ent-Clavilactone J and Its Quinone Derivative, Meroterpenoids from the Fungus Resupinatus sp.

Metabolites 1 and 2, isolated from cultures of the basidiomycete Resupinatus sp. BCC84615, collected in a tropical forest in northeastern Thailand, showed weak antibiotic activity against Bacillus subtilis and Staphylococcus aureus and cytotoxicity against cancer cell lines. Their planar structures were elucidated by high-resolution electrospray ionization mass spectrometry and NMR spectroscopy as clavilactone J, known from the basidiomycete Ampulloclitocybe clavipes, and its new 1,4-benzoquinone derivative. A detailed analysis of the ROESY correlations in 1 confirmed the recent revision of the relative configuration of clavilactone J. However, specific rotation and Cotton effects observed by electronic circular dichroism were contrary to those of the clavilactones; thus, we assigned a rare antipodal absolute configuration.

Drimane-Type Sesquiterpenoids Derived from the Tropical Basidiomycetes Perenniporia centrali-africana and Cerrena sp. nov.

Five new drimane-type sesquiterpenoids were isolated from cultures of the tropical basidiomycetes, Perenniporia centrali-africana (originating from Kenya) and Cerrena sp. nov. (originating from Thailand). A new pereniporin A derivative (1), a new drimane-type sesquiterpene lactam (2), and the new 6,7-Dehydro-isodrimenediol (3) were isolated from P. centrali-africana. In parallel, the two new drimane-type sesquiterpene lactams 5 and 6 were isolated together with known isodrimenediol (4) from Cerrena sp. This is the first report of drimane-type sesquiterpene lactams from basidiomycetes. The structures were elucidated based on 1D and 2D nuclear magnetic resonance (NMR) spectroscopic data, in combination with high-resolution electrospray mass spectrometric (HR-ESIMS) data. The compounds were devoid of significant antimicrobial and cytotoxic activities.

Improved yeast-screening assay for phytoestrogens.

A yeast estrogen screening (YES) assay was improved to increase sensitivity for detection of phytoestrogens. New yeast strains minus one or the other of transporters Pdr5 or Snq2 and harboring yEGFP as a reporter gene were developed. The new strains showed 2-100-fold improvement in sensitivity for detection of standard estrogens and antiestrogens. In addition, the assay time (1 h) using the newly developed strains was shorter than that (4 h) previously reported. Furthermore, the snq2-minus strains were most effective for detection of estrogenic activity while the pdr5-minus strains were most effective for detection of antiestrogenic activity. The efficacy of the new methods was evaluated and confirmed by testing with 23 Thai medicinal plant species. The new strains were also tested for detection of xenoestrogens. The results revealed that the newly developed YES methods were specific and rapid and suitable for simple high-throughput screening or detection of estrogen-like compounds.

A sensitive and specific SYBR Green-based qPCR assay for detecting scale drop disease virus (SDDV) in Asian sea bass.

Scale drop disease virus (SDDV) is a megalocytivirus known to cause disease in Asian sea bass in Southeast Asia. To support SDDV diagnosis and surveillance, we report on a sensitive and specific SYBR Green qPCR assay. The qPCR primers were designed to target a 135 bp fragment of the SDDV ATPase gene. The optimized SDDV qPCR assay reliably detected 2 copies of a plasmid dsDNA control and did not cross-amplify DNA to any of 12 viral or bacterial pathogens commonly found in aquatic animals. When assessed with 86 field samples, the assay detected SDDV in DNA extracted from each of 34 scale drop disease-affected fish collected from 5 affected farms. The qPCR also detected SDDV in DNA from 30 of 52 overtly healthy fish collected from 9 farms where SDDV had not been detected previously, using a semi-nested conventional PCR. The higher sensitivity of our SDDV qPCR assay can thus be useful in detecting fish with subclinical/chronic infections. However, the qPCR showed that SDDV DNA loads varied from 8.0 × 102 to 6.8 × 104 viral DNA copies per 200 ng DNA template among the 8 organ tissue types sampled from 3 diseased fish. In circumstances requiring SDDV to be detected unequivocally in subclinical carriers with lower-level infection, qPCR testing of more than one type of tissue is advisable.

High level accumulation of soluble diphtheria toxin mutant (CRM197) with co-expression of chaperones in recombinant Escherichia coli.

CRM197 is the diphtheria toxin mutant used in many conjugate vaccines. A fusion CRM197 (fCRM197) containing all the tags conferred by the pET32a vector was produced as a soluble protein in Escherichia coli co-expressing several chaperone proteins in conjunction with low temperature cultivation. Trigger factor (Tf) enhanced formation of soluble fCRM197 (150.69 ± 8.95 µg/mL) to a greater degree than other chaperones when fCRM197 expression was induced at 25 °C for 12 h. However, prolonged cultivation resulted in a progressive reduction of fCRM197 accumulation. In contrast, at 15 °C cells, with or without Tf, fCRM197 accumulated to the highest level at 48 h (153.70 ± 13.14 µg/mL and 150.07 ± 8.13 µg/mL, respectively). Transmission electron microscopy (TEM) demonstrated that the formation of inclusion protein as well as cell lysis was reduced in cultures grown at 15 °C. Cell viability was substantially reduced in cells expressing Tf, compared to cultures without Tf, when fCRM197 was induced at 25 °C. The viability of Tf-expressing cells was enhanced when cultured at 15 °C. Both purified fCRM197 and CRM197 efficiently digested lambda DNA (λDNA) at 37 °C (92.78 and 97.45 %, respectively). Digestion efficiency of fCRM197 and CRM197 was reduced at 25 °C (80.80 and 62.73 %, respectively) and at 15 °C (7.34 and 24.79 %, respectively). These results demonstrating nuclease activity, enhanced cell lysis, and reduced cell viability are consistent with the finding of lower fCRM197 yield when cultivation and induction times were prolonged at 25 °C. The present work provides a procedure for the high-level production of soluble fCRM197 using E. coli as a heterologous host.

Two new anti-apoptotic proteins of white spot syndrome virus that bind to an effector caspase (PmCasp) of the giant tiger shrimp Penaeus (Penaeus) monodon.

White spot syndrome virus proteins WSSV134 and WSSV322 have been shown to bind with the p20 domain (residues 55-214) of Penaeus monodon caspase (PmCasp) protein through yeast two-hybrid screening. Binding was confirmed for the p20 domain and the full-length caspase by co-immunoprecipitation. WSSV134 is also known as the WSSV structural protein VP36A, but no function or conserved domains have been ascribed to WSSV322. Discovery of the caspase binding activity of these two proteins led to an investigation of their possible anti-apoptotic roles. Full-length PmCasp was confirmed to be an effector caspase by inducing apoptosis in transfected Sf-9 cells as assessed by DAPI staining. Using the same cell model, comparison of cells co-transfected with PmCasp and either WSSV134 or WSSV322 revealed that both of the binding proteins had anti-apoptotic activity. However, using the same Sf-9 protocol with anti-apoptosis protein-1 (AAP-1; also called WSSV449) previously shown to bind and inactivate a different effector caspase from P. monodon (Pm caspase) did not block apoptosis induced by PmCasp. The results revealed diversity in effector caspases and their viral protein inhibitors in P. monodon.

Pueraria mirifica leaves, an alternative potential isoflavonoid source.

We investigated the major leaf isoflavonoid contents of Pueraria mirifica from three different cultivars (PM-III, PM-IV, and PM-V) using reverse RP-HPLC analysis. The proportions and net levels of puerarin, daidzin, genistin, and daidzein in P. mirifica leaves were found to depend on the plant cultivar and to correlate with cultivation temperature and rainfall amount. The crude leaf-extracts were tested using the Yeast Estrogen Screen (YES) assay with both human estrogen receptors (hERα and hERß). Their estrogenic activity was higher when determined by the YES system containing hERß than that with hERα and was also higher when the Δsnq2 than the wildtype yeast was employed. The results open the possibility of selecting and cultivating certain P. mirifica cultivars at a farm scale to produce a sufficient supply of leaf material to act as a starting source for the commercial scale extraction of these major isoflavonoids.

Persistence of Penaeus stylirostris densovirus delays mortality caused by white spot syndrome virus infection in black tiger shrimp (Penaeus monodon).

BACKGROUND: Persistent infection of Penaeus stylirostris densovirus (PstDNV) (also called IHHNV) and its non-infectious inserts in the black tiger shrimp, Penaeus monodon (P. monodon) genome are commonly found without apparent disease. Here, we introduced the method of multiplex PCR in order to differentiate shrimp with viral inserts from ones with the infectious virus. The method allowed us to study the effect of pre-infection of IHHNV, in comparison to IHHNV inserts, on WSSV resistance in P. monodon. RESULTS: A multiplex PCR system was developed to amplify the entire IHHNV genome, ensuring the accurate diagnosis. Field samples containing IHHNV DNA templates as low as 20 pg or equivalent 150 viral copies can be detected by this method. By challenging the two groups of diagnosed shrimp with WSSV, we found that shrimp with IHHNV infection and those with viral inserts responded to WSSV differently. Considering cumulative mortality, average time to death of shrimp in IHHNV-infected group (day 14) was significantly delayed relative to that (day 10) of IHHNV-inserted group. Real-time PCR analysis of WSSV copy number indicated the lower amount of WSSV in the IHHNV-infected group than the virus-inserted group. The ratio of IHHNV: WSSV copy number in all determined IHHNV-infected samples ranged from approximately 4 to 300-fold. CONCLUSION: The multiplex PCR assay developed herein proved optimal for convenient differentiation of shrimp specimens with real IHHNV infection and those with insert types. Diagnosed shrimp were also found to exhibit different WSSV tolerance. After exposed to WSSV, the naturally pre-infected IHHNV P. monodon were less susceptible to WSSV and, consequently, survived longer than the IHHNV-inserted shrimp.

Construction of Bordetella pertussis strains with enhanced production of genetically-inactivated Pertussis Toxin and Pertactin by unmarked allelic exchange.

BACKGROUND: Acellular Pertussis vaccines against whooping cough caused by Bordetella pertussis present a much-improved safety profile compared to the original vaccine of killed whole cells. The principal antigen of acellular Pertussis vaccine, Pertussis Toxin (PT), must be chemically inactivated to obtain the corresponding toxoid (PTd). This process, however, results in extensive denaturation of the antigen. The development of acellular Pertussis vaccines containing PTd or recombinant PT (rPT) with inactivated S1, Filamentous Hemagglutinin (FHA), and Pertactin (PRN) has shown that the yield of PRN was limiting, whereas FHA was overproduced. To improve antigen yields and process economics, we have constructed strains of Bordetella pertussis that produce enhanced levels of both rPT and PRN. RESULTS: Three recombinant strains of Bordetella pertussis were obtained by homologous recombination using an allelic exchange vector, pSS4245. In the first construct, the segment encoding PT subunit S1 was replaced by two mutations (R9K and E129G) that removed PT toxicity and Bp-WWC strain was obtained. In the second construct, a second copy of the whole cluster of PT structural genes containing the above mutations was inserted elsewhere into the chromosome of Bp-WWC and the Bp-WWD strain was obtained. This strain generated increased amounts of rPT (3.77 ± 0.53 µg/mL) compared to Bp-WWC (2.61 ± 0.16 µg/mL) and wild type strain (2.2 µg/mL). In the third construct, a second copy of the prn gene was inserted into the chromosome of Bp-WWD to obtain Bp-WWE. Strain Bp-WWE produced PRN at 4.18 ± 1.02 µg/mL in the cell extract which was about two-fold higher than Bp-WWC (2.48 ± 0.10 µg/mL) and Bp-WWD (2.31 ± 0.17 µg/mL). Purified PTd from Bp-WWD at 0.8-1.6 µg/well did not show any toxicity against Chinese hamster ovary (CHO) cell whereas purified PT from WT demonstrated a cell clustering endpoint at 2.6 pg/well. CONCLUSIONS: We have constructed Bordetella pertussis strains expressing increased amounts of the antigens, rPT or rPT and PRN. Expression of the third antigen, FHA was unchanged (always in excess). These strains will be useful for the manufacture of affordable acellular Pertussis vaccines.

