An efficient conversion of xylose along with glucose is needed for the sustainable production of lignocellulosic biofuels. However, the assimilation of xylose is still a major loophole for economical 2G ethanol production. Evolutionary adaptation is known to provide stability to the strains in a challenging environment. As an extension of an earlier study, the strains Kluyveromyces marxianus NIRE-K1.1 and K. marxianus NIRE-K3.1 (KmNIRE-K3.1) were subjected to secondary adaptation on optimized minimal media (MM) with the aim to enhance xylose utilization for ethanol production. Both the strains were adapted till a saturated improvement in xylose uptake, that is, 54 generations on MM medium containing xylose. Xylose utilization increased from 14.21% to 45.80% and 10.55% to 45.31%, in evolved strains KmNIRE-K1.2 and KmNIRE-K3.2, respectively. Specific xylose reductase activity also increased 2.04- and 3.36-folds in KmNIRE-K1.2 and KmNIRE-K3.2, respectively. Xylitol dehydrogenase activity was also increased by 2.82- and 1.35-folds in KmNIRE-K1.2 and KmNIRE-K3.2, respectively. Further, a decrease in redox imbalance was also observed in evolved strains, and hence, reduced in xylitol production was observed during growth and fermentation. Xylose uptake rate increased by 2.53- and 1.5-folds in KmNIRE-K1.2 and KmNIRE-K3.2, respectively with 2.20- and 6.46-folds higher ethanol concentrations, with 2.25- and 5.86-folds higher volumetric productivity, respectively. This study has demonstrated the role of evolutionary adaptation in developing robust yeast strains. KmNIRE-K1.2 and KmNIRE-K3.2 have shown enhanced ethanol production, enzyme activities, and less by-product formation such as xylitol during xylose metabolism.
Get full access to this article
View all access options for this article.
References
1.
AroraR, BeheraS, SharmaNK, et al.Evaluating the pathway for co-fermentation of glucose and xylose for enhanced bioethanol production using flux balance analysis. Biotechnol Bioproc E, 2019; 24(6):924–933; doi: 10.1007/s12257-019-0026-5
2.
SharmaNK, BibraM. Molecular biology based innovations in lignocellulose biorefinery. In: Lignocellulose bioconversion through white biotechnology. (ChandelAK. . ed.) Wiley Professional. 2022; pp. 177–202.
3.
AchinasS, HorjusJ, AchinasV, et al.A PESTLE analysis of biofuels energy industry in Europe. Sustainability, 2019; 11(21):5981; doi: 10.3390/su11215981
4.
RodrussameeN, SattayawatP, YamadaM. Highly efficient conversion of xylose to ethanol without glucose repression by newly isolated thermotolerant Spathaspora passalidarum CMUWF1–2. BMC Microbiol, 2018; 18(1):73–11; doi: 10.1186/s12866-018-1218-4
5.
ZhangJ, ZhangB, WangD, et al.Rapid ethanol production at elevated temperatures by engineered thermotolerant Kluyveromyces marxianus via the NADP (H)-preferring xylose reductase-xylitol dehydrogenase pathway. Metab Eng, 2015; 31:140–152; doi: 10.1016/j.ymben.2015.07.008
6.
HuCH, CaiX, XuWD, et al.Cellulosic Ethanol Production from High-Solids Corncob Residues by Simultaneous Saccharification and Fermentation on a Pilot Scale. ACS Sustainable Resour Manage, 2024; 1(8):1845–1854; doi: 10.1021/acssusresmgt.4c00208
7.
SharmaNK, BeheraS, AroraR, et al.Enhancement in xylose utilization using Kluyveromyces marxianus NIRE-K1 through evolutionary adaptation approach. Bioprocess Biosyst Eng, 2016; 39(5):835–843; doi: 10.1007/s00449-016-1563-3
8.
SharmaNK, BeheraS, AroraR, et al.Evolutionary adaptation of Kluyveromyces marxianus NIRE-K3 for enhanced xylose utilization. Front Energy Res, 2017; 5(32):1–8; doi: 10.3389/fenrg.2017.00032
9.
