Construction and application of multi-host integrative vector system for xylose-fermenting yeast.

The xylose-fermenting yeasts (CTG clade yeasts, e.g. Scheffersomyces stipitis, Spathaspora passalidarum, Candida amazonensis and Candida jeffriesii) have the potential to be superior platforms for the conversion of lignocellulosic hydrolysate into fuel-grade ethanol and other chemical products. Here, a genetic expression system compatible with the genetic coding characteristics of CTG clade yeasts was constructed for use in xylose-fermenting yeasts. The pRACTH-gfpm plasmid based on an 18S rDNA shuttle vector was capable of stable integration into the genomes of a wide range of heterologous hosts. Green fluorescent protein was transformed and functionally expressed in S. stipitis, S. passalidarum, C. jeffriesii, C. amazonensis and Saccharomyces cerevisiae under control of the SpADH1 promoter and SpCYC1 terminator. Finally, the expression system was useful in multiple yeast hosts for construction of the plasmid pRACTH-ldh. Scheffersomyces stipitis, S. passalidarum, C. jeffriesii, C. amazonensis and S. cerevisiae were enabled to produce lactate from glucose or xylose by pRACTH-based expression of a heterologous lactate dehydrogenase. Among them, C. amazonensis (pRACTH-ldh) exhibited the highest lactate fermentation capacity, which reached a maximum of 44 g L-1 of lactate with a yield of 0.85 g lactate/g xylose.

Metabolic engineering of Escherichia coli W3110 for the production of L-methionine.

In this study, we constructed an L-methionine-producing recombinant strain from wild-type Escherichia coli W3110 by metabolic engineering. To enhance the carbon flux to methionine and derepression met regulon, thrBC, lysA, and metJ were deleted in turn. Methionine biosynthesis obstacles were overcome by overexpression of metA Fbr (Fbr, Feedback resistance), metB, and malY under control of promoter pN25. Recombinant strain growth and methionine production were further improved by attenuation of metK gene expression through replacing native promoter by metK84p. Blocking the threonine pathway by deletion of thrBC or thrC was compared. Deletion of thrC showed faster growth rate and higher methionine production. Finally, metE, metF, and metH were overexpressed to enhance methylation efficiency. Compared with the original strain E. coli W3110, the finally obtained Me05 (pETMAFbr-B-Y/pKKmetH) improved methionine production from 0 to 0.65 and 5.62 g/L in a flask and a 15-L fermenter, respectively.

Targeting PSG1 to enhance chemotherapeutic efficacy: new application for anti-coagulant the dicumarol.

Chemotherapeutic response is critical for the successful treatment and good prognosis in cancer patients. In this study, we analysed the gene expression profiles of preoperative samples from oestrogen receptor (ER)-negative breast cancer patients with different responses to taxane-anthracycline-based (TA-based) chemotherapy, and identified a group of genes that was predictive. Pregnancy specific beta-1-glycoprotein 1 (PSG1) played a central role within signalling pathways of these genes. Inhibiting PSG1 can effectively reduce chemoresistance via a transforming growth factor-ß (TGF-ß)-related pathway in ER-negative breast cancer cells. Drug screening then identified dicumarol (DCM) to target the PSG1 and inhibit chemoresistance to TA-based chemotherapy in vitro, in vivo, and in clinical samples. Taken together, this study highlights PSG1 as an important mediator of chemoresistance, whose effect could be diminished by DCM.

Improved Production and Antitumor Properties of Triterpene Acids from Submerged Culture of Ganoderma lingzhi.

Triterpene acids (TAs) are the major bioactive constituents in the medicinal fungus Ganoderma lingzhi. However, fermentative production of TAs has not been optimized for commercial use, and whether the TAs isolated from G. lingzhi submerged culture mycelia possess antitumor activity needs to be further proven. In this study, enhanced TA yield and productivity were attained with G. lingzhi using response surface methodology. The interactions of three variables were studied using a Box-Benhnken design, namely initial pH, dissolved oxygen (DO) and fermentation temperature. The optimum conditions were an initial pH of 5.9, 20.0% DO and 28.6 °C. These conditions resulted in a TA yield of 308.1 mg/L in a 5-L stirred bioreactor. Furthermore, the optimized conditions were then successfully scaled up to a production scale of 200 L, and maximum TA production and productivity of 295.3 mg/L and 49.2 mg/L/day were achieved, which represented 80.9% and 111.5% increases, respectively, compared with the non-optimized conditions. Additionally, the triterpene acid extract (TAE) from G. lingzhi mycelia was found to be cytotoxic to the SMMC-7721 and SW620 cell lines in vitro, and the TAE exhibited dose-dependent antitumor activity against the solid tumor sarcoma 180 in vivo. Chemical analysis revealed that the key active triterpene compounds, ganoderic acid T and ganoderic acid Me, predominated in the extract.

Metabolic engineering of Escherichia coli for the production of phenylpyruvate derivatives.

Phenylpyruvate derivatives (PPD), such as phenylpropanoids, DL-phenylglycine, dl-phenylalanine, and styrene, are biosynthesized using phenylpyruvate as the precursor. They are widely used in human health and nutrition products. Recently, metabolic engineering provides effective strategies to develop PPD producers. Based on phenylpyruvate-producing chassis, genetically defined PPD producers have been successfully constructed. In this work, the most recent information on genetics and on the molecular mechanisms regulating phenylpyruvate synthesis pathways in Escherichia coli are summarized, and the engineering strategies to construct the PPD producers are also discussed. The enzymes and pathways are proposed for PPD-producer constructions, and potential difficulties in strain construction are also identified and discussed. With respect to recent advances in synthetic biology, future strategies to construct efficiently producers are discussed.

A multifactorial analysis of the pregnancy outcomes in cytomegalovirus-infected women.

AIMS: To investigate the impacts of cytomegalovirus (CMV) viral load, TORCH (toxoplasmosis, others, rubella, CMV and herpes) coinfections, CMV glycoprotein B (gB) genotypes and maternal genetic polymorphisms on pregnancy outcomes among CMV-infected women. METHODS: A total of 731 CMV-infected pregnant women (634 and 97 with normal and adverse pregnancy outcomes, respectively) were recruited. CMV load quantification and screening of TORCH coinfections were performed by using real-time polymerase chain reaction (PCR) and immunodetection techniques, respectively. Genotyping of CMV gB and maternal NFKB1 -94 ins/del, NFKBIA -826C/T and -881A/G polymorphisms was performed by using PCR-restriction fragment length polymorphism. RESULTS: We found that the mean CMV viral load in women with adverse pregnancy outcomes was significantly higher than that in women with normal outcomes at all pregnancy stages (p < 0.01). We also found that TORCH coinfections resulted in a 1.65-fold (95% CI = 1.00-2.73) increase in the risk of adverse pregnancy outcomes (p = 0.05). Additionally, we noticed no significant difference in the distribution of CMV gB genotypes between women with normal and adverse pregnancy outcomes (p = 0.42). We also observed that the ins/ins variant genotype of the NFKB1 polymorphism could reduce the risk of adverse pregnancy outcomes (OR = 0.38, 95% CI = 0.15-0.98; p = 0.04). CONCLUSION: CMV viral load, TORCH coinfections and maternal NFKB1 polymorphism could influence pregnancy outcomes among CMV-infected women.

Overexpression of KPNA2 correlates with poor prognosis in patients with gastric adenocarcinoma.

This study aims to investigate the expression and significance of KPNA2 in human gastric adenocarcinoma progression and prognosis. Using immunohistochemistry and real-time reverse transcriptase polymerase chain reaction assay, we identified abnormally elevated expression of KPNA2 in gastric adenocarcinoma tissues compared to paired normal stomach mucosa tissues in 30 patients (p < 0.05). In order to investigate the correlations between KPNA2 and the clinicopathological features of gastric adenocarcinoma, the expression of KPNA2 in 142 patients with gastric adenocarcinoma was detected by immunohistochemistry, and the results showed that overexpression of KPNA2 was associated with the size of tumor (p < 0.001), histological grade (p < 0.001), lymph node involvement (p = 0.001), and tumor node metastasis stage (p < 0.001). Kaplan-Meier survival analysis showed that patients with high KPNA2 expression showed a significantly shorter overall survival time compared with patients with low KPNA2 expression. Multivariate analysis suggested that KPNA2 expression might be an independent prognostic indicator (p < 0.001) for the survival of patients with gastric adenocarcinoma. In conclusion, overexpression of KPNA2 is closely related to progression of gastric adenocarcinoma and might be regarded as an independent predictor of poor prognosis for gastric adenocarcinoma.

