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Characterization of the relevant genes and development of salt-tolerant yeast strains by transposon mutagenesis
New Biotechnology · Volume 31S · July 2014
PZ-04 Characterization of the relevant genes and development of salt-tolerant yeast strains by transposon mutagenesis Hyun-Soo Kim ∗ , Hye-Min Kang Department of Food Science and Industry, Jungwon University, South Korea Kimchi is a traditional, fermented Korean food prepared with different vegetables, spices, and ingredients and is an important dietary source of vitamins, minerals, and other nutrients. The waste brine from manufacturing process of kimchi is released and eventually results in serious environmental pollution. Saccharomyces cerevisiae strains tolerant to salt stress are important for the production of single cell protein by using kimchi waste brine and removal of potential pollutants from the waste water. In this study, approximately 3000 transformants of the mTn3mutagenized genome library were selected as leucine prototrophs and replica-plated to both YPD agar and YPD agar containing 10% NaCl. Three strains (Tn 1–3) tolerant to up to 10% NaCl were isolated by screening a transposon-mediated mutant library. Three transposon mutants showed higher ethanol production and grew faster than control strain when cultured in rich media containing 5%, 7.5%, and 10% NaCl respectively. The determination of transposon insertion sites and Northern blot analysis identified putative genes and revealed simultaneous down-regulations and disruptions of the genes indicating that salt tolerance can be conferred. The genes identified in this study may provide a basis for the application in developing industrial yeast strains. http://dx.doi.org/10.1016/j.nbt.2014.05.1007
PZ-05 Epicoccum purpurascens (Didymellaceae, Ascomycota) as a non-model source of novel bioactive compounds: biotechnological perspectives Mikhail Fokin 1,∗ , Bevan Weir 2 , Ting-Li Han 3 , Neethu Arun 3 , Lydia Yamaguchi 4 , Massuo Jorge Kato 4 , Silas Villas-Boas 3 1
University of Auckland, New Zealand Landcare Research, New Zealand 3 Centre for Microbial Innovation, School of Biological Sciences, The University of Auckland, New Zealand 4 Chemistry Institute, University of São Paulo, Brazil 2
Filamentous fungi have been widely used as a source of valuable bioactive compounds for medicine, agriculture and white biotechnology. Recently, genomic studies have significantly improved our knowledge of the functional background for known compounds and revealed a vast amount of novel secondary metabolites not commonly expressed in the laboratory. Among fungi these studies have been focused mostly on a few model species. Epicoccum purpurascens is a widespread member of Dothideomycetes class, which consists of over 20 thousands species. E. purpurascens has been isolated from different substrates such as plants, soil, and marine invertebrates. Several bioactive compounds were
YEAST AND FILAMENTOUS FUNGI
isolated from different E. purpurascens strains, including epicoccaene, a broad-range fungicide, discovered and patented by our group. It is remarkable that the biology of this fungal species has not been much studied, and we have very little knowledge on its genetic and phenotypic diversity. Only recently the genus Epicoccum was placed in the newly described family Didymellaceae, and great intraspecific diversity of E. purpurascens has been reported. We determined the genetic and metabolite profiles of different E. purpurascens strains isolated in New Zealand, and we have found that metabolite profile was closely associated with the ability of each strain to produce biologically active compounds, which did not match their similarities based on their genetic profile. We have now de novo sequenced and assembled the draft genome of our working E. purpurascens strain. Further annotation of the genome and specific mining for secondary metabolite gene clusters will strongly facilitate biotechnological use of E. purpurascens. http://dx.doi.org/10.1016/j.nbt.2014.05.1008
PZ-06 The effect of carbon sources on upstream regulatory regions controlling the expression of the Candida utilis maltase gene ˇ Veronika Ján Krahulec ∗ , Hanka Bonková, ˇ Osadská, Stanislav Stuchlík, Ján Turna
Liˇsková, Michaela
Faculty of Natural Sciences, Comenius University in Bratislava, Slovakia In the last few years it has been demonstrated that the industrially important yeast system Candida utilis represents a promising expression host, generating relatively high levels of recombinant proteins. Nevertheless, basic knowledge of its gene structure and regulation of gene expression, which is needed to allow more extensive use of this organism, is lacking. The current study presents preliminary characterization of the carbon sourcedependent expression of the C. utilis maltase gene in order to compare its regulation. Our results showed that C. utilis maltase is able to hydrolyze cellobiose and soluble starch, and its affinity for cellobiose is even three times higher than for maltose. To further investigate the function of maltase and the use of the maltase promoter for the heterologous protein expression, we successfully applied the Cre-loxP system to acquire a null mutant strain with disrupted maltase gene and promoter in the polyploid yeast C. utilis. The first step was to introduce a mutagenesis cassette harboring a marker gene between two loxP sites into the target cells. After identification of a partial mutant strain, the same mutagenesis cassette was repeatedly used and the null mutant strain was selected on higher antibiotic concentration. The last step was to introduce a Cre-expression plasmid and screen for marker rescue strains. Acknowledgements: This work is result of project implementation: “Production of biologically active agents based on recombinant proteins” (ITMS 26240220048) supported by the
