Overexpression of <Emphasis Type="Italic">ARO10</Emphasis> in <Emphasis Type="Italic">pdc5</Emphasis>Δmutant resulted in higher isobutanol titers in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

To investigate effects of different pyruvate decarboxylases on isobutanol titers in Saccharomyces cerevisiae, single-gene deletion of the three PDCs genes encoding pyruvate decarboxylases were constructed in this study. In addition, we over-expressed Ilv2, which catalyzed the first step in the valine synthetic pathway, and Bat2, which was the cytoplasmic branched-chain amino-acid aminotransferase that catalyzed L-valine to 2-ketoisovalerate, to increase isobutanol production in the genetically modified strains. Our results showed that knockout of PDC5 were one of the main factors among the three PDC genes for improving isobutanol titers in S. cerevisiae. Additionally, we found that deletion of PDC5 in strain carrying overexpressed ILV2 and ARO10 resulted in 8-fold higher isobutanol productivity as compared to the control strain in micro-aerobic fermentations. Our results also suggested that engineered strain pdc5ΔpILV2 pARO10 generated lower ethanol titers and higher acetate acid titers than the control strain, while the growth rate and glucose consumption rate of engineered strain pdc5ΔpILV2 pARO10 were slightly lower than that of the control strain. Meanwhile, the biomass concentration of pdc5ΔpILV2 pARO10 decreased dramatically than that of the control strain.     

Overexpressing <Emphasis Type="Italic">GLT1</Emphasis> in <Emphasis Type="Italic">gpd1</Emphasis>Δ mutant to improve the production of ethanol of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

To improve ethanol production in Saccharomyces cerevisiae, two yeast strains were constructed. In the mutant, KAM-4, the GPD1 gene, which encodes a glycerol 3-phosphate dehydrogenase of S. cerevisiae to synthesize glycerol, was deleted. The mutant KAM-12 had the GLT1 gene (encodes glutamate synthase) placed under the PGK1 promoter while harboring the GPD1 deletion. Notably, overexpression of GLT1 by the PGK1 promoter along with GPD1 deletion resulted in a 10.8% higher ethanol production and a 25.0% lower glycerol formation compared to the wild type in anaerobic fermentations. The growth rate of KAM-4 was slightly lower than that of the wild type under the exponential phase whereas KAM-12 and the wild type were indistinguishable in the biomass concentration at the end of growth period. Meanwhile, dramatic reduction of formation of acetate and pyruvic acid was observed in all the mutants compared to the wild type.     

Functional expression of the lipase gene Lip2 of <Emphasis Type="Italic">Pleurotus</Emphasis> <Emphasis Type="Italic">sapidus</Emphasis> in <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis>

The lipase Lip2 of the edible basidiomycete, Pleurotus sapidus, is an extracellular enzyme capable of hydrolysing xanthophyll esters with high efficiency. The gene encoding Lip2 was expressed in Escherichia coli TOP10 using the gene III signal sequence to accumulate proteins in the periplasmatic space. The heterologous expression under control of the araBAD promoter led to the high level production of recombinant protein, mainly as inclusion bodies, but partially in a soluble and active form. A fusion with a C-terminal His tag was used for purification and immunochemical detection of the target protein. This is the first example of a heterologous expression and periplasmatic accumulation of a catalytically active lipase from a basidiomycete fungus.     

Heterologous expression of two <Emphasis Type="Italic">Glycine max</Emphasis> ω-3 fatty acid desaturases in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

