Recombinant Saccharomyces Cerevisiae Research Articles

An efficient xylose-fermenting recombinant Saccharomyces cerevisiae strain obtained through adaptive evolution and its global transcription profile

Factors related to ethanol production from xylose in engineered Saccharomyces cerevisiae that contain an exogenous initial metabolic pathway are still to be elucidated. In the present study, a strain that expresses the xylose isomerase gene of Piromyces sp. Pi-xylA and overexpresses XKS1, RPE1, RKI1, TAL1, and TKL1, with deleted GRE3 and COX4 genes was constructed. The xylose utilization capacity of the respiratory deficiency strain was poor but improved via adaptive evolution in xylose. The μ (max) of the evolved strain in 20 g l(-1) xylose is 0.11 ± 0.00 h(-1), and the evolved strain consumed 17.83 g l(-1) xylose within 72 h, with an ethanol yield of 0.43 g g(-1) total consumed sugars during glucose-xylose cofermentation. Global transcriptional changes and effect of several specific genes were studied. The result revealed that the increased xylose isomerase acivity, the upregulation of enzymes involved in glycolysis and glutamate synthesis, and the downregulation of trehalose and glycogen synthesis, may have contributed to the improved xylose utilization of the strain. Furthermore, the deletion of PHO13 decreased the xylose growth in the respiration deficiency strain although deleting PHO13 can improve the xylose metabolism in other strains.

Reduction of furan derivatives by overexpressing NADH-dependent Adh1 improves ethanol fermentation using xylose as sole carbon source with Saccharomyces cerevisiae harboring XR–XDH pathway

Several alcohol dehydrogenase (ADH)-related genes have been identified as enzymes for reducing levels of toxic compounds, such as, furfural and/or 5-hydroxymethylfurfural (5-HMF), in hydrolysates of pretreated lignocelluloses. To date, overexpression of these ADH genes in yeast cells have aided ethanol production from glucose or glucose/xylose mixture in the presence of furfural or 5-HMF. However, the effects of these ADH isozymes on ethanol production from xylose as a sole carbon source remain uncertain. We showed that overexpression of mutant NADH-dependent ADH1 derived from TMB3000 strain in the recombinant Saccharomyces cerevisiae, into which xylose reductase (XR) and xylitol dehydrogenase (XDH) pathway of Pichia stipitis has been introduced, improved ethanol production from xylose as a sole carbon source in the presence of 5-HMF. Enhanced furan-reducing activity is able to regenerate NAD(+) to relieve redox imbalance, resulting in increased ethanol yield arising from decreased xylitol accumulation. In addition, we found that overexpression of wild-type ADH1 prevented the more severe inhibitory effects of furfural in xylose fermentation as well as overexpression of TMB3000-derived mutant. After 120h of fermentation, the recombinant strains overexpressing wild-type and mutant ADH1 completely consumed 50g/L xylose in the presence of 40mM furfural and most efficiently produced ethanol (15.70g/L and 15.24g/L) when compared with any other test conditions. This is the first report describing the improvement of ethanol production from xylose as the sole carbon source in the presence of furan derivatives with xylose-utilizing recombinant yeast strains via the overexpression of ADH-related genes.

Overexpression of HAC1 gene increased levels of both intracellular and secreted human kringle fragment in Saccharomyces cerevisiae

Effects of overexpression of an unfolded protein response (UPR) gene were investigated in recombinant Saccharomyces cerevisiae strains harboring 16 copies of the gene for human kringle protein, LK8. In S. cerevisiae, Hac1p plays a crucial role as a transcription activator and Ire1p as endonuclease acting on the HAC1 transcript to remove an intron and induce the UPR. The disruption of the two genes was detrimental to LK8 expression, and interestingly, the hac1Δ strain was not able to utilize galactose as a carbon source and concomitantly delayed cell growth compared with the wild type and the ire1Δ strains. In a complementation test, the growth defect was partially recovered by the overexpression of the HAC1 gene controlled by the GAL1 promoter. Additional activation of UPR was mediated by the GAL1 promoter driven coexpression of the HAC1 gene and enhanced the intracellular and secreted levels of LK8 by 4- and 1.6-fold, respectively. The UPR was essentially required for the heterologous production of LK8. Furthermore, Hac1p is the factor promoting LK8 protein production as well as cell growth in recombinant S. cerevisiae strains. This result indicates that the additional activation of UPR might be a good option for overproduction of heterologous proteins in S. cerevisiae.

Sm-like protein enhanced tolerance of recombinant Saccharomyces cerevisiae to inhibitors in hemicellulosic hydrolysate

A current challenge of the cellulosic ethanol industry is to improve the resistance of inhibitors present in biomass hydrolysates. RNA-binding protein gene lsm6 was cloned from industrial Saccharomyces cerevisiae ZU-E8, which is able to conferment glucose and xylose, and transformed into ZU-E8 via expression vector pRS426. The positive transformant ZU-910 with over-expressing lsm6 was identified on the culture plates using high concentration of acetate and re-screened by fermentation test. Fermentation by the recombinants was performed in a medium containing 80g/L xylose and 2g/L acetic acid or 20g/L NH4Ac/NaAc. After 96h shaking-flask fermentation, ZU-910 utilized 90.2% xylose with an ethanol yield of 26.9g/L, which was 8.5- and 10-fold higher than ZU-E8. Further, in the corn stover hemicellulosic hydrolysate fermentation, both the xylose conversion and ethanol production by ZU-910 was larger by 50% and 40% than ZU-E8. ZU-910 has also enhanced tolerance against furfural and SO42−.

Implementation of a transhydrogenase-like shunt to counter redox imbalance during xylose fermentation in Saccharomyces cerevisiae

Three enzymes responsible for the transhydrogenase-like shunt, including malic enzyme (encoded by MAE1), malate dehydrogenase (MDH2), and pyruvate carboxylase (PYC2), were overexpressed to regulate the redox state in xylose-fermenting recombinant Saccharomyces cerevisiae. The YPH499XU/MAE1 strain was constructed by overexpressing native Mae1p in the YPH499XU strain expressing xylose reductase and xylitol dehydrogenase from Scheffersomyces stipitis, and native xylulokinase. Analysis of the xylose fermentation profile under semi-anaerobic conditions revealed that the ethanol yield in the YPH499XU/MAE1 strain (0.38 ± 0.01 g g⁻¹ xylose consumed) was improved from that of the control strain (0.31 ± 0.01 g g⁻¹ xylose consumed). Reduced xylitol production was also observed in YPH499XU/MAE1, suggesting that the redox balance was altered by Mae1p overexpression. Analysis of intracellular metabolites showed that the redox imbalance during xylose fermentation was partly relieved in the transformant. The specific ethanol production rate in the YPH499XU/MAE1-MDH2 strain was 1.25-fold higher than that of YPH499XU/MAE1 due to the additional overexpression of Mdh2p, whereas the ethanol yield was identical to that of YPH499XU/MAE1. The specific xylose consumption rate was drastically increased in the YPH499XU/MAE1-MDH2-PYC2 strain. However, poor ethanol yield as well as increased production of xylitol was observed. These results demonstrate that the transhydrogenase function implemented in S. cerevisiae can regulate the redox state of yeast cells.

O-0002 A Phase 2 Adjuvant Trial of GI-4000 Plus Gemcitabine vs. Gemcitabine Alone in Ras+ Patients with Resected Pancreas Cancer: R1 Subgroup Analysis

ABSTRACT Introduction Patients with resected pancreas cancer treated with standard of care gemcitabine have a median overall survival of 22 months (vs 20 months with observation). Activating mutations in ras occur in > 90% of pancreas cancer cases. GI-4000 is a proprietary immunotherapy designed to target cells with activating ras mutations using whole, heat-killed recombinant Saccharomyces cerevisiae yeast (called Tarmogens = Tar geted Mo lecular Immuno gens ). Tarmogens have demonstrated selective killing of target cells expressing a number of cancer antigens including mutated Ras in vivo by activating an antigen-specific T cell mediated response. This trial is designed to evaluate the efficacy, immunogenicity, and safety of GI-4000 plus gemcitabine in patients with Ras mutant positive resected pancreas cancer. Methods The study enrolled 176 subjects with Ras mutant positive adenocarcinoma of the pancreas post resection randomized 1:1 to GI-4000 plus gemcitabine or placebo plus gemcitabine (stratified by resection status; R0 or R1). Three weekly injections of GI-4000 or placebo were followed by 6 cycles of gemcitabine 1000 mg/m2 iv (day 1, 8, 15 every 28 days). Monthly GI-4000 or placebo were administered on the gemcitabine off-weeks and continued monthly until disease recurrence, intolerable toxicity, or death. The primary endpoint is recurrence-free survival. Data for the 39 R1 subjects (GI-4000 N = 19, Placebo N = 20) have been unblinded and analyzed. Results The GI-4000 group had an 11.4 week advantage in median overall survival (524 days vs 444 days), 16% advantage in 1 year survival (72% vs 56%), and a 4.6 week advantage in median RFS (287 days vs 255 days). The GI-4000 group showed a significantly higher rate of mutation specific T cell response to Ras by ELISpot assay; 7/15 (47%) vs 1/12 (8%), p = 0.032,with a more pronounced survival benefit in GI-4000 treated immune responders; 21.7 week advantage in median survival (596 Days vs 444 Days) compared to placebo. No significant novel toxicities have been observed to date. Conclusion GI-4000 in combination with adjuvant gemcitabine showed a clinically meaningful point estimate for the treatment effect on survival in R1 subjects with Ras mutant positive adenocarcinoma of the pancreas. GI-4000 was immunogenic and well tolerated. Ras specific immune response was associated with a more pronounced benefit in median survival. These data warrant further study in a definitively powered clinical trial for GI-4000 in the adjuvant setting in R1 subjects. The results for the R0 cohort will be reported when the data are mature.

A pilot trial of a combination of therapeutic vaccines (GI-4000 and GI-6207) as adjunctive therapy with first-line therapy with bevacizumab plus either FOLFOX or FOLFIRI in stage IV patients with newly diagnosed Ras-mutant positive or negative metastatic colorectal cancer.

TPS3638 Background: A promising approach for the treatment of cancer is the development of vaccines that target specific tumor antigens. In the metastatic CRC patient population, targeted and active immunotherapy may inhibit cancer progression and improve survival. This trial is designed to evaluate the efficacy, immunogenicity, and safety of GI-4000 plus standard therapy in patients with metastatic colorectal cancer. GI-4000 is a proprietary immunotherapy that uses whole, heat-killed recombinant Saccharomyces cerevisiae yeast (called Tarmogens = Targeted Molecular Immunogens). GI-4000 is designed to activate a cellular immune response to target cells with activating ras mutations. Tarmogens have been shown to elicit selective killing of target cells that express a number of cancer antigens, including mutated Ras, by activation of antigenspecific T cell mediated responses. Methods: The study population consists of subjects with metastatic colorectal cancer with an activating mutation in ras. Newly diagnosed subjects receive FOLFOX (or FOLFIRI) + bevacizumab (Bev) + GI- 4000; 3 weekly injections of GI-4000 are followed by 8 cycles of Bev + FOLFOX (or FOLFIRI); day 1 and 2 every 14 days. Doses of GI-4000 are administered on day 8 of each cycle. Upon completion of chemotherapy, GI-4000 continues along with Bev maintenance every 2 weeks for up to 5 years or until subjects experience intolerance, disease recurrence, or death. If Bev is stopped, GI-4000 may continue on the same maintenance schedule alone. Subjects that have already completed standard chemotherapy (FOLFOX or FOLFIRI) may enter the study and receive Bev + GI-4000 every 2 weeks for up to 5 years. Enrollment is ongoing and will continue up to 52 subjects.

Effects of Trx2p and Sec23p expression on stable production of hepatitis B surface antigen S domain in recombinant Saccharomyces cerevisiae

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.

The metabolic burden of cellulase expression by recombinant Saccharomyces cerevisiae Y294 in aerobic batch culture

Two recombinant strains of Saccharomyces cerevisiae Y294 producing cellulase using different expression strategies were compared to a reference strain in aerobic culture to evaluate the potential metabolic burden that cellulase expression imposed on the yeast metabolism. In a chemically defined mineral medium with glucose as carbon source, S. cerevisiae strain Y294[CEL5] with plasmid-borne cellulase genes produced endoglucanase and β-glucosidase activities of 0.038 and 0.30 U mg dry cell weight(-1), respectively. Chromosomal expression of these two cellulases in strain Y294[Y118p] resulted in no detectable activity, although low levels of episomally co-expressed cellobiohydrolase (CBH) activity were detected. Whereas the biomass concentration of strain Y294[CEL5] was slightly greater than that of a reference strain, CBH expression by Y294[Y118p] resulted in a 1.4-fold lower maximum specific growth rate than that of the reference. Supplementation of the growth medium with amino acids significantly improved culture growth and enzyme production, but only partially mitigated the physiological effects and metabolic burden of cellulase expression. Glycerol production was decreased significantly, up to threefold, in amino acid-supplemented cultures, apparently due to redox balancing. Disproportionately higher levels of glycerol production by Y294[CEL5] indicated a potential correlation between the redox balance of anabolism and the physiological stress of cellulase production. With the reliance on cellulase expression in yeast for the development of consolidated bioprocesses for bioethanol production, this work demonstrates the need for development of yeasts that are physiologically robust in response to burdens imposed by heterologous enzyme production.

Characterization of non-oxidative transaldolase and transketolase enzymes in the pentose phosphate pathway with regard to xylose utilization by recombinant Saccharomyces cerevisiae

The activity of transaldolase and transketolase, key enzymes in the non-oxidative pentose phosphate pathway, is rate-limiting for xylose utilization in recombinant Saccharomyces cerevisiae. Overexpression of TAL1 and TKL1, the major transaldolase and transketolase genes, increases the flux from the pentose phosphate pathway into the glycolytic pathway. However, the functional roles of NQM1 and TKL2, the secondary transaldolase and transketolase genes, especially in xylose utilization, remain unclear. This study focused on characterization of NQM1 and TKL2, together with TAL1 and TKL1, regarding their roles in xylose utilization and fermentation. Knockout or overexpression of these four genes on the phenotype of xylose-utilizing S. cerevisiae strains was also examined. Transcriptional analysis indicated that the expression of TAL1, NQM1, and TKL1 was up-regulated in the presence of xylose. A significant decrease in both growth on xylose and xylose-fermenting ability in tal1Δ and tkl1Δ mutants confirmed that TAL1 and TKL1 are essential for xylose assimilation and fermentation. Gene disruption analysis using a tkl1Δ mutant revealed that TKL1 is also required for utilization of glucose. Growth on xylose and xylose-fermenting ability were slightly influenced by deletion of NQM1 or TKL2 when xylose was used as the sole carbon source. Moreover, the rate of xylose consumption and ethanol production was slightly impaired in TKL1- and TKL2-overexpressing strains. NQM1 and TKL2 may thus play a physiological role via an effect on the non-oxidative pentose phosphate pathway in the xylose metabolic pathway, although their roles in xylose utilization and fermentation are less important than those of TAL1 and TKL1.

Direct Conversion of Xylan to Ethanol by Recombinant Saccharomyces cerevisiae Strains Displaying an Engineered Minihemicellulosome

Arabinoxylan is a heteropolymeric chain of a β-1,4-linked xylose backbone substituted with arabinose residues, representing a principal component of plant cell walls. Here we developed recombinant Saccharomyces cerevisiae strains as whole-cell biocatalysts capable of combining hemicellulase production, xylan hydrolysis, and hydrolysate fermentation into a single step. These strains displayed a series of uni-, bi-, and trifunctional minihemicellulosomes that consisted of a miniscaffoldin (CipA3/CipA1) and up to three chimeric enzymes. The miniscaffoldin derived from Clostridium thermocellum contained one or three cohesin modules and was tethered to the cell surface through the S. cerevisiae a-agglutinin adhesion receptor. Up to three types of hemicellulases, an endoxylanase (XynII), an arabinofuranosidase (AbfB), and a β-xylosidase (XlnD), each bearing a C-terminal dockerin, were assembled onto the miniscaffoldin by high-affinity cohesin-dockerin interactions. Compared to uni- and bifunctional minihemicellulosomes, the resulting quaternary trifunctional complexes exhibited an enhanced rate of hydrolysis of arabinoxylan. Furthermore, with an integrated d-xylose-utilizing pathway, the recombinant yeast displaying the bifunctional minihemicellulosome CipA3-XynII-XlnD could simultaneously hydrolyze and ferment birchwood xylan to ethanol with a yield of 0.31 g per g of sugar consumed.

