<Emphasis Type="Italic">PGM2</Emphasis> overexpression improves anaerobic galactose fermentation in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Background  

In Saccharomyces cerevisiae galactose is initially metabolized through the Leloir pathway after which glucose 6-phosphate enters glycolysis. Galactose is controlled both by glucose repression and by galactose induction. The gene PGM2 encodes the last enzyme of the Leloir pathway, phosphoglucomutase 2 (Pgm2p), which catalyses the reversible conversion of glucose 1-phosphate to glucose 6-phosphate. Overexpression of PGM2 has previously been shown to enhance aerobic growth of S. cerevisiae in galactose medium.     

Anaerobic xylose fermentation by <Emphasis Type="Italic">Spathaspora passalidarum</Emphasis>

A cost-effective conversion of lignocellulosic biomass into bioethanol requires that the xylose released from the hemicellulose fraction (20–40% of biomass) can be fermented. Baker’s yeast, Saccharomyces cerevisiae, efficiently ferments glucose but it lacks the ability to ferment xylose. Xylose-fermenting yeast such as Pichia stipitis requires accurately controlled microaerophilic conditions during the xylose fermentation, rendering the process technically difficult and expensive. In this study, it is demonstrated that under anaerobic conditions Spathaspora passalidarum showed high ethanol production yield, fast cell growth, and rapid sugar consumption with xylose being consumed after glucose depletion, while P. stipitis was almost unable to utilize xylose under these conditions. It is further demonstrated that for S. passalidarum, the xylose conversion takes place by means of NADH-preferred xylose reductase (XR) and NAD⁺-dependent xylitol dehydrogenase (XDH). Thus, the capacity of S. passalidarum to utilize xylose under anaerobic conditions is possibly due to the balance between the cofactor’s supply and demand through this XR–XDH pathway. Only few XRs with NADH preference have been reported so far. 2-Deoxy glucose completely inhibited the conversion of xylose by S. passalidarum under anaerobic conditions, but only partially did that under aerobic conditions. Thus, xylose uptake by S. passalidarum may be carried out by different xylose transport systems under anaerobic and aerobic conditions. The presence of glucose also repressed the enzymatic activity of XR and XDH from S. passalidarum as well as the activities of those enzymes from P. stipitis.     

Expression of a lipid-inducible,self-regulating form of <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> lipase <Emphasis Type="Italic">LIP2</Emphasis> in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Saccharomyces cerevisiae is frequently used as a bioreactor for conversion of exogenously acquired metabolites into value-added products, but has not been utilized for bioconversion of low-cost lipids such as triacylglycerols (TAGs) because the cells are typically unable to acquire these lipid substrates from the growth media. To help circumvent this limitation, the Yarrowia lipolytica lipase 2 (LIP2) gene was cloned into S. cerevisiae expression vectors and used to generate S. cerevisiae strains that secrete active Lip2 lipase (Lip2p) enzyme into the growth media. Specifically, LIP2 expression was driven by the S. cerevisiae PEX11 promoter, which maintains basal transgene expression levels in the presence of sugars in the culture medium but is rapidly upregulated by fatty acids. Northern blotting, lipase enzyme activity assays, and gas chromatographic measurements of cellular fatty acid composition after lipid feeding all confirmed that cells transformed with the PEX11 promoter–LIP2 construct were responsive to lipids in the media, i.e., cells expressing LIP2 responded rapidly to either free fatty acids or TAGs and accumulated high levels of the corresponding fatty acids in intracellular lipids. These data provided evidence of the creation of a self-regulating positive control feedback loop that allows the cells to upregulate Lip2p production only when lipids are present in the media. Regulated, autonomous production of extracellular lipase activity is a necessary step towards the generation of yeast strains that can serve as biocatalysts for conversion of low-value lipids to value-added TAGs and other novel lipid products.     

