New molecular genetic tools for the non-conventional yeasts Candida sonorensis and Candida methanosorbosa

Marja Ilmen, Kari Koivuranta, Pirkko Suominen, V. Rajgarhia, Laura Ruohonen, Merja Penttilä

Research output: Chapter in Book/Report/Conference proceedingConference abstract in proceedingsScientific

Abstract

New molecular genetic tools were developed for Candida sonorensis and Candida methanosorbosa enabling their genetic engineering for the first time. Transformation was based on antibiotic markers and, alternatively, a dominant non-antibiotic marker, the MEL5 gene from Saccharomyces cerevisiae, enabling growth on melibiose as sole carbon source. Endogenous promoters were cloned from C. sonorensis and used to control the expression of the heterologous genes in both hosts. Introduction of the lactate dehydrogenase gene (ldhL) of Lactobacillus helveticus into C. sonorensis and C. methanosorbosa resulted in moderate (~10 g/l, ~20% yield) production of lactic acid with co-production of ethanol. Targeted gene deletions were accomplished in C. sonorensis. Two genes encoding pyruvate decarboxylase (PDC) were isolated. Strains deleted of PDC1, PDC2, or both PDC genes were constructed generating C. sonorensis strains producing reduced amounts or no ethanol. The PDC2 encoded enzyme was identified as the major activity contributing to ethanol production. Integration of the PDC1 and PDC2 replacement constructs into both homologous and non-homologous sites occurred in C. sonorensis. This work clearly demonstrates the feasibility of genetically engineering novel, non-conventional yeast species, and illustrates the usefulness of the molecular tools for analysing gene function.
Original languageEnglish
Title of host publication3rd European Federation of Biotechnology Conference
Subtitle of host publicationPhysiology of Yeasts and Filamentous Fungi PYFF3
Place of PublicationEspoo
PublisherVTT Technical Research Centre of Finland
Pages165
ISBN (Electronic)978-951-38-6314-2
ISBN (Print)978-951-38-6313-5
Publication statusPublished - 2007
Event3rd European Federation of Biotechnology Conference : Physiology of Yeasts and Filamentous Fungi - Helsinki, Finland
Duration: 13 Jun 200716 Jun 2007

Publication series

NameVTT Symposium
PublisherVTT
Number245
ISSN (Print)0357-9387
ISSN (Electronic)1455-0873

Conference

Conference3rd European Federation of Biotechnology Conference
Abbreviated titlePYFF3
CountryFinland
CityHelsinki
Period13/06/0716/06/07

Fingerprint

Candida
molecular genetics
pyruvate decarboxylase
yeasts
ethanol production
genes
melibiose
Lactobacillus helveticus
gene deletion
lactate dehydrogenase
genetic engineering
lactic acid
Saccharomyces cerevisiae
engineering
antibiotics
ethanol
promoter regions
genetic markers
carbon
enzymes

Cite this

Ilmen, M., Koivuranta, K., Suominen, P., Rajgarhia, V., Ruohonen, L., & Penttilä, M. (2007). New molecular genetic tools for the non-conventional yeasts Candida sonorensis and Candida methanosorbosa. In 3rd European Federation of Biotechnology Conference: Physiology of Yeasts and Filamentous Fungi PYFF3 (pp. 165). [P107] Espoo: VTT Technical Research Centre of Finland. VTT Symposium, No. 245
Ilmen, Marja ; Koivuranta, Kari ; Suominen, Pirkko ; Rajgarhia, V. ; Ruohonen, Laura ; Penttilä, Merja. / New molecular genetic tools for the non-conventional yeasts Candida sonorensis and Candida methanosorbosa. 3rd European Federation of Biotechnology Conference: Physiology of Yeasts and Filamentous Fungi PYFF3. Espoo : VTT Technical Research Centre of Finland, 2007. pp. 165 (VTT Symposium; No. 245).
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Ilmen, M, Koivuranta, K, Suominen, P, Rajgarhia, V, Ruohonen, L & Penttilä, M 2007, New molecular genetic tools for the non-conventional yeasts Candida sonorensis and Candida methanosorbosa. in 3rd European Federation of Biotechnology Conference: Physiology of Yeasts and Filamentous Fungi PYFF3., P107, VTT Technical Research Centre of Finland, Espoo, VTT Symposium, no. 245, pp. 165, 3rd European Federation of Biotechnology Conference , Helsinki, Finland, 13/06/07.

New molecular genetic tools for the non-conventional yeasts Candida sonorensis and Candida methanosorbosa. / Ilmen, Marja; Koivuranta, Kari; Suominen, Pirkko; Rajgarhