Recombinant Saccharomyces Cerevisiae Strain Research Articles

Saccharomyces cerevisiae oral immunization in mice using multi-antigen of the African swine fever virus elicits a robust immune response.

African swine fever virus (ASFV) is one of the most complex viruses. ASFV is a serious threat to the global swine industry because no commercial vaccines against this virus are currently available except in Vietnam. Moreover, ASFV is highly stable in the environment and can survive in water, feed, and aerosols for a long time. ASFV is transmitted through the digestive and respiratory tract. Mucosal immunity is the first line of defense against ASFV. Saccharomyces cerevisiae (SC), which has been certified by the U.S. Food and Drug Administration and has a generally recognized as safe status in the food industry, was used for oral immunization in this study. ASFV antigens were effectively expressed in recombinant SC strains with high DNA copy numbers and stable growth though surface display technology and chromosome engineering (δ-integration). The recombinant SC strains containing eight ASFV antigens-KP177R, E183L, E199L, CP204L, E248R, EP402R, B602L, and B646L- induced strong humoral and mucosal immune responses in mice. There was no antigenic competition, and these antigens induced Th1 and Th2 cellular immune responses. Therefore, the oral immunization strategy using recombinant SC strains containing multiple ASFV antigens demonstrate potential for future testing in swine, including challenge studies to evaluate its efficacy as a vaccine against ASFV.

CYPArm2 is a CYP450 Monooxygenase with Protoilludene 13-Hydroxylase Activity Involved in the Biosynthesis of Armillyl Orsellinate-Type Sesquiterpenoids.

The melleolides are a family of structurally and functionally diverse sesquiterpenoids with potential applications as fungicides, antimicrobials, and cancer therapeutics. The initial and terminal steps of the biosynthesis pathway in Armillaria spp. have been characterized, but the intermediate steps are unclear. Biosynthetic gene clusters in A. mellea and A. gallica were shown to encode a terpene cyclase, a polyketide synthase, and four CYP450 monooxygenases. We have characterized CYPArm3, which is responsible for the hydroxylation of Δ-6-protoilludene, but the functions of the other CYP450s remain to be determined. Here we describe CYPArm2, which accepts Δ-6-protoilludene and 8α-hydroxy-6-protoilludene as substrates. To investigate the products in more detail, we generated recombinant Saccharomyces cerevisiae strains overexpressing CYPArm2 in combination with the previously characterized protoilludene synthase and 8α-hydroxylase. Using this total biosynthesis approach, sufficient quantities of product were obtained for NMR spectroscopy. This allowed the identification of 8α,13-dihydroxy-protoilludene, confirming that CYPArm2 is a protoilludene 13-hydroxylase.

Clorgyline Analogs Synergize with Azoles against Drug Efflux in Candida auris

Concern about the global emergence of multidrug-resistant fungal pathogens led us to explore the use of combination therapy to combat azole resistance in Candida auris. Clorgyline had previously been shown to be a multi-target inhibitor of Cdr1 and Mdr1 efflux pumps of Candida albicans and Candida glabrata. A screen for antifungal sensitizers among synthetic analogs of Clorgyline detected interactions with the C. auris efflux pump azole substrates Posaconazole and Voriconazole. Of six Clorgyline analogs, M19 and M25 were identified as potential sensitizers of azole resistance. M19 and M25 were found to act synergistically with azoles against resistant C. auris clade I isolates and recombinant Saccharomyces cerevisiae strains overexpressing C. auris efflux pumps. Nile Red assays with the recombinant strains showed M19 and M25 inhibited the activity of Cdr1 and Mdr1 efflux pumps that are known to play key roles in azole resistance in C. auris clades I, III, and IV. While Clorgyline, M19 and M25 uncoupled the Oligomycin-sensitive ATPase activity of Cdr1 from C. albicans and C. auris, their mode of action is yet to be fully elucidated. The experimental combinations described herein provides a starting point to combat azole resistance dominated by overexpression of CauCdr1 in C. auris clades I and IV and CauMdr1 in C. auris clade III.

