Enhanced pyruvate production in <Emphasis Type="Italic">Candida glabrata</Emphasis> by overexpressing the <Emphasis Type="Italic">CgAMD1</Emphasis> gene to improve acid tolerance

Objectives

To enhance acid tolerance of Candida glabrata for pyruvate production by engineering AMP metabolism.

Results

The physiological function of AMP deaminase in AMP metabolism from C. glabrata was investigated by deleting or overexpresseing the corresponding gene, CgAMD1. At pH 4, CgAMD1 overexpression resulted in 59 and 51% increases in biomass and cell viability compared to those of wild type strain, respectively. In addition, the intracellular ATP level of strain Cgamd1Δ/CgAMD1 was down-regulated by 22%, which led to a 94% increase in pyruvate production. Further, various strengths of CgAMD1 expression cassettes were designed, thus resulting in a 59% increase in pyruvate production at pH 4. Strain Cgamd1Δ/CgAMD1 _(H) was grown in a 30 l batch bioreactor at pH 4, and pyruvate reached 46.1 g/l.

Conclusion

CgAMD1 overexpression plays an active role in improving acid tolerance and pyruvate fermentation performance of C. glabrata at pH 4.

     

The promoter of filamentation (POF1) protein from <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> is an ATPase involved in the protein quality control process

Background

The gene YCL047C, which has been renamed promoter of filamentation gene (POF1), has recently been described as a cell component involved in yeast filamentous growth. The objective of this work is to understand the molecular and biological function of this gene.

Results

Here, we report that the protein encoded by the POF1 gene, Pof1p, is an ATPase that may be part of the Saccharomyces cerevisiae protein quality control pathway. According to the results, Δpof1 cells showed increased sensitivity to hydrogen peroxide, tert-butyl hydroperoxide, heat shock and protein unfolding agents, such as dithiothreitol and tunicamycin. Besides, the overexpression of POF1 suppressed the sensitivity of Δpct1, a strain that lacks a gene that encodes a phosphocholine cytidylyltransferase, to heat shock. In vitro analysis showed, however, that the purified Pof1p enzyme had no cytidylyltransferase activity but does have ATPase activity, with catalytic efficiency comparable to other ATPases involved in endoplasmic reticulum-associated degradation of proteins (ERAD). Supporting these findings, co-immunoprecipitation experiments showed a physical interaction between Pof1p and Ubc7p (an ubiquitin conjugating enzyme) in vivo.

Conclusions

Taken together, the results strongly suggest that the biological function of Pof1p is related to the regulation of protein degradation.

     

An Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter gene from wheat plays an important role in stress tolerance

A vacuole Na⁺/H⁺ antiporter gene TaNHX2 was obtained by screening the wheat cDNA library and by the 5′-RACE method. The expression of TaNHX2 was induced in roots and leaves by treatment with NaCl, polyethylene glycol (PEG), cold and abscisic acid (ABA). When expressed in a yeast mutant (Δnhx1), TaNHX2 suppressed the salt sensitivity of the mutant, which was deficient in vacuolar Na⁺/H⁺ antiporter, and caused partial recovery of growth of Δnhx1 in NaCl and LiC1 media. The survival rate of yeast cells was improved by overexpressing the TaNHX2 gene under NaCl, KCl, sorbitol and freezing stresses when compared with the control. The results imply that TaNHX2 might play an important role in salt and osmotic stress tolerance in plant cells.     

A putative <Emphasis Type="Italic">NEM1</Emphasis> homologue regulates lipid droplet biogenesis <Emphasis Type="Italic">via PAH1</Emphasis> in <Emphasis Type="Italic">Tetrahymena thermophila</Emphasis>

Nuclear envelope morphology protein 1 (NEM1) along with a phosphatidate phosphatase (PAH1) regulates lipid homeostasis and membrane biogenesis in yeast and mammals. We investigated four putative NEM1 homologues (TtNEM1A, TtNEM1B, TtNEM1C and TtNEM1D) in the Tetrahymena thermophila genome. Disruption of TtNEM1B, TtNEM1C or TtNEM1D did not compromise normal cell growth. In contrast, we were unable to generate knockout strain of TtNEM1A under the same conditions, indicating that TtNEM1A is essential for Tetrahymena growth. Interestingly, loss of TtNEM1B but not TtNEM1C or TtNEM1D caused a reduction in lipid droplet number. Similar to yeast and mammals, TtNem1B of Tetrahymena exerts its function via Pah1, since we found that PAH1 overexpression rescued loss of Nem1 function. However, unlike NEM1 in other organisms, TtNEM1B does not regulate ER/nuclear morphology. Similarly, neither TtNEM1C nor TtNEM1D is required to maintain normal ER morphology. While Tetrahymena PAH1 was shown to functionally replace yeast PAH1 earlier, we observed that Tetrahymena NEM1 homologues did not functionally replace yeast NEM1. Overall, our results suggest the presence of a conserved cascade for regulation of lipid homeostasis and membrane biogenesis in Tetrahymena. Our results also suggest a Nem1-independent function of Pah1 in the regulation of ER morphology in Tetrahymena.     

A <Emphasis Type="Italic">pqr2</Emphasis> mutant encodes a defective polyamine transporter and is negatively affected by ABA for paraquat resistance in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Despite the paraquat-resistant mutants that have been reported in plants, this study identified a novel A. thaliana mutant (pqr2) from an XVE inducible activation library based on its resistance to 2 μM paraquat. The pqr2 mutant exhibited a termination mutation in the exon of AT1G31830/PAR1/PQR2, encoded a polyamine uptake transporter AtPUT2/PAR1/PQR2. The PQR2 mutation could largely reduce superoxide accumulation and cell death in the pqr2 plants under paraquat treatment. Moreover, compared with wild type, the pqr2 mutant exhibited much reduced tolerance to putrescine, a classic polyamine compound, which confirmed that PQR2 encoded a defective polyamine transporter. Notably, co-treated with ABA and paraquat, both pqr2 mutant and wild type exhibited a lethal phenotype from seed germination, but the wild type like pqr2 mutant, could remain paraquat-resistance while co-treated with high dosage of Na₂WO₄, an ABA synthesis inhibitor. Gene expression analysis suggested that ABA signaling should widely regulate paraquat-responsive genes distinctively in wild type and pqr2 mutant. Hence, this study has for the first time reported about ABA negative effect on paraquat-resistance in A. thaliana, providing insight into the ABA signaling involved in the oxidative stress responses induced by paraquat in plants.     

Phylogenetic and Expression Analysis of Pear Yellow Stripe-Like Transporters and Functional Verification of <Emphasis Type="Italic">PbrYSL4</Emphasis> in Pear Pollen

Yellow stripe-like (YSL) family transporters, belonging to the oligopeptide transporter family, are significant iron transport proteins. In this study, we provided a genome-wide identification and analysis of the YSL gene family in Pyrus bretschneideri. We found eight YSL gene members in pear, clustered into four main groups in the phylogenetic tree. Segmental duplication has played a key role in the expansion of the pear YSL family. The pollen activity analysis indicated that the low concentration of iron ion was beneficial to both pear pollen germination and pollen tube growth. Among the eight YSL genes, PbrYSL4 had particularly high expression in all pear tissues; it was significantly responsive to change in the external iron ion supply in the pollen cultivation in vitro. Moreover, expression of PbrYSL4 in yeast mutant Δccc1 (Ca ²⁺ -sensitive cross-complementer 1 mutant) made Δccc1 restore growth in high iron medium. These data together suggest that PbrYSL4 was involved in the movement of iron in the pear pollen tube growth.     