Lappe-OliverasP, AvitiaM, Sánchez-RobledoSD, et al.Genotypic and phenotypic diversity of Kluyveromyces marxianus isolates obtained from the elaboration process of two traditional Mexican alcoholic beverages derived from agave: Pulque and henequen (Agave fourcroydes) mescal. JoF, 2023; 9(8):795; doi: 10.3390/jof9080795
10.
WangR, LiL, ZhangB, et al.Improved xylose fermentation of Kluyveromyces marxianus at elevated temperature through construction of a xylose isomerase pathway. J Ind Microbiol Biotechnol, 2013; 40(8):841–854; doi: 10.1007/s10295-013-1294-2
11.
AroraR, BeheraS, SharmaNK, et al.Augmentation of ethanol production through statistically designed growth and fermentation medium using novel thermotolerant yeast isolates. Renew Energy, 2017; 109:406–421; doi: 10.1016/j.renene.2017.03.059
12.
IkeuchiT, KiritaniR, AzumaM, et al.Effect of D‐glucose on induction of xylose reductase and xylitol dehydrogenase in Candida tropicalis in the presence of NaCl. J Basic Microbiol, 2000; 40(3):167–175.
13.
BaileyJE, OllisDF. Biochemical engineering fundamentals. McGraw-Hill, New York; 1986.
14.
LiuE, HuY. Construction of a xylose-fermenting Saccharomyces cerevisiae strain by combined approaches of genetic engineering, chemical mutagenesis and evolutionary adaptation. Biochem Eng J, 2010; 48(2):204–210; doi: 10.1016/j.bej.2009.10.011
15.
MartínC, MarcetM, AlmazánO, et al.Adaptation of a recombinant xylose-utilizing Saccharomyces cerevisiae strain to a sugarcane bagasse hydrolysate with high content of fermentation inhibitors. Bioresour Technol, 2007; 98(9):1767–1773; doi: 10.1016/j.biortech.2006.07.021
16.
MavrommatiM, PapanikolaouS, AggelisG. Improving ethanol tolerance of Saccharomyces cerevisiae through adaptive laboratory evolution using high ethanol concentrations as a selective pressure. Process Biochem, 2023; 124:280–289; doi: 10.1016/j.procbio.2022.11.027
DhaliwalSS, OberoiHS, SandhuSK, et al.Enhanced ethanol production from sugarcane juice by galactose adaptation of a newly isolated thermotolerant strain of Pichia kudriavzevii. Bioresour Technol, 2011; 102(10):5968–5975; doi: 10.1016/j.biortech.2011.02.015
19.
García-RíosE, Lairon-PerisM, Muniz-CalvoS, et al.Thermo-adaptive evolution to generate improved Saccharomyces cerevisiae strains for cocoa pulp fermentations. Int J Food Microbiol, 2021; 342:109077; doi: 10.1016/j.ijfoodmicro.2021.109077
20.
SondereggerM, SauerU. Evolutionary engineering of Saccharomyces cerevisiae for anaerobic growth on xylose. Appl Environ Microbiol, 2003; 69(4):1990–1998; doi: 10.1128/AEM.69.4.1990-1998.2003
21.
SharmaNK, BeheraS, AroraR, et al.Xylose transport in yeast for lignocellulosic ethanol production: Current status. J Biosci Bioeng, 2018; 125(3):259–267; doi: 10.1016/j.jbiosc.2017.10.006
22.
JeppssonM, TräffK, JohanssonB, et al.Effect of enhanced xylose reductase activity on xylose consumption and product distribution in xylose-fermenting recombinant Saccharomyces cerevisiae. FEMS Yeast Res, 2003; 3(2):167–175; doi: 10.1016/S1567-1356(02)00186-1
23.