Shrimp protected from a virus by feed containing yeast with a surface-displayed viral binding protein.

Recombinant Pichia pastoris biomass surface-expressing the viral binding protein PmRab7 (YSD-PmRab7) was prepared by fed-batch, aerobic fermentation with methanol induction for 48 h. By cell based ELISA assay, immunofluorescence and flow cytometry, 45% of the YSD-PmRab7 cells were positive for PmRab7. Freeze dried YSD-PmRab7 cells were added to formulated shrimp feed pellets at 0.25 g and 0.5 g per g feed and fed to 2 shrimp groups for 7 days prior to challenge with white spot syndrome virus (WSSV). Controls consisted of 1 shrimp group fed normal pellets and one fed pellets containing P. pastoris carrying an empty gene cassette. At 10 days post challenge, survival in the two control groups was 6.7 ± 6.6%, while it was 26.7 ± 6.6% in the 0.25 g YSD-PmRab7 group and significantly higher (p < 0.05) in the 0.5 g YSD-PmRab7 group at 46.7 ± 10.1%. Nested PCR assays and histopathological analysis revealed significantly lower WSSV replication levels in the 0.5 g YSD-PmRab7 group. The results indicated potential for development of YSD-PmRab7 cells as an oral prophylactic against WSSV in shrimp.

Ethanol production from biomass by repetitive solid-state fed-batch fermentation with continuous recovery of ethanol.

To save cost and input energy for bioethanol production, a consolidated continuous solid-state fermentation system composed of a rotating drum reactor, a humidifier, and a condenser was developed. Biomass, saccharifying enzymes, yeast, and a minimum amount of water are introduced into the system. Ethanol produced by simultaneous saccharification and fermentation is continuously recovered as vapor from the headspace of the reactor, while the humidifier compensates for the water loss. From raw corn starch as a biomass model, 95 +/- 3, 226 +/- 9, 458 +/- 26, and 509 +/- 64 g l(-1) of ethanol solutions were recovered continuously when the ethanol content in reactor was controlled at 10-20, 30-50, 50-70 and 75-85 g kg-mixture(-1), respectively. The residue showed a lesser volume and higher solid content than that obtained by conventional liquid fermentation. The cost and energy for intensive waste water treatment are decreased, and the continuous fermentation enabled the sustainability of enzyme activity and yeast in the system.

Estrogenic and anti-estrogenic activities of the Thai traditional herb, Butea superba Roxb.

This study evaluated the estrogenic and antiestrogenic activities of native and in vitro hepatic metabolized tuberous extracts of wild Butea superba collected from 23 out of the 76 provinces in Thailand by yeast estrogen screening (YES). The YES screen used consisted of the human estrogen receptors hERα and hERß and the human transcriptional intermediary factor 2 or human steroid receptor coactivator 1, respectively, together with the ß-galactosidase expression cassette as the reporter. The relative potency, effectiveness and relative inductive efficiency were evaluated by determining the ß-galactosidase activity (EC(50)) of each tuberous extract in relation to that induced by 17ß-estradiol. Six pure compounds isolated from B. superba were tested in parallel and exhibited a maximum relative potency compared to 17ß-estradiol of 15.5% and 5.27% in the respective hERα and hERß assays. Eighteen and seventeen plant extracts were respectively found to interact with the hERα and hERß receptors in the YES assays with higher relative potency and relative inductive efficiency with hERß than with hERα. The selected plant extracts tested exhibited antiestrogenic activity. Coincubation with the rat liver S9 mixture also elevated the estrogenic potency of these plant extracts.