Improvements in ethanol production from xylose by mating recombinant xylose-fermenting Saccharomyces cerevisiae strains

To improve the ability of recombinant Saccharomyces cerevisiae strains to utilize the hemicellulose components of lignocellulosic feedstocks, the efficiency of xylose conversion to ethanol needs to be increased. In the present study, xylose-fermenting, haploid, yeast cells of the opposite mating type were hybridized to produce a diploid strain harboring two sets of xylose-assimilating genes encoding xylose reductase, xylitol dehydrogenase, and xylulokinase. The hybrid strain MN8140XX showed a 1.3- and 1.9-fold improvement in ethanol production compared to its parent strains MT8-1X405 and NBRC1440X, respectively. The rate of xylose consumption and ethanol production was also improved by the hybridization. This study revealed that the resulting improvements in fermentation ability arose due to chromosome doubling as well as the increase in the copy number of xylose assimilation genes. Moreover, compared to the parent strain, the MN8140XX strain exhibited higher ethanol production under elevated temperatures (38 °C) and acidic conditions (pH 3.8). Thus, the simple hybridization technique facilitated an increase in the xylose fermentation activity.

Physiological characterization of recombinant Saccharomyces cerevisiae expressing the Aspergillus nidulans phosphoketolase pathway: validation of activity through 13C-based metabolic flux analysis

Several bacterial species and filamentous fungi utilize the phosphoketolase pathway (PHK) for glucose dissimilation as an alternative to the Embden-Meyerhof-Parnas pathway. In Aspergillus nidulans, the utilization of this metabolic pathway leads to increased carbon flow towards acetate and acetyl CoA. In the first step of the PHK, the pentose phosphate pathway intermediate xylulose-5-phosphate is converted into acetylphosphate and glyceraldehyde-3-phosphate through the action of xylulose-5-phosphate phosphoketolase, and successively acetylphosphate is converted into acetate by the action of acetate kinase. In the present work, we describe a metabolic engineering strategy used to express the fungal genes of the phosphoketolase pathway in Saccharomyces cerevisiae and the effects of the expression of this recombinant route in yeast. The phenotype of the engineered yeast strain MP003 was studied during batch and chemostat cultivations, showing a reduced biomass yield and an increased acetate yield during batch cultures. To establish whether the observed effects in the recombinant strain MP003 were due directly or indirectly to the expression of the phosphoketolase pathway, we resolved the intracellular flux distribution based on (13)C labeling during chemostat cultivations. From flux analysis it is possible to conclude that yeast is able to use the recombinant pathway. Our work indicates that the utilization of the phosphoketolase pathway does not interfere with glucose assimilation through the Embden-Meyerhof-Parnas pathway and that the expression of this route can contribute to increase the acetyl CoA supply, therefore holding potential for future metabolic engineering strategies having acetyl CoA as precursor for the biosynthesis of industrially relevant compounds.

Competition between pentoses and glucose during uptake and catabolism in recombinant Saccharomyces cerevisiae

BackgroundIn mixed sugar fermentations with recombinant Saccharomyces cerevisiae strains able to ferment D-xylose and L-arabinose the pentose sugars are normally only utilized after depletion of D-glucose. This has been attributed to competitive inhibition of pentose uptake by D-glucose as pentose sugars are taken up into yeast cells by individual members of the yeast hexose transporter family. We wanted to investigate whether D-glucose inhibits pentose utilization only by blocking its uptake or also by interfering with its further metabolism.ResultsTo distinguish between inhibitory effects of D-glucose on pentose uptake and pentose catabolism, maltose was used as an alternative carbon source in maltose-pentose co-consumption experiments. Maltose is taken up by a specific maltose transport system and hydrolyzed only intracellularly into two D-glucose molecules. Pentose consumption decreased by about 20 - 30% during the simultaneous utilization of maltose indicating that hexose catabolism can impede pentose utilization. To test whether intracellular D-glucose might impair pentose utilization, hexo-/glucokinase deletion mutants were constructed. Those mutants are known to accumulate intracellular D-glucose when incubated with maltose. However, pentose utilization was not effected in the presence of maltose. Addition of increasing concentrations of D-glucose to the hexo-/glucokinase mutants finally completely blocked D-xylose as well as L-arabinose consumption, indicating a pronounced inhibitory effect of D-glucose on pentose uptake. Nevertheless, constitutive overexpression of pentose-transporting hexose transporters like Hxt7 and Gal2 could improve pentose consumption in the presence of D-glucose.ConclusionOur results confirm that D-glucose impairs the simultaneous utilization of pentoses mainly due to inhibition of pentose uptake. Whereas intracellular D-glucose does not seem to have an inhibitory effect on pentose utilization, further catabolism of D-glucose can also impede pentose utilization. Nevertheless, the results suggest that co-fermentation of pentoses in the presence of D-glucose can significantly be improved by the overexpression of pentose transporters, especially if they are not inhibited by D-glucose.

