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Borun Zhang - One of the best experts on this subject based on the ideXlab platform.
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Uricase production by a recombinant Hansenula polymorpha strain harboring Candida Utilis uricase gene
2013Co-Authors: Xuena Guo, Borun Zhang, Zhaoyue Wang, Zhiyu ChenAbstract:Abstract Uricase is an important medical enzyme which can be used to determine urate in clinical analysis, to therapy gout, hyperuricemia, and tumor lysis syndrome. Uricase of Candida Utilis was successfully expressed in Hansenula polymorpha under the control of methanol oxidase promoter using Saccharomyces cerevisiae α-factor signal peptide as the secretory sequence. Recombinant H. polymorpha MU200 with the highest extracellular uricase production was characterized with three copies of expression cassette and selected for process optimization for the production of recombinant enzyme. Among the parameters investigated in shaking flask cultures, the pH value of medium and inoculum size had great influence on the recombinant uricase production. The maximum extracellular uricase yield of 2.6 U/ml was obtained in shaking flask culture. The yield of recombinant uricase was significantly improved by the combined use of a high cell-density cultivation technique and a pH control strategy of switching culture pH from 5.5 to 6.5 in the induction phase. After induction for 58 h, the production of recombinant uricase reached 52.3 U/ml (about 2.1 g/l of protein) extracellularly and 60.3 U/ml (about 2.4 g/l) intracellularly in fed-batch fermentation, which are much higher than those expresse
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uricase production by a recombinant hansenula polymorpha strain harboring Candida Utilis uricase gene
Applied Microbiology and Biotechnology, 2008Co-Authors: Zhiyu Chen, Xuena Guo, Zhaoyue Wang, Borun ZhangAbstract:Uricase is an important medical enzyme which can be used to determine urate in clinical analysis, to therapy gout, hyperuricemia, and tumor lysis syndrome. Uricase of Candida Utilis was successfully expressed in Hansenula polymorpha under the control of methanol oxidase promoter using Saccharomyces cerevisiae α-factor signal peptide as the secretory sequence. Recombinant H. polymorpha MU200 with the highest extracellular uricase production was characterized with three copies of expression cassette and selected for process optimization for the production of recombinant enzyme. Among the parameters investigated in shaking flask cultures, the pH value of medium and inoculum size had great influence on the recombinant uricase production. The maximum extracellular uricase yield of 2.6 U/ml was obtained in shaking flask culture. The yield of recombinant uricase was significantly improved by the combined use of a high cell-density cultivation technique and a pH control strategy of switching culture pH from 5.5 to 6.5 in the induction phase. After induction for 58 h, the production of recombinant uricase reached 52.3 U/ml (about 2.1 g/l of protein) extracellularly and 60.3 U/ml (about 2.4 g/l) intracellularly in fed-batch fermentation, which are much higher than those expressed in other expression systems. To our knowledge, this is the first report about the heterologous expression of uricase in H. polymorpha.
Zhiyu Chen - One of the best experts on this subject based on the ideXlab platform.
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Uricase production by a recombinant Hansenula polymorpha strain harboring Candida Utilis uricase gene
2013Co-Authors: Xuena Guo, Borun Zhang, Zhaoyue Wang, Zhiyu ChenAbstract:Abstract Uricase is an important medical enzyme which can be used to determine urate in clinical analysis, to therapy gout, hyperuricemia, and tumor lysis syndrome. Uricase of Candida Utilis was successfully expressed in Hansenula polymorpha under the control of methanol oxidase promoter using Saccharomyces cerevisiae α-factor signal peptide as the secretory sequence. Recombinant H. polymorpha MU200 with the highest extracellular uricase production was characterized with three copies of expression cassette and selected for process optimization for the production of recombinant enzyme. Among the parameters investigated in shaking flask cultures, the pH value of medium and inoculum size had great influence on the recombinant uricase production. The maximum extracellular uricase yield of 2.6 U/ml was obtained in shaking flask culture. The yield of recombinant uricase was significantly improved by the combined use of a high cell-density cultivation technique and a pH control strategy of switching culture pH from 5.5 to 6.5 in the induction phase. After induction for 58 h, the production of recombinant uricase reached 52.3 U/ml (about 2.1 g/l of protein) extracellularly and 60.3 U/ml (about 2.4 g/l) intracellularly in fed-batch fermentation, which are much higher than those expresse
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uricase production by a recombinant hansenula polymorpha strain harboring Candida Utilis uricase gene
Applied Microbiology and Biotechnology, 2008Co-Authors: Zhiyu Chen, Xuena Guo, Zhaoyue Wang, Borun ZhangAbstract:Uricase is an important medical enzyme which can be used to determine urate in clinical analysis, to therapy gout, hyperuricemia, and tumor lysis syndrome. Uricase of Candida Utilis was successfully expressed in Hansenula polymorpha under the control of methanol oxidase promoter using Saccharomyces cerevisiae α-factor signal peptide as the secretory sequence. Recombinant H. polymorpha MU200 with the highest extracellular uricase production was characterized with three copies of expression cassette and selected for process optimization for the production of recombinant enzyme. Among the parameters investigated in shaking flask cultures, the pH value of medium and inoculum size had great influence on the recombinant uricase production. The maximum extracellular uricase yield of 2.6 U/ml was obtained in shaking flask culture. The yield of recombinant uricase was significantly improved by the combined use of a high cell-density cultivation technique and a pH control strategy of switching culture pH from 5.5 to 6.5 in the induction phase. After induction for 58 h, the production of recombinant uricase reached 52.3 U/ml (about 2.1 g/l of protein) extracellularly and 60.3 U/ml (about 2.4 g/l) intracellularly in fed-batch fermentation, which are much higher than those expressed in other expression systems. To our knowledge, this is the first report about the heterologous expression of uricase in H. polymorpha.
Shigehito Ikushima - One of the best experts on this subject based on the ideXlab platform.
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genome and transcriptome analysis of the food yeast Candida Utilis
PLOS ONE, 2012Co-Authors: Yasuyuki Tomita, Hideyuki Tamakawa, Kazuho Ikeo, Takashi Gojobori, Shigehito IkushimaAbstract:The industrially important food-yeast Candida Utilis is a Crabtree effect-negative yeast used to produce valuable chemicals and recombinant proteins. In the present study, we conducted whole genome sequencing and phylogenetic analysis of C. Utilis, which showed that this yeast diverged long before the formation of the CUG and Saccharomyces/Kluyveromyces clades. In addition, we performed comparative genome and transcriptome analyses using next-generation sequencing, which resulted in the identification of genes important for characteristic phenotypes of C. Utilis such as those involved in nitrate assimilation, in addition to the gene encoding the functional hexose transporter. We also found that an antisense transcript of the alcohol dehydrogenase gene, which in silico analysis did not predict to be a functional gene, was transcribed in the stationary-phase, suggesting a novel system of repression of ethanol production. These findings should facilitate the development of more sophisticated systems for the production of useful reagents using C. Utilis.
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efficient production of l lactic acid from xylose by a recombinant Candida Utilis strain
Journal of Bioscience and Bioengineering, 2012Co-Authors: Hideyuki Tamakawa, Shigehito Ikushima, Satoshi YoshidaAbstract:Abstract Efficient l -lactic acid production from xylose was achieved using a pyruvate decarboxylase-deficient Candida Utilis strain expressing an l -lactate dehydrogenase, an NADH-preferring mutated xylose reductase (XR), a xylitol dehydrogenase and a xylulokinase. The recombinant strain showed 53% increased l -lactic acid production compared with the reference strain expressing native XR (NADPH-preferring).
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Ethanol Production from Xylose by a Recombinant Candida Utilis Strain Expressing Protein-Engineered Xylose Reductase and Xylitol Dehydrogenase
Bioscience biotechnology and biochemistry, 2011Co-Authors: Hideyuki Tamakawa, Shigehito Ikushima, Satoshi YoshidaAbstract:The industrial yeast Candida Utilis can grow on media containing xylose as sole carbon source, but cannot ferment it to ethanol. The deficiency might be due to the low activity of NADPH-preferring xylose reductase (XR) and NAD(+)-dependent xylitol dehydogenase (XDH), which convert xylose to xylulose, because C. Utilis can ferment xylulose. We introduced multiple site-directed mutations in the coenzyme binding sites of XR and XDH derived from the xylose-fermenting yeast Candida shehatae to alter their coenzyme specificities. Several combinations of recombinant and native XRs and XDHs were tested. Highest productivity was observed in a strain expressing CsheXR K275R/N277D (NADH-preferring) and native CsheXDH (NAD(+)-dependent), which produced 17.4 g/L of ethanol from 50 g/L of xylose in 20 h. Analysis of the genes responsible for ethanol production from the xylose capacity of C. Utilis indicated that the introduction of CsheXDH was essential, while overexpression of CsheXR K275R/N277D improved efficiency of ethanol production.
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genetic engineering of Candida Utilis yeast for efficient production of l lactic acid
Bioscience Biotechnology and Biochemistry, 2009Co-Authors: Shigehito Ikushima, Satoshi Yoshida, Toshio Fujii, Osamu Kobayashi, Aruto YoshidaAbstract:Polylactic acid is receiving increasing attention as a renewable alternative for conventional petroleum-based plastics. In the present study, we constructed a metabolically-engineered Candida Utilis strain that produces L-lactic acid with the highest efficiency yet reported in yeasts. Initially, the gene encoding pyruvate decarboxylase (CuPDC1) was identified, followed by four CuPDC1 disruption events in order to obtain a null mutant that produced little ethanol (a by-product of L-lactic acid). Two copies of the L-lactate dehydrogenase (L-LDH) gene derived from Bos taurus under the control of the CuPDC1 promoter were then integrated into the genome of the CuPdc1-null deletant. The resulting strain produced 103.3 g/l of L-lactic acid from 108.7 g/l of glucose in 33 h, representing a 95.1% conversion. The maximum production rate of L-lactic acid was 4.9 g/l/h. The optical purity of the L-lactic acid was found to be more than 99.9% e.e.
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efficient gene disruption in the high ploidy yeast Candida Utilis using the cre loxp system
Bioscience Biotechnology and Biochemistry, 2009Co-Authors: Shigehito Ikushima, Toshio Fujii, Osamu KobayashiAbstract:In order to take full advantage of the industrially important yeast Candida Utilis, we developed a practical recombinant DNA tool for multiple gene disruption in C. Utilis based on the Cre-loxP system, which makes possible the reuse of selection markers. For this purpose, two plasmids were constructed: one harbored a heterologous loxP-flanked selection marker cassette carrying the gene responsible for hygromycin B-resistance, and the other had an autonomous replication sequence (ARS) and a Cre-recombinase expression module. Multiple disruption of C. Utilis NBRC0988 URA3 genes (CuURA3), encoding orotidine-5′-phosphate decarboxylase, validated the efficiency of the system. The fourth round of deletion yielded a null mutant, i.e., a uracil auxotroph, giving some support to the possibility that C. Utilis NBRC0988 is a tetraploid. This agreed very well with the outcomes of FACS analysis, which showed that various strains of this yeast contained 3–5 times more DNA than a Saccharomyces cerevisiae haploid.
Joachim F. Ernst - One of the best experts on this subject based on the ideXlab platform.