Minimization of glycerol synthesis in industrial ethanol yeast without influencing its fermentation performance.

To synthesize glycerol, a major by-product during anaerobic production of ethanol, the yeast Saccharomyces cerevisiae would consume up to 4% of the sugar feedstock in typical industrial ethanol processes. The present study was dedicated to decreasing the glycerol production mostly in industrial ethanol producing yeast without affecting its desirable fermentation properties including high osmotic and ethanol tolerance, natural robustness in industrial processes. In the present study, the GPD1 gene, encoding NAD+-dependent glycerol-3-phosphate dehydrogenase in an industrial ethanol producing strain of S. cerevisiae, was deleted. Simultaneously, a non-phosphorylating NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPN) from Bacillus cereus was expressed in the mutant deletion of GPD1. Although the resultant strain AG1A (gpd1△ P(PGK)-gapN) exhibited a 48.7±0.3% (relative to the amount of substrate consumed) lower glycerol yield and a 7.6±0.1% (relative to the amount of substrate consumed) higher ethanol yield compared to the wild-type strain, it was sensitive to osmotic stress and failed to ferment on 25% glucose. However, when trehalose synthesis genes TPS1 and TPS2 were over-expressed in the above recombinant strain AG1A, its high osmotic stress tolerance was not only restored but also improved. In addition, this new recombinant yeast strain displayed further reduced glycerol yield, indistinguishable maximum specific growth rate (µ(max)) and fermentation ability compared to the wild type in anaerobic batch fermentations. This study provides a promising strategy to improve ethanol yields by minimization of glycerol production.

Improving the ethanol yield by reducing glycerol formation using cofactor regulation in Saccharomyces cerevisiae.

To increase ethanol yield and decrease glycerol production in Saccharomyces cerevisiae, the strategies of direct cofactor-regulation were explored. During anaerobic batch fermentations, the yeast expressing Bacillus cereus gapN gene, encoding non-phosphorylating NADP(+)-dependent glyceraldehyde-3-phosphate dehydrognease, produced 73.8 g ethanol l(-1), corresponding to 96% of theoretical maximum yield compared to 92% for the wild type. The yeast expressing Escherichia coli frdA gene encoding the NAD(+)-dependent fumarate reductase, exhibited a 22% (relative to the amount of substrate consumed) increase in glycerol yield in medium containing 2 g fumarate l(-1). The yeast expressing mhpF gene, encoding acetylating NAD(+)-dependent acetaldehyde dehydrogenase, produced 74.5 g ethanol l(-1), corresponding to 97.4% of theoretical maximum yield while glycerol decreased by 40% when acetic acid was added before inoculation. This strain represents a promising alternative for ethanol production with lignocellulosic hydrolysates where acetate is available at significant amounts.

Improving ethanol productivity by modification of glycolytic redox factor generation in glycerol-3-phosphate dehydrogenase mutants of an industrial ethanol yeast.

The GPD2 gene, encoding NAD(+)-dependent glycerol-3-phosphate dehydrogenase in an industrial ethanol-producing strain of Saccharomyces cerevisiae, was deleted. And then, either the non-phosphorylating NADP(+)-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPN) from Bacillus cereus, or the NADP(+)-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Kluyveromyces lactis, was expressed in the obtained mutant AG2 deletion of GPD2, respectively. The resultant recombinant strain AG2A (gpd2Δ P (PGK)-gapN) exhibited a 48.70 ± 0.34% (relative to the amount of substrate consumed) decrease in glycerol production and a 7.60 ± 0.12% (relative to the amount of substrate consumed) increase in ethanol yield, while recombinant AG2B (gpd2Δ P (PGK)-GAPDH) exhibited a 52.90 ± 0.45% (relative to the amount of substrate consumed) decrease in glycerol production and a 7.34 ± 0.15% (relative to the amount of substrate consumed) increase in ethanol yield compared with the wild-type strain. More importantly, the maximum specific growth rates (µ (max)) of the recombinant AG2A and AG2B were higher than that of the mutant gpd2Δ and were indistinguishable compared with the wild-type strain in anaerobic batch fermentations. The results indicated that the redox imbalance of the mutant could be partially solved by expressing the heterologous genes.

Improving the performance of industrial ethanol-producing yeast by expressing the aspartyl protease on the cell surface.