α-Linolenic acid (ALA, C 18:3^Δ9,12,15) has many important biological functions. ω-3 Fatty acid desaturase (FAD3), existing in cytosolic and plastidic compartments of higher plants, catalyzes linoleic acid (LA) desaturation to produce ALA. GmFAD3A-2 and GmFAD3C genes encoding cytosolic FAD3 from Qihuang 29 soybean were cloned and inserted into p416 vector and expressed in K601 yeast strain. Gas chromatography showed that the transformed yeast strains could produce ALA. The ALA accumulation levels for the strains transformed with GmFAD3A-2 or GmFAD3C genes were 0.77 ± 0.1 and 4.13 ± 0.4% of total fatty acids, respectively, while, as compared with that of the control, the contents of LA decreased from 14.34 ± 0.8 to 10.93 ± 0.0 and 7.85 ± 0.1%, respectively, implying that the GmFAD3C enzyme is more vigorous or stable, than GmFAD3A-2. Published in Russian in Fiziologiya Rastenii, 2009, Vol. 56, No. 4, pp. 629–634. This text was submitted by the authors in English.     

Over-expressing <Emphasis Type="Italic">GLT1</Emphasis> in a <Emphasis Type="Italic">gpd2</Emphasis>Δ mutant of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> to improve ethanol production

We constructed two recombinant strains of Saccharomyces cerevisiae in which the GPD2 gene was deleted using a one-step gene replacement method to minimize formation of glycerol and improve ethanol production. In addition, we also over-expressed the GLT1 gene by a two-step gene replacement method to overcome the redox-imbalancing problem in the genetically modified strains. The result of anaerobic batch fermentations showed that the rate of growth and glucose consumption of the KAM-5 (MATα ura3 gpd2Δ::RPT) strain were slower than the original strain, and the KAM-13 (MATα ura3 gpd2Δ::RPT P _PGK -GLT1) strain, however, was indistinguishable compared to the original strain using the same criteria, as analyzed. On the other hand, when compared to the original strain, there were 32 and 38% reduction in glycerol formation for KAM-5 and KAM-13, respectively. Ethanol production increased by 8.6% for KAM-5 and 13.4% for KAM-13. Dramatic reduction in acetate and pyruvic acid was also observed in both mutants compared to the original strains. Although gene GPD2 is responsible for the glycerol synthesis, the mutant KAM-13, in which glycerol formation was substantially reduced, was able to cope and maintain osmoregulation and redox balance and have increased ethanol production under anaerobic fermentations. The result verified the proposed concept of increasing ethanol production in S. cerevisiae by genetic engineering of glycerol synthesis and over-expressing the GLT1 gene along with reconstituted nicotinamide adenine dinucleotide metabolism.     

Production of <Emphasis Type="Italic">N</Emphasis><Superscript><Emphasis Type="Italic">α</Emphasis></Superscript>-acetylated thymosin α1 in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Thymosin α1 (Tα1), a 28-amino acid N ^α -acetylated peptide, has a powerful general immunostimulating activity. Although biosynthesis is an attractive means of large-scale manufacture, to date, Tα1 can only be chemosynthesized because of two obstacles to its biosynthesis: the difficulties in expressing small peptides and obtaining N ^α -acetylation. In this study, we describe a novel production process for N ^α -acetylated Tα1 in Escherichia coli.     

The heterologous expression potential of an acid-tolerant <Emphasis Type="Italic">Talaromyces pinophilus</Emphasis> β-glucosidase in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

A filamentous fungus displaying high cellulase activity was isolated from a compost heap with triticale (a wheat-rye hybrid) as the main constituent. It was preliminarily identified as a Talaromyces pinophilus species. A 2577 base pair β-glucosidase gene was cloned from complementary DNA and heterologously expressed in Saccharomyces cerevisiae. The recombinant β-glucosidase production profile was assessed and compared to that of the Saccharomycopsis fibuligera β-glucosidase which served as a benchmark. The enzyme was also characterised in terms of pH and temperature tolerance as well as response to inhibitors. Maximal extracellular β-glucosidase activity of 0.56 nkat/mg total protein was measured using p-nitrophenyl-β-D-glucopyranoside as substrate. The recombinant protein displayed a pH optimum of 4.0, and good thermostability as 70% of maximal enzyme activity was retained after 1 h at 60 °C. Activity of the recombinant β-glucosidase was adversely affected by the presence of glucose and ethanol at higher concentrations while xylose had no effect. The expression of the T. pinophilus β-glucosidase did not reach the same titres as for the benchmark; however, in the context of constructing a yeast strain for bioethanol production in a consolidated bioprocess, the enzyme may still show good potential.     