Millerozyma phetchabunensis sp. nov., a novel ascomycetous yeast species isolated from Nam Nao forest soil in Thailand, and the transfer of Pichia koratensis to the genus Millerozyma.

A total of 4 strains (DR2-4, DW2-1, DS2-1 and DS3-1) of undescribed ascomycetous yeast, isolated from Nam Nao forest soil, were identified as a novel species in the genus Millerozyma. The D1/D2 sequences of the strains differed from the closely related species Millerozyma acaciae and Pichia koratensis by 1.2% (7 nucleotide substitutions) and 1.4% (8 nucleotide substitutions). Phenotypically, all the novel strains were identical, but differed from M. acaciae and P. koratensis by a variety of phenotypic characteristics. Based on phenotypic and phylogenetic data, these four yeast strains were assigned to a single novel species in the genus Millerozyma and the name Millerozyma phetchabunensis sp. nov. is proposed. In addition, we also propose the transfer of P. koratensis, which was placed in the Millerozyma clade based on the analysis of the D1/D2 and ITS sequences, to the genus Millerozyma as M. koratensis comb. nov.

Enhancement of ethanol production in very high gravity fermentation by reducing fermentation-induced oxidative stress in Saccharomyces cerevisiae.

During fermentation, yeast cells encounter a number of stresses, including hyperosmolarity, high ethanol concentration, and high temperature. Previous deletome analysis in the yeast Saccharomyces cerevisiae has revealed that SOD1 gene encoding cytosolic Cu/Zn-superoxide dismutase (SOD), a major antioxidant enzyme, was required for tolerances to not only oxidative stress but also other stresses present during fermentation such as osmotic, ethanol, and heat stresses. It is therefore possible that these fermentation-associated stresses may also induce endogenous oxidative stress. In this study, we show that osmotic, ethanol, and heat stresses promoted generation of intracellular reactive oxygen species (ROS) such as superoxide anion in the cytosol through a mitochondria-independent mechanism. Consistent with this finding, cytosolic Cu/Zn-SOD, but not mitochondrial Mn-SOD, was required for protection against oxidative stress induced by these fermentation-associated stresses. Furthermore, supplementation of ROS scavengers such as N-acetyl-L-cysteine (NAC) alleviated oxidative stress induced during very high gravity (VHG) fermentation and enhanced fermentation performance at both normal and high temperatures. In addition, NAC also plays an important role in maintaining the Cu/Zn-SOD activity during VHG fermentation. These findings suggest the potential role of ROS scavengers for application in industrial-scale VHG ethanol fermentation.

Phytohormone priming elevates the accumulation of defense-related gene transcripts and enhances bacterial blight disease resistance in cassava.