Ethanol production from non-detoxified hardwood spent sulfite liquor in submerged fed-batch culture using advanced yeasts

AbstractTo improve process feasibility, it is essential to use hardwood spent sulfite liquor (HSSL) as the main feedstock for bioethanol production, without prior detoxification. In addition, operating at large-scale under cost-effective conditions such as a small inoculum size (< 1 g/L), pH 5, using industrially acceptable nutrients, and without sugar addition, will require the use of harsh, concentrated HSSL streams. The potential of non-detoxified HSSL as a feedstock for ethanol production using two recombinant Saccharomyces cerevisiae strains, CelluX™4 and TFA7, was assessed. The inhibitory effect of non-detoxified HSSL was mitigated, and the ethanol titer increased from 4.1 to 7.9 g/L when pulse fed-batch was used instead of batch production, with CelluX™4 performing best. Both strains made use of the xylose isomerase (XI) pathway, with strain TFA7 engineered for increased tolerance against inhibitors. By administering concentrated HSSL in pulses to shake-flask cultures, the ethanol titer could be increased by approximately 50–90% when compared to simple batch cultures supplemented with 20%, 40%, and 60% (v/v) dilutions of HSSL. CelluX™4 was used in non-aerated, non-sterile 5-L bioreactor fermentations with a low cell concentration (< 1 g/L), pH 5, and 5 g/L corn steep liquor (CSL) as the nitrogen source. In comparison, undiluted HSSL was fed continuously to obtain a final 65% (v/v) HSSL supplementation, which corresponded to a total sugar concentration of 70.8–80.8 g/L. Despite the use of harsher, concentrated feedstock and inexpensive process conditions, the reactor fed-batch fermentations obtained ethanol yields of 0.35–0.43 g/g, which, based on a maximum theoretical ethanol yield of 0.51 g/g of hexoses or pentoses, corresponds to yield efficiencies of 68.6 and 84.3%. This illustrates an improvement on the highest titers reported in the literature for non-detoxified HSSL. The use of the advanced industrial S. cerevisiae strain, CelluX™4, combined with a fed-batch strategy, offers an inexpensive and straightforward process with real upscaling potential for industrial HSSL fermentations.

Promoter-proximal introns impact recombinant amylase expression in Saccharomyces cerevisiae.

Consolidated bioprocessing (CBP) of starch requires recombinant Saccharomyces cerevisiae strains that produce raw starch-degrading enzymes and ferment the resultant sugars to ethanol in a single step. In this study, the native S. cerevisiae COX4 and RPS25A promoter-proximal introns were evaluated for enhanced expression of amylase genes (ateA, temA or temG_Opt) under the control of an S. cerevisiae promoter (ENO1P, TEF1P, TDH3P or HXT7P). The results showed that different promoters and promoter-intron combinations differentially affected recombinant amylase production: ENO1P-COX4i and TDH3P-RPS25Ai were the best promoters for AteA, followed closely by HXT7P. The latter was also the best promoter for TemA and TemG production, followed closely by TDH3P-RPS25Ai for both these enzymes. Introducing promoter-proximal introns increased amylase activity up to 62% in Y294[ENO-COX-AteA] and Y294[TDH3-RPS-TemA], a significant improvement relative to the intron-less promoters. Strains co-expressing both an α-amylase and glucoamylase genes yielded up to 56 g/L ethanol from 20% w/v raw starch, with a higher carbon conversion observed with strains co-expressing TDH3P-RPS25Ai-temG_Opt than HXT7P-temG_Opt. The study showed that promoter-proximal introns can enhance amylase activity in S. cerevisiae and suggest that these alternative cassettes may also be considered for expression in more efficient ethanol-producing industrial yeast strains for raw starch CBP.

Comparison of Spathaspora passalidarum and recombinant Saccharomyces cerevisiae for integration of first- and second-generation ethanol production.