Surface-<Emphasis Type="Italic">engineered Saccharomyces cerevisiae</Emphasis> displaying α-acetolactate decarboxylase from <Emphasis Type="Italic">Acetobacter aceti</Emphasis> ssp <Emphasis Type="Italic">xylinum</Emphasis>

Objectives

To convert α-acetolactate into acetoin by an α-acetolactate decarboxylase (ALDC) to prevent its conversion into diacetyl that gives beer an unfavourable buttery flavour.

Results

We constructed a whole Saccharomyces cerevisiae cell catalyst with a truncated active ALDC from Acetobacter aceti ssp xylinum attached to the cell wall using the C-terminal anchoring domain of α-agglutinin. ALDC variants in which 43 and 69 N-terminal residues were absent performed equally well and had significantly decreased amounts of diacetyl during fermentation. With these cells, the highest concentrations of diacetyl observed during fermentation were 30 % less than those in wort fermented with control yeasts displaying only the anchoring domain and, unlike the control, virtually no diacetyl was present in wort after 7 days of fermentation.

Conclusions

Since modification of yeasts with ALDC variants did not affect their fermentation performance, the display of α-acetolactate decarboxylase activity is an effective approach to decrease the formation of diacetyl during beer fermentation.

     

Expression and purification of <Emphasis Type="Italic">Zantedeschia aethiopica</Emphasis> agglutinin in <Emphasis Type="Italic">Escherichia </Emphasis><Emphasis Type="Italic">coli</Emphasis>

Recombinant Zantedeschia aethiopica agglutinin (ZAA) was expressed in Escherichia coli as N-terminal His-tagged fusion. After induction with isopropylthio-β-d-galactoside (IPTG), the recombinant ZAA was purified by metal-affinity chromatography. The purified ZAA protein was applied in anti-fungal assay and the result showed that recombinant ZAA had anti-fungal activity towards leaf mold (Fulvia fulva), one of the most serious phytopathogenic fungi causing significant yield loss of crops. This study suggests that ZAA could be an effective candidate in genetic engineering of plants for the control of leaf mold.     

Interaction of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>–<Emphasis Type="Italic">Lactobacillus fermentum</Emphasis>–<Emphasis Type="Italic">Dekkera bruxellensis</Emphasis> and feedstock on fuel ethanol fermentation

The alcoholic fermentation for fuel ethanol production in Brazil occurs in the presence of several microorganisms present with the starter strain of Saccharomyces cerevisiae in sugarcane musts. It is expected that a multitude of microbial interactions may exist and impact on the fermentation yield. The yeast Dekkera bruxellensis and the bacterium Lactobacillus fermentum are important and frequent contaminants of industrial processes, although reports on the effects of both microorganisms simultaneously in ethanolic fermentation are scarce. The aim of this work was to determine the effects and interactions of both contaminants on the ethanolic fermentation carried out by the industrial yeast S. cerevisiae PE-2 in two different feedstocks (sugarcane juice and molasses) by running multiple batch fermentations with the starter yeast in pure or co-cultures with D. bruxellensis and/or L. fermentum. The fermentations contaminated with D. bruxellensis or L. fermentum or both together resulted in a lower average yield of ethanol, but it was higher in molasses than that of sugarcane juice. The decrease in the CFU number of S. cerevisiae was verified only in co-cultures with both D. bruxellensis and L. fermentum concomitant with higher residual sucrose concentration, lower glycerol and organic acid production in spite of a high reduction in the medium pH in both feedstocks. The growth of D. bruxellensis was stimulated in the presence of L. fermentum resulting in a more pronounced effect on the fermentation parameters than the effects of contamination by each microorganism individually.     