The mechanism of action of reactivating factor from <Emphasis Type="Italic">Luteococcus japonicus</Emphasis> subsp. <Emphasis Type="Italic">casei</Emphasis>

Reactivating factor (RF) from Luteococcus japonicus subsp. casei was shown to be constitutively synthesized and to act a by one-step mechanism, being activated independently from stress. Cell reactivation (reversion of a cell’s ability to form macrocolonies) might be ensured by the membrane mechanism of RF action, which is proved with the dependence of antistress activity from the condition of the cytoplasmic membrane and with the form of concentration dependence. The incubation of UV-treated L. casei suspension with RF increased the number of cells with intact barrier membrane (1.6–1.8-fold increase compared to RF-untreated cells) and the number of colony-forming cells. Cross defensive and reactivating RF effects on both L. casei and yeast Saccharomyces cerevisiae cells were described. Bacterial and yeast’s RF compete for membrane receptors. Matrix Assisted Laser Desorption/Ionization time-of-flight (MALDI-TOF) spectrometry revealed that RF of L. casei contained two major peptides of 5.8 and 7.6 kDa, while RF of S. cerevisiae was represented by a single peptide of 5.8 kDa. The presence of 5.8 kDa peptide in RF from bacteria and yeasts might ensure cross responses in these organisms.     

Construction of flocculent industrial yeast by the yeast flocculation gene <Emphasis Type="Italic">FLO1</Emphasis>

The structure gene FLO1 from Saccharomyces cerevisiae W303-1A encoding a flocculation protein and the G418 resistance gene kanMX from plasmid pUG6 were amplified by PCR method. The expression vector pYX212 harboring FLO1 gene and kanMX gene was transformed into Angel yeast. The transformant Angel yeast F6 was obtained and showed strong and stable flocculation ability during 20 batches inoculation. And the flocculation ability of the transformant Angel yeast F6 showed no difference in the medium with the initial pH ranging from 3.5 to 6.0. Noteworthily, the flocculation onset of the transformant strain was in the early stationary growth phase, not coincident with the glucose depletion in the cultural medium. And in the experiment the ethanol yield and other properties of the transformant Angel yeast F6 were similar to those of the wild-type strain, although its fermentation time was a little slower comparing with the wild-type strain. Those would be potential application for yeast cells to separate and recycle in the fuel ethanol industry.     

<Emphasis Type="Italic">MPK1</Emphasis> gene is required for filamentous growth induced by isoamyl alcohol in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains from the alcoholic fermentation

The aim of this study was to evaluate the MPK1 (SLT2) gene deletion upon filamentous growth induced by isoamyl alcohol (IAA) in two haploid industrial strains of Saccharomyces cerevisiae using oligonucleotides especially designed for a laboratory S. cerevisiae strain. The gene deletion was performed by replacing part of the open reading frames from the target gene with the KanMX gene. The recombinant strains were selected by their resistance to G418, and after deletion confirmation by polymerase chain reaction, they were cultivated in a yeast extract peptone dextrose medium + 0.5% IAA to evaluate the filamentous growth in comparison to wild strains. Mpk1 derivatives were obtained for both industrial yeasts showing the feasibility of the oligonucleotides especially designed for a laboratory strain (Σ1278b) by Martinez-Anaya et al. (In yeast, the pseudohyphal phenotype induced by isoamyl alcohol results from the operation of the morphogenesis checkpoint. J Cell Sci 116:3423–3431, 2003). The filamentation rate in these derivatives was significantly lower for both strains, as induced by IAA. This drastic reduction in the filamentation ability in the deleted strains suggests that the gene MPK1 is required for IAA-induced filamentation response. The growth curves of wild and derivative strains did not differ substantially. It is not known yet whether the switch to filamentous growth affects the fermentative characteristics of the yeast or other physiological traits. A genetically modified strain for nonfilamentous growth would be useful for these studies, and the gene MPK1 could be a target gene. The feasibility of designed oligonucleotides for this deletion in industrial yeast strains is shown.     