Dynamic metabolomics differentiates between carbon and energy starvation in recombinant Saccharomyces cerevisiae fermenting xylose

BackgroundThe concerted effects of changes in gene expression due to changes in the environment are ultimately reflected in the metabolome. Dynamics of metabolite concentrations under a certain condition can therefore give a description of the cellular state with a high degree of functional information. We used this potential to evaluate the metabolic status of two recombinant strains of Saccharomyces cerevisiae during anaerobic batch fermentation of a glucose/xylose mixture. Two isogenic strains were studied, differing only in the pathways used for xylose assimilation: the oxidoreductive pathway with xylose reductase (XR) and xylitol dehydrogenase (XDH) or the isomerization pathway with xylose isomerase (XI). The isogenic relationship between the two strains ascertains that the observed responses are a result of the particular xylose pathway and not due to unknown changes in regulatory systems. An increased understanding of the physiological state of these strains is important for further development of efficient pentose-utilizing strains for bioethanol production.ResultsUsing LC-MS/MS we determined the dynamics in the concentrations of intracellular metabolites in central carbon metabolism, nine amino acids, the purine nucleotides and redox cofactors. The general response to the transition from glucose to xylose was increased concentrations of amino acids and TCA-cycle intermediates, and decreased concentrations of sugar phosphates and redox cofactors. The two strains investigated had significantly different uptake rates of xylose which led to an enhanced response in the XI-strain. Despite the difference in xylose uptake rate, the adenylate energy charge remained high and stable around 0.8 in both strains. In contrast to the adenylate pool, large changes were observed in the guanylate pool.ConclusionsThe low uptake of xylose by the XI-strain led to several distinguished responses: depletion of key metabolites in glycolysis and NADPH, a reduced GTP/GDP ratio and accumulation of PEP and aromatic amino acids. These changes are strong indicators of carbon starvation. The XR/XDH-strain displayed few such traits. The coexistence of these traits and a stable adenylate charge indicates that xylose supplies energy to the cells but does not suppress a response similar to carbon starvation. Particular signals may play a role in the latter, of which the GTP/GMP ratio could be a candidate as it decreased significantly in both strains.

Reduction of Glucose Uptake through Inhibition of Hexose Transporters and Enhancement of Their Endocytosis by Methylglyoxal in Saccharomyces cerevisiae

Diabetes mellitus is characterized by an impairment of glucose uptake even though blood glucose levels are increased. Methylglyoxal is derived from glycolysis and has been implicated in the development of diabetes mellitus, because methylglyoxal levels in blood and tissues are higher in diabetic patients than in healthy individuals. However, it remains to be elucidated whether such factors are a cause, or consequence, of diabetes. Here, we show that methylglyoxal inhibits the activity of mammalian glucose transporters using recombinant Saccharomyces cerevisiae cells genetically lacking all hexose transporters but carrying cDNA for human GLUT1 or rat GLUT4. We found that methylglyoxal inhibits yeast hexose transporters also. Glucose uptake was reduced in a stepwise manner following treatment with methylglyoxal, i.e. a rapid reduction within 5 min, followed by a slow and gradual reduction. The rapid reduction was due to the inhibitory effect of methylglyoxal on hexose transporters, whereas the slow and gradual reduction seemed due to endocytosis, which leads to a decrease in the amount of hexose transporters on the plasma membrane. We found that Rsp5, a HECT-type ubiquitin ligase, is responsible for the ubiquitination of hexose transporters. Intriguingly, Plc1 (phospholipase C) negatively regulated the endocytosis of hexose transporters in an Rsp5-dependent manner, although the methylglyoxal-induced endocytosis of hexose transporters occurred irrespective of Plc1. Meanwhile, the internalization of hexose transporters following treatment with methylglyoxal was delayed in a mutant defective in protein kinase C.

Balance of XYL1 and XYL2 expression in different yeast chassis for improved xylose fermentation

Reducing xylitol formation is necessary in engineering xylose utilization in recombinant Saccharomyces cerevisiae for ethanol production through xylose reductase/xylitol dehydrogenase pathway. To balance the expression of XYL1 and mutant XYL2 encoding xylose reductase (XR) and NADP+-dependent xylitol dehydrogenase (XDH), respectively, we utilized a strategy combining chassis selection and direct fine-tuning of XYL1 and XYL2 expression in this study. A XYL1 gene under the control of various promoters of ADH1, truncated ADH1 and PGK1, and a mutated XYL2 with different copy numbers were constructed into different xylose-utilizing modules, which were then expressed in two yeast chassises W303a and L2612. The strategy enabled an improved L2612-derived recombinant strain with XYL1 controlled by promoter PGK1 and with two copies of XYL2. The strain exhibited a 21.3% lower xylitol yield and a 40.0% higher ethanol yield. The results demonstrate the feasibility of the combinatorial strategy for construction of an efficient xylose-fermenting S. cerevisiae.