Heterologous production of secondary metabolites as pharmaceuticals in <Emphasis Type="Italic">Saccharomyces</Emphasis> <Emphasis Type="Italic">cerevisiae</Emphasis>

Heterologous expression of genes involved in the biosynthesis of various products is of increasing interest in biotechnology and in drug research and development. Microbial cells are most appropriate for this purpose. Availability of more microbial genomic sequences in recent years has greatly facilitated the elucidation of metabolic and regulatory networks and helped gain overproduction of desired metabolites or create novel production of commercially important compounds. Saccharomyces cerevisiae, as one of the most intensely studied eukaryotic model organisms with a rich density of knowledge detailing its genetics, biochemistry, physiology, and large-scale fermentation performance, can be capitalized upon to enable a substantial increase in the industrial application of this yeast. In this review, we describe recent efforts made to produce commercial secondary metabolites in Saccharomyces cerevisiae as pharmaceuticals. As natural products are increasingly becoming the center of attention of the pharmaceutical and nutraceutical industries, such as naringenin, coumarate, artemisinin, taxol, amorphadiene and vitamin C, the use of S. cerevisiae for their production is only expected to expand in the future, further allowing the biosynthesis of novel molecular structures with unique properties.     

Sequence Analysis of the <Emphasis Type="Italic">Lactoferrin</Emphasis> Gene and Variation of <Emphasis Type="Italic">g</Emphasis>.<Emphasis Type="Italic">7605C</Emphasis>→<Emphasis Type="Italic">T</Emphasis> in 10 Chinese Indigenous Goat Breeds

Much attention has been focused on the study of lactoferrin at the protein or nucleotide level in mice, humans, and cattle, but little is known about it in goats. The goat LF gene from 5' UTR to exon 17 was amplified, and the variation of g.7605C→T in 10 Chinese indigenous goat breeds was analyzed. Among the three ruminant species (cattle, sheep, and goats), the intron-exon distribution pattern was similar, and all the exons had the same length, but the length of introns varied greatly due to insertions or deletions. The frequency of allele T at g.7605C→T (50.12%) was a little higher than that of allele C (49.88%), and the genotype distribution differed greatly between goat populations. The g.7605C→T site showed higher genetic diversity in goat populations. The genetic differentiation was 0.0783, and gene flow was 2.9433 among the 10 Chinese indigenous goat populations.     

Expression of an endoglucanase–cellobiohydrolase fusion protein in <Emphasis Type="Italic">Saccharomyces cerevisiae,Yarrowia lipolytica,</Emphasis> and <Emphasis Type="Italic">Lipomyces starkeyi</Emphasis>

The low secretion levels of cellobiohydrolase I (CBHI) in yeasts are one of the key barriers preventing yeast from directly degrading and utilizing lignocellulose. To overcome this obstacle, we have explored the approach of genetically linking an easily secreted protein to CBHI, with CBHI being the last to be folded. The Trichoderma reesei eg2 (TrEGII) gene was selected as the leading gene due to its previously demonstrated outstanding secretion in yeast. To comprehensively characterize the effects of this fusion protein, we tested this hypothesis in three industrially relevant yeasts: Saccharomyces cerevisiae, Yarrowia lipolytica, and Lipomyces starkeyi. Our initial assays with the L. starkeyi secretome expressing differing TrEGII domains fused to a chimeric Talaromyces emersonii–T. reesei CBHI (TeTrCBHI) showed that the complete TrEGII enzyme, including the glycoside hydrolase (GH) 5 domain is required for increased expression level of the fusion protein when linked to CBHI. We found that this new construct (TrEGII–TeTrCBHI, Fusion 3) had an increased secretion level of at least threefold in L. starkeyi compared to the expression level of the chimeric TeTrCBHI. However, the same improvements were not observed when Fusion 3 construct was expressed in S. cerevisiae and Y. lipolytica. Digestion of pretreated corn stover with the secretomes of Y. lipolytica and L. starkeyi showed that conversion was much better using Y. lipolytica secretomes (50% versus 29%, respectively). In Y. lipolytica, TeTrCBHI performed better than the fusion construct. Furthermore, S. cerevisiae expression of Fusion 3 construct was poor and only minimal activity was observed when acting on the substrate, pNP-cellobiose. No activity was observed for the pNP-lactose substrate. Clearly, this approach is not universally applicable to all yeasts, but works in specific cases. With purified protein and soluble substrates, the exoglucanase activity of the GH7 domain embedded in the Fusion 3 construct in L. starkeyi was significantly higher than that of the GH7 domain in TeTrCBHI expressed alone. It is probable that a higher fraction of fusion construct CBHI is in an active form in Fusion 3 compared to just TeTrCBHI. We conclude that the strategy of leading TeTrCBHI expression with a linked TrEGII module significantly improved the expression of active CBHI in L. starkeyi.     

