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Hyper-thermal (HT) acid hydrolysis of red seaweed Gelidium amansii was performed using 12% (w/v) slurry and an acid mix concentration of 180 mM at 150°C for 10 min. Enzymatic saccharification when using a combination of Celluclast 1.5 L and CTec2 at a dose of 16 U/ml led to the production of 12.0 g/l of reducing sugar with an efficiency of enzymatic saccharification of 13.2%. After the enzymatic saccharification, 2,3-butanediol (2,3-BD) fermentation was carried out using an engineered S. cerevisiae strain. The use of HT acid-hydrolyzed medium with 1.9 g/l of 5-hydroxymethylfurfural showed a reduction in the lag time from 48 to 24 h. The 2,3-BD concentration and yield coefficient at 72 h were 14.8 g/l and 0.30, respectively. Therefore, HT acid hydrolysis and the use of the engineered S. cerevisiae strain can enhance the overall 2,3-BD yields from G. amansii seaweed.

2,3-Butanediol (2,3-BD) can be produced at high titers by engineered Saccharomyces cerevisiae by abolishing the ethanol biosynthetic pathway and introducing the bacterial butanediol-producing pathway. However, production of 2,3-BD instead of ethanol by engineered S. cerevisiae has resulted in glycerol production because of surplus NADH accumulation caused by a lower degree of reduction (γ = 5.5) of 2,3-BD than that (γ = 6) of ethanol. In order to eliminate glycerol production and resolve redox imbalance during 2,3-BD production, both GPD1 and GPD2 coding for glycerol-3-phosphate dehydrogenases were disrupted after overexpressing NADH oxidase from Lactococcus lactis. As disruption of the GPD genes caused growth defects due to limited supply of C 2 compounds, Candida tropicalis PDC1 was additionally introduced to provide a necessary amount of C 2 compounds while minimizing ethanol production. The resulting strain (BD5_T2 nox_dGPD1,2_CtPDC1) produced 99.4 g/L of 2,3-BD with 0.5 g/L glycerol accumulation in a batch culture. The fed-batch fermentation led to production of 108.6 g/L 2,3-BD with a negligible amount of glycerol production, resulting in a high BD yield (0.462 g2,3-BD/gglucose) corresponding to 92.4 % of the theoretical yield. These results demonstrate that glycerol-free production of 2,3-BD by engineered yeast is feasible., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Saccharomyces cerevisiae has a natural ability to produce higher alcohols, making it a promising candidate for production of isobutanol. However, the several pathways competing with isobutanol biosynthesis lead to production of substantial amounts of l-valine and l-isoleucine in mitochondria and isobutyrate, l-leucine, and ethanol in cytosol. To increase flux to isobutanol by removing by-product formation, the genes associated with formation of l-valine (BAT1), l-isoleucine (ILV1), isobutyrate (ALD6), l-leucine (LEU1), and ethanol (ADH1) were disrupted to construct the S. cerevisiae WΔGBIALA1_2vec strain. This strain showed 8.9 and 8.6 folds increases in isobutanol concentration and yield, respectively, relative the corresponding values of the background strain on glucose medium. In a bioreactor fermentation with a gas trapping system, the WΔGBIALA1_2vec strain produced 662 mg/L isobutanol concentration with a yield of 6.71 mg isobutanol /g glucose . With elimination of the competing pathways, the WΔGBIALA1_2vec strain would serve as a platform strain for isobutanol production., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The aim of this study was to produce 2,3-butanediol (2,3-BDO) from xylose efficiently by modulation of the xylose metabolic pathway in engineered Saccharomyces cerevisiae. Expression of the Scheffersomyces stipitis transaldolase and NADH-preferring xylose reductase in S. cerevisiae improved xylose consumption rate by a 2.1-fold and 2,3-BDO productivity by a 1.8-fold. Expression of the Lactococcus lactis noxE gene encoding NADH oxidase also increased 2,3-BDO yield by decreasing glycerol accumulation. Additionally, the disadvantage of C 2 -dependent growth of pyruvate decarboxylase-deficient (Pdc - ) S. cerevisiae was overcome by expression of the Candida tropicalis PDC1 gene. A fed-batch fermentation of the BD5X-TXmNP strain resulted in 96.8g/L 2,3-BDO and 0.58g/L-h productivity from xylose, which were 15.6- and 2-fold increases compared with the corresponding values of the BD5X strain. It was concluded that facilitation of the xylose metabolic pathway, oxidation of NADH and relief of C 2 -dependency synergistically triggered 2,3-BDO production from xylose in Pdc - S. cerevisiae., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Engineered Saccharomyces cerevisiae has been used for ethanol production from xylose, the abundant sugar in lignocellulosic hydrolyzates. Development of engineered S. cerevisiae able to utilize xylose effectively is crucial for economical and sustainable production of fuels. To this end, the xylose-metabolic genes (XYL1, XYL2 and XYL3) from Scheffersomyces stipitis have been introduced into S. cerevisiae. The resulting engineered S. cerevisiae strains, however, often exhibit undesirable phenotypes such as slow xylose assimilation and xylitol accumulation. This work was undertaken to construct an improved xylose-fermenting strain by developing a synthetic isozyme system of xylose reductase (XR). The DXS strain having both wild XR and mutant XR showed low xylitol accumulation and fast xylose consumption compared to the engineered strains expressing only one type of XRs, resulting in improved ethanol yield and productivity. These results suggest that the introduction of the XR-based synthetic isozyme system is a promising strategy to develop efficient xylose-fermenting strains., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Spermidine is a polyamine compound exhibiting important biological activities, such as increasing lifespan, inflammation reduction, and plant growth control. As such, many applications of spermidine as a bio-modulating agent are anticipated. However, sustainable and scalable production of spermidine has not been achieved yet. Therefore, construction of a spermidine production system using Saccharomyces cerevisiae was attempted in this study. In order to secrete spermidine into fermentation broth, TPO1 coding for the polyamine transporter was overexpressed in an engineered S. cerevisiae strain capable of accumulating high concentrations of spermidine. Through optimization of fermentation conditions, the resulting strain (OS123/pTPO1) produced 63.6mg/l spermidine with a yield of 1.3mg spermidine/g glucose. However, we observed that spermidine production was repressed in the presence of glucose. To circumvent this problem, the genetic modifications for overproducing spermidine were introduced into an engineered S. cerevisiae capable of fermenting xylose. In a fed-batch fermentation using a mixture of glucose and xylose, the resulting strain (SR8 OS123/pTPO1) produced 224mg/l spermidine with a yield of 2.2mg spermidine/g sugars. These results suggest that engineered yeast constructed in this study can be employed for the production of spermidine., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Construction of robust and efficient yeast strains is a prerequisite for commercializing a biofuel production process. We have demonstrated that high intracellular spermidine (SPD) contents in Saccharomyces cerevisiae can lead to improved tolerance against various fermentation inhibitors, including furan derivatives and acetic acid. In this study, we examined the potential applicability of the S. cerevisiae strains with high SPD contents under two cases of ethanol fermentation: glucose fermentation in repeated-batch fermentations and xylose fermentation in the presence of fermentation inhibitors. During the sixteen times of repeated-batch fermentations using glucose as a sole carbon source, the S. cerevisiae strains with high SPD contents maintained higher cell viability and ethanol productivities than a control strain with lower SPD contents. Specifically, at the sixteenth fermentation, the ethanol productivity of a S. cerevisiae strain with twofold higher SPD content was 31% higher than that of the control strain. When the SPD content was elevated in an engineered S. cerevisiae capable of fermenting xylose, the resulting S. cerevisiae strain exhibited much 40-50% higher ethanol productivities than the control strain during the fermentations of synthetic hydrolysate containing high concentrations of fermentation inhibitors. These results suggest that the strain engineering strategy to increase SPD content is broadly applicable for engineering yeast strains for robust and efficient production of ethanol.

