Abstract
Second-generation ethanol, a promising biofuel for reducing greenhouse gas emissions, faces challenges due to the inefficient metabolism of xylose, a pentose sugar. Overcoming this hurdle requires exploration of genes, pathways, and organisms capable of fermenting xylose. Thermoanaerobacterium saccharolyticum is an organism capable of naturally fermenting compounds of industrial interest, such as xylose, and understanding evolutionary adaptations may help to bring novel genes and information that can be used for industrial yeast, increasing production of current bio-platforms. This study presents a deep evolutionary study of members of the firmicutes clade, focusing on adaptations in Thermoanaerobacterium saccharolyticum that may be related to overall fermentation metabolism, especially for xylose fermentation. One highlight is the finding of positive selection on a xylose-binding protein of the xylFGH operon, close to the annotated sugar binding site, with this protein already being found to be expressed in xylose fermenting conditions in a previous study. Results from this study can serve as basis for searching for candidate genes to use in industrial strains or to improve Thermoanaerobacterium saccharolyticum as a new microbial cell factory, which may help to solve current problems found in the biofuels’ industry.
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Data generated for this work have been made available publicly at https://zenodo.org/record/7708328
References
Adler M, Anjum M, Berg OG et al (2014) High fitness costs and instability of gene duplications reduce rates of evolution of new genes by duplication-divergence mechanisms. Mol Biol Evol 31:1526–1535. https://doi.org/10.1093/molbev/msu111
Albalat R, Cañestro C (2016) Evolution by gene loss. Nat Rev Genet 17:379–391. https://doi.org/10.1038/nrg.2016.39
Borelli G, Fiamenghi MB, Dos Santos LV et al (2019) Positive selection evidence in xylose-related genes suggests methylglyoxal reductase as a target for the improvement of yeasts’ fermentation in industry. Genome Biol Evol 11:1923–1938. https://doi.org/10.1093/gbe/evz036
Bratlie MS, Johansen J, Sherman BT et al (2010) Gene duplications in prokaryotes can be associated with environmental adaptation. BMC Genom. https://doi.org/10.1186/1471-2164-11-588
Buchfink B, Xie C, Huson DH (2014) Fast and sensitive protein alignment using DIAMOND. Nat Methods 12:59–60. https://doi.org/10.1038/nmeth.3176
Bueno JGR, Borelli G, Corrêa TLR et al (2020) Novel xylose transporter Cs4130 expands the sugar uptake repertoire in recombinant Saccharomyces cerevisiae strains at high xylose concentrations. Biotechnol Biofuels 13:145. https://doi.org/10.1186/s13068-020-01782-0
Burroughs AM, Allen KN, Dunaway-Mariano D, Aravind L (2006) Evolutionary genomics of the HAD Superfamily: understanding the structural adaptations and catalytic diversity in a superfamily of phosphoesterases and allied enzymes. J Mol Biol 361:1003–1034. https://doi.org/10.1016/j.jmb.2006.06.049
Cui J, Davidson AL (2011) ABC solute importers in bacteria. Essays Biochem 50:85–99. https://doi.org/10.1042/BSE0500085
Cui J, Maloney MI, Olson DG, Lynd LR (2020) Conversion of phosphoenolpyruvate to pyruvate in Thermoanaerobacterium saccharolyticum. Metab Eng Commun 10:e00122. https://doi.org/10.1016/j.mec.2020.e00122