Expression and characterization of <Emphasis Type="Italic">Trichoderma virens</Emphasis> UKM-1 endochitinase in <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis>

A gene encoding endochitinase from Trichoderma virens UKM-1 was cloned and expressed in E. coli BL21 (DE3). Both the endochitinase gene and its cDNA sequences were obtained. The endochitinase gene encodes 430 amino acids from an open reading frame comprising of 1,690 bp nucleotide sequence with three introns. The endochitinase was expressed as soluble and active enzyme at 20°C when induced with 1 mM IPTG. Maximum activity was observed at 4 h of post-induction time. SDS-PAGE showed that the purified endochitinase exhibited a single band with molecular weight of 42 kDa. Biochemical characterization of the enzyme displayed a near neutral pH characteristic with an optimum pH at 6.0 and optimum temperature at 50°C. The enzyme is stable between pH 3.0–7.0 and is able to retain its activity from 30 to 60°C. The presence of Mg²⁺ and Ca²⁺ ions increased the enzyme activity up to 20%. The purified enzyme has a strong affinity towards colloidal chitin and low effect on ethyl cellulose and D-cellubiose which are non-chitin related substrates. HPLC analysis from the chitin hydrolysis showed the release of (GlcNAc)₃, (GlcNAc)₂ and GlcNAc, in which (GlcNAc)₂ was the main product.     

Employing oxygen pulses to modulate <Emphasis Type="Italic">Lachancea thermotolerans</Emphasis>–<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> Chardonnay fermentations

Oxygen is sometimes deliberately introduced in winemaking at various stages to enhance yeast biomass formation and prevent stuck fermentation. However, there is limited information on how such interventions affect the dynamics of yeast populations. Our previous study in synthetic grape juice showed that oxygen supply enhances the persistence of Lachancea thermotolerans, Torulaspora delbrueckii and Metschnikowia pulcherrima. The three non-Saccharomyces yeasts showed differences in growth as a function of oxygen. The present study focused on evaluating the influence of short oxygen pulses on population dynamics and the aroma profile of Chardonnay wine inoculated with L. thermotolerans and Saccharomyces cerevisiae. The results confirmed a positive effect of oxygen on the relative performance of L. thermotolerans. The mixed culture fermentation with L. thermotolerans with S. cerevisiae developed a distinct aroma profile when compared to monoculture S. cerevisiae. Specifically, a high concentration of esters, medium chain fatty acids and higher alcohols was detected in the mixed culture fermentation. The data also showed that the longer persistence of L. thermotolerans due to addition of oxygen pulses influenced the formation of major volatile compounds such as ethyl acetate, ethyl butyrate, ethyl hexanoate, ethyl caprylate, ethyl caprate, ethyl-3-hydroxybutanoate, ethyl phenylacetate, propanol, isobutanol, butanol, isoamyl alcohol, hexanol, isobutyric acid, butyric acid, iso-valeric acid, hexanoic acid, octanoic acid, and decanoic acid.     

