves the ethanol yield from xylose. Appl. Environ. Microbiol. 68:1604-1609.
- Miskovic L, Alff-Tuomala S, Soh KC, Barth D, Salusjarvi L, Pitkanen JP, et al. 2017. A design-build-test cycle using modeling and experiments reveals interdependencies between upper glycolysis and xylose uptake in recombinant S. cerevisiae and improves predictive capabilities of large-scale kinetic models. Biotechnol. Biofuels 10: 166.
- Matsushika A, Nagashima A, Goshima T, Hoshino T. 2013. Fermentation of xylose causes inefficient metabolic state due to carbon/energy starvation and reduced glycolytic flux in recombinant industrial Saccharomyces cerevisiae. PLoS One 8: e69005.
- Hortschansky P, Eisendle M, Al-Abdallah Q, Schmidt AD, Bergmann S, Thon M, et al. 2007. Interaction of HapX with the CCAATbinding complex—a novel mechanism of gene regulation by iron. EMBO J. 26: 3157-3168.
- Zeilinger S, Ebner A, Marosits T, Mach R, Kubicek CP. 2001. The Hypocrea jecorina HAP 2/3/5 protein complex binds to the inverted CCAAT-box (ATTGG) within the cbh2 (cellobiohydrolase II-gene) activating element. Mol. Genet. Genomics 266: 56-63.
- Buschlen S, Amillet JM, Guiard B, Fournier A, Marcireau C, Bolotin-Fukuhara M. 2003. The S. cerevisiae HAP complex, a key regulator of mitochondrial function, coordinates nuclear and mitochondrial gene expression. Comp. Funct. Genomics 4: 37-46.
- Kato M. 2014. An overview of the CCAAT-Box binding factor in filamentous fungi: assembly, nuclear translocation, and transcriptional enhancement. Biosci. Biotechnol. Biochem. 69: 663-672.
- Young EM, Tong A, Bui H, Spofford C, Alper HS. 2014. Rewiring yeast sugar transporter preference through modifying a conserved protein motif. Proc. Natl. Acad. Sci. USA 111: 131-136.
- Colabardini AC, Ries LNA, Brown NA, Reis TFd, Savoldi M, Goldman MHS, et al. 2014. Functional characterization of a xylose transporter in Aspergillus nidulans. Biotechnol. Biofuels. 7: 46.
- Ozcan S, Johnston M. 1999. Function and regulation of yeast hexose transporters. Microbiol. Mol. Biol. Rev. 63: 554-569.
- MH Saier Jr, Beatty JT, Goffeau A, Harley KT, Heijne WHM, Huang SC, et al. 1999. The major facilitator superfamily. J. Mol. Microbiol. Biotechnol. 1: 257-279.
- Sharma NK, Behera S, Arora R, Kumar S, Sani RK. 2018. Xylose transport in yeast for lignocellulosic ethanol production: current status. J. Biosci. Bioeng. 125: 259-267.
- Du J, Li S, Zhao H. 2010. Discovery and characterization of novel d-xylose-specific transporters from Neurospora crassa and Pichia stipitis. Mol. Biosyst. 6: 2150-2156.
- Sloothaak J, Tamayo-Ramos JA, Odoni DI, Laothanachareon T, Derntl C, Mach-Aigner AR, et al. 2016. Identification and functional characterization of novel xylose transporters from the cell factories Aspergillus niger and Trichoderma reesei. Biotechnol. Biofuels 9: 148.