Currie DH, Raman B, Gowen CM et al (2015) Genome-scale resources for Thermoanaerobacterium saccharolyticum. BMC Syst Biol 9:1–15. https://doi.org/10.1186/s12918-015-0159-x
Dai L, Chang Z, Yang J et al (2021) Structural investigation of a thermostable 1,2-β-mannobiose phosphorylase from Thermoanaerobacter sp. X-514. Biochem Biophys Res Commun 579:54–61. https://doi.org/10.1016/j.bbrc.2021.09.046
De Bie T, Cristianini N, Demuth JP, Hahn MW (2006) CAFE: A computational tool for the study of gene family evolution. Bioinformatics 22:1269–1271. https://doi.org/10.1093/bioinformatics/btl097
Dideberg O, Charlier P, Dive G et al (1982) Structure of a Zn 2+ -containing D -alanyl- D -alanine-cleaving carboxypeptidase at 2.5 Å resolution. Nature 299(5882):469–470. https://doi.org/10.1038/299469a0
Emms DM, Kelly S (2015) OrthoFinder: solving fundamental biases in whole genome comparisons dramatically improves orthogroup inference accuracy. Genome Biol 16:157. https://doi.org/10.1186/s13059-015-0721-2
Fan C, Wu YH, Decker CM et al (2019) Defensive function of transposable elements in bacteria. ACS Synth Biol 8:2141–2151. https://doi.org/10.1021/acssynbio.9b00218
Farwick A, Bruder S, Schadeweg V et al (2014) Engineering of yeast hexose transporters to transport D-xylose without inhibition by D-glucose. Proc Natl Acad Sci USA 111:5159–5164. https://doi.org/10.1073/pnas.1323464111
Fiamenghi MB, Bueno JGR, Camargo AP et al (2022) Machine learning and comparative genomics approaches for the discovery of xylose transporters in yeast. Biotechnol Biofuels Bioprod 15:57. https://doi.org/10.1186/s13068-022-02153-7
Foster SJ (1991) Cloning, expression, sequence analysis and biochemical characterization of an autolytic amidase of Bacillus subtilis 168 trpC2. Microbiology 137:1987–1998. https://doi.org/10.1099/00221287-137-8-1987
Furdui C, Ragsdale SW (2000) The role of pyruvate ferredoxin oxidoreductase in pyruvate synthesis during autotrophic growth by the Wood-Ljungdahl pathway. J Biol Chem 275:28494–28499. https://doi.org/10.1074/jbc.M003291200
Galhardo JP, Piffer AP, Fiamenghi MB et al (2023) Wide distribution of D-xylose dehydrogenase in yeasts reveals a new element in the D-xylose metabolism for bioethanol production. FEMS Yeast Res 23:foad003. https://doi.org/10.1093/femsyr/foad003
Hall BG (1998) Activation of the bgl operon by adaptive mutation. Mol Biol Evol 15:1–5. https://doi.org/10.1093/oxfordjournals.molbev.a025842
Heider J, Mai X, Adams MWW (1996) Characterization of 2-ketoisovalerate ferredoxin oxidoreductase, a new and reversible coenzyme A-dependent enzyme involved in peptide fermentation by hyperthermophilic archaea. J Bacteriol 178:780–787. https://doi.org/10.1128/jb.178.3.780-787.1996
Hernández SB, Dörr T, Waldor MK, Cava F (2020) Modulation of peptidoglycan synthesis by recycled cell wall tetrapeptides. Cell Rep. https://doi.org/10.1016/J.CELREP.2020.107578
Hsu J-L, Peng H-L, Chang H-Y (2008) The ATP-binding motif in AcoK is required for regulation of acetoin catabolism in Klebsiella pneumoniae CG43. Biochem Biophys Res Commun 376:121–127. https://doi.org/10.1016/j.bbrc.2008.08.103
Huang M, Oppermann-Sanio FB, Steinbüchel A (1999) Biochemical and molecular characterization of the Bacillus subtilis acetoin catabolic pathway. J Bacteriol 181:3837–3841. https://doi.org/10.1128/JB.181.12.3837-3841.1999