Cassava bacterial blight (CBB) disease caused by Xanthomonas axonopodis pv. manihotis (Xam) is a severe disease in cassava worldwide. In addition to causing significant cassava yield loss, CBB disease has not been extensively studied, especially in terms of CBB resistance genes. The present research demonstrated the molecular mechanisms underlining the defense response during Xam infection in two cassava cultivars exhibiting different degrees of disease resistance, Huay Bong60 (HB60) and Hanatee (HN). Based on gene expression analysis, ten of twelve putative defense-related genes including, leucine-rich repeat receptor-like kinases (LRR-RLKs), resistance (R), WRKY and pathogenesis-related (PR) genes, were differentially expressed between these two cassava cultivars during Xam infection. The up-regulation of defense-related genes observed in HB60 may be the mechanism required for the reduction of disease severity in the resistant cultivar. Interestingly, priming with salicylic acid (SA) or methyl jasmonate (MeJA) for 24 h before Xam inoculation could enhance the defense response in both cassava cultivars. The disease severity was decreased 10% in the resistant cultivar (HB60) and was remarkably reduced 21% in the susceptible cultivar (HN) by SA/MeJA priming. Priming with Xam inoculation modulated cassava4.1_013417, cassava4.1_030866 and cassava4.1_020555 (highest similarity to MeWRKY59, MePR1 and AtPDF2.2, respectively) expression and led to enhanced resistance of the susceptible cultivar in the second infection. The putative cis-regulatory elements were predicted in an upstream region of these three defense-related genes. The different gene expression levels in these genes between the two cultivars were due to the differences in cis-regulatory elements in their promoter regions. Taken together, our study strongly suggested that the induction of defense-related genes correlated with defense resistance against Xam infection, and exogenous application of SA or MeJA could elevate the defense response in both cultivars of cassava. This finding should pave the way for management to reduce yield loss from disease and genetic improvement in cassava.

Functional expression of the rat organic anion transporter 1 (rOAT1) in Saccharomyces cerevisiae.

Organic anion transporter 1 (OAT1) is localized in the basolateral membrane of the proximal tubule in the kidney and plays an essential role in eliminating a wide range of organic anions, preventing their toxic effects on the body. Structural and functional studies of the transporter would be greatly assisted by inexpensive and rapid expression in the yeast Saccharomyces cerevisiae. The gene encoding rat OAT1 (rOAT1) contains many yeast non-preferred codons at the N-terminus and so was modified by fusion of the favored codon sequence of a hemagglutinin (HA) epitope preceding the start codon. The modified gene was cloned into several yeast expression plasmids, both integrative and multicopy, with either ADH1 promoter or GAL1 promoter in order to find a suitable expression system. Compared with the wild type gene, a substantial increase in rOAT1 expression was achieved by modification in the translational initiation region, suggesting that the codon chosen at the N-terminus influenced its expression. The highest inducible expression of rOAT1 was obtained under GAL1 promoter in 2 mu plasmid. A large fraction of rOAT1 was glycosylated in yeast, unaffected by growth temperature. The recombinant yeast expressing rOAT1 showed an increase in the uptake of p-aminohippurate (PAH) and this showed a positive correlation with rOAT1 expression level. Location of rOAT1 predominantly in the yeast plasma membrane confirmed correct processing. The importance of glycosylation for rOAT1 targeting was also shown. To our knowledge, this is the first successful functional expression of rOAT1 in the yeast S. cerevisiae.

Cellular mechanisms contributing to multiple stress tolerance in Saccharomyces cerevisiae strains with potential use in high-temperature ethanol fermentation.

High-temperature ethanol fermentation has several benefits including a reduction in cooling cost, minimizing risk of bacterial contamination, and enabling simultaneous saccharification and fermentation. To achieve the efficient ethanol fermentation at high temperature, yeast strain that tolerates to not only high temperature but also the other stresses present during fermentation, e.g., ethanol, osmotic, and oxidative stresses, is indispensable. The C3253, C3751, and C4377 Saccharomyces cerevisiae strains, which have been previously isolated as thermotolerant yeasts, were found to be multiple stress-tolerant. In these strains, continuous expression of heat shock protein genes and intracellular trehalose accumulation were induced in response to stresses causing protein denaturation. Compared to the control strains, these multiple stress-tolerant strains displayed low intracellular reactive oxygen species levels and effective cell wall remodeling upon exposures to almost all stresses tested. In response to simultaneous multi-stress mimicking fermentation stress, cell wall remodeling and redox homeostasis seem to be the primary mechanisms required for protection against cell damage. Moreover, these strains showed better performances of ethanol production than the control strains at both optimal and high temperatures, suggesting their potential use in high-temperature ethanol fermentation.