First-generation ethanol (E1G) is based on the fermentation of sugars released from saccharine or starch sources, while second-generation ethanol (E2G) is focused on the fermentation of sugars released from lignocellulosic feedstocks. During the fractionation process to release sugars from hemicelluloses (mainly xylose), some inhibitor compounds are released hindering fermentation. Thus, the biggest challenge of using hemicellulosic hydrolysate is selecting strains and processes able to efficiently ferment xylose and tolerate inhibitors. With the aim of diluting inhibitors, sugarcane molasses (80% of sucrose content) can be mixed to hemicellulosic hydrolysate in an integrated E1G-E2G process. Cofermentations of xylose and sucrose were evaluated for the native xylose consumer Spathaspora passalidarum and a recombinant Saccharomyces cerevisiae strain. The industrial S. cerevisiae strain CAT-1 was modified to overexpress the XYL1, XYL2 and XKS1 genes and a mutant ([4-59Δ]HXT1) version of the low-affinity HXT1 permease, generating strain MP-C5H1. Although S. passalidarum showed better results for xylose fermentation, this yeast showed intracellular sucrose hydrolysis and low sucrose consumption in microaerobic conditions. Recombinant S. cerevisiae showed the best performance for cofermentation, and a batch strategy at high cell density in bioreactor achieved unprecedented results of ethanol yield, titer and volumetric productivity in E1G-E2G production process.

Porcine Immunoglobulin Fc Fused P30/P54 Protein of African Swine Fever Virus Displaying on Surface of S. cerevisiae Elicit Strong Antibody Production in Swine.

African swine fever virus (ASFV) infects domestic pigs and European wild boars with strong, hemorrhagic and high mortality. The primary cellular targets of ASFV is the porcine macrophages. Up to now, no commercial vaccine or effective treatment available to control the disease. In this study, three recombinant Saccharomyces cerevisiae (S. cerevisiae) strains expressing fused ASFV proteins-porcine Ig heavy chains were constructed and the immunogenicity of the S. cerevisiae-vectored cocktail ASFV feeding vaccine was further evaluated. To be specific, the P30-Fcγ and P54-Fcα fusion proteins displaying on surface of S. cerevisiae cells were produced by fusing the Fc fragment of porcine immunoglobulin IgG1 or IgA1 with p30 or p54 gene of ASFV respectively. The recombinant P30-Fcγ and P54-Fcα fusion proteins expressed by S. cerevisiae were verified by Western blotting, flow cytometry and immunofluorescence assay. Porcine immunoglobulin Fc fragment fused P30/P54 proteins elicited P30/P54-specific antibody production and induced higher mucosal immunity in swine. The absorption and phagocytosis of recombinant S. cerevisiae strains in IPEC-J2 cells or porcine alveolar macrophage (PAM) cells were significantly enhanced, too. Here, we introduce a kind of cheap and safe oral S. cerevisiae-vectored vaccine, which could activate the specific mucosal immunity for controlling ASFV infection.

Cyclodextrins facilitate the efficient secretion of an anti-tumor triterpenoid ganoderic acid HLDOA by Saccharomyces cerevisiae

As a large group of natural product with significant biological activities, triterpenoid secretion is of particular importance towards its bioproduction. Due to the lack of specific transporters, most triterpenoids are naturally accumulated inside the cells. In this study, by taking an antitumor triterpenoid ganoderic acid 3-hydroxy-lanosta-8,24-dien-26 oic acid (GA-HLDOA) as example, we discovered that addition of 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) or 2,6-dimethyl-β-cyclodextrin (DM-β-CD) enable the fast and sufficient secretion of GA-HLDOA by the recombinant Saccharomyces cerevisiae strain as constructed in our previous study. In addition, these cyclodextrins (CDs) could not enter into cells, while no significant change of the cell membrane fluidity was observed after CDs treatment. This discovery provides a potential generally applicable method for triterpenoid secretion.

Simulating Extracellular Glucose Signals Enhances Xylose Metabolism in Recombinant Saccharomyces cerevisiae.