Immunohistochemical Cross-Reactivity Between <Emphasis Type="Italic">Paracoccidioides</Emphasis> sp. from Dolphins and <Emphasis Type="Italic">Histoplasma capsulatum</Emphasis>

Paracoccidioidomycosis ceti is a cutaneous disease of cetaceans caused by uncultivated Paracoccidioides brasiliensis or Paracoccidioides spp. Serological cross-reactions between paracoccidioidomycosis ceti and paracoccidioidomycosis, paracoccidioidomycosis and histoplasmosis, and paracoccidioidomycosis and coccidioidomycosis have been reported before. The present study aimed to detect immunohistochemical cross-reaction between antibodies to Paracoccidioides sp. and Histoplasma capsulatum, and vice versa. Thirty murine sera, obtained from experimental infections of 6 isolates of H. capsulatum, were reacted with paraffin-embedded yeast-form cells of Paracoccidioides sp. derived from a case of paracoccidioidomycosis ceti in Japan. The murine sera were also reacted with human isolates of H. capsulatum yeast cells, with P. brasiliensis yeast cells, and with fungal cells of Coccidioides posadasii. Three dolphins’ sera from cases of paracoccidioidomycosis ceti, two human sera from patients with paracoccidioidomycosis, and a serum from a healthy person with a history of coccidioidomycosis were used in order to determine that the tested fungal cells reacted properly. Sera derived from mice infected with an isolate of H. capsulatum reacted positively against yeast cells of Paracoccidioides sp., yeast cells of P. brasiliensis, and fungal cells of C. posadasii, while those derived from other strains were negative. The present study recorded for the first time the cross-reaction between the yeast cells of H. capsulatum and antibodies against Paracoccidioides spp., the yeast cells of Paracoccidioides sp. and antibodies against H. capsulatum, the yeast cells of Paracoccidioides sp. and antibodies against Coccidioides sp., and fungal cells of C. posadasii and antibodies against Paracoccidioides spp.     

Identification of new cell size control genes in <Emphasis Type="Italic">S. cerevisiae</Emphasis>

Cell size homeostasis is a conserved attribute in many eukaryotic species involving a tight regulation between the processes of growth and proliferation. In budding yeast S. cerevisiae, growth to a “critical cell size” must be achieved before a cell can progress past START and commit to cell division. Numerous studies have shown that progression past START is actively regulated by cell size control genes, many of which have implications in cell cycle control and cancer. Two initial screens identified genes that strongly modulate cell size in yeast. Since a second generation yeast gene knockout collection has been generated, we screened an additional 779 yeast knockouts containing 435 new ORFs (~7% of the yeast genome) to supplement previous cell size screens. Upon completion, 10 new strong size mutants were identified: nine in log-phase cells and one in saturation-phase cells, and 97% of the yeast genome has now been screened for cell size mutations. The majority of the logarithmic phase size mutants have functions associated with translation further implicating the central role of growth control in the cell division process. Genetic analyses suggest ECM9 is directly associated with the START transition. Further, the small (whi) mutants mrpl49Δ and cbs1Δ are dependent on CLN3 for cell size effects. In depth analyses of new size mutants may facilitate a better understanding of the processes that govern cell size homeostasis.     

Overexpression of <Emphasis Type="Italic">SNF4</Emphasis> and deletions of <Emphasis Type="Italic">REG1</Emphasis>- and <Emphasis Type="Italic">REG2</Emphasis>-enhanced maltose metabolism and leavening ability of baker’s yeast in lean dough

Maltose metabolism of baker’s yeast (Saccharomyces cerevisiae) in lean dough is suppressed by the glucose effect, which negatively affects dough fermentation. In this study, differences and interactions among SNF4 (encoding for the regulatory subunit of Snf1 kinase) overexpression and REG1 and REG2 (which encodes for the regulatory subunits of the type I protein phosphatase) deletions in maltose metabolism of baker’s yeast were investigated using various mutants. Results revealed that SNF4 overexpression and REG1 and REG2 deletions effectively alleviated glucose repression at different levels, thereby enhancing maltose metabolism and leavening ability to varying degrees. SNF4 overexpression combined with REG1/REG2 deletions further enhanced the increases in glucose derepression and maltose metabolism. The overexpressed SNF4 with deleted REG1 and REG2 mutant ΔREG1ΔREG2?+?SNF4 displayed the highest maltose metabolism and strongest leavening ability under the test conditions. Such baker’s yeast strains had excellent potential applications.     

