Construction of flavocytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>2</Subscript>-overproducing strains of the thermotolerant methylotrophic yeast <Emphasis Type="Italic">Hansenula polymorpha</Emphasis> (<Emphasis Type="Italic">Pichia angusta</Emphasis>)

L-Lactate cytochrome c oxidoreductase (flavocytochrome b ₂, FC b ₂) from the thermotolerant methylotrophic yeast Hansenula polymorpha (Pichia angusta) is, unlike the enzyme form baker’s yeast, a thermostable enzyme potentially important for bioanalytical technologies for highly selective assays of L-lactate in biological fluids and foods. This paper describes the construction of flavocytochrome b ₂ producers with over-expression of the H. polymorpha CYB2 gene, encoding FC b ₂. The HpCYB2 gene under the control of the strong H. polymorpha alcohol oxidase promoter in a plasmid for multicopy integration was transformed into the recipient strain H. polymorpha C-105 (grc1 catX), impaired in glucose repression and devoid of catalase activity. A method was developed for preliminary screening of the transformants with increased FC b ₂ activity in permeabilized yeast cells. The optimal cultivation conditions providing for the maximal yield of the target enzyme were found. The constructed strain is a promising FC b ₂ producer characterized by a sixfold increased (to 3 μmol min^?1 mg^?1 protein in cell-free extract) activity of the enzyme.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation: an efficient tool for targeted gene disruption in <Emphasis Type="Italic">Talaromyces marneffei</Emphasis>

Talaromyces marneffei causes life-threatening infections in immunocompromised hosts. An efficient tool for genetic manipulation of T. marneffei will allow for increased understanding of this thermally dimorphic fungus. Agrobacterium tumefaciens-mediated transformation (ATMT) was optimized for targeted gene disruption in T. marneffei using the plasmid pDHt/acuD::pyrG. Molecular analyses of transformants were performed by PCR, Southern blot and semi-quantitative RT-PCR. A. tumefaciens strain EHA105 was more efficient at transformation than strain AGL-1 in ATMT via solid co-cultivation. An A. tumefaciens:T. marneffei ratio of 1000:1 in an ATMT liquid co-cultivation led to a relatively high transformation efficiency of 90 transformants per 10⁶ yeast cells. Using ATMT-mediated knockout mutagenesis, we successfully deleted the acuD gene in T. marneffei. PCR and Southern blot analysis confirmed that acuD was disrupted and that the foreign pyrG gene was integrated into T. marneffei. Semi-quantitative RT-PCR analysis further confirmed that pyrG was expressed normally. These results suggest that ATMT can be a potential platform for targeted gene disruption in T. marneffei and that liquid co-cultivation may provide new opportunities to develop clinical treatments.     

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.     

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.

     

Development of <Emphasis Type="Italic">mazF</Emphasis>-based markerless genome editing system and metabolic pathway engineering in <Emphasis Type="Italic">Candida tropicalis</Emphasis> for producing long-chain dicarboxylic acids

Candida tropicalis can grow with alkanes or plant oils as the sole carbon source, and its industrial application thus has great potential. However, the choice of a suitable genetic operating system can effectively increase the speed of metabolic engineering. MazF functions as an mRNA interferase that preferentially cleaves single-stranded mRNAs at ACA sequences to inhibit protein synthesis, leading to cell growth arrest. Here, we constructed a suicide plasmid named pPICPJ-mazF that uses the mazF gene of Escherichia coli as a counterselectable marker for the markerless editing of C. tropicalis genes to increase the rate of conversion of oils into long-chain dicarboxylic acids. To reduce the β-oxidation of fatty acids, the carnitine acetyltransferase gene (CART) was deleted using the gene editing system, and the yield of long-chain acids from the strain was increased to 8.27 g/L. By two homologous single exchanges, the promoters of both the cytochrome P450 gene and the NADPH–cytochrome P450 reductase gene were subsequently replaced by the constitutively expressed promoter pGAP, and the production of long-chain dicarboxylic acids by the generated strain (C. tropicalis PJPP1702) reached 11.39 g/L. The results of fed-batch fermentation showed that the yield of long-chain acids from the strain was further increased to 32.84 g/L, which was 11.4 times higher than that from the original strain. The results also showed that the pPICPJ-mazF-based markerless editing system may be more suited for completing the genetic editing of C. tropicalis.     