Huerta-Cepas J, Forslund K, Coelho LP et al (2017) Fast genome-wide functional annotation through orthology assignment by eggNOG-mapper. Mol Biol Evol 34:2115–2122. https://doi.org/10.1093/molbev/msx148
Joe Shaw A, Jenney FE, Adams MWWW, Lynd LR (2008) End-product pathways in the xylose fermenting bacterium, Thermoanaerobacterium saccharolyticum. Enzyme Microb Technol 42:453–458. https://doi.org/10.1016/j.enzmictec.2008.01.005
Kaster AK, Moll J, Parey K, Thauer RK (2011) Coupling of ferredoxin and heterodisulfide reduction via electron bifurcation in hydrogenotrophic methanogenic archaea. Proc Natl Acad Sci USA 108:2981–2986. https://doi.org/10.1073/pnas.1016761108
Katoh K, Standley DM (2013) MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol 30:772–780. https://doi.org/10.1093/molbev/mst010
Kohut P, Wüstner D, Hronska L et al (2011) The role of ABC proteins Aus1p and Pdr11p in the uptake of external sterols in yeast: dehydroergosterol fluorescence study. Biochem Biophys Res Commun 404:233–238. https://doi.org/10.1016/j.bbrc.2010.11.099
Kominek J, Doering DT, Opulente DA et al (2019) Eukaryotic acquisition of a bacterial operon. Cell 176:1356-1366.e10. https://doi.org/10.1016/j.cell.2019.01.034
Kück P, Longo GC (2014) FASconCAT-G: Extensive functions for multiple sequence alignment preparations concerning phylogenetic studies. Front Zool 11:81. https://doi.org/10.1186/s12983-014-0081-x
Kumari S, Kumar M, Gaur NA, Prasad R (2021) Multiple roles of ABC transporters in yeast. Fungal Genet Biol 150:103550. https://doi.org/10.1016/j.fgb.2021.103550
Kwak S, Jin Y-S (2017) Production of fuels and chemicals from xylose by engineered Saccharomyces cerevisiae: a review and perspective. Microb Cell Fact 16:82. https://doi.org/10.1186/s12934-017-0694-9
Lee YE, Jain MK, Lee C et al (1993) Taxonomic distinction of saccharolytic thermophilic anaerobes. Int J Syst Bacteriol 43:41–51. https://doi.org/10.1099/00207713-43-1-41
Lewinson O, Livnat-Levanon N (2017) Mechanism of action of ABC importers: conservation, divergence, and physiological adaptations. J Mol Biol 429:606–619. https://doi.org/10.1016/j.jmb.2017.01.010
Lin Z, Li WH (2011) Expansion of hexose transporter genes was associated with the evolution of aerobic fermentation in yeasts. Mol Biol Evol 28:131–142. https://doi.org/10.1093/MOLBEV/MSQ184
Lin L, Song H, Tu Q et al (2011) The Thermoanaerobacter glycobiome reveals mechanisms of pentose and hexose co-utilization in bacteria. PLoS Genet 7:e1002318. https://doi.org/10.1371/journal.pgen.1002318
Liu S-Y, Rainey FA, Morgan HW et al (1996) Thermoanaerobacterium aotearoense sp. nov., a slightly acidophilic, anaerobic thermophile isolated from various hot springs in New Zealand, and emendation of the genus Thermoanaerobacterium. Int J Syst Bacteriol 46:388–396. https://doi.org/10.1099/00207713-46-2-388
Mai V, Wiegel J (2000) Advances in development of a genetic system for Thermoanaerobacterium spp.: Expression of genes encoding hydrolytic enzymes, development of a second shuttle vector, and integration of genes into the chromosome. Appl Environ Microbiol 66:4817–4821. https://doi.org/10.1128/AEM.66.11.4817-4821.2000
Mai V, Lorenz WW, Wiegel J (2006) Transformation of Thermoanaerobacterium sp. strain JW/SL-YS485 with plasmid pIKM1 conferring kanamycin resistance. FEMS Microbiol Lett 148:163–167. https://doi.org/10.1111/j.1574-6968.1997.tb10283.x