KhattabSM, SaimuraM, KodakiT. Boost in bioethanol production using recombinant Saccharomyces cerevisiae with mutated strictly NADPH-dependent xylose reductase and NADP+-dependent xylitol dehydrogenase. J Biotechnol, 2013; 165(3–4):153–156; doi: 10.1016/j.jbiotec.2013.03.009
24.
DasguptaD, GhoshD, BandhuS, et al.Purification, characterization and molecular docking study of NADPH dependent xylose reductase from thermotolerant Kluyveromyces sp. IIPE453. Process Biochem, 2016; 51(1):124–133; doi: 10.1016/j.procbio.2015.11.007
25.
YablochkovaEN, BolotnikovaOI, MikhailovaNP, et al.The activity of xylose reductase and xylitol dehydrogenase in yeasts. Microbiol, 2003; 72:414–417.
26.
GoshimaT, NegiK, TsujiM, et al.Ethanol fermentation from xylose by metabolically engineered strains of Kluyveromyces marxianus. J Biosci Bioeng, 2013; 116(5):551–554; doi: 10.1016/j.jbiosc.2013.05.010
27.
ZhangB, SunH, LiJ, et al.High-titer-ethanol production from cellulosic hydrolysate by an engineered strain of Saccharomyces cerevisiae during an in situ removal process reducing the inhibition of ethanol on xylose metabolism. Process Biochem, 2016; 51(8):967–972; doi: 10.1016/j.procbio.2016.04.019
28.
RegmiP, KnesebeckM, BolesE, et al.A comparative analysis of NADPH supply strategies in Saccharomyces cerevisiae: Production of d-xylitol from d-xylose as a case study. Metab Eng Commun, 2024; 19:e00245; doi: 10.1016/j.mec.2024.e00245
29.
WeiN, XuH, KimSR, et al.Deletion of FPS1, encoding aquaglyceroporin Fps1p, improves xylose fermentation by engineered Saccharomyces cerevisiae. Appl Environ Microbiol, 2013; 79(10):3193–3201; doi: 10.1128/AEM.00490-13
30.
LiF, BaiW, ZhangY, et al.Construction of an economical xylose-utilizing Saccharomyces cerevisiae and its ethanol fermentation. FEMS Yeast Res, 2024; 24:foae001; doi: 10.1093/femsyr/foae001
31.
QiX, ZhaJ, LiuGG, et al.Heterologous xylose isomerase pathway and evolutionary engineering improve xylose utilization in Saccharomyces cerevisiae. Front Microbiol, 2015; 6:1165; doi: 10.3389/fmicb.2015.01165
32.
BiswasD, DattM, AggarwalM, et al.Molecular cloning, characterization, and engineering of xylitol dehydrogenase from Debaryomyces hansenii. Appl Microbiol Biotechnol, 2013; 97(4):1613–1623.
33.
Yamasaki-YashikiS, KomedaH, HoshinoK, et al.Molecular analysis of NAD+-dependent xylitol dehydrogenase from the zygomycetous fungus Rhizomucor pusillus and reversal of the coenzyme preference. Biosci Biotechnol Biochem, 2014; 78(11):1943–1953.
34.
KomedaH, Yamasaki-YashikiS, HoshinoK, et al.Identification and characterization of d-xylose reductase involved in pentose catabolism of the zygomycetous fungus Rhizomucor pusillus. J Biosci Bioeng, 2015; 119(1):57–64; doi: 10.1016/j.jbiosc.2014.06.012
35.
HamillPG, StevensonA, McMullanPE, et al.Microbial lag phase can be indicative of, or independent from, cellular stress. Sci Rep, 2020; 10(1):5948; doi: 10.1038/s41598-020-62552-4
36.
LaPanseAJ, KrishnanA, PosewitzMC. Adaptive Laboratory Evolution for algal strain improvement: Methodologies and applications. Algal Res, 2021; 53:102122; doi: 10.1016/j.algal.2020.102122
37.
NewAM, CerulusB, GoversSK, et al.Different levels of catabolite repression optimize growth in stable and variable environments. PLoS Biol, 2014; 12(1):e1001764; doi: 10.1371/journal.pbio.1001764
38.