<Emphasis Type="Italic">Zunongwangia flava</Emphasis> sp. nov., belonging to the family <Emphasis Type="Italic">Flavobacteriaceae</Emphasis>, isolated from <Emphasis Type="Italic">Salicornia europaea</Emphasis>

A yellow pigmented bacterium designated strain MBLN094^T within the family Flavobacteriaceae was isolated from a halophyte Salicornia europaea on the coast of the Yellow Sea. This strain was a Gram-stain negative, aerobic, non-spore forming, rod-shaped bacterium. Phylogenetic analysis of the 16S rRNA gene sequence of strain MBLN094^T was found to be related to the genus Zunongwangia, exhibiting 16S rRNA gene sequence similarity values of 97.0, 96.8, 96.4, and 96.3% to Zunongwangia mangrovi P2E16^T, Z. profunda SM-A87^T, Z. atlantica 22II14-10F7^T, and Z. endophytica CPA58^T, respectively. Strain MBLN094^T grew at 20?37°C (optimum, 25?30°C), at pH 6.0?10.0 (optimum, 7.0?8.0), and with 0.5?15.0% (w/v) NaCl (optimum, 2.0?5.0%). Menaquinone MK-6 was the sole respiratory quinone. The polar lipids were phosphatidylethanolamine, two unidentified aminolipids, and four unidentified lipids. Major fatty acids were iso-C_17:0 3-OH, summed feature 3 (C_16:1ω6c and/or C16:1 ω7c), and iso-C_15:0. The genomic DNA G + C content was 37.4 mol%. Based on these polyphasic taxonomic data, strain MBLN094^T is considered to represent a novel species of the genus Zunongwangia, for which the name Zunongwangia flava sp. nov. is proposed. The type strain is MBLN094^T (= KCTC 62279^T = JCM 32262^T).     

The diversity of rhizobia nodulating the <Emphasis Type="Italic">Medicago</Emphasis>, <Emphasis Type="Italic">Melilotus</Emphasis> and <Emphasis Type="Italic">Trigonella</Emphasis> inoculation group in Egypt is marked by the dominance of two genetic types

Twenty four rhizobial strains were isolated from root nodules of Melilotus, Medicago and Trigonella plants growing wild in soils throughout Egypt. The nearly complete 16S rRNA gene sequence from each strain showed that 12 strains (50 %) were closely related to the Ensifer meliloti LMG6133^T type strain with identity values higher than 99.0 %, that 9 (37.5 %) strains were more than 99 % identical to the E. medicae WSM419^T type strain, and that 3 (12.5 %) strains showed 100 % identity with the type strain of N. huautlense S02^T. Accordingly, the diversity of rhizobial strains nodulating wild Melilotus, Medicago and Trigonella species in Egypt is marked by predominance of two genetic types, E. meliloti and E. medicae, although the frequency of isolation was slightly higher in E. meliloti. Sequencing of the symbiotic nodC gene from selected Medicago and Melilotus strains revealed that they were all similar to those of the E. meliloti LMG6133^T and E. medicae WSM419^T type strains, respectively. Similarly, nodC sequences of strains identified as members of the genus Neorhizobium were more than 99 % identical to that of N. galegae symbiovar officinalis HAMBI 114.     

Regulatory activity of heterologous gene-activator <Emphasis Type="Italic">xlnR</Emphasis> of <Emphasis Type="Italic">Aspergillus niger</Emphasis> in <Emphasis Type="Italic">Penicillium canescens</Emphasis>

The gene encoding the xlnR xylanolytic activator of the heterologous fungus Aspergillus niger was incorporated into the Penicillium canescens genome. Integration of the xlnR gene resulted in the increase in a number of activities, i.e. endoxylanase, β-xylosidase, α-L-arabinofuranosidase, α-galactosidase, and feruloyl esterase, compared to the host P. canescens PCA 10 strain, while β-galactosidase, β-glucosidase, endoglucanase, and CMCase activities remained constant. Two different expression constructs were developed. The first consisted of the nucleotide sequence containing the mature P. canescens phytase gene under control of the axhA promoter region gene encoding A. niger (1,4)-β-D-arabinoxylan-arabinofuranohydrolase. The second construct combined the P. canescens phytase gene and the bgaS promoter region encoding homologous β-galactosidase. Both expression cassettes were transformed into P. canescens host strain containing xlnR. Phytase synthesis was observed only for strains with the bgaS promoter on arabinose-containing culture media. In conclusion, the bgaS and axhA promoters were regulated by different inducers and activators in the P. canescens strain containing a structural tandem of the axhA promoter and the gene of the xlnR xylanolytic activator.     

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.     

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.

