| Bioethanol is one of the most promising and popular alternatives to renewable fuels,with potential for energy security and environmental safety relative to fossil fuels To date,many biomass resources have been investigated for bioethanol production.which can be broadly classified into sugar.starch,lignocellulose.and algal biomass Compared with the first-generation fuel ethanol produced from starch or saccharide raw materials,the lignocellulosic material composed of cellulose,hemicellulose and lignin has the advantages of low cost,large-scale acquisition,and no competition with food The second-generation fuel ethanol produced by lignocellulosic material shows high energy,economic and environmental advantages,and is the direction of large-scale and sustainable development of fuel ethanolThe process of lignocellulosic fuel ethanol production is the physical and chemical factor pretreatment to change the complex structure of cellulosic biomass,cellulase degradation releases monosaccharides(mainly glucose,xylose)and a small amount of oligosaccharides.which are then subjected to microbial fermentation to complete sugar alcohol conversion and recovery of the ethanol product.Therefore,in order to achieve economically viable cellulosic ethanol production,it is necessary to reduce the cost of raw materials such as raw material collection,pretreatment,cellulase.and optimization of the microbial fermentation process to efficiently perform sugar alcohol conversionSaccharomvces cerevisicae is a fermenting microorganism commonly used in ethanol production with high ethanol productivity,ethanol and inhibitor tolerance,and a wide range of fermentation substrates.S.cerevisiae can efficiently ferment glucose to produce ethanol,but natural S.cerevisiae can hardly use xylose.Although many studies have realized the co-utilization of glucose and xylose by S.cerevisiae.however,there are still many technical problems in the industrial application of second-generation fuel ethanol using recombinant S.cerevisiae strains.For example:1.Improve the level of efficient simultaneous co-fermentation of xylose/glucose:2.Improve the tolerance to inhibitors in the upstream process such as pretreatment:3.Improve the conversion efficiency of residual oligosaccharides in lignocellulosic feedstock.Recombinant S.cerevisiae can ferment monosaccharides(glucose.xylose,etc.)to produce ethanol,but use o f oligosaccharides(Xylo-oligosaccharides,cello-oligosaccharides,etc.)is often overlooked.These are also the main challenges faced by fermenting microorganisms including S.cerevisiae to produce biobased products from lignocellulosic biomass.In order to further improve the industrialization performance of the second-generation fuel ethanol strains,we will continue our research on the basis of the highly efficient simultaneous fermentation of xylose/glucose engineering S.cerevisiae strain LF2(pre-laboratory work).including:(1)Comprehensive evaluation and screening for highly resistant S.cerevisiaeThe fuel ethanol production process is inherently present and produces a variety of inhibitors,and S.cereivisiae strains with a higher tolerance phenotype for these inhibitors are essential for achieving cost-effective production of fuel ethanol.Considering that all the excellent phenotypes are not concentrated in the same strain,this paper adopts different evaluatioin measures for screening:Including the determination of trehalose content.GSH/GSSG determination,single factor tolerance analysis of a variety of inhibitors(specifically,high temperature stress,high osmotic pressure,oxidative stress,and furfural,acetic acid,vanillin,ethanol stress that S.cerevisiae may encounter in the fermentation process of lignocellulosic raw materials).According to the screening results,RC212 with good comprehensive tolerance was selected as the starting strain for further study.(2)Heterologous expression of xylosidase in xylose-metabolizing laboratory strains to achieve utilization of xylo-oligosaccharide substratesXylose isonmerase(derived from bovine rumen fluid metagenomics)and ?-xylosidasc gene(derived from Penicillium oxalicum)were heterologously expressed into the haploid laboratory strain BSPX042(a genetically modified xylose metabolic pathway that was passaged and lost a plasmid containing xylose isomerase)by episomal plasmid.which made it theoretically capable of fermenting xylo-oligosaccharides(XOS).And replacing the self-signaling peptide of the xylosidase gene derived from Penicillium oxalicum with the S.cererisiae-derived signal peptides MF?.SUC2.PHO5 and Kluyveromyces INU signal peptide.The xylosidase recombinant strain BSGIBX with the extracellular enzyme activity with the INU signal peptide was obtained,and the fermentation with XOS as the sole carbon source showed that the S.cerevisiae strain with high expression ?-xylosidase could metabolize xylo-oligosaccharide to produce ethanol.(3)?-glucosidase and ?-xylosidase confer the ability of industrial strains to metabolize oligosaccharidesThe pretreatment and enzymatic hydrolysis of lignocellulosic feedstock produces glucose and xylose which can be directly fermented by recombinant S.cerevisiae.In addition,some oligosaccharides are present in the fermentation substrate,such as cellobiose and xylo-oligosaccharide.This is related to the insufficient activity of ?-glucosidase(BGL)and ?-xylosidase(XYL)in some natural cellulose degrading enzymes(such as cellulase mixtures derived from Trichoderma reesei).In order to achieve the utilization of the full sugar component in the lignocellulosic material as much as possible,it is often necessary to additionally add BGL and XYL.which undoubtedly increases the cost of fuel ethanol production.In the preliminary work of the research group,recombinant S.cerevisiae strain 102SB was constructed by heterologous expression of the ?-glucosidase gene derived from Saccharomycopsis fibuligera.which has high ?-glucosidase activity and can better metabolize cellobiose.The corresponding(3-glucosidase gene and ?-xylosidase gene expression cassette were amplified by strain 102SB and the above-mentioned strained strain BSGIBX with higher xvlosidase activity.The above gene was integrated into the chromosome of strain RC212 by ?-sequence homologous recombination to obtain recombinant S.cerevisiae strain BLN04.Bv enzyme activity assay and fermentation test.BLN04 has corresponding BGL and XYL enzyme activities,and it is capable of metabolizing cellobiose and xylo-oligosaccharide substrates to produce ethanol.