www.elsevier.com/locate/nbt S217
PZ-04 Characterization of the relevant genes and development of salt-tolerant yeast strains by transposon mutagenesis Hyun-Soo Kim ∗ , Hye-Min Kang Department of Food Science and Industry, Jungwon University, South Korea Kimchi is a traditional, fermented Korean food prepared with different vegetables, spices, and ingredients and is an important dietary source of vitamins, minerals, and other nutrients. The waste brine from manufacturing process of kimchi is released and eventually results in serious environmental pollution. Saccharomyces cerevisiae strains tolerant to salt stress are important for the production of single cell protein by using kimchi waste brine and removal of potential pollutants from the waste water. In this study, approximately 3000 transformants of the mTn3mutagenized genome library were selected as leucine prototrophs and replica-plated to both YPD agar and YPD agar containing 10% NaCl. Three strains (Tn 1–3) tolerant to up to 10% NaCl were isolated by screening a transposon-mediated mutant library. Three transposon mutants showed higher ethanol production and grew faster than control strain when cultured in rich media containing 5%, 7.5%, and 10% NaCl respectively. The determination of transposon insertion sites and Northern blot analysis identified putative genes and revealed simultaneous down-regulations and disruptions of the genes indicating that salt tolerance can be conferred. The genes identified in this study may provide a basis for the application in developing industrial yeast strains. http://dx.doi.org/10.1016/j.nbt.2014.05.1007
PZ-05 Epicoccum purpurascens (Didymellaceae, Ascomycota) as a non-model source of novel bioactive compounds: biotechnological perspectives Mikhail Fokin 1,∗ , Bevan Weir 2 , Ting-Li Han 3 , Neethu Arun 3 , Lydia Yamaguchi 4 , Massuo Jorge Kato 4 , Silas Villas-Boas 3 1
University of Auckland, New Zealand Landcare Research, New Zealand 3 Centre for Microbial Innovation, School of Biological Sciences, The University of Auckland, New Zealand 4 Chemistry Institute, University of São Paulo, Brazil 2
Filamentous fungi have been widely used as a source of valuable bioactive compounds for medicine, agriculture and white biotechnology. Recently, genomic studies have significantly improved our knowledge of the functional background for known compounds and revealed a vast amount of novel secondary metabolites not commonly expressed in the laboratory. Among fungi these studies have been focused mostly on a few model species. Epicoccum purpurascens is a widespread member of Dothideomycetes class, which consists of over 20 thousands species. E. purpurascens has been isolated from different substrates such as plants, soil, and marine invertebrates. Several bioactive compounds were
YEAST AND FILAMENTOUS FUNGI
isolated from different E. purpurascens strains, including epicoccaene, a broad-range fungicide, discovered and patented by our group. It is remarkable that the biology of this fungal species has not been much studied, and we have very little knowledge on its genetic and phenotypic diversity. Only recently the genus Epicoccum was placed in the newly described family Didymellaceae, and great intraspecific diversity of E. purpurascens has been reported. We determined the genetic and metabolite profiles of different E. purpurascens strains isolated in New Zealand, and we have found that metabolite profile was closely associated with the ability of each strain to produce biologically active compounds, which did not match their similarities based on their genetic profile. We have now de novo sequenced and assembled the draft genome of our working E. purpurascens strain. Further annotation of the genome and specific mining for secondary metabolite gene clusters will strongly facilitate biotechnological use of E. purpurascens. http://dx.doi.org/10.1016/j.nbt.2014.05.1008
PZ-06 The effect of carbon sources on upstream regulatory regions controlling the expression of the Candida utilis maltase gene ˇ Veronika Ján Krahulec ∗ , Hanka Bonková, ˇ Osadská, Stanislav Stuchlík, Ján Turna
Liˇsková, Michaela
Faculty of Natural Sciences, Comenius University in Bratislava, Slovakia In the last few years it has been demonstrated that the industrially important yeast system Candida utilis represents a promising expression host, generating relatively high levels of recombinant proteins. Nevertheless, basic knowledge of its gene structure and regulation of gene expression, which is needed to allow more extensive use of this organism, is lacking. The current study presents preliminary characterization of the carbon sourcedependent expression of the C. utilis maltase gene in order to compare its regulation. Our results showed that C. utilis maltase is able to hydrolyze cellobiose and soluble starch, and its affinity for cellobiose is even three times higher than for maltose. To further investigate the function of maltase and the use of the maltase promoter for the heterologous protein expression, we successfully applied the Cre-loxP system to acquire a null mutant strain with disrupted maltase gene and promoter in the polyploid yeast C. utilis. The first step was to introduce a mutagenesis cassette harboring a marker gene between two loxP sites into the target cells. After identification of a partial mutant strain, the same mutagenesis cassette was repeatedly used and the null mutant strain was selected on higher antibiotic concentration. The last step was to introduce a Cre-expression plasmid and screen for marker rescue strains. Acknowledgements: This work is result of project implementation: “Production of biologically active agents based on recombinant proteins” (ITMS 26240220048) supported by the
www.elsevier.com/locate/nbt S217