Metabolic engineering of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> for linalool production

Objectives

To engineer the yeast Saccharomyces cerevisiae for the heterologous production of linalool.

Results

Expression of linalool synthase gene from Lavandula angustifolia enabled heterologous production of linalool in S. cerevisiae. Downregulation of ERG9 gene, that encodes squalene synthase, by replacing its native promoter with the repressible MET3 promoter in the presence of methionine resulted in accumulation of 78 µg linalool l^?1 in the culture medium. This was more than twice that produced by the control strain. The highest linalool titer was obtained by combined repression of ERG9 and overexpression of tHMG1. The yeast strain harboring both modifications produced 95 μg linalool l^?1.

Conclusions

Although overexpression of tHMG1 and downregulation of ERG9 enhanced linalool titers threefold in the engineered yeast strain, alleviating linalool toxicity is necessary for further improvement of linalool biosynthesis in yeast.

     

Production of optically pure 2,3-butanediol from <Emphasis Type="Italic">Miscanthus floridulus</Emphasis> hydrolysate using engineered <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> strains

2,3-Butanediol (2,3-BD) can be produced by fermentation of natural resources like Miscanthus. Bacillus licheniformis mutants, WX-02ΔbudC and WX-02ΔgldA, were elucidated for the potential to use Miscanthus as a cost-effective biomass to produce optically pure 2,3-BD. Both WX-02ΔbudC and WX-02ΔgldA could efficiently use xylose as well as mixed sugars of glucose and xylose to produce optically pure 2,3-BD. Batch fermentation of M. floridulus hydrolysate could produce 21.6 g/L d-2,3-BD and 23.9 g/L meso-2,3-BD in flask, and 13.8 g/L d-2,3-BD and 13.2 g/L meso-2,3-BD in bioreactor for WX-02ΔbudC and WX-02ΔgldA, respectively. Further fed-batch fermentation of hydrolysate in bioreactor showed both of two strains could produce optically pure 2,3-BD, with 32.2 g/L d-2,3-BD for WX-02ΔbudC and 48.5 g/L meso-2,3-BD for WX-02ΔgldA, respectively. Collectively, WX-02ΔbudC and WX-02ΔgldA can efficiently produce optically pure 2,3-BD with M. floridulus hydrolysate, and these two strains are candidates for industrial production of optical purity of 2,3-BD with M. floridulus hydrolysate.     

Heterochromatic DNA repeats in <Emphasis Type="Italic">Drosophila</Emphasis> and unusual gene silencing in yeast cells

The 1.25-kb heterochromatic Stellate repeats of Drosophila melanogaster are capable of stably persisting in transgenic constructs and silencing the white reporter gene (mosaic position effect variegation). This system reveals an unusual form of silencing, which is insensitive to known modifiers of position effect variegation. The unusual form of silencing was studied with yeast Saccharomyces cerevisiae, a simple eukaryotic model. To be transferred into yeast cells, the D. melanogaster Stellate repeats were cloned in the pYAC4 centromeric vector (CEN4, URA3, TRP1, HIS3). The HIS3 and/or URA3 genes could be inactive in plasmids consisting of pYAC4 and the Stellate insert in yeast cells. Deletion of D. melanogaster DNA from the plasmid was found to activate the URA3 and HIS3 genes. It was assumed that the genes were repressed rather than damaged in the presence of the Stellate repeats and that a new form of gene silencing was revealed in.     