(4)Co-cultu re system(mixed fermentation)to achieve the common use of a variety of mixed sugarPreviously,many metabolic engineering works had been made to extend the substrate range of S.cerevisiae to a variety of cellulose sugars,and to integrate multiple substrate utilization pathways into a single strain enabling it to simultaneously metabolize multiple monosaccharides and oligosaccharides.However,studies have shown that simultaneous integration and expression of multiple heterologous metabolic pathways in a single fermenting microorganism can cause an adverse metabolic burden on the strain,particularly at high sugar concentrations.Co-culture systems could decrease the metabolic burden of each particular strain and reduce inhibition during metabolic processes compared to common single-strain fermentations.Moreover,the various sugar components in the lignocellulosic feedstock hydrolysate fluctuate greatly with different pretreatment and enzymatic hydrolysis,but the proportion of specific strains in the co-culture system can be easily adjusted to fit in the changes of sugar in lignocellulosic hydrolysate.Although there was also a report of fermentation of mixed sugar by a single strain,mixed-sugar fermentation cannot be achieved without a proper substrate ratio.There,the mixed sugar limited oxygen fermentation in glucose,xylose,cellobiose and xylo-oligosaccharide(G X.C.O)were taken by adjusting the initial inoculation ratio(total OD600=1)of the cellulase-secreting strain BLN04 and the co-fermented glucose/xylose strain LF2 in the co-culture system.The metabolic processes in YPGXC and YPGXCO(all sugar contents were 2%)were compared when the ratio of LF2:BLN04 = 5:5(OD600)and 3:7.The results showed that whether during the fermentation of YPGXC or YPGXCO,when the inoculation ratio at 3:7,the total biomass(DCW),the total consumption of various oligosaccharides and the ethanol yield were higher than the ratio of 5:5.However,the glucose and xylose utilization rates did not change much,because the early monosaccharides could be directly utilized to produce ethanol.but the oligosaccharides required to be hydrolyzed by the secretase of grown strains before being further utilized.In addition,at the 48th hour of fermentation,most of the cellobiose in the system had been utilized,and the xylo-oligosaccharide has not been fully converted.It is considered that the commercial xylo-oligosaccharide had a higher degree of polymerization and the xylosidase enzyme activity not enough to degrade,limiting conversion efficiency.Therefore,we supplemented the YPGXCO fermentation of commercial XOS(mainly xylobiose and a small amount of xylotriose and xylose after hydrolysis)after 16 hours of hydrolvsis by self-made xvlanse as co-culture substrate.The results showed that at a 3:7 ratio,the consumption ofcellobiose and xvlo-oligosaccharide in the system increases,and more ethanol was produced,and the consumption rate of xvlobiose was also relatively increased.This showed that with a lower deguree of polymerization of xylo-oligosaccharide as a fermentation substrate,the substrate component could be more fully utilized to increase ethanol production.The above results indicated that by adjusting the composition co-culture system,the consumption rate of substrates and biomass were changed.And it was proved that the problem of second-generation ethanol could be solved to some extent by adjusting the composition of the co-cultured microorganisms to adapt to different lignocellulose hydrolyzate.(5)Xylose-specific transporter enhances the xylose transport capacity of strains for protoplast fusionXylose transporter mutant Mgt05196N360F.was derived from the genus Meyerozyma guilliermondii,a xylose-specific transporter mutant obtained by our group that completely eliminated glucose inhibition.Mgt05196pN360F was introduced in the xylose utilization strain in which all hexose transporters were knocked out.It was shown that the recombinant strain could not grow on the medium with glucose as the sole carbon source,and the growth ability on xylose was not inhibited by the presence of glucose.The presence of glucose increased the xylose growth ability of the strain to some extent.Therefore.Mat05196pN360F can specifically transport xylose without being inhibited by the presence of glucose.Therefore,we selected the xylose-specific transporter mutant Mgt05196N360F and integrated it into the strain BLN04 to obtain the recombinant strain BLN26.It is expected that through protoplast fusion with the strain LF2.an effective fusion agent with increased xylose consumption rate but no inhibition by glucose could be obtained,then to compensate for the deficiency of xylose metabolism rate lower than the glucose metabolism rate of the strain LF2.(6)Preliminary study on the protoplast preparation and fusion process of strains LF2 and BLN26Genetically rich fusions can be obtained by protoplast fusion,which highlights some of the superior features of the two related parental strains from different species.In this paper,a protoplast fusion of strains LF2 and BLN26 was carried out to obtain a fusion strain with high trehalose content.good tolerance,high-efficiency co-fermentation of glucose and xylose,and simultaneous use of cellobiose and XOS.which could be used in the production of second-generation fuel ethanol.The conditions for the preparation and regeneration of the protoplasts of the parents were preliminarily explored,and the suitable protoplast preparation rate and regeneration rate were obtained,which is the prerequisite for the effective fusion of the parents.After PEG-smelting,strains LF2 and BLN26 were subjected to fusion screening through a selective plate(YPX+G41 8+HygB).However,all single colonies on the selective plate cannot grow on the medium with xylose as the sole carbon source even after the recovery culture process of non-selective medium(YPD).And the characteristic genes(XI and HygB)of the LF2 parent were also not amplified in the genome of the fusion.Throuth the analysis of the cause of this phenomenon,we ruled out the possibility that the parent strain BLN26 itself could grow on selective plates:and there is also no effective fusion for changing the type of osmotic pressure stabilizer.Other possible causes are genetic heterocaryon state or chromosome exchange and loss caused by genetic recombination between two sets of homologous chromosomes from two parents:and the damage to the cell wall or cell membrane of strain LF2 after multiple transformations may lead to the effective cell fusion cannot be performed. |
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