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Candida Utilis and Cyberlindnera (Pichia) jadinii: yeast relatives with expanding applications
Applied Microbiology and Biotechnology, 2016Co-Authors: Christoph Buerth, Denis Tielker, Joachim F. ErnstAbstract:The yeast Candida Utilis is used as a food additive and as a host for heterologous gene expression to produce various metabolites and proteins. Reliable protocols for intracellular production of recombinant proteins are available for C . Utilis and have now been expanded to secrete proteins into the growth medium or to achieve surface display by linkage to a cell wall protein. A recombinant C . Utilis strain was recently shown to induce oral tolerance in a mouse model of multiple sclerosis suggesting future applications in autoimmune therapy. Whole genome sequencing of C . Utilis and its presumed parent Cyberlindnera ( Pichia ) jadinii demonstrated different ploidy but high sequence identity, consistent with identical recombinant technologies for both yeasts. C . jadinii was recently described as an antagonist to the important human fungal pathogen Candida albicans suggesting its use as a probiotic agent. The review summarizes the status of recombinant protein production in C . Utilis , as well as current and future biotechnological and medical applications of C . Utilis and C . jadinii .
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The structure of the Cyberlindnera jadinii genome and its relation to Candida Utilis analyzed by the occurrence of single nucleotide polymorphisms.
Journal of biotechnology, 2015Co-Authors: Oliver Rupp, Karina Brinkrolf, Christoph Buerth, Maya Kunigo, Jessica Schneider, Sebastian Jaenicke, Alexander Goesmann, Alfred Pühler, Karl-erich Jaeger, Joachim F. ErnstAbstract:The yeast Cyberlindnera jadinii is a close relative of Candida Utilis that is being used in the food and feed industries. Here, we present the 12.7Mb genome sequence of C. jadinii strain CBS 1600 generated by next generation sequencing. The deduced draft genome sequence consists of seven large scaffolds analogous to the seven largest chromosomes of C. Utilis. An automated annotation of the C. jadinii genome identified 6147 protein-coding sequences. The level of ploidy for both genomes was analyzed by calling single nucleotide polymorphisms (SNPs) and was verified measuring nuclear DNA contents by florescence activated cell sorting (FACS). Both analyses determined the level of ploidy to diploid for C. jadinii and to triploid for C. Utilis. However, SNP calling for C. jadinii also identified scaffold regions that seem to be haploid, triploid or tetraploid.
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heterologous protein secretion by Candida Utilis
Applied Microbiology and Biotechnology, 2013Co-Authors: Maya Kunigo, Christoph Buerth, Denis Tielker, Joachim F. ErnstAbstract:The yeast Candida Utilis (also referred to as Torula) is used as a whole-cell food additive and as a recombinant host for production of intracellular molecules. Here, we report recombinant C. Utilis strains secreting significant amounts of Candida antarctica lipase B (CalB). Native and heterologous secretion signals led to secretion of CalB into the growth medium; CalB was enzymatically active and it carried a short N-glycosyl chain lacking extensive mannosylation. Furthermore, CalB fusions to the C. Utilis Gas1 cell wall protein led to effective surface display of enzymatically active CalB and of β-galactosidase. Secretory production in C. Utilis was achieved using a novel set of expression vectors containing sat1 conferring nourseothricin resistance, which could be transformed into C. Utilis, Pichia jadinii, Candida albicans, and Saccharomyces cerevisiae; C. Utilis promoters including the constitutive TDH3 and the highly xylose-inducible GXS1 promoters allowed efficient gene expression. These results establish C. Utilis as a promising host for the secretory production of proteins.