Murphy CL, Youssef NH, Hanafy RA et al (2019) Horizontal gene transfer as an indispensable driver for evolution of Neocallimastigomycota into a distinct gut-dwelling fungal lineage. Appl Environ Microbiol 85:e00988-e1019. https://doi.org/10.1128/AEM.00988-19
Murrell B, Wertheim JO, Moola S et al (2012) Detecting individual sites subject to episodic diversifying selection. PLoS Genet 8:1002764. https://doi.org/10.1371/journal.pgen.1002764
Parks DH, Chuvochina M, Chaumeil PA et al (2020) A complete domain-to-species taxonomy for bacteria and archaea. Nat Biotechnol 38:1079–1086. https://doi.org/10.1038/s41587-020-0501-8
Peng HL, Yang YH, Deng WL, Chang HY (1997) Identification and characterization of acoK, a regulatory gene of the Klebsiella pneumoniae acoABCD operon. J Bacteriol 179:1497–1504. https://doi.org/10.1128/jb.179.5.1497-1504.1997
Reider Apel A, Ouellet M, Szmidt-Middleton H et al (2016) Evolved hexose transporter enhances xylose uptake and glucose/xylose co-utilization in Saccharomyces cerevisiae. Sci Rep 6:19512. https://doi.org/10.1038/srep19512
Riley R, Haridas S, Wolfe KH et al (2016) Comparative genomics of biotechnologically important yeasts. Proc Natl Acad Sci 113:9882–9887. https://doi.org/10.1073/pnas.1603941113
Ronquist F, Teslenko M, van der Mark P et al (2012) MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst Biol 61:539–542. https://doi.org/10.1093/sysbio/sys029
Rosewich UL, Kistler HC (2000) Role of horizontal gene transfer in the evolution of fungi. Annu Rev Phytopathol 38:325–363. https://doi.org/10.1146/annurev.phyto.38.1.325
Shaw AJ, Podkaminer KK, Desai SG et al (2008) Metabolic engineering of a thermophilic bacterium to produce ethanol at high yield. Proc Natl Acad Sci 105:13769–13774. https://doi.org/10.1073/pnas.0801266105
Shaw AJ, Hogsett DA, Lynd LR (2009) Identification of the [FeFe]-hydrogenase responsible for hydrogen generation in Thermoanaerobacterium saccharolyticum and demonstration of increased ethanol yield via hydrogenase knockout. J Bacteriol 191:6457–6464. https://doi.org/10.1128/JB.00497-09
Szklarczyk D, Gable AL, Lyon D et al (2019) STRING v11: Protein-protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets. Nucleic Acids Res 47:D607–D613. https://doi.org/10.1093/nar/gky1131
Tan M-F, Gao T, Liu W-Q et al (2015) MsmK, an ATPase, contributes to utilization of multiple carbohydrates and host colonization of Streptococcus suis. PLoS One 10:e0130792. https://doi.org/10.1371/journal.pone.0130792
Tanaka KJ, Song S, Mason K, Pinkett HW (2018) Selective substrate uptake: The role of ATP-binding cassette (ABC) importers in pathogenesis. Biochim Biophys Acta (BBA) Biomembr 1860:868–877. https://doi.org/10.1016/j.bbamem.2017.08.011
Tian L, Lo J, Shao X et al (2016) Ferredoxin: NAD+ oxidoreductase of Thermoanaerobacterium saccharolyticum and its role in ethanol formation. Appl Environ Microbiol 82:7134–7141. https://doi.org/10.1128/AEM.02130-16
Törönen P, Medlar A, Holm L (2018) PANNZER2: A rapid functional annotation web server. Nucleic Acids Res 46:W84–W88. https://doi.org/10.1093/nar/gky350
Treangen TJ, Rocha EPC (2011) Horizontal transfer, not duplication, drives the expansion of protein families in prokaryotes. PLoS Genet 7:e1001284. https://doi.org/10.1371/journal.pgen.1001284
Trifinopoulos J, Nguyen L-T, von Haeseler A, Minh BQ (2016) W-IQ-TREE: a fast online phylogenetic tool for maximum likelihood analysis. Nucleic Acids Res 44:1–4. https://doi.org/10.1093/nar/gkw256