Yeast Surface Display of Two Proteins Previously Shown to Be Protective Against White Spot Syndrome Virus (WSSV) in Shrimp.

Cell surface display using the yeasts Saccharomyces cerevisiae and Pichia pastoris has been extensively developed for application in bioindustrial processes. Due to the rigid structure of their cell walls, a number of proteins have been successfully displayed on their cell surfaces. It was previously reported that the viral binding protein Rab7 from the giant tiger shrimp Penaeus monodon (PmRab7) and its binding partner envelope protein VP28 of white spot syndrome virus (WSSV) could independently protect shrimp against WSSV infection. Thus, we aimed to display these two proteins independently on the cell surfaces of 2 yeast clones with the ultimate goal of using a mixture of the two clones as an orally deliverable, antiviral agent to protect shrimp against WSSV infection. PmRab7 and VP28 were modified by N-terminal tagging to the C-terminal half of S. cerevisiae α-agglutinin. DNA fragments, harboring fused-gene expression cassettes under control of an alcohol oxidase I (AOX1) promoter were constructed and used to transform the yeast cells. Immunofluorescence microscopy with antibodies specific to both proteins demonstrated that mutated PmRab7 (mPmRab7) and partial VP28 (pVP28) were localized on the cell surfaces of the respective clones, and fluorescence intensity for each was significantly higher than that of control cells by flow cytometry. Enzyme-linked immunosorbant assay (ELISA) using cells displaying mPmRab7 or pVP28 revealed that the binding of specific antibodies for each was dose-dependent, and could be saturated. In addition, the binding of mPmRab7-expressing cells with free VP28, and vice versa was dose dependent. Binding between the two surface-expressed proteins was confirmed by an assay showing agglutination between cells expressing complementary mPmRab7 and pVP28. In summary, our genetically engineered P. pastoris can display biologically active mPmRab7 and pVP28 and is now ready for evaluation of efficacy in protecting shrimp against WSSV by oral administration.

Enhanced bio-ethanol production from cellulosic materials by semi-simultaneous saccharification and fermentation using high temperature resistant Saccharomyces cerevisiae TJ14.

The capability of multi-stress-tolerant Saccharomyces cerevisiae diploid strain TJ14 for the production of cellulosic bio-ethanol by semi-simultaneous saccharification and fermentation (SSSF) technology was evaluated under high-temperature conditions. At 39°C, the TJ14 produced 45 g/l ethanol by SSSF of 100 g (w/v)/l cellulose - a significantly higher concentration than reported in prevailing literature.

CDC19 encoding pyruvate kinase is important for high-temperature tolerance in Saccharomyces cerevisiae.

Use of thermotolerant strains is a promising way to reduce the cost of maintaining optimum temperatures in the fermentation process. Here we investigated genetically a Saccharomyces cerevisiae strain showing a high-temperature (41°C) growth (Htg(+)) phenotype and the result suggested that the Htg(+) phenotype of this Htg(+) strain is dominant and under the control of most probably six genes, designated HTG1 to HTG6. As compared with a Htg(-) strain, the Htg(+) strain showed a higher survival rate after exposure to heat shock at 48°C. Moreover, the Htg(+) strain exhibited a significantly high content of trehalose when cultured at high temperature and stronger resistance to Congo Red, an agent that interferes with cell wall construction. These results suggest that a strengthened cell wall in combination with increased trehalose accumulation can support growth at high temperature. The gene CDC19, encoding pyruvate kinase, was cloned as the HTG2 gene. The CDC19 allele from the Htg(+) strain possessed five base changes in its upstream region, and two base changes resulting in silent mutations in its coding region. Interestingly, the latter base changes are probably responsible for the increased pyruvate kinase activity of the Htg(+) strain. The possible mechanism leading to this increased activity and to the Htg(+) phenotype, which may lead to the activation of energy metabolism to maintain cellular homeostasis, is discussed.