Flower-specific expression of <Emphasis Type="Italic">Arabidopsis</Emphasis><Emphasis Type="Italic">PCS1</Emphasis> driven by <Emphasis Type="Italic">AGAMOUS</Emphasis> second intron in tobacco decreases the fertility of transgenic plants

The PROMOTION OF CELL SURVIVAL 1 (PCS1) gene, encoding an aspartic protease, has an important role in determining the fate of cells in embryonic development and reproduction processes in Arabidopsis. To explore the potential function of the PCS1 gene in generating reproductive sterility, we placed the PCS1 gene under the control of an 1,869-bp nucleotide sequence from the 3′ end of the second intron (AG-I) of Arabidopsis AGAMOUS and CaMV 35S (–60) minimal promoter [AG-I-35S (–60)::PCS1], and introduced it into tobacco. RT–PCR results demonstrated that the PCS1 gene driven by AG-I-35S (–60) chimeric promoter was expressed only in anthers and carpels in the reproductive tissues of transgenic tobacco. Compared to wild-type plants, all AG-I-35S (–60) and AG-I-35S (–60)::PCS1 transgenic lines showed a normal phenotype throughout the vegetative growth phase. However, during the reproductive stage, most AG-I-35S (–60)::PCS1 transgenic plant anthers displayed delayed dehiscence, failed dehiscence, petalody and hypoplasia, and the pollen grains had different shapes and sizes with a distorted, shrunken, or collapsed morphology. Moreover, three transgenic lines, PCS1-1, PCS1-3 and PCS1-4, showed higher sterility than wild-type and AG-I-35S (–60) transgenic plants, respectively. These results showed that the construct of AG-I-35S (–60)::PCS1 was partially effective at preventing seed set and provided a novel sterility strategy.     

One-pot synthesis of genistein from tyrosine by coincubation of genetically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> cells

For production of genistein from N-acetylcysteamine-attached p-coumarate (p-coumaroyl-NAC) supplemented to the medium, a chalcone synthase (CHS) gene from Glycyrrhiza echinata, a chalcone isomerase (CHI) gene from Pueraria lobata, and an isoflavone synthase (IFS) gene from G. echinata were placed under the control of the galactose-inducible GAL promoters in pESC vector and were introduced in Saccharomyces cerevisiae. When the recombinant yeast cells (0.5 g wet weight) were used as “enzyme bags” and incubated at 30°C for 48 h in 100 ml of the buffer containing galactose and 1 mM (265 mg/l) p-coumaroyl-NAC, ca. 340 μg genistein/l was produced. Another system consisting of two enzyme bags was also generated for the purpose of production of genistein from tyrosine. One enzyme bag was an Escherichia coli cell containing a phenylalanine ammonia-lyase gene from a yeast, a 4-coumarate/cinnamate:CoA ligase gene from the actinomycete Streptomyces coelicolor A3(2), the CHS gene, and the CHI gene, in addition to the acetyl-CoA carboxylase gene from Corynebacterium glutamicum, all of which were under the control of the isopropyl-β-d-thiogalactopyranoside-inducible T7 promoter, and thus producing (S)-naringenin from tyrosine. The other enzyme bag was a S. cerevisiae cell containing the IFS gene. Coincubation of the E. coli cells (0.5 g wet weight) and S. cerevisiae cells (0.5 g wet weight) at 26°C for 60 h in 20 ml of the buffer containing 3 mM (543 mg/l) tyrosine as the starting substrate yielded ca. 6 mg genistein/l.     