Even though industrial yeast strains exhibit numerous advantageous traits for the production of bioethanol, their genetic manipulation has been limited. This study demonstrates that an industrial polyploidy Saccharomyces cerevisiae JHS200 can be engineered through Cas9 (CRISPR associated protein 9)-based genome editing. Specifically, we generated auxotrophic mutants and introduced a xylose metabolic pathway into the auxotrophic mutants. As expected, the engineered strain (JX123) enhanced ethanol production from cellulosic hydrolysates as compared to other engineered haploid strains. However, the JX123 strain produced substantial amounts of xylitol as a by-product during xylose fermentation. Hypothesizing that the xylitol accumulation might be caused by intracellular redox imbalance from cofactor difference, the NADH oxidase from Lactococcus lactis was introduced into the JX123 strain. The resulting strain (JX123_noxE) not only produced more ethanol, but also produced xylitol less than the JX123 strain. These results suggest that industrial polyploidy yeast can be modified for producing biofuels and chemicals., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Saccharomyces cerevisiae is a work horse for production of valuable biofuels and biochemicals including 2,3-butanediol (2,3-BDO), a platform chemical with wide industrial applications for synthetic rubber, biosolvents and food additives. Recently, a cutting-edge technology of metabolic engineering has enabled S. cerevisiae to produce 2,3-BDO with high yield and productivity. These include (i) amplification of the 2,3-BDO biosynthetic pathway, (ii) redirection of carbon flux from ethanol or glycerol toward 2,3-BDO, and (iii) 2,3-BDO production from sugars derived from renewable biomass. These breakthroughs enforced S. cerevisiae to become a promising microbial host for production of 2,3-BDO.

Furfural, one of the most common inhibitors in pre-treatment hydrolysates, reduces the cell growth and ethanol production of yeast. Evolutionary engineering has been used as a selection scheme to obtain yeast strains that exhibit furfural tolerance. However, the response of Saccharomyces cerevisiae to furfural at the metabolite level during evolution remains unknown. In this study, evolutionary engineering and metabolomic analyses were applied to determine the effects of furfural on yeasts and their metabolic response to continuous exposure to furfural. After 50 serial transfers of cultures in the presence of furfural, the evolved strains acquired the ability to stably manage its physiological status under the furfural stress. A total of 98 metabolites were identified, and their abundance profiles implied that yeast metabolism was globally regulated. Under the furfural stress, stress-protective molecules and cofactor-related mechanisms were mainly induced in the parental strain. However, during evolution under the furfural stress, S. cerevisiae underwent global metabolic allocations to quickly overcome the stress, particularly by maintaining higher levels of metabolites related to energy generation, cofactor regeneration and recovery from cellular damage. Mapping the mechanisms of furfural tolerance conferred by evolutionary engineering in the present study will be led to rational design of metabolically engineered yeasts., (© 2016 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

Ethanol, the major fermentation product of yeast, is a stress factor in yeast. We previously constructed an ethanol-tolerant mutant yeast iETS3 by using the global transcriptional machinery engineering. However, the ethanol-tolerance mechanism has not been systematically investigated. In this study, global metabolite profiling was carried out, mainly by gas chromatography/time-of-flight mass spectrometry (GC/TOF MS), to investigate the mechanisms of ethanol tolerance in iETS3. A total of 108 intracellular metabolites were identified by GC/TOF MS and high performance liquid chromatography, and these metabolites were mostly intermediates of the central carbon metabolism. The metabolite profiles of iETS3 and BY4741, cultured with or without ethanol, were significantly different based on principal component and hierarchical clustering analyses. Our metabolomic analyses identified the compositional changes in cell membranes and the activation of glutamate metabolism and the trehalose synthetic pathway as the possible mechanisms for the ethanol tolerance. These metabolic traits can be considered possible targets for further improvement of ethanol tolerance in the mutant. For example, the KGD1 deletion mutant, with up-regulated glutamate metabolism, showed increased tolerance to ethanol. This study has demonstrated that metabolomics can be a useful tool for strain improvement and phenotypic analysis of microorganisms under stress., (Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Xylitol, a natural sweetener, can be produced by hydrogenation of xylose in hemicelluloses. In microbial processes, utilization of only NADPH cofactor limited commercialization of xylitol biosynthesis. To overcome this drawback, Saccharomyces cerevisiae D452-2 was engineered to express two types of xylose reductase (XR) with either NADPH-dependence or NADH-preference. Engineered S. cerevisiae DWM expressing both the XRs exhibited higher xylitol productivity than the yeast strain expressing NADPH-dependent XR only (DWW) in both batch and glucose-limited fed-batch cultures. Furthermore, the coexpression of S. cerevisiae ZWF1 and ACS1 genes in the DWM strain increased intracellular concentrations of NADPH and NADH and improved maximum xylitol productivity by 17%, relative to that for the DWM strain. Finally, the optimized fed-batch fermentation of S. cerevisiae DWM-ZWF1-ACS1 resulted in 196.2 g/L xylitol concentration, 4.27 g/L h productivity and almost the theoretical yield. Expression of the two types of XR utilizing both NADPH and NADH is a promising strategy to meet the industrial demands for microbial xylitol production., (Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