     

Influence of <Emphasis Type="Italic">UPF</Emphasis> genes on severity of <Emphasis Type="Italic">SUP45</Emphasis> mutations

Eukaryotic cells possess a special mechanism for the degradation of mRNAs containing premature termination codons (PTCs), referred to as NMD (nonsense-mediated mRNA decay). The strength of this pathway depends on the recognition of the PTCs by translational machinery and the interaction of translation termination factors eRF1 and eRF3 with Upf1, Upf2 and Upf3 proteins in Sachromyces cerevisiae yeast. Previously, we have shown that the decrease of eRF1 protein amounts in sup45 nonsense mutants leads to the impairment of NMD. Here we show that the deletion of UPF1 or UPF2 genes leads to an increase in the viability of sup45 mutants, while the effect of UPF3 gene deletion is allele-specific. Two-hybrid data have shown that amino acid residues 1–555 of Upf1 protein interact with eRF1. Any UPF gene deletion leads to allosupression of the adel1-14 mutation without a change in eRF1 content. The Upf1 depletion does not influence the synthetic lethality of sup45 mutations and the [PSI ⁺] prion. It is possible that the absence of Upf1 (or its activator Upf2) leads to a more effective formation of the translation termination complex and consequently to the increased viability of the cells containing mutant termination factors.     

<Emphasis Type="Italic">Streptomyces tunisialbus</Emphasis> sp. nov., a novel <Emphasis Type="Italic">Streptomyces</Emphasis> species with antimicrobial activity

A novel actinomycete strain designated S2^T was isolated from Tunisian rhizosphere soil of Lavandula officinalis. This isolate exhibited broad spectrum antibacterial activity against several Gram-positive and Gram-negative bacteria and also antifungal activity against yeast and filamentous fungi. The isolate S2^T presents morphological and chemotaxonomic characteristics typical of the members of the genus Streptomyces. Whole cell hydrolysates of S2^T were found to contain LL-diaminopimelic acid. The major fatty acids were identified as C16:0, anteiso-C15:0 and iso-C16:0 whereas the predominant menaquinones were found to be MK-9(H6) and MK-9(H8). The polar lipids were identified as diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside and three unidentified compounds. The G+C content of the genomic DNA was determined to be 71.8 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain S2^T belongs to the genus Streptomyces and is closely related to Streptomyces netropsis DSM 40259^T with 99.86% sequence similarity. Multi-locus sequence analysis (MLSA) based on four house-keeping gene alleles (gyrB, recA, trpB, rpoB) showed that isolate S2^T is closely related to S. netropsis, with an MLSA distance greater than 0.007. The DNA–DNA relatedness between strain S2^T and its near phylogenetic neighbour was 63.6 ± 2.3%, which is lower than the 70% threshold value for delineation of genomic prokaryotic species. This isolate was also distinguished from the type strain S. netropsis DSM 40259^T, using a combination of morphological and physiological features. Based on its phenotypic and molecular properties, strain S2^T is considered to represent a novel species of the genus Streptomyces, for which the name Streptomyces tunisialbus sp. nov. is proposed. The type strain is S2^T (= JCM 32165^T = DSM 105760^T).     

<Emphasis Type="Italic">In silico</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis> analysis reveal a novel gene in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> trehalose metabolism

Background

The ability to respond rapidly to fluctuations in environmental changes is decisive for cell survival. Under these conditions trehalose has an essential protective function and its concentration increases in response to enhanced expression of trehalose synthase genes, TPS1, TPS2, TPS3 and TSL1. Intriguingly, the NTH1 gene, which encodes neutral trehalase, is highly expressed at the same time. We have previously shown that trehalase remains in its inactive non-phosphorylated form by the action of an endogenous inhibitor. Recently, a comprehensive two-hybrid analysis revealed a 41-kDa protein encoded by the YLR270w ORF, which interacts with NTH1p.

Results

In this work we investigate the correlation of this Trehalase Associated Protein, in trehalase activity regulation. The neutral trehalase activity in the ylr270w mutant strain was about 4-fold higher than in the control strain. After in vitro activation by PKA the ylr270w mutant total trehalase activity increased 3-fold when compared to a control strain. The expression of the NTH1 gene promoter fused to the heterologous reporter lacZ gene was evaluated. The mutant strain lacking YLR270w exhibited a 2-fold increase in the NTH1-lacZ basal expression when compared to the wild type strain.

Conclusions

These results strongly indicate a central role for Ylr270p in inhibiting trehalase activity, as well as in the regulation of its expression preventing a wasteful futile cycle of synthesis-degradation of trehalose.