Efficient utilization of both glucose and xylose from lignocellulosic biomass would be economically beneficial for biofuel production. Recombinant Saccharomyces cerevisiae strains with essential genes and metabolic networks for xylose metabolism can ferment xylose; however, the efficiency of xylose fermentation is much lower than that of glucose, the preferred carbon source of yeast. Implications from our previous work suggest that activation of the glucose sensing system may benefit xylose metabolism. Here, we show that deleting cAMP phosphodiesterase genes PDE1 and PDE2 increased PKA activity of strains, and consequently, increased xylose utilization. Compared to the wild type strain, the specific xylose consumption rate (rxylose) of the pde1Δ pde2Δ mutant strains increased by 50%; the specific ethanol-producing rate (rethanol) of the strain increased by 70%. We also show that HXT1 and HXT2 transcription levels slightly increased when xylose was present. We also show that HXT1 and HXT2 transcription levels slightly increased when xylose was present. Deletion of either RGT2 or SNF3 reduced expression of HXT1 in strains cultured in 1 g L−1 xylose, which suggests that xylose can bind both Snf3 and Rgt2 and slightly alter their conformations. Deletion of SNF3 significantly weakened the expression of HXT2 in the yeast cultured in 40 g L−1 xylose, while deletion of RGT2 did not weaken expression of HXT2, suggesting that S. cerevisiae mainly depends on Snf3 to sense a high concentration of xylose (40 g L−1). Finally, we show that deletion of Rgt1, increased rxylose by 24% from that of the control. Our findings indicate how S. cerevisiae may respond to xylose and this study provides novel targets for further engineering of xylose-fermenting strains.

Characterization of two Vitis vinifera carotenoid cleavage dioxygenases by heterologous expression in Saccharomyces cerevisiae

Norisoprenoids are produced from carotenoids under oxidative degradation mediated by carotenoid cleavage dioxygenases (CCDs) and contribute to floral and fruity notes in grape berries and wine. The diversity of CCD substrates and products has been demonstrated by in vitro recombinant proteins extracted from Escherichia coli expressing CCD genes and of in vivo proteins in an E. coli system co-expressing genes for carotenoid synthesis and cleavage. In the current study, VvCCD1 and VvCCD4b were isolated from the cDNA library of Vitis vinifera L. cv. Cabernet Sauvignon and then transformed into carotenoid-accumulating recombinant Saccharomyces cerevisiae strains. The expression of the target genes was monitored during the yeast growth period, and the accumulation of carotenoids and norisoprenoids in the recombinant strains was measured. The results indicated that both of the VvCCDs cleaved β-carotene at the 7, 8 (7', 8') position into β-cyclocitral for the first time. Additionally, the two enzymes also degraded β-carotene at the 9, 10 (9', 10') position to generate β-ionone and cleaved lycopene at the 5, 6 (5', 6') position into 6-methyl-5-hepten-2-one. These findings suggested that the VvCCDs may possess more cleavage characteristics under the eukaryotic expression system in S. cerevisiae than the prokaryotic system in E. coli, which could better explain the biochemical functions of VvCCDs in grape berries.

Build Your Bioprocess on a Solid Strain-β-Carotene Production in Recombinant Saccharomyces cerevisiae.