To minimize glycerol production during 2,3-BD fermentation by the engineered Saccharomyces cerevisiae, the Lactococcus lactis water-forming NADH oxidase gene (noxE) was expressed at five different levels. The expression of NADH oxidase substantially decreased the intracellular NADH/NAD(+) ratio. The S. cerevisiae BD5_T2nox strain expressing noxE produced 2,3-BD with yield of 0.359 g 2,3-BD/gglucose and glycerol with 0.069gglycerol/gglucose, which are 23.8% higher and 65.3% lower than those of the isogenic strain without noxE. These results demonstrate that the carbon flux could be redirected from glycerol to 2,3-BD through alteration of the NADH/NAD(+) ratio by the expression of NADH oxidase., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The aim of this work was to apply the evolutionary engineering to construct a mutant Saccharomyces cerevisiae HJ7-14 resistant on 2-deoxy-D-glucose and with an enhanced ability of bioethanol production from galactose, a mono-sugar in red algae. In batch and repeated-batch fermentations, HJ7-14 metabolized 5-fold more galactose and produced ethanol 2.1-fold faster than the parental D452-2 strain. Transcriptional analysis of genes involved in the galactose metabolism revealed that moderate relief from the glucose-mediated repression of the transcription of the GAL genes might enable HJ7-14 to metabolize galactose rapidly. HJ7-14 produced 7.4 g/L ethanol from hydrolysates of the red alga Gelidium amansii within 12 h, which was 1.5-times faster than that observed with D452-2. We demonstrate conclusively that evolutionary engineering is a promising tool to manipulate the complex galactose metabolism in S. cerevisiae to produce bioethanol from red alga., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Fermentation inhibitors present in lignocellulose hydrolysates are inevitable obstacles for achieving economic production of biofuels and biochemicals by industrial microorganisms. Here we show that spermidine (SPD) functions as a chemical elicitor for enhanced tolerance of Saccharomyces cerevisiae against major fermentation inhibitors. In addition, the feasibility of constructing an engineered S. cerevisiae strain capable of tolerating toxic levels of the major inhibitors without exogenous addition of SPD was explored. Specifically, we altered expression levels of the genes in the SPD biosynthetic pathway. Also, OAZ1 coding for ornithine decarboxylase (ODC) antizyme and TPO1 coding for the polyamine transport protein were disrupted to increase intracellular SPD levels through alleviation of feedback inhibition on ODC and prevention of SPD excretion, respectively. Especially, the strain with combination of OAZ1 and TPO1 double disruption and overexpression of SPE3 not only contained spermidine content of 1.1mg SPD/g cell, which was 171% higher than that of the control strain, but also exhibited 60% and 33% shorter lag-phase period than that of the control strain under the medium containing furan derivatives and acetic acid, respectively. While we observed a positive correlation between intracellular SPD contents and tolerance phenotypes among the engineered strains accumulating different amounts of intracellular SPD, too much SPD accumulation is likely to cause metabolic burden. Therefore, genetic perturbations for intracellular SPD levels should be optimized in terms of metabolic burden and SPD contents to construct inhibitor tolerant yeast strains. We also found that the genes involved in purine biosynthesis and cell wall and chromatin stability were related to the enhanced tolerance phenotypes to furfural. The robust strains constructed in this study can be applied for producing chemicals and advanced biofuels from cellulosic hydrolysates., (Copyright © 2015 International Metabolic Engineering Society. All rights reserved.)

Inulin is a polyfructan that is abundant in plants such as Jerusalem artichoke, chicory and dahlia. Inulinase can easily hydrolyze inulin to fructose, which is consumed by microorganisms. Generally, Saccharomyces cerevisiae, an industrial workhorse strain for bioethanol production, is known for not having inulinase activity. The inulinase gene from Kluyveromyces marxianus (KmINU), with the ability of converting inulin to fructose, was introduced into S. cerevisiae D452-2. The inulinase gene was fused to three different types of promoter (GPD, PGK1, truncated HXT7) and secretory signal sequence (KmINU, MFα1, SUC2) to generate nine expression cassettes. The inulin fermentation performance of the nine transformants containing different promoter and signal sequence combinations for inulinase production were compared to select an optimized expression system for efficient inulin fermentation. Among the nine inulinase-producing transformants, the S. cerevisiae carrying the PGK1 promoter and MFα1 signal sequence (S. cerevisiae D452-2/p426PM) showed not only the highest specific KmINU activity, but also the best inulin fermentation capability. Finally, a batch fermentation of the selected S. cerevisiae D452-2/p426PM in a bioreactor with 188.2 g/L inulin was performed to produce 80.2 g/L ethanol with 0.43 g ethanol/g inulin of ethanol yield and 1.22 g/L h of ethanol productivity.

2,3-Butanediol (2,3-BD) production from xylose that is abundant in lignocellulosic hydrolyzate would make the production of 2,3-BD more sustainable and economical. Saccharomyces cerevisiae can produce only trace amounts of 2,3-BD, but also cannot ferment xylose. Therefore, it is necessary to introduce both 2,3-BD production and xylose assimilation pathways into S. cerevisiae for producing 2,3-BD from xylose. A pyruvate decarboxylase (Pdc)-deficient mutant (SOS4) was used as a host in order to increase carbon flux toward 2,3-BD instead of ethanol. The XYL1, XYL2, and XYL3 genes coding for xylose assimilating enzymes derived from Scheffersomyces stipitis were introduced into the SOS4 strain to enable xylose utilization. Additionally, the alsS and alsD genes from Bacillus subtilis and endogenous BDH1 gene were overexpressed to increase 2,3-BD production from xylose. As a result, the resulting strain (BD4X) produced 20.7g/L of 2,3-BD from xylose with a yield of 0.27g 2,3-BD/g xylose. The titer of 2,3-BD from xylose increased up to 43.6g/L under a fed-batch fermentation. The BD4X strain produced (R, R)-2,3-BD dominantly (>97% of the total 2,3-BD) with trace amounts of meso-2,3-BD. These results suggest that S. cerevisiae might be a promising host for producing 2,3-BD from lignocellulosic biomass for industrial applications., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Production of renewable fuels and chemicals from cellulosic biomass is a critical step towards energy sustainability and reduced greenhouse gas emissions. Microbial cells have been engineered for producing chemicals from cellulosic sugars. Among these chemicals, 2,3-butanediol (2,3-BDO) is a compound of interest due to its diverse applications. While microbial production of 2,3-BDO with high yields and productivities has been reported, there are concerns associated with utilization of potential pathogenic bacteria and inefficient utilization of cellulosic sugars. To address these problems, we engineered 2,3-BDO production in Saccharomyces cerevisiae, especially from cellobiose, a prevalent sugar in cellulosic hydrolysates. Specifically, we overexpressed alsS and alsD from Bacillus subtilis to convert pyruvate into 2,3-BDO via α-acetolactate and acetoin in an engineered cellobiose fermenting S. cerevisiae. Under oxygen-limited conditions, the resulting strain was able to produce 2,3-BDO. Still, major carbon flux went to ethanol, resulting in substantial amounts of ethanol produced as a byproduct. To enhance pyruvate flux to 2,3-BDO through elimination of the pyruvate decarboxylation reaction, we employed a deletion mutant of both PDC1 and PDC5 for producing 2,3-BDO. When a cellobiose utilization pathway, consisting of a cellobiose transporter and intracellular β-glucosidase, and the 2,3-BDO producing pathway were introduced in a pyruvate decarboxylase deletion mutant, the resulting strain produced 2,3-BDO without ethanol production from cellobiose under oxygen-limited conditions. A titer of 5.29 g/l 2,3-BDO with a productivity of 0.22 g/l h and yield of 0.29 g 2,3-BDO/g cellobiose was attained. These results suggest the possibility of producing 2,3-BDO safely and sustainably from cellulosic hydrolysates.