The yeasts used in fuel ethanol manufacture are unable to metabolize soluble proteins. The PEP4 gene, encoding a vacuolar aspartyl protease in Saccharomyces cerevisiae, was either secretively or cell-surface anchored expressed in industrial ethanol-producing S. cerevisiae. The obtained recombinant strains APA (expressing the protease secretively) and APB (expressing the protease on the cell wall) were studied under ethanol fermentation conditions in feed barley cultures. The effects of expression of the protease on product formation, growth and cell protein content were measured. The biomass yield of the wild-type was clearly lower than that of the recombinant strains (0.578 ± 0.12 g biomass/g glucose for APA and 0.582 ± 0.08 g biomass/g glucose for APB). In addition, nearly 98-99% of the theoretical maximum level of ethanol yield was achieved (relative to the amount of substrate consumed) for the recombinant strains, while limiting the nitrogen source resulted in dissatisfactory fermentation for the wild-type and more than 30 g/l residual sugar was detected at the end of fermentation. In addition, higher growth rate, viability and lower yields of byproducts such as glycerol and pyruvic acid for recombinant strains were observed. Expressing acid protease can be expected to lead to a significant increase in ethanol productivity.

Interruption of glycerol pathway in industrial alcoholic yeasts to improve the ethanol production.

The two homologous genes GPD1 and GPD2, encoding two isoenzymes of NAD(+)-dependent glycerol-3-phosphate dehydrogenase in industrial yeast Saccharomyces cerevisiae CICIMY0086, had been deleted. The obtained two kinds of mutants gpd1Delta and gpd2Delta were studied under alcoholic fermentation conditions. gpd1Delta mutants exhibited a 4.29% (relative to the amount of substrate consumed) decrease in glycerol production and 6.83% (relative to the amount of substrate consumed) increased ethanol yield while gpd2Delta mutants exhibited a 7.95% (relative to the amount of substrate consumed) decrease in glycerol production and 7.41% (relative to the amount of substrate consumed) increased ethanol yield compared with the parental strain. The growth rate of the two mutants were slightly lower than that of the wild type under the exponential phase whereas ANG1 (gpd1Delta) and the decrease in glycerol production was not accompanied by any decline in the protein content of the strain ANG1 (gpd1Delta) but a slight decrease in the strain ANG2 (gpd2Delta). Meanwhile, dramatic decrease of acetate acid formation was observed in strain ANG1 (gpd1Delta) and ANG2 (gpd2Delta) compared to the parental strain. Therefore, it is possible to improve the ethanol yield by interruption of glycerol pathway in industrial alcoholic yeast.

Efficient Accumulation and In Vitro Antitumor Activities of Triterpene Acids from Submerged Batch--Cultured Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Agaricomycetes).

Triterpene acids are among the major bioactive constituents of lucidum. However, submerged fermentation techniques for isolating triterpene acids from G. lucidum have not been optimized for commercial use, and the antitumor activity of the mycelial triterpene acids needs to be further proven. The aim of this work was to optimize the conditions for G. lucidum culture with respect to triterpene acid production, scaling up the process, and examining the in vitro antitumor activity of mycelial triterpene acids. The key conditions (i.e., initial pH, fermentation temperature, and rotation speed) were optimized using response surface methodology, and the in vitro antitumor activity was evaluated using the MTT method. The optimum key fermentation conditions for triterpene acid production were pH 6.0; rotation speed, 161.9 rpm; and temperature, 30.1°C, resulting in a triterpene acid yield of 291.0 mg/L in the validation experiment in a 5-L stirred bioreactor; this yield represented a 70.8% increase in titer compared with the nonoptimized conditions. Furthermore, the optimized conditions were then successfully scaled up to a production scale of 200 L, and a triterpene productivity of 47.9 mg/L/day was achieved, which is, to our knowledge, the highest reported in the large-scale fermentation of G. lucidum. In addition, the mycelial triterpene acids were found to be cytotoxic to the SMMC-7721 and SW620 cell lines in vitro. Chemical analysis showed that the key active triterpene acid compounds, ganoderic acids T and Me, predominated in the extract, at 69.2 and 41.6 mg/g, respectively. Thus, this work develops a simple and feasible batch fermentation technique for the large-scale production of antitumor triterpene acids from G. lucidum.

Silencing of hypoxia-inducible factor-1α promotes thyroid cancer cell apoptosis and inhibits invasion by downregulating WWP2, WWP9, VEGF and VEGFR2.