Robust fermentation performance of microbial cell factories is critical for successful scaling of a biotechnological process. From shake flask cultivations to industrial-scale bioreactors, consistent strain behavior is fundamental to achieve the production targets. To assert the importance of this feature, we evaluated the impact of the yeast strain design and construction method on process scalability -from shake flasks to bench-scale fed-batch fermentations- using two recombinant Saccharomyces cerevisiae strains capable of producing β-carotene; SM14 and βcar1.2 strains. SM14 strain, obtained previously from adaptive evolution experiments, was capable to accumulate up to 21 mg/gDCW of β-carotene in 72 h shake flask cultures; while the βcar1.2, constructed by overexpression of carotenogenic genes, only accumulated 5.8 mg/gDCW of carotene. Surprisingly, fed-batch cultivation of these strains in 1L bioreactors resulted in opposite performances. βcar1.2 strain reached much higher biomass and β-carotene productivities (1.57 g/L/h and 10.9 mg/L/h, respectively) than SM14 strain (0.48 g/L/h and 3.1 mg/L/h, respectively). Final β-carotene titers were 210 and 750 mg/L after 80 h cultivation for SM14 and βcar1.2 strains, respectively. Our results indicate that these substantial differences in fermentation parameters are mainly a consequence of the exacerbated Crabtree effect of the SM14 strain. We also found that the strategy used to integrate the carotenogenic genes into the chromosomes affected the genetic stability of strains, although the impact was significantly minor. Overall, our results indicate that shake flasks fermentation parameters are poor predictors of the fermentation performance under industrial-like conditions, and that appropriate construction designs and performance tests must be conducted to properly assess the scalability of the strain and the bioprocess.

A Thi2p Regulatory Network Controls the Post-glucose Effect of Xylose Utilization in Saccharomyces cerevisiae

The complete and efficient utilization of both glucose and xylose is necessary for the economically viable production of biofuels and chemicals using lignocellulosic feedstocks. Although recently obtained recombinant Saccharomyces cerevisiae strains metabolize xylose well when xylose is the sole carbon source in the medium (henceforth referred to as “X stage”), their xylose consumption rate is significantly reduced during the xylose-only consumption phase of glucose-xylose co-fermentation (“GX stage”). This post-glucose effect seriously decreases overall fermentation efficiency. We showed in previous work that THI2 deletion can alleviate this post-glucose effect, but the underlying mechanisms were ill-defined. In the present study, we profiled the transcriptome of a thi2Δ strain growing at the GX stage. Thi2p in GX stage cells regulates genes involved in the cell cycle, stress tolerance, and cell viability. Importantly, the regulation of Thi2p differs from a previous regulatory network that functions when glucose is the sole carbon source, which suggests that the function of Thi2p depends on the carbon source. Modeling research seeking to optimize metabolic engineering via TFs should account for this important carbon source difference. Building on our initial study, we confirmed that several identified factors did indeed increase fermentation efficiency. Specifically, overexpressing STT4, RGI2, and TFC3 increases specific xylose utilization rate of the strain by 36.9, 29.7, 42.8%, respectively, in the GX stage of anaerobic fermentation. Our study thus illustrates a promising strategy for the rational engineering of yeast for lignocellulosic ethanol production.

Identification of modifications procuring growth on xylose in recombinant Saccharomyces cerevisiae strains carrying the Weimberg pathway

The most prevalent xylose-assimilating pathways in recombinant Saccharomyces cerevisiae, i.e. the xylose isomerase (XI) and the xylose reductase/xylitol dehydrogenase (XR/XDH) pathways, channel the carbon flux through the pentose phosphate pathway and further into glycolysis. In contrast, the oxidative and non-phosphorylative bacterial Weimberg pathway channels the xylose carbon through five steps into the metabolic node α-ketoglutarate (αKG) that can be utilized for growth or diverted into production of various metabolites.In the present study, steps preventing the establishment of a functional Weimberg pathway in S. cerevisiae were identified. Using an original design where a S. cerevisiae strain was expressing the essential four genes of the Caulobacter crescentus pathway (xylB, xylD, xylX, xylA) together with a deletion of FRA2 gene to upregulate the iron-sulfur metabolism, it was shown that the C. crescentus αKG semialdehyde dehydrogenase, XylA was not functional in S. cerevisiae. When replaced by the recently described analog from Corynebacterium glutamicum, KsaD, significantly higher in vitro activity was observed but the strain did not grow on xylose. Adaptive laboratory evolution (ALE) on a xylose/glucose medium on this strain led to a loss of XylB, the first step of the Weimberg pathway, suggesting that ALE favored minimizing the inhibiting xylonate accumulation by restricting the upper part of the pathway. Therefore three additional gene copies of the lower Weimberg pathway (XylD, XylX and KsaD) were introduced. The resulting S. cerevisiae strain (ΔΔfra2, xylB, 4x (xylD-xylX-ksaD)) was able to generate biomass from xylose and Weimberg pathway intermediates were detected. To our knowledge this is the first report of a functional complete Weimberg pathway expressed in fungi. When optimized this pathway has the potential to channel xylose towards value-added specialty chemicals such as dicarboxylic acids and diols.