Carboxypeptidase Y (CPY) is a yeast vacuolar protease with useful applications including C-terminal sequencing of peptides and terminal modification of target proteins. To overexpress CPY with the pro-sequence (proCPY) encoded by the Saccharomyces cerevisiae PRC1 gene in recombinant S. cerevisiae, the proCPY gene was combined with the gene coding for a signal sequence of S. cerevisiae mating factor α (MFα), invertase (SUC2), or Kluyveromyces marxianus inulinase (INU1). Among the three constructs, the MFα signal sequence gave the best specific activity of extracellular CPY. To enhance the CPY expression level, folding accessory proteins of Kar2p, Pdi1p and Ero1p located in the S. cerevisiae endoplasmic reticulum were expressed individually and combinatorially. A single expression of Kar2p led to a 28 % enhancement in extracellular CPY activity, relative to the control strain of S. cerevisiae CEN.PK2-1D/p426Gal1-MFαCPY. Coexpression of Kar2p, Pdi1p and Ero1p gave a synergistic effect on CPY expression, of which activity was 1.7 times higher than that of the control strain. This work showed that engineering of signal sequences and protein-folding proteins would be helpful to overexpress yeast proteins of interest.

Cellobiose was once regarded as a byproduct that should be removed from biomass hydrolysates because of its inhibitory activity to cellulases. It was revealed, however, that cellobiose could serve as a co-substrate for xylose fermentation by engineered Saccharomyces cerevisiae. Despite its advantages, to date, little is known about cellodextrin transporters that endow S. cerevisiae with cellobiose transporting ability. In this study, engineered S. cerevisiae strains capable of fermenting cellobiose were constructed by expressing various fungal cellobiose transporters and intracellular β-glucosidases. Among them, the strain expressing a putative sugar transporter from Penicillium chrysogenum (Pc_ST) and β-glucosidase from Thielavia terrestris (Tt_BG) showed an improved cellobiose fermentation performance compared to the strain expressing a cellodextrin transporter from Neurospora crassa (Nc_CDT-1) and β-glucosidase from N. crassa (Nc_GH1-1). Cellobiose fermentation by S. cerevisiae Pc_ST/Tt_BG under microaerobic conditions resulted in 14.5±0.5g/L of final ethanol concentration with a yield of 0.37±0.01g ethanol/g cellobiose, which are 22% and 26% higher than the corresponding values of S. cerevisiae Nc_CDT-1/Nc_GH1-1. These results suggest that the yield and rate of cellobiose fermentation can be improved by adopting optimal pairs of cellobiose transporters and β-glucosidase., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Efficient and rapid fermentation of all sugars present in cellulosic hydrolysates is essential for economic conversion of renewable biomass into fuels and chemicals. Xylose is one of the most abundant sugars in cellulosic biomass but it cannot be utilized by wild type Saccharomyces cerevisiae, which has been used for industrial ethanol production. Therefore, numerous technologies for strain development have been employed to engineer S. cerevisiae capable of fermenting xylose rapidly and efficiently. These include i) optimization of xylose-assimilating pathways, ii) perturbation of gene targets for reconfiguring yeast metabolism, and iii) simultaneous co-fermentation of xylose and cellobiose. In addition, the genetic and physiological background of host strains is an important determinant to construct efficient and rapid xylose-fermenting S. cerevisiae. Vibrant and persistent researches in this field for the last two decades not only led to the development of engineered S. cerevisiae strains ready for industrial fermentation of cellulosic hydrolysates, but also deepened our understanding of operational principles underlying yeast metabolism., (Copyright © 2013 Elsevier Inc. All rights reserved.)