<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>, the Baker’s Yeast,suppresses the growth of Ehrlich carcinoma-bearing mice

This study was undertaken to evaluate the effectiveness and mechanisms of anti-tumor activity of Baker’s yeast, Saccharomyces cerevisiae, in immunocompetent mice. Swiss albino mice were inoculated intramuscularly in the right thigh with Ehrlich Ascites Carcinoma (EAC) cells. At day 8, mice bearing Solid Ehrlich Carcinoma tumor (SEC) were intratumorally (IT) injected with killed S. cerevisiae (10 × 10⁶ and 20 × 10⁶ cells) for 35 days. Histopathology of yeast-treated mice showed extensive tumor degeneration, apoptosis, and ischemic (coagulative) and liquefactive necrosis. These changes are associated with a tumor growth curve that demonstrates a significant antitumor response that peaked at 35 days. Yeast treatment (20 × 10⁶ cells) three times a week resulted in a significant decrease in tumor volume (TV) (67.1%, P < 0.01) as compared to PBS-treated mice. The effect was determined to be dependent on dose and frequency. Yeast administered three and two times per week induced significant decrease in TV as early as 9 and 25 days post-treatment, respectively. Administration of yeast significantly enhanced the recruitment of leukocytes, including macrophages, into the tumors and triggered apoptosis in SEC cells as determined by flow cytometry (78.6%, P < 0.01) at 20 × 10⁶ cells, as compared to PBS-treated mice (42.6%). In addition, yeast treatment elevated TNF-α and IFN-γ plasma levels and lowered the elevated IL-10 levels. No adverse side effects from the yeast treatment were observed, including feeding/drinking cycle and life activity patterns. Indeed, yeast-treated mice showed significant final body weight gain (+21.5%, P < 0.01) at day 35. These data may have clinical implications for the treatment of solid cancer with yeast, which is known to be safe for human consumption.     

Expression and Functional Characterization of the Human Ether-à-go-go-Related Gene (<Emphasis Type="Italic">HERG</Emphasis>) K<Superscript>+</Superscript> Channel Cardiac Splice Variant in <Emphasis Type="Italic">Xenopus laevis</Emphasis> Oocytes

HERG C_Cardiac, a C-terminal splice variant of the human ether-à-go-go-related gene (HERG A), was identified and found to be 100% homologous to HERG_USO. Real-time polymerase chain reaction data indicated that in the human heart HERG C_Cardiac mRNA was expressed eight times more than HERG A, whereas in human ventricular tissue it was expressed six times more than HERG A. A HERG C_Cardiac-green fluorescence protein (GFP) construct was heterologously expressed in Xenopus oocytes. Confocal micrographs revealed that HERG C_Cardiac was mainly expressed in the plasma membrane. HERG C_Cardiac channel expressed in oocytes produced slower inactivating outward currents and faster deactivating tail currents than those of HERG A channel. Equal amounts of HERG A and HERG C_Cardiac cRNA coinjected into oocytes formed intermediate HERG A + HERG C_Cardiac heteromultimers, which was reconfirmed by immunoprecipitation experiments with a HERG A N-terminal antibody. These heteromultimers had different inactivation, deactivation and activation kinetics from those of HERG A and HERG C_Cardiac channels. HERG A + HERG C_Cardiac heteromultimers significantly reduced the model action potential mean amplitude and increased the fast and slow inactivation τ values of the action potential repolarization phase, suggesting involvement of HERG A and HERG C_Cardiac heteromultimers in modulation of the refractory interval.     

<Emphasis Type="Italic">Carex</Emphasis> × <Emphasis Type="Italic">involuta</Emphasis> and <Emphasis Type="Italic">Carex juncella</Emphasis> in the flora of Slovakia

The paper brings information on an isolated occurrence and morphological characters of Carex × involuta and C. juncella populations in the Vel’ká Fatra Mts. Their presence has been known neither from the territory of Slovakia nor from the whole Western Carpathians till now.