Genetically engineered probiotic Saccharomyces cerevisiae strains mature human dendritic cells and stimulate Gag-specific memory CD8+ T cells ex vivo.

Recombinant Saccharomyces cerevisiae strains expressing HIV antigens have shown promising pre-clinical results. Probiotic S. cerevisiae strains naturally induce gut immunity; thus, genetically engineered probiotic strains could be used to stimulate immune responses against HIV in the mucosa. Probiotic strains have a higher rate of heterologous protein production, meaning higher antigen's epitope expression levels per yeast cell. We expressed HIV-1 Gag protein in the probiotic yeasts' surface, which was eagerly phagocytosed by and induced type 1 polarization of human monocyte-derived dendritic cells (DCs) from healthy donors in vitro. We further matured DCs derived from HIV-1+ donors with transformed yeasts and incubated them with autologous T cells. Only DCs matured with Gag-expressing probiotic strains were able to efficiently present antigen to CD8+ T cells and induced their clonal expansion. Our results show that genetically engineered probiotic S. cerevisiae strains are a promising vaccination strategy against HIV.

Co-expression of cellulase and xylanase genes in Saccharomyces cerevisiae toward enhanced bioethanol production from corn stover

ABSTRACT Lignocellulose is considered as a good resource for producing renewable energy. Previous in vitro studies have shown the synergistic action between cellulase and xylanase during lignocellulose biohydrolysis. In order to achieve the same effect in S. cerevisiae to enhance the practical biotransformation, two recombinant Saccharomyces cerevisiae strains (INVSc1-CBH-CA and INVSc1-CBH-TS) with co-expressed cellulase and xylanase were constructed. The cellulase and xylanase activities in INVSc1-CBH-CA and INVSc1-CBH-TS were 716.43 U/mL and 205.13 U/mL, 931.27 U/mL and 413.70 U/mL, respectively. The recombinant S. cerevisiae can use the partly delignified corn stover (PDCS) more efficiently and more ethanol producted than S. cerevisiae only expressing cellulase. Fermentation with INVSc1-CBH-CA and INVSc1-CBH-TS using PDCS ethanol yields increased by 1.7 and 2.1 folds higher than INVSc1-CBH, 2.8 and 3.4 folds higher than the wild type S. cerevisiae. The strategy of co-expression cellulase and xylanase in saccharomyces cerevisiae is effective and can be a foundation to research the mechanism of synergy effect of cellulose and xylanase.

Effect of Trehalose and Glycerol on the Resistance of Recombinant Saccharomyces cerevisiae Strains to Desiccation, Freeze-Thaw and Osmotic Stresses

This research is supported by the National Academy of Sciences of Ukraine (Grant 27—17) and our partners from ENZYM COMPANY PJSC (Lviv). The authors appreciate very much the contribution of Yuri Pynyaha and Olena Krasovska from ENZYM COMPANY PJSC.

Effect of Trehalose and Glycerol on the Resistance of Recombinant Saccharomyces cerevisiae Strains to Desiccation, Freeze-Thaw and Osmotic Stresses

This research is supported by the National Academy of Sciences of Ukraine (Grant 27—17) and our partners from ENZYM COMPANY PJSC (Lviv). The authors appreciate very much the contribution of Yuri Pynyaha and Olena Krasovska from ENZYM COMPANY PJSC.