In order to produce 2,3-butanediol (2,3-BD) with a high titer, it is necessary to engineer Saccharomyces cerevisiae by deleting the competing pathway and overexpressing the 2,3-BD biosynthetic pathway. A pyruvate decarboxylase (Pdc)-deficient mutant was constructed and evolved for rapid glucose consumption without ethanol production. Genome re-sequencing of the evolved strain (SOS4) revealed a point mutation (A81P) in MTH1 coding for a transcriptional regulator involved in glucose sensing, unlike the previously reported Pdc-deficient mutant which had internal deletion in MTH1. When alsS and alsD genes from Bacillus subtilis, and endogenous BDH1 gene were overexpressed in SOS4, the resulting strain (BD4) not only produced 2,3-BD efficiently, but also consumed glucose faster than the parental strain. In fed-batch fermentation with optimum aeration, 2,3-BD concentration increased up to 96.2 g/L. These results suggest that S. cerevisiae might be a promising host for producing 2,3-BD for industrial applications., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Saccharomyces cerevisiae can be engineered for xylose fermentation through introduction of wild type or mutant genes (XYL1/XYL1 (R276H), XYL2, and XYL3) coding for xylose metabolic enzymes from Scheffersomyces stipitis. The resulting engineered strains, however, often yielded undesirable phenotypes such as slow xylose assimilation and xylitol accumulation. In this study, we performed the mating of two engineered strains that exhibit suboptimal xylose-fermenting phenotypes in order to develop an improved xylose-fermenting diploid strain. Specifically, we obtained two engineered haploid strains (YSX3 and SX3). The YSX3 strain consumed xylose rapidly and produced a lot of xylitol. On the contrary, the SX3 strain consumed xylose slowly with little xylitol production. After converting the mating type of SX3 from alpha to a, the resulting strain (SX3-2) was mated with YSX3 to construct a heterozygous diploid strain (KSM). The KSM strain assimilated xylose (0.25gxyloseh(-1)gcells(-1)) as fast as YSX3 and accumulated a small amount of xylitol (0.03ggxylose(-1)) as low as SX3, resulting in an improved ethanol yield (0.27ggxylose(-1)). We found that the improvement in xylose fermentation by the KSM strain was not because of heterozygosity or genome duplication but because of the complementation of the two xylose-metabolic pathways. This result suggested that mating of suboptimal haploid strains is a promising strategy to develop engineered yeast strains with improved xylose fermenting capability., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Kluyveromyces marxianus is now considered one of the best choices of option for industrial applications of yeast because the strain is able to grow at high temperature, utilizes various carbon sources, and grows fast. However, the use of K. marxianus as a host for industrial applications is still limited. This limitation is largely due to a lack of knowledge on the characteristics of the promoters since the time and amount of protein expression is strongly dependent on the promoter employed. In this study, four well-known constitutive promoters (P(CYC), P(TEF), P(GPD), and P(ADH)) of Saccharomyces cerevisiae were characterized in K. marxianus in terms of protein expression level and their stochastic behavior. After constructing five URA3-auxotrophic K. marxianus strains and a plasmid vector, four cassettes each comprising one of the promoters--the gene for the green fluorescence protein (GFP)--CYC1 terminator (T(CYC)) were inserted into the vector. GFP expression under the control of each one of the promoters was analyzed by reverse transcription PCR, fluorescence microscopy, and flow cytometer. Using these combined methods, the promoter strength was determined to be in the order of P(GPD) > P(ADH) ∼ P(TEF) >> P(CYC). All promoters except for the P(CYC) exhibited three distinctive populations, including non-expressing cells, weakly expressing cells, and strongly expressing cells. The relative ratios between populations were strongly dependent on the promoter and culture time. Forward scattering was independent of GFP fluorescence intensity, indicating that the different fluorescence intensities were not just due to different cell sizes derived from budding. It also excluded the possibility that the non-expressing cells resulted from plasmid loss because plasmid stability was maintained at almost 100 % over the culture time. The same cassettes, cloned into a single copy plasmid pRS416 and transformed into S. cerevisiae, showed only one population. When the cassettes were integrated into the chromosome, the stochastic behavior was markedly reduced. These combined results imply that the gene expression stochasticity should be overcome in order to use this strain for delicate metabolic engineering, which would require the co-expression of several genes.

Oil palm fronds are the most abundant lignocellulosic biomass in Malaysia. In this study, fronds were tested as the potential renewable biomass for ethanol production. The soaking in aqueous ammonia pretreatment was applied, and the fermentability of pretreated fronds was evaluated using simultaneous saccharification and fermentation. The optimal pretreatment conditions were 7 % (w/w) ammonia, 80 °C, 20 h of pretreatment, and 1:12 S/L ratio, where the enzymatic digestibility was 41.4 % with cellulase of 60 FPU/g-glucan. When increasing the cellulase loading in the hydrolysis of pretreated fronds, the enzymatic digestibility increased until the enzyme loading reached 60 FPU/g-glucan. With 3 % glucan loading in the SSF of pretreated fronds, the ethanol concentration and yield based on the theoretical maximum after 12 and 48 h of the SSF were 7.5 and 9.7 g/L and 43.8 and 56.8 %, respectively. The ethanol productivities found at 12 and 24 h from pretreated fronds were 0.62 and 0.36 g/L/h, respectively.

Engineering of Saccharomyces cerevisiae to produce advanced biofuels such as isobutanol has received much attention because this yeast has a natural capacity to produce higher alcohols. In this study, construction of isobutanol production systems was attempted by overexpression of effective 2-keto acid decarboxylase (KDC) and combinatorial overexpression of valine biosynthetic enzymes in S. cerevisiae D452-2. Among the six putative KDC enzymes from various microorganisms, 2-ketoisovalerate decarboxylase (Kivd) from L. lactis subsp. lactis KACC 13877 was identified as the most suitable KDC for isobutanol production in the yeast. Isobutanol production by the engineered S. cerevisiae was assessed in micro-aerobic batch fermentations using glucose as a sole carbon source. 93 mg/L isobutanol was produced in the Kivd overexpressing strain, which corresponds to a fourfold improvement as compared with the control strain. Isobutanol production was further enhanced to 151 mg/L by additional overexpression of acetolactate synthase (Ilv2p), acetohydroxyacid reductoisomerase (Ilv5p), and dihydroxyacid dehydratase (Ilv3p) in the cytosol.