Alcoholic fermentation by wild-type <Emphasis Type="Italic">Hansenula polymorpha</Emphasis> and <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> versus recombinant strains with an elevated level of intracellular glutathione

The ability of baker’s yeast Saccharomyces cerevisiae and of the thermotolerant methylotrophic yeast Hansenula polymorpha to produce ethanol during alcoholic fermentation of glucose was compared between wild-type strains and recombinant strains possessing an elevated level of intracellular glutathione (GSH) due to overexpression of the first gene of GSH biosynthesis, gamma-glutamylcysteine synthetase, or of the central regulatory gene of sulfur metabolism, MET4. The analyzed strains of H. polymorpha with an elevated pool of intracellular GSH were found to accumulate almost twice as much ethanol as the wild-type strain during glucose fermentation, in contrast to GSH1-overexpressing S. cerevisiae strains, which also possessed an elevated pool of GSH. The ethanol tolerance of the GSH-overproducing strains was also determined. For this, the wild-type strain and transformants with an elevated GSH pool were compared for their viability upon exposure to exogenous ethanol. Unexpectedly, both S. cerevisiae and H. polymorpha transformants with a high GSH pool proved more sensitive to exogenous ethanol than the corresponding wild-type strains.     

Comparison of engineered <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> and engineered <Emphasis Type="Italic">Escherichia coli</Emphasis> for the production of an optically pure keto alcohol

In this study, the production of enantiomerically pure (1R,4S,6S)-6-hydroxy-bicyclo[2.2.2]octane-2-one ((−)-2) through stereoselective bioreduction was used as a model reaction for the comparison of engineered Saccharomyces cerevisiae and engineered Escherichia coli as biocatalysts. For both microorganisms, over-expression of the gene encoding the NADPH-dependent aldo-keto reductase YPR1 resulted in high purity of the keto alcohol (−)-2 (>99% ee, 97–98% de). E. coli had three times higher initial reduction rate but S. cerevisiae continued the reduction reaction for a longer time period, thus reaching a higher conversion of the substrate (95%). S. cerevisiae was also more robust than E. coli, as demonstrated by higher viability during bioreduction. It was also investigated whether the NADPH regeneration rate was sufficient to supply the over-expressed reductase with NADPH. Five strains of each microorganism with varied carbon flux through the NADPH regenerating pentose phosphate pathway were genetically constructed and compared. S. cerevisiae required an increased NADPH regeneration rate to supply YPR1 with co-enzyme while the native NADPH regeneration rate was sufficient for E. coli. Nádia Skorupa Parachin and Magnus Carlquist have contributed equally to the paper.     

Biosorption of manganese by <Emphasis Type="Italic">Aspergillus niger</Emphasis> and <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Summary Biosorption of manganese from its aqueous solution using yeast biomass Saccharomyces cerevisiae and fungal biomass Aspergillus niger was carried out. Manganese biosorption equilibration time for A. niger and S. cerevisiae were found to be 60 and 20 min, with uptakes of 19.34 and 18.95 mg/g, respectively. Biosorption increased with rise in pH, biomass, and manganese concentration. The biosorption equilibrium data fitted with the Freundlich isotherm model revealed that A. niger was a better biosorbent of manganese than S. cerevisiae.     

The Sulfur Oxidation Operon Repressor Function is Influenced by the Product of its Adjacent Upstream ORF in <Emphasis Type="Italic">Pseudaminobacter salicylatoxidans</Emphasis> KCT001

The repressor of sulfur-oxidizing (sox) operon regulates expression of genes encoding a multienzyme complex that governs the chemolithotrophic sulfur oxidation in Pseudaminobacter salycylatoxidans KCT001. The inducer of sox operon viz., thiosulfate and other sulfur anions had no impact on in vitro repressor–operator interaction which indicates an atypical derepression mechanism. The reduced repressor has higher affinity for its operator DNA. The sulfur oxidation repressor binds with operator regions and led to efficient repression in trans, however, increased repressor concentration resulted in higher gene expression. Using a reporter system in E. coli, the present study established that the thioredoxin-like protein, encoded in immediate upstream ORF, could nullify the observed reversal of the repression at higher repressor concentration. In this context, the involvement of the upstream gene product in the regulation of the sulfur oxidation gene expression has been reported.     