Deletion of <Emphasis Type="Italic">ldh</Emphasis>A and <Emphasis Type="Italic">ald</Emphasis>H genes in <Emphasis Type="Italic">Klebsiella</Emphasis><Emphasis Type="Italic">pneumoniae</Emphasis> to enhance 1,3-propanediol production

Objectives

To improve 1,3-propanediol (1,3-PD) production and reduce byproduct concentration during the fermentation of Klebsiella pneumonia.

Results

Klebsiella. pneumonia 2-1ΔldhA, K. pneumonia 2-1ΔaldH and K. pneumonia 2-1ΔldhAΔaldH mutant strains were obtained through deletion of the ldhA gene encoding lactate dehydrogenase required for lactate synthesis and the aldH gene encoding acetaldehyde dehydrogenase involved in the synthesis of ethanol. After fed-batch fermentation, the production of 1,3-PD from glycerol was enhanced and the concentrations of byproducts were reduced compared with the original strain K. pneumonia 2-1. The maximum yields of 1,3-PD were 85.7, 82.5 and 87.5 g/l in the respective mutant strains.

Conclusion

Deletion of either aldH or ldhA promoted 1,3-PD production in K. pneumonia.

     

The impact of heterologous catalase expression and superoxide dismutase overexpression on enhancing the oxidative resistance in <Emphasis Type="Italic">Lactobacillus casei</Emphasis>

Two heme-dependent catalase genes were amplified from genomic DNA of Lactobacillus plantarum WCFS1 (KatE1) and Lactobacillus brevis ATCC 367 (KatE2), respectively, and a manganese-containing superoxide dismutase from Lactobacillus casei MCJΔ1 (MnSOD) were cloned into plasmid pELX1, yielding pELX1-KatE1, pELX1-KatE2 and pELX1-MnSOD, then the recombinant plasmids were transferred into L. casei MCJΔ1. The strains of L. casei MCJΔ1/pELX1-KatE1 and L. casei MCJΔ1/pELX1-KatE2 were tolerant at 2 mM H₂O₂. The survival rates of L. casei MCJΔ1/pELX1-KatE1 and L. casei MCJΔ1/pELX1-KatE2 were 270-fold and 300-fold higher than that of the control strain on a short-term H₂O₂ exposure, and in aerated condition, the survival cells counts were 146- and 190-fold higher than that of the control strain after 96 h of incubation. Furthermore, L. casei MCJΔ1/pELX1-MnSOD was the best in three recombinants which was superior in the living cell viability during storage when co-storage with Lactobacillus delbrueckii subsp. lactis LBCH-1.     

The yeast polyubiquitin gene is essential for resistance to high temperatures, starvation, and other stresses

Conjugation of ubiquitin to intracellular proteins mediates their selective degradation in eukaryotes. In the yeast Saccharomyces cerevisiae, four distinct ubiquitin-coding loci have been described. UBI1, UBI2, and UBI3 each encode hybrid proteins in which ubiquitin is fused to unrelated sequences. The fourth gene, UBI4, contains five ubiquitin-coding elements in a head-to-tail arrangement, and thus encodes a polyubiquitin precursor protein. A precise, oligonucleotide-directed deletion of UBI4 was constructed in vitro and substituted in the yeast genome in place of the wild-type allele. ubi4 deletion mutants are viable as vegetative cells, grow at wild-type rates, and contain wild-type levels of free ubiquitin under exponential growth conditions. However, although ubi4/UBI4 diploids can form four initially viable spores, the two ubi4 spores within the ascus lose viability extremely rapidly, apparently a novel phenotype in yeast. Furthermore, ubi4/ubi4 diploids are sporulation-defective. ubi4 mutants are also hypersensitive to high temperatures, starvation, and amino acid analogs. These three conditions, while diverse in nature, are all known to induce stress proteins. Expression of the UBI4 gene is similarly induced by either heat stress or starvation. These results indicate that UBI4 is specifically required for the resistance of cells to stress, and that ubiquitin is an essential component of the stress response system.