Resveratrol, a polyphenol compound found in grape skins, has been proposed to account for the beneficial effects of red wine against heart disease. To produce resveratrol in Saccharomyces cerevisiae, four heterologous genes were introduced: the phenylalanine ammonia lyase gene from Rhodosporidium toruloides, the cinnamic acid 4-hydroxylase and 4-coumarate:coenzyme A ligase genes both from Arabidopsis thaliana, and the stilbene synthase gene from Arachis hypogaea. When this recombinant yeast was cultivated by batch fermentation in YP medium containing 2% galactose, it produced 2.6 mg/L p-coumaric acid and 3.3 mg/L resveratrol. In order to increase the pool of malonyl-CoA, a key precursor in resveratrol biosynthesis, the acetyl-CoA carboxylase (ACC1) gene was additionally overexpressed in the yeast by replacing the native promoter of the ACC1 gene with the stronger GAL1 promoter and this resulted in enhanced production of resveratrol (4.3 mg/L). Furthermore, when tyrosine was supplemented in the medium, the concentration of resveratrol increased up to 5.8 mg/L. This result illustrates a possible strategy for developing metabolically engineered yeast strain for the economical production of resveratrol from cheap amino acids., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The S domain of hepatitis B virus surface antigen (sHBsAg) is the primary component for vaccine development against virus infection. For stable expression of sHBsAg in recombinant Saccharomyces cerevisiae, new accessory genes necessary for foreign protein expression were screened by DNA microarray. Among 600 genes of interest, genes coding for an activating protein of ATPase in Hsp90 (Aha1p), S. cerevisiae DnaJ (Scj1p), thioredoxin 2 (Trx2p) and a GTPase-activator specific for Sar1 (Sec23p) as well as Pdi1p were selected in transcriptome analysis, which are known to facilitate disulfide bond formation or induce protein transport in the secretion pathway. Individual and combinatorial expression of SEC23, TRX2 and PDI1 increased total sHBsAg concentration by 1.9-6.5-fold, relative to the control strain expressing sHBsAg only. Additionally, moderate expression of Kex2p protease able to cut off the signal peptide enhanced the portion of the authentic sHBsAg to total sHBsAg. Fed-batch fermentation of the S. cerevisiae 2805 strain coexpressing the sHBsAg, SEC23, PDI1 and KEX2 genes resulted in 70.6mg/L final sHBsAg concentration which was 5.6 times higher than that of the control. Transmission electron microscopic analysis of the yeast cells elucidated the effects of the accessory gene coexpression on the intracellular localization of sHBsAg. Like PDI1, coexpression of both SEC23 and/or TRX2 newly isolated in this study is expected to improve the target protein expression in S. cerevisiae., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Efficient conversion of xylose to ethanol is an essential factor for commercialization of lignocellulosic ethanol. To minimize production of xylitol, a major by-product in xylose metabolism and concomitantly improve ethanol production, Saccharomyces cerevisiae D452-2 was engineered to overexpress NADH-preferable xylose reductase mutant (XR(MUT)) and NAD⁺-dependent xylitol dehydrogenase (XDH) from Pichia stipitis and endogenous xylulokinase (XK). In vitro enzyme assay confirmed the functional expression of XR(MUT), XDH and XK in recombinant S. cerevisiae strains. The change of wild type XR to XR(MUT) along with XK overexpression led to reduction of xylitol accumulation in microaerobic culture. More modulation of the xylose metabolism including overexpression of XR(MUT) and transaldolase, and disruption of the chromosomal ALD6 gene encoding aldehyde dehydrogenase (SX6(MUT)) improved the performance of ethanol production from xylose remarkably. Finally, oxygen-limited fermentation of S. cerevisiae SX6(MUT) resulted in 0.64 g l⁻¹ h⁻¹ xylose consumption rate, 0.25 g l⁻¹ h⁻¹ ethanol productivity and 39% ethanol yield based on the xylose consumed, which were 1.8, 4.2 and 2.2 times higher than the corresponding values of recombinant S. cerevisiae expressing XR(MUT), XDH and XK only., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Bioethanol is currently used as an alternative fuel for gasoline worldwide. For economic production of bioethanol by Saccharomyces cerevisiae, formation of a main by-product, glycerol, should be prevented or minimized in order to reduce a separation cost of ethanol from fermentation broth. In this study, S. cerevisiae was engineered to investigate the effects of the sole and double disruption of NADH-dependent glycerol-3-phosphate dehydrogenase 1 (GPD1) and NADPH-requiring glutamate dehydrogenase 1 (GDH1) on the production of glycerol and ethanol from glucose. Even though sole deletion of GPD1 or GDH1 reduced glycerol production, double deletion of GPD1 and GDH1 resulted in the lowest glycerol concentration of 2.31 g/L, which was 46.4% lower than the wild-type strain. Interestingly, the recombinant S. cerevisiae ∆GPD1∆GDH1 strain showed a slight improvement in ethanol yield (0.414 g/g) compared with the wild-type strain (0.406 g/g). Genetic engineering of the glycerol and glutamate metabolic pathways modified NAD(P)H-requiring metabolic pathways and exerted a positive effect on glycerol reduction without affecting ethanol production.

Yeast dehydrogenases and reductases were overexpressed in Saccharomyces cerevisiae D452-2 to detoxify 2-furaldehyde (furfural) and 5-hydroxymethyl furaldehyde (HMF), two potent toxic chemicals present in acid-hydrolyzed cellulosic biomass, and hence improve cell growth and ethanol production. Among those enzymes, aldehyde dehydrogenase 6 (ALD6) played the dual roles of direct oxidation of furan derivatives and supply of NADPH cofactor to their reduction reactions. Batch fermentation of S. cerevisiae D452-2/pH-ALD6 in the presence of 2g/L furfural and 0.5 g/L HMF resulted in 20-30% increases in specific growth rate, ethanol concentration and ethanol productivity, compared with those of the wild type strain. It was proposed that overexpression of ALD6 could recover the yeast cell metabolism and hence increase ethanol production from lignocellulosic biomass containing furan-derived inhibitors., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Although Saccharomyces cerevisiae is capable of fermenting galactose into ethanol, ethanol yield and productivity from galactose are significantly lower than those from glucose. An inverse metabolic engineering approach was undertaken to improve ethanol yield and productivity from galactose in S. cerevisiae. A genome-wide perturbation library was introduced into S. cerevisiae, and then fast galactose-fermenting transformants were screened using three different enrichment methods. The characterization of genetic perturbations in the isolated transformants revealed three target genes whose overexpression elicited enhanced galactose utilization. One confirmatory (SEC53 coding for phosphomannomutase) and two novel targets (SNR84 coding for a small nuclear RNA and a truncated form of TUP1 coding for a general repressor of transcription) were identified as overexpression targets that potentially improve galactose fermentation. Beneficial effects of overexpression of SEC53 may be similar to the mechanisms exerted by overexpression of PGM2 coding for phosphoglucomutase. While the mechanism is largely unknown, overexpression of SNR84, improved both growth and ethanol production from galactose. The most remarkable improvement of galactose fermentation was achieved by overexpression of the truncated TUP1 (tTUP1) gene, resulting in unrivalled galactose fermentation capability, that is 250% higher in both galactose consumption rate and ethanol productivity compared to the control strain. Moreover, the overexpression of tTUP1 significantly shortened lag periods that occurs when substrate is changed from glucose to galactose. Based on these results we proposed a hypothesis that the mutant Tup1 without C-terminal repression domain might bring in earlier and higher expression of GAL genes through partial alleviation of glucose repression. mRNA levels of GAL genes (GAL1, GAL4, and GAL80) indeed increased upon overexpression of tTUP. The results presented in this study illustrate that alteration of global regulatory networks through overexpression of the identified targets (SNR84 and tTUP1) is as effective as overexpression of a rate limiting metabolic gene (PGM2) in the galactose assimilation pathway for efficient galactose fermentation in S. cerevisiae. In addition, these results will be industrially useful in the biofuels area as galactose is one of the abundant sugars in marine plant biomass such as red seaweed as well as cheese whey and molasses., (Copyright © 2010 Wiley Periodicals, Inc.)