Heterologous interferons synthesis in yeast <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The HuIFNA16, HuIFNB1, and BoIFNG genes encoding human α16, β-interferons and bovine γ-interferon were cloned under the control of the yeast Pichia pastoris AOX1 gene promoter. The yeast strains producing heterologous interferons intracellularly and extracellularly were constructed. There was no effect of high level of heterologous protein synthesis on the yeast P. pastoris cell growth, unlike yeast Saccharomyces cerevisiae. The considerable part of the heterologous interferons was detected in the yeast P. pastoris soluble protein fraction but not in the “inclusion bodies.” The treatment of human β-interferon with endoglycosidase H showed that protein was expressed in glycosylated and unglycosylated forms. On the strength of these data, the hypothesis was suggested that the more effective heterologous gene expression in yeast P. pastoris and enhanced resistance of the methylotrophic yeast to negative effects of recombinant proteins was due to the special features of its metabolism.     

Inactivation of the transcription factor <Emphasis Type="Italic">mig1</Emphasis> (<Emphasis Type="Italic">YGL035C</Emphasis>) in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> improves tolerance towards monocarboxylic weak acids: acetic,formic and levulinic acid

Toxic concentrations of monocarboxylic weak acids present in lignocellulosic hydrolyzates affect cell integrity and fermentative performance of Saccharomyces cerevisiae. In this work, we report the deletion of the general catabolite repressor Mig1p as a strategy to improve the tolerance of S. cerevisiae towards inhibitory concentrations of acetic, formic or levulinic acid. In contrast with the wt yeast, where the growth and ethanol production were ceased in presence of acetic acid 5 g/L or formic acid 1.75 g/L (initial pH not adjusted), the m9 strain (Δmig1::kan) produced 4.06?±?0.14 and 3.87?±?0.06 g/L of ethanol, respectively. Also, m9 strain tolerated a higher concentration of 12.5 g/L acetic acid (initial pH adjusted to 4.5) without affecting its fermentative performance. Moreover, m9 strain produced 33% less acetic acid and 50–70% less glycerol in presence of weak acids, and consumed acetate and formate as carbon sources under aerobic conditions. Our results show that the deletion of Mig1p provides a single gene deletion target for improving the acid tolerance of yeast strains significantly.     

Cloning and expression of bacteriophage FMV lysocyme gene in cells of yeasts <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> and <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Cloning, sequencing, and expression of the gene for soluble lysozyme of bacteriophage FMV from Gram-negative Pseudomonas aeruginosa bacteria were conducted in yeast cells. Comparable efficiency of two lysozyme expression variants (as intracellular or secreted proteins) was estimated in cells of Saccharomyces cerevisiae and Pichia pastoris. Under laboratory conditions, yeast S. cerevisiae proved to be more effective producer of phage lysozyme than P. pastoris, the yield of the enzyme in the secreted form being significantly higher than that produced in the intracellular form.     

<Emphasis Type="Italic">Starmerella bombicola</Emphasis> influences the metabolism of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> at pyruvate decarboxylase and alcohol dehydrogenase level during mixed wine fermentation

Background  

The use of a multistarter fermentation process with Saccharomyces cerevisiae and non-Saccharomyces wine yeasts has been proposed to simulate natural must fermentation and to confer greater complexity and specificity to wine. In this context, the combined use of S. cerevisiae and immobilized Starmerella bombicola cells (formerly Candida stellata) was assayed to enhance glycerol concentration, reduce ethanol content and to improve the analytical composition of wine. In order to investigate yeast metabolic interaction during controlled mixed fermentation and to evaluate the influence of S. bombicola on S. cerevisiae, the gene expression and enzymatic activity of two key enzymes of the alcoholic fermentation pathway such as pyruvate decarboxylase (Pdc1) and alcohol dehydrogenase (Adh1) were studied.