Tsakraklides V, Shaw AJ, Miller BB et al (2012) Carbon catabolite repression in Thermoanaerobacterium saccharolyticum. Biotechnol Biofuels 5:85. https://doi.org/10.1186/1754-6834-5-85
Wang W, Zhou H, Ma B et al (2016) Divergent evolutionary pattern of sugar transporter genes is associated with the difference in sugar accumulation between grasses and eudicots. Sci Rep 6:29153. https://doi.org/10.1038/srep29153
Webb AJ, Homer KA, Hosie AHF (2008) Two closely related ABC transporters in Streptococcus mutans are involved in disaccharide and/or oligosaccharide uptake. J Bacteriol 190:168–178. https://doi.org/10.1128/JB.01509-07
Wilkens S (2015) Structure and mechanism of ABC transporters. F1000Prime Rep. https://doi.org/10.12703/P7-14
Williams OB, Morrow MB (1928) The bacterial destruction of acetyl-methyl-carbinol. J Bacteriol 16:43–48. https://doi.org/10.1128/jb.16.1.43-48.1928
Wohlbach DJ, Kuo A, Sato TK et al (2011) Comparative genomics of xylose-fermenting fungi for enhanced biofuel production. Proc Natl Acad Sci USA 108:13212–13217. https://doi.org/10.1073/pnas.1103039108
Xiao Z, Xu P (2007) Acetoin metabolism in bacteria. Crit Rev Microbiol 33:127–140. https://doi.org/10.1080/10408410701364604
Xu X, Zeng W, Li Z et al (2022) Genome-wide identification and expression profiling of sugar transporter genes in tobacco. Gene 835:146652. https://doi.org/10.1016/j.gene.2022.146652
Yang Z (2007) PAML 4: phylogenetic analysis by maximum likelihood. Mol Biol Evol 24:1586–1591. https://doi.org/10.1093/molbev/msm088
Zhang J, Nielsen R, Yang Z (2005) Evaluation of an improved branch-site likelihood method for detecting positive selection at the molecular level. Mol Biol Evol 22:2472–2479. https://doi.org/10.1093/molbev/msi237
Zhao Z, Xian M, Liu M, Zhao G (2020) Biochemical routes for uptake and conversion of xylose by microorganisms. Biotechnol Biofuels 13:21. https://doi.org/10.1186/s13068-020-1662-x
Zheng T, Lanahan AA, Lynd LR, Olson DG (2018) The redox-sensing protein Rex modulates ethanol production in Thermoanaerobacterium saccharolyticum. PLoS One 13:e0195143. https://doi.org/10.1371/journal.pone.0195143
Zhou J, Olson DG, Lanahan AA et al (2015) Physiological roles of pyruvate ferredoxin oxidoreductase and pyruvate formate-lyase in Thermoanaerobacterium saccharolyticum JW/SL-YS485. Biotechnol Biofuels 8:138. https://doi.org/10.1186/s13068-015-0304-1
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The authors are thankful for the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-Brasil (CAPES) for funding this study, through Grant Nos. 88882.329486/2019-01 and 88887.502245/2020-00)
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MBF: conceived, designed the research conducted experiments and analysis, and wrote the manuscript; JSP: conducted experiments and analysis; GB: conceived the research; JJ: designed the research and wrote the manuscript; MFC, GAG, and JJ: supervised the study. All authors revised the manuscript.
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Fiamenghi, M.B., Prodonoff, J.S., Borelli, G. et al. Comparative genomics reveals probable adaptations for xylose use in Thermoanaerobacterium saccharolyticum. Extremophiles 28, 9 (2024). https://doi.org/10.1007/s00792-023-01327-x
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DOI: https://doi.org/10.1007/s00792-023-01327-x