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growth dependent secretome of Candida Utilis
Microbiology, 2011Co-Authors: Christoph Buerth, Joachim F. Ernst, Clemens J Heilmann, Frans M Klis, Chris G De Koster, Denis TielkerAbstract:Recently, the food yeast Candida Utilis has emerged as an excellent host for production of heterologous proteins. Since secretion of the recombinant product is advantageous for its purification, we characterized the secreted proteome of C. Utilis. Cells were cultivated to the exponential or stationary growth phase, and the proteins in the medium were identified by MS. In parallel, a draft genome sequence of C. Utilis strain DSM 2361 was determined by massively parallel sequencing. Comparisons of protein and coding sequences established that C. Utilis is not a member of the CUG clade of Candida species. In total, we identified 37 proteins in the culture solution, 17 of which were exclusively present in the stationary phase, whereas three proteins were specific to the exponential growth phase. Identified proteins represented mostly carbohydrate-active enzymes associated with cell wall organization, while no proteolytic enzymes and only a few cytoplasmic proteins were detected. Remarkably, cultivation in xylose-based medium generated a protein pattern that diverged significantly from glucose-grown cells, containing the invertase Inv1 as the major extracellular protein, particularly in its highly glycosylated S-form (slow-migrating). Furthermore, cultivation without ammonium sulfate induced the secretion of the asparaginase Asp3. Comparisons of the secretome of C. Utilis with those of Kluyveromyces lactis and Pichia pastoris, as well as with those of the human fungal pathogens Candida albicans and Candida glabrata, revealed a conserved set of 10 and six secretory proteins, respectively.
Zhaoyue Wang - One of the best experts on this subject based on the ideXlab platform.
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Uricase production by a recombinant Hansenula polymorpha strain harboring Candida Utilis uricase gene
2013Co-Authors: Xuena Guo, Borun Zhang, Zhaoyue Wang, Zhiyu ChenAbstract:Abstract Uricase is an important medical enzyme which can be used to determine urate in clinical analysis, to therapy gout, hyperuricemia, and tumor lysis syndrome. Uricase of Candida Utilis was successfully expressed in Hansenula polymorpha under the control of methanol oxidase promoter using Saccharomyces cerevisiae α-factor signal peptide as the secretory sequence. Recombinant H. polymorpha MU200 with the highest extracellular uricase production was characterized with three copies of expression cassette and selected for process optimization for the production of recombinant enzyme. Among the parameters investigated in shaking flask cultures, the pH value of medium and inoculum size had great influence on the recombinant uricase production. The maximum extracellular uricase yield of 2.6 U/ml was obtained in shaking flask culture. The yield of recombinant uricase was significantly improved by the combined use of a high cell-density cultivation technique and a pH control strategy of switching culture pH from 5.5 to 6.5 in the induction phase. After induction for 58 h, the production of recombinant uricase reached 52.3 U/ml (about 2.1 g/l of protein) extracellularly and 60.3 U/ml (about 2.4 g/l) intracellularly in fed-batch fermentation, which are much higher than those expresse
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uricase production by a recombinant hansenula polymorpha strain harboring Candida Utilis uricase gene
Applied Microbiology and Biotechnology, 2008Co-Authors: Zhiyu Chen, Xuena Guo, Zhaoyue Wang, Borun ZhangAbstract:Uricase is an important medical enzyme which can be used to determine urate in clinical analysis, to therapy gout, hyperuricemia, and tumor lysis syndrome. Uricase of Candida Utilis was successfully expressed in Hansenula polymorpha under the control of methanol oxidase promoter using Saccharomyces cerevisiae α-factor signal peptide as the secretory sequence. Recombinant H. polymorpha MU200 with the highest extracellular uricase production was characterized with three copies of expression cassette and selected for process optimization for the production of recombinant enzyme. Among the parameters investigated in shaking flask cultures, the pH value of medium and inoculum size had great influence on the recombinant uricase production. The maximum extracellular uricase yield of 2.6 U/ml was obtained in shaking flask culture. The yield of recombinant uricase was significantly improved by the combined use of a high cell-density cultivation technique and a pH control strategy of switching culture pH from 5.5 to 6.5 in the induction phase. After induction for 58 h, the production of recombinant uricase reached 52.3 U/ml (about 2.1 g/l of protein) extracellularly and 60.3 U/ml (about 2.4 g/l) intracellularly in fed-batch fermentation, which are much higher than those expressed in other expression systems. To our knowledge, this is the first report about the heterologous expression of uricase in H. polymorpha.