A monolith purification process for virus-like particles from yeast homogenate

Monoliths are an alternative stationary phase format to conventional particle based media for large biomolecules. Conventional resins suffer from limited capacities and flow rates when used for viruses, virus-like particles (VLP) and other nanoplex materials. The monolith structure provides a more open pore structure to improve accessibility for these materials and better mass transport from convective flow and reduced pressure drops. To examine the performance of this format for bioprocessing we selected the challenging capture of a VLP from clarified yeast homogenate. Using a recombinant Saccharomyces cerevisiae host it was found hydrophobic interaction based separation using a hydroxyl derivatised monolith had the best performance. The monolith was then compared to a known beaded resin method, where the dynamic binding capacity was shown to be three-fold superior for the monolith with equivalent 90% recovery of the VLP. To understand the impact of the crude feed material confocal microscopy was used to visualise lipid contaminants, deriving from the homogenised yeast. It was seen that the lipid formed a layer on top of the column, even after regeneration of the column with isopropanol, resulting in increasing pressure drops with the number of operational cycles. Removal of the lipid pre-column significantly reduces the amount and rate of this fouling process. Using Amberlite/XAD-4 beads around 70% of the lipid was removed, with a loss of VLP around 20%. Applying a reduced lipid feed versus an untreated feed further increased the dynamic binding capacity of the monolith from 0.11mg/mL column to 0.25mg/mL column.

Enhancement of β-Carotene Production by Over-Expression of HMG-CoA Reductase Coupled with Addition of Ergosterol Biosynthesis Inhibitors in Recombinant Saccharomyces cerevisiae

In this study, the synergistic effect of overexpressing the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene and adding ergosterol synthesis inhibitor, ketoconazole, on β-carotene production in the recombinant Saccharomyces cerevisiae was investigated. The results showed that the over-expression of HMG-CoA reductase gene and adding 100 mg/l ketoconazole alone can result in 135.1 and 15.6% increment of β-carotene concentration compared with that of the control (2.05 mg/g dry weight of cells), respectively. However, the combination of overexpressing HMG-CoA reductase gene and adding ketoconazole can achieve a 206.8% increment of pigment content (6.29 mg/g dry weight of cells) compared with that of the control. Due to the fact that over-expression of the HMG-CoA reductase gene can simultaneously improve the flux of the sterol and carotenoid biosynthetic pathway, it can be concluded that under the circumstances of blocking sterol biosynthesis, increasing the activity of HMG-CoA reductase can result in more precursors FPP fluxing into carotenoid branch and obtain a high increment of β-carotene production. The results of this study collectively suggest that the combination of overexpressing HMG-CoA reductase gene and supplying ergosterol synthesis inhibitor is an effective strategy to improve the production of desirable isoprenoid compounds such as carotenoids.

Enhanced Production of β-Carotene by Recombinant Industrial Wine Yeast Using Grape Juice as Substrate

In this study, both recombinant Saccharomyces cerevisiae T73-63 and FY-09 derived from the industrial wine yeast T73-4 and laboratory yeast FY1679-01B, respectively, were constructed and compared for their β-carotene production in real grape juice. The results showed that highest β-carotene content (5.89mg/g) was found in strain T73-63, which was 2.1 fold higher than that of strain FY-09. Although the cell growth was inhibited by the metabolic burden induced by the production of heterogeneous β-carotene, the pigment yield in T73-63 was still 1.7 fold higher than that of FY-09. Furthermore, high contents of ergosterol and fatty acid were also observed in T73-63. These results suggest that industrial wine yeast has highly active metabolic flux in mevalonate pathway, which leads to more carbon flux into carotenoid branch compared to that of laboratory yeast. The results of this study collectively suggest that in the application of recombinant strains to produce carotenoid using agro-industrial by-products as substrate, the suitable host strains should have active mevalonate pathway. For this purpose, the industrial wine yeast is a suitable candidate.