Adaptation to hypoxia is an important process physiologically and pathologically. Hypoxia-inducible factor-1α (HIF-1α) participates in the cancer biology of numerous endocrine tumors, including their proliferation and differentiation. In the present study, the hypothesis that HIF-1α promotes tumorigenesis in thyroid cancer via upregulating angiogenesis-associated markers is investigated. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis were used to examine the expression of HIF-1α in thyroid cancer cell lines, and to detect the expression of WW domain containing E3 ubiquitin protein ligase (WWP)2, WWP9, vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2) in MZ-CRC-1 and TT thyroid cancer cells. Cell proliferation was measured using a Cell Count Kit-8. Cell apoptosis and cell cycle was assessed by flow cytometry. Cell invasive ability was examined by Matrigel transwell analysis. RT-qPCR and western blot analyses demonstrated that the mRNA and protein expression levels of HIF-1α were significant higher in MZ-CRC-1 and TT thyroid cancer cells than in another three thyroid cancer cells (P<0.01). HIF-1α knockdown cells demonstrated inhibition of cell proliferation and invasion, arrested cell cycle at the G1 phase, and induction of cell apoptosis. The protein expression levels of WWP2, WWP9, VEGF and VEGFR2 were decreased in HIF-1α knockdown MZ-CRC-1 and TT cells. In conclusion, HIF-1α may be important in cell apoptosis and invasion of thyroid cancer cells, likely through regulating WWP2, WWP9, VEGF and VEGFR2 expression.

Inhibitor or promoter? The performance of polysaccharides from Ganoderma lucidum on human tumor cells with different p53 statuses.

Polysaccharides from Ganoderma lucidum (GLPs) have been taken as effective supplements by both healthy people and cancer patients for many years. However, this short survey indicates that instead of inhibiting cancer cell growth, both submerge-cultured intracellular GLP and fruiting body GLP can stimulate the growth of human carcinoma cell lines lacking functional p53, such as HCT-116 p53(-/-), Saos-2, H1299, HL-60, MDA-MB-157. Conversely, the two GLPs inhibit all other assayed cells with functional p53. These results could be an alert since mutational inactivation of the tumor suppressor protein p53 is the most frequent genetic alteration found in human tumors.

Expression characteristics of CDC20 in gastric cancer and its correlation with poor prognosis.

The cell division cycle 20 homolog (CDC20) expression is increased in diverse human cancers and plays a vital role in tumorigenesis and progression. However, the clinical significance of CDC20 expression in gastric cancer (GC) remains largely unknown. The aim of this study was to investigate the clinicopathologic features and prognostic significance of CDC20 in GC. The CDC20 mRNA expression was measured by quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR). Immunohistochemistry (IHC) was used to detect the expression of CDC20 protein in 131 clinicopathologically characterized GC cases. The relationship between CDC20 expression and clinicopathological features was analyzed by appropriate statistics. Kaplan-Meier analysis and Cox proportional hazards regression models were used to investigate the correlation between CDC20 expression and prognosis of GC patients. The relative mRNA expression of CDC20 were significantly higher in GC tumor tissues than in the corresponding noncancerous tissues (P<0.001). Simultaneously, CDC20 protein expression was positively correlated with tumor size (P=0.02), histological grade (P=0.037), lymph node involvement (P=0.009), and TNM stage (P=0.015). Furthermore, Kaplan-Meier analysis indicated that patients with high CDC20 expression had poor overall survival (P<0.001). Multivariate analysis showed that high CDC20 expression was an independent predictor of overall survival. In conclusion, our data indicated that CDC20 upregulation was associated with aggressive progression and poor prognosis in GC. CDC20 was identified for the first time as an independent marker for predicting the clinical outcome of GC patients.

An antioxidative galactomannan-protein complex isolated from fermentation broth of a medicinal fungus Cs-HK1.

A protein-containing polysaccharide, EPS2BW, was fractionated from the exopolysaccharide (EPS) produced by a medicinal fungus Cordyceps sinensis (Cs-HK1). EPS2BW was mainly composed of galactomannan with about 16% (w/w) protein and 50 kDa average molecular weight. The galactomannan part consisted of mannose and galactose at 1.7:1.0 molar ratio, and the protein segments were composed of sixteen amino acids with 12.5% proline and 16.6% threonine (mol%) being the most abundant. Based on analytical results from NMR, methylation analysis, partial acid hydrolysis and GC-MS, the galactomannan structure was elucidated as a (1 → 2)-α-D-mannopyranosyl (Manp) backbone with O-6-linked galactopyranosyl (Galp) branches. EPS2BW exhibited a high antioxidant capacity in both chemical and cell culture assays, with a Trolox equivalent radical scavenging activity of 44.7 µmol Trolox/mg, a Fe(3+) reducing power of 38.9 µmol Fe(2+)/mg, and significant cytoprotective effect against H2O2-induced PC12 cell death at 50-250 µg/mL.