The use of plant biomass for biofuel production will require efficient utilization of the sugars in lignocellulose, primarily glucose and xylose. However, strains of Saccharomyces cerevisiae presently used in bioethanol production ferment glucose but not xylose. Yeasts engineered to ferment xylose do so slowly, and cannot utilize xylose until glucose is completely consumed. To overcome these bottlenecks, we engineered yeasts to coferment mixtures of xylose and cellobiose. In these yeast strains, hydrolysis of cellobiose takes place inside yeast cells through the action of an intracellular β-glucosidase following import by a high-affinity cellodextrin transporter. Intracellular hydrolysis of cellobiose minimizes glucose repression of xylose fermentation allowing coconsumption of cellobiose and xylose. The resulting yeast strains, cofermented cellobiose and xylose simultaneously and exhibited improved ethanol yield when compared to fermentation with either cellobiose or xylose as sole carbon sources. We also observed improved yields and productivities from cofermentation experiments performed with simulated cellulosic hydrolyzates, suggesting this is a promising cofermentation strategy for cellulosic biofuel production. The successful integration of cellobiose and xylose fermentation pathways in yeast is a critical step towards enabling economic biofuel production.

Resveratrol is a well-known polyphenol present in red wine and exerts antioxidative and anti-carcinogenic effects on the human body. To produce resveratrol in a food-grade yeast, the 4-coumarate:coenzyme A ligase gene (4CL1) from Arabidopsis thaliana and stilbene synthase gene (STS) from Arachis hypogaea were cloned and transformed into Saccharomyces cerevisiae W303-1A. The resveratrol produced was unglycosylated and secreted into the culture medium. A batch culture with 15.3mg/l p-coumaric acid used as precursor resulted in the production of 3.1mg/l resveratrol with 14.4 mol% yield. Deletion of the putative phenyl acrylic acid decarboxylase gene (PAD1) did not enhance resveratrol production., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Xylose-fermenting Saccharomyces strains are needed for commercialization of ethanol production from lignocellulosic biomass. Engineered Saccharomyces cerevisiae strains expressing XYL1, XYL2 and XYL3 from Pichia stipitis, however, utilize xylose in an oxidative manner, which results in significantly lower ethanol yields from xylose as compared to glucose. As such, we hypothesized that reconfiguration of xylose metabolism from oxidative into fermentative manner might lead to efficient ethanol production from xylose. To this end, we generated a respiration-deficient (RD) mutant in order to enforce engineered S. cerevisiae to utilize xylose only through fermentative metabolic routes. Three different repeated-batch fermentations were performed to characterize characteristics of the respiration-deficient mutant. When fermenting glucose as a sole carbon source, the RD mutant exhibited near theoretical ethanol yields (0.46 g g(-1)) during repeated-batch fermentations by recycling the cells. As the repeated-batch fermentation progressed, the volumetric ethanol productivity increased (from 7.5 to 8.3 g L(-1)h(-1)) because of the increased biomass from previous cultures. On the contrary, the mutant showed decreasing volumetric ethanol productivities during the repeated-batch fermentations using xylose as sole carbon source (from 0.4 to 0.3 g L(-1)h(-1)). The mutant did not grow on xylose and lost fermenting ability gradually, indicating that the RD mutant cannot maintain a good fermenting ability on xylose as a sole carbon source. However, the RD mutant was capable of fermenting a mixture of glucose and xylose with stable yields (0.35 g g(-1)) and productivities (0.52 g L(-1)h(-1)) during the repeated-batch fermentation. In addition, ethanol yields from xylose during the mixed sugar fermentation (0.30 g g(-1)) were higher than ethanol yields from xylose as a sole carbon source (0.21 g g(-1)). These results suggest that a strategy for increasing ethanol yield through respiration-deficiency can be applied for the fermentation of lignocellulosic hydrolyzates containing glucose and xylose., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Hepatitis B virus, a member of the hepadnavirus family, causes the acute and chronic diseases of the human liver. The S domain of hepatitis B virus surface antigen (sHBsAg) was expressed under the control of the galactose-inducible GAL1 promoter in recombinant Saccharomyces cerevisiae. Batch fermentation of S. cerevisiae 2805/pdeltaMFalpha-sHBsAg resulted in 4.92gl(-1) dry cell mass and 2.21mgl(-1) sHBsAg concentration. To improve the expression level of sHBsAg, the pdi1 gene encoding protein disulfide isomerase was coexpressed in recombinant S. cerevisiae. Batch fermentation of S. cerevisiae 2805/pdeltaMFalpha-sHBsAg+pPDI using 21gl(-1) glucose and 33gl(-1) galactose resulted in 9.75gl(-1) dry cell mass and 13.3mgl(-1) sHBsAg concentration, which were 2 and 6 times higher than those for S. cerevisiae 2805/pdeltaMFalpha-sHBsAg, respectively. Appearance of three sHBsAgs with different molecular sizes of 24kDa, 34kDa and 40kDa in immunoblot assay and their endoglycosidase treatment indicated that sHBsAg might be expressed in three types of the authentic, MFalpha signal sequence-containing and N-glycosylated MFalpha signal sequence-containing forms. Fed-batch fermentation of recombinant S. cerevisiae 2805 coexpressing the sHBsAg and pdi1 genes was carried out by feeding 600gl(-1) glucose continuously and controlling galactose concentration at around 20gl(-1). As a result, 20.2gl(-1) dry cell mass and 74.4mgl(-1) maximum sHBsAg concentration were obtained, which were 4.1 and 33.7 times higher than those for the batch fermentation of S. cerevisiae 2805/pdeltaMFalpha-sHBsAg, respectively.

2-Phenylethanol is a widely used aroma compound with rose-like fragrance and L-homophenylalanine is a building block of angiotensin-converting enzyme (ACE) inhibitor. 2-phenylethanol and L-homophenylalanine were synthesized simultaneously with high yield from 2-oxo-4-phenylbutyric acid and L-phenylalanine, respectively. A recombinant Escherichia coli harboring a coupled reaction pathway comprising of aromatic transaminase, phenylpyruvate decarboxylase, carbonyl reductase, and glucose dehydrogenase (GDH) was constructed. In the coupled reaction pathway, the transaminase reaction was coupled with the Ehrlich pathway of yeast; (1) a phenylpyruvate decarboxylase (YDR380W) as the enzyme to generate the substrate for the carbonyl reductase from phenylpyruvate (i.e., byproduct of the transaminase reaction) and to shift the reaction equilibrium of the transaminase reaction, and (2) a carbonyl reductase (YGL157W) to produce the 2-phenylethanol. Selecting the right carbonyl reductase showing the highest activity on phenylacetaldehyde with narrow substrate specificity was the key to success of the constructing the coupling reaction. In addition, NADPH regeneration was achieved by incorporating the GDH from Bacillus subtilis in the coupled reaction pathway. Based on 40 mM of L-phenylalanine used, about 96% final product conversion yield of 2-phenylethanol was achieved using the recombinant E. coli.