     

Structural organization characteristics of the <Emphasis Type="Italic">DIP1</Emphasis> gene in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> strains mutant for the <Emphasis Type="Italic">flamenco</Emphasis> gene

Molecular cloning of the DIP1 gene located in the 20A4-5 region has been performed from the following strains with the flamenco phenotype: flam ^SS (SS) and flam ^MS (MS) characterized by a high transposition rate of retrotransposon gypsy (mdg4), flam ^{py + (P)} carrying the insertion of a construction based on the P element into the region of the flamenco gene, and flamenco ⁺. The results of restriction analysis and sequencing cloned DNA fragments has shown that strains flam ^SS , flam ^MS considerably differ from flam ^{py + (P)}, and flamenco ⁺ in the structure of DIP1. Strains flam ^SS and flam ^MS have no DraI restriction site at position 1765 in the coding region of the gene, specifically, in the domain determining the signal of the nuclear localization of the DIP1 protein. This mutation has been found to consist in a nucleotide substitution in the recognition site of DraI restriction endonuclease, which is transformed from TTTAAA into TTTAAG and, hence, is not recognized by the enzyme. This substitution changes codon AAA into AAG and is translationally insignificant, because both triplets encode the same amino acid, lysine. The DIP1 gene of strains flam ^SS and flam ^MS has been found to contain a 182-bp insertion denoted IdSS (insertion in DIP1 strain SS); it is located in the second intron of the gene. The IdSS sequence is part of the open reading frame encoding the putative transposase of the mobile genetic element HB1 belonging to the Tc1/mariner family. This insertion is presumed to disturb the conformations of DNA and the chromosome, in particular, by forming loops, which alters the expression of DIP1 and, probably, neighboring genes. In strains flamenco ⁺ and flam ^{py + (P)}, the IdSS insertion within the HB1 sequence is deleted. The deletion encompasses five C-terminal amino acid residues of the conserved domain and the entire C-terminal region of the putative HB1 transposase. The obtained data suggest that DIP1 is involved in the control of gypsy transpositions either directly or through interaction with other elements of the genome.     

Enhanced succinic acid productivity by expression of <Emphasis Type="Italic">mgtCB</Emphasis> gene in <Emphasis Type="Italic">Escherichia coli</Emphasis> mutant

In this study, a novel engineering Escherichia coli strain (CBMG111) with the expression of mgtCB gene was constructed for the enhanced fermentative production of succinic acid by utilizing the synergetic effect of mgtC gene to improve the growth of strains at the environment of low Mg²⁺ concentration and mgtB to enhance the transport of Mg²⁺ into cells. After the effect of the expression of the individual genes (mgtA, mgtB, mgtC) on the growth of E. coli was clarified, the fermentative production of succinic acid by CBMG111 was studied with the low-price mixture of Mg(OH)₂ and NH₃·H₂O as the alkaline neutralizer and the biomass hydrolysates as the carbon sources, which demonstrated that the expression of mgtCB gene can significantly increase the productivity of succinic acid (2.97 g L^?1 h^?1) compared with that by using the engineering strain with the overexpression of mgtA gene.     

Characterization of <Emphasis Type="Italic">lpaH2</Emphasis> gene corresponding to lipopeptide synthesis in <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> HAB-2

Background

Bacillus spp. have prominent ability to suppress plant pathogens and corresponding diseases. Previous analyses of Bacillus spp. revealed numerous gene clusters involved in nonribosomal synthesis of cyclic lipopeptides with distinct antimicrobial action. The 4′-phosphopantetheinyl transferase (PPTase) encoded by sfp gene is a key factor in lipopeptide synthesis in Bacillus spp. In previous study, B. amyloliquefaciens strain HAB-2 was found to inhibit a broad range of plant pathogens, which was attributed to its secondary metabolite lipopeptide.

Results

A sfp homologue lpaH2 which encoded phosphopantetheinyl transferase but shared 71% sequence similarity was detected in strain HAB-2. Disruption of lpaH2 gene resulted in losing the ability of strain HAB-2 to produce lipopeptide, as well as antifungal and hemolytic activities. When lpaH2 replaced sfp gene of B. subtilis strain 168, a non-lipopeptide producer, the genetically engineered strain 168 could produced lipopeptides and recovered antifungal activity. Quantitative PCR assays indicated that, the expression level of lpaH2 in B. subtilis 168 strain decrease to 0.27-fold compared that of the wild type B. amyloliquefaciens strain HAB-2.

Conclusion

Few studies have reported about lpa gene which can replace sfp gene in the different species. Taken together, our study showed for the first time that lpaH2 from B. amyloliquefaciens could replace sfp gene.