Cow manure as a lignocellulosic substrate for fungal cellulase expression and bioethanol production

Conversion of various lignocellulosic materials into bioethanol is growing in demand but greatly depends on feedstock availability. Dairy cow manure is an agricultural waste widely distributed worldwide. This study investigated the induction of cellulases by cow manure and the conversion of cow manure materials into lignocellulosic ethanol. Alkaline NaOH pretreatment improved the accessibility of cow manure lignocellulose to enzymes followed by enzymatic hydrolysis using Penicillium oxalicum cellulases. The ethanol yields from pretreated cow manure and anaerobically digested cow manure were 0.19 and 0.13 g/g-raw biomass, respectively, using recombinant Saccharomyces cerevisiae strain LF1 designed for lignocellulosic ethanol production through simultaneous saccharification and fermentation. Fed-batch supplementation with cellulolytic enzymes and substrates after initial enzymatic hydrolysis also contributed to ethanol production up to 25.65 g/L. These results demonstrate that cow manure is a potential feedstock for inducing fungal cellulase expression and converting lignocellulose into bioethanol.

Cellobiose fermentation by Saccharomyces cerevisiae: Comparative analysis of intra versus extracellular sugar hydrolysis

Abstract A prevalent procedure for the production of second generation bioethanol makes use of engineered yeast strains capable to hydrolyze cellobiose either in the cytosol or extracellularly. These two approaches have been compared in this study. For intracellular cellobiose hydrolysis, we initially tested three recombinant Saccharomyces cerevisiae strains that produced different cytosolic β-glucosidases and the cellodextrin transporter Po_CdtC from Penicillium oxalicum . The strain coexpressing Po_CdtC and the β-glucosidase from Neurospora crassa (NcBgl) showed the highest cellobiase activity but its growth in cellobiose was limited by sugar intake. A search of alternative cellobiose permeases was carried out among putative sugar transporters from Trichoderma reesei . Ten candidates were selected by sequence similarity with previously characterized fungal cellobiose permeases. Only one of them (Tr_StrC) was able to support yeast growth in cellobiose. Strains NcBgl/PoCdtC and NcBgl/TrStrC were compared to a yeast transformant (T500) that produced the extracellular β-glucosidase from Saccharomycopsis fibuligera , in terms of cellobiose fermentation. Extracellular hydrolysis resulted in faster cellobiose fermentation rates and higher ethanol yields. The strain producing extracellular β-glucosidase was also more efficient for simultaneous saccharification and fermentation of cellulose. The main targets to improve cellobiose fermentation through both strategies are discussed.

Recombinant Diploid Saccharomyces cerevisiae Strain Development for Rapid Glucose and Xylose Co-Fermentation

Cost-effective production of cellulosic ethanol requires robust microorganisms for rapid co-fermentation of glucose and xylose. This study aims to develop a recombinant diploid xylose-fermenting Saccharomyces cerevisiae strain for efficient conversion of lignocellulosic biomass sugars to ethanol. Episomal plasmids harboring codon-optimized Piromyces sp. E2 xylose isomerase (PirXylA) and Orpinomyces sp. ukk1 xylose (OrpXylA) genes were constructed and transformed into S. cerevisiae. The strain harboring plasmids with tandem PirXylA was favorable for xylose utilization when xylose was used as the sole carbon source, while the strain harboring plasmids with tandem OrpXylA was beneficial for glucose and xylose cofermentation. PirXylA and OrpXylA genes were also individually integrated into the genome of yeast strains in multiple copies. Such integration was beneficial for xylose alcoholic fermentation. The respiration-deficient strain carrying episomal or integrated OrpXylA genes exhibited the best performance for glucose and xylose co-fermentation. This was partly attributed to the high expression levels and activities of xylose isomerase. Mating a respiration-efficient strain carrying the integrated PirXylA gene with a respiration-deficient strain harboring integrated OrpXylA generated a diploid recombinant xylose-fermenting yeast strain STXQ with enhanced cell growth and xylose fermentation. Co-fermentation of 162 g L−1 glucose and 95 g L−1 xylose generated 120.6 g L−1 ethanol in 23 h, with sugar conversion higher than 99%, ethanol yield of 0.47 g g−1, and ethanol productivity of 5.26 g L−1·h−1.