Heterologous pathway for the production of L-phenylglycine from glucose by E. coli.

The aproteinogenic amino acid L-phenylglycine (L-Phg) is an important side chain building block for the preparation of several antibiotics and taxol. To biosynthesis L-Phg from glucose, an engineered Escherichia coli containing L-Phg synthetic genes was firstly developed by an L-phenylalanine producing chassis supplying phenylpyruvate. The enzymes HmaS (L-4-hydroxymandelate synthase), Hmo (L-4-hydroxymandelate oxidase) and HpgT (L-4-hydroxyphenylglycine transaminase) from Amycolatopsis orientalis as well as Streptomyces coelicolor were heterologously expressed in E. coli and purified to evaluate their abilities on L-Phg formation. HpgT conversing phenylglyoxylate to L-Phg uses an unusual amino donor L-phenylalanine, which releases another phenylpyruvate as the substrate of HmaS. Thus, a recycle reaction was developed to maximize the utilization of precursor phenylpyruvate. To amplify the accumulation of L-Phg, the effects of attenuating L-phenylalanine transamination was investigated. After deletion of tyrB and aspC, L-Phg yield increased by 12.6-fold. The limiting step in the L-Phg biosynthesis was also studied; the L-Phg yield was further improved by 14.9-fold after enhancing hmaS expression. Finally, by optimizing expression of hmaS, hmo and hpgT and attenuation of L-phenylalanine transamination, the L-Phg yield was increased by 224-fold comparing with the original strain.

Development of an industrial ethanol-producing yeast strain for efficient utilization of cellobiose.

The BGL1 gene, encoding ß-glucosidase in Saccharomycopsis fibuligera, was intracellular, secreted or cell-wall associated expressed in an industrial strain of Saccharomyces cerevisiae. The obtained recombinant strains were studied under aerobic and anaerobic conditions. The results indicated that both the wild type and recombinant strain expressing intracellular ß-glucosidase cannot grow in medium using cellobiose as sole carbon source. As for the recombinant EB1 expressing secreted enzyme and WB1 expressing cell-wall associated enzyme, the maximum specific growth rates (µ(max)) could reach 0.03 and 0.05 h(-1) under anaerobic conditions, respectively. Meanwhile, the surface-engineered S. cerevisiae utilized 5.2 g cellobioseL(-1) and produced 2.3 g ethanol L(-1) in 48 h, while S. cerevisiae secreting ß-glucosidase into culture broth used 3.6 g cellobiose L(-1) and produced 1.5 g ethanolL(-1) over the same period, but no-full depletion of cellobiose were observed for both the used recombinant strains. The results suggest that S. cerevisiae used in industrial ethanol production is deficient in cellobiose transporter. However, when ß-glucoside permease and ß-glucosidase were co-expressed in this strain, it could uptake cellobiose and showed higher growth rate (0.11h(-1)) on cellobiose.

Expression of aspartic protease from Neurospora crassa in industrial ethanol-producing yeast and its application in ethanol production.

In order to further improve the utilization rate of raw materials and increase ethanol productivity, the gene Asp, encoding aspartic protease in Neurospora crassa was cloned and expressed in industrial ethanol-producing yeast. To promote secretion of the acid protease, the gene was fused to signal sequence of the yeast α-factor gene and constitutively expressed under transcriptional control of the Saccharomyces cerevisiae PGK1 promoter. The resultant recombinant enzyme was characterized with respect to pH and temperature optimum. Then, this acid protease was anchored to the yeast cell wall by fusing the mature protein to the α-agglutinin peptides. The resultant strain was evaluated in clarifying corn mash and very high gravity (VHG) raw starch fermentation. The present results demonstrated that expression of the acid protease increased both growth rate and viable yeast counts and the recombinant strain of S. cerevisiae exhibited a higher ethanol yield as compared to the parent strain.