The endoxylanase (GenBank Access No. U51675) of Bacillus spp. and endoglucanase (GenBank Access No. AY466436) of Trichoderma spp. were separately inserted downstream of the yeast constitutive ADH1 promoter, resulting in three different plasmids (pAGX1, pAGX2, and pAGX3) according to the transcription direction of two genes. When the yeast transformants, S. cerevisiae SEY2102 harboring each expression plasmid, were grown on YPD medium, the total activities of the enzymes were approximately 3.01 unit/ml, 3.24 unit/ml, and 7.56 unit/ml for endoxylanase and 0.60 unit/ml, 0.54 unit/ml, and 0.39 unit/ ml for endoglucanase, in the following order: the pAGX1, pAGX2, and pAGX3. More than 70% of the endoxylanase and endoglucanase activities was found in the extracellular media.

The GAL1 gene encoding galactokinase was disrupted in a recombinant Saccharomyces cerevisiae strain in which production of LK8 protein, a kringle fragment of human apolipoprotein, is under the control of GAL1 promoter. Null mutation of the HXK2 gene was introduced further in the gal1Delta strain to relieve glucose repression. A pattern for LK8 expression was compared for the two recombinant S. cerevisiae systems in continuous and fed-batch cultivations. A critical dilution rate in continuous cultivation that repressed LK8 expression was significantly higher for the gal1Deltahxk2Delta strain than that for the gal1Delta strain to sustain the LK8 production even at high glucose consumption rate. Expressed LK8 for the gal1Delta strain was not detectable when the dilution rate exceeded 0.05 h(-1). Maximum LK8 concentration of 57 mgl(-1) was obtained in glucose-limited fed-batch cultivation of the gal1Deltahxk2Delta strain, corresponding to a 13.8-fold enhancement compared with the gal1Delta strain grown under the same conditions.

A gratuitous strain was developed by disrupting the GAL1 gene (galactokinase) of recombinant Saccharomyces cerevisiae harboring the antithrombotic hirudin gene in the chromosome under the control of the GAL10 promoter. A series of glucose-limited fed-batch cultures were carried out to examine the effects of glucose supply on hirudin expression in the gratuitous strain. Controlled feeding of glucose successfully supported both cell growth and hirudin expression in the gratuitous strain. The optimum fed-batch culture done by feeding glucose at a rate of 0.3 g h(-1) produced a maximum hirudin concentration of 62.1 mg l(-1), which corresponded to a 4.5-fold increase when compared with a simple batch culture done with the same strain.

In order to increase a production level of antithrombotic hirudin, BiP was simultaneously expressed in recombinant Saccharomyces cerevisiae strains carrying ten and 15 copies of the hirudin expression cassette integrated in the chromosome. Coexpression of BiP greatly enhanced both cell growth and hirudin production in recombinant S. cerevisiae. Maximum hirudin concentration of 36 mg l(-1) was obtained from batch culture of the ten copy-number transformant concomitantly harboring an episomal copy of the BiP gene under the control of the GAL1 promoter, which is corresponding to a 2.5-fold increase compared with the control strain carrying the genomic BiP gene only. The mean size of the recombinant yeast cells expressing the BiP gene remained at a relatively constant level compared with the control strains of which size increased after the onset of hirudin expression by the GAL10 promoter.

Aims: Recombinant Saccharomyces cerevisiae strains harbouring different levels of xylulokinase (XK) activity and effects of XK activity on utilization of xylulose were studied in batch and fed-batch cultures., Methods and Results: The cloned xylulokinase gene (XKS1) from S. cerevisiae was expressed under the control of the glyceraldehyde 3-phosphate dehydrogenase promoter and terminator. Specific xylulose consumption rate was enhanced by the increased specific XK activity, resulting from the introduction of the XKS1 into S. cerevisiae. In batch and fed-batch cultivations, the recombinant strains resulted in twofold higher ethanol concentration and 5.3- to six-fold improvement in the ethanol production rate compared with the host strain S. cerevisiae., Conclusions: An effective conversion of xylulose to xylulose 5-phosphate catalysed by XK in S. cerevisiae was considered to be essential for the development of an efficient and accelerated ethanol fermentation process from xylulose., Significance and Impact of the Study: Overexpression of the XKS1 gene made xylulose fermentation process accelerated to produce ethanol through the pentose phosphate pathway.

Zero trans-influx assays of glucose and xylose were performed using Saccharomyces cerevisiae to investigate transport characteristics under high and low glucose conditions. Under high glucose conditions, most glucose was transported by the low-affinity transporter. The high-affinity transporter was expressed under low glucose conditions, transporting over 50% glucose. Inhibition kinetics revealed that xylose was transported by both high- and low-affinity glucose transporters. Affinities of both glucose transporters for xylose were very low under high glucose condition but increased to a similar level to glucose under low glucose condition. The maximum rate of xylose transport increased by 85%, while an overall maximum glucose transport rate decreased by 42% under low glucose condition, indicating the presence of other transport system for sugars except for glucose. It was suggested that expression of the high-affinity transporter and increased affinity of glucose transporters for xylose under low glucose condition would provide a fermentation strategy for enhancing the productivity of xylitol by recombinant S. cerevisiae harboring the xylose reductase gene.

Recombinant Saccharomyces cerevisiae strains were developed to overproduce an anticoagulant hirudin. The delta-sequences of the yeast retrotransposon Ty1 and URA3 were used as target sites for a hirudin expression cassette. High copy-number transformants were successfully selected using a dominant selection antibiotic, G418. The copy numbers of the hirudin expression cassette integrated into delta-sequences of the yeast chromosome ranged from five to ten copies per cell. Production of hirudin in the delta-integrated recombinant S. cerevisiae system increased over two-fold compared with the YEp-based episomal hirudin expression system. A linear relationship between the copy number of the hirudin expression cassette and hirudin expression level was observed up to 10 copies. The hirudin expression cassettes integrated into the yeast chromosome were stably maintained in non-selective culture conditions.