KoppramR, AlbersE, OlssonL. Evolutionary engineering strategies to enhance tolerance of xylose utilizing recombinant yeast to inhibitors derived from spruce biomass. Biotechnol Biofuels, 2012; 5(1):32–12; doi: 10.1186/1754-6834-5-32
39.
LandaetaR, ArocaG, AcevedoF, et al.Adaptation of a flocculent Saccharomyces cerevisiae strain to lignocellulosic inhibitors by cell recycle batch fermentation. Appl Energy, 2013; 102:124–130; doi: 10.1016/j.apenergy.2012.06.048
40.
SliningerPJ, Shea-AndershMA, ThompsonSR, et al.Evolved strains of Scheffersomyces stipitis achieving high ethanol productivity on acid-and base-pretreated biomass hydrolyzate at high solids loading. Biotechnol Biofuels, 2015; 8(60):60–27; doi: 10.1186/s13068-015-0239-6
41.
HamacherT, BeckerJ, GárdonyiM, et al.Characterization of the xylose-transporting properties of yeast hexose transporters and their influence on xylose utilization. Microbiology (Reading), 2002; 148(Pt 9):2783–2788; doi: 10.1099/00221287-148-9-2783
42.
JangYS, YangJ, KimJK, et al.Adaptive laboratory evolution and transcriptomics‐guided engineering of Escherichia coli for increased isobutanol tolerance. Biotechnol J, 2024; 19(1):2300270; doi: 10.1002/biot.202300270
43.
AroraR, BeheraS, SharmaNK, et al.A new search for thermotolerant yeasts, its characterization and optimization using response surface methodology for ethanol production. Front Microbiol, 2015; 6:889; doi: 10.3389/fmicb.2015.00889
44.
ZhouH, ChengJS, WangBL, et al.Xylose isomerase overexpression along with engineering of the pentose phosphate pathway and evolutionary engineering enable rapid xylose utilization and ethanol production by Saccharomyces cerevisiae. Metab Eng, 2012; 14(6):611–622; doi: 10.1016/j.ymben.2012.07.011
45.
AgbogboFK, HaagensenFD, MilamD, et al.Fermentation of acid-pretreated corn stover to ethanol without detoxification using Pichia stipitis. Appl Biochem Biotechnol, 2008; 145(1–3):53–58; doi: 10.1007/s12010-007-8056-4
46.
SilvaC, RobertoI. Improvement of xylitol production by Candida guilliermondii FTI 20037 previously adapted to rice straw hemicellulosic hydrolysate. Lett Appl Microbiol, 2001; 32(4):248–252; doi: 10.1046/j.1472-765x.2001.00899.x
47.
PereiraSR, Sànchez I NoguéV, FrazãoCJR, et al.Adaptation of Scheffersomyces stipitis to hardwood spent sulfite liquor by evolutionary engineering. Biotechnol Biofuels, 2015; 8:50–51-8; doi: 10.1186/s13068-015-0234-y
48.
CadièreA, Ortiz-JulienA, CamarasaC, et al.Evolutionary engineered Saccharomyces cerevisiae wine yeast strains with increased in vivo flux through the pentose phosphate pathway. Metab Eng, 2011; 13(3):263–271; doi: 10.1016/j.ymben.2011.01.008
49.
SaengphingT, SattayawatP, KalawilT, et al.Improving furfural tolerance in a xylose-fermenting yeast Spathaspora passalidarum CMUWF1–2 via adaptive laboratory evolution. Microb Cell Fact, 2024; 23(1):80; doi: 10.1186/s12934-024-02352-x
50.
NielsenF, Tomás-PejóE, OlssonL, et al.Short-term adaptation during propagation improves the performance of xylose-fermenting Saccharomyces cerevisiae in simultaneous saccharification and co-fermentation. Biotechnol Biofuels, 2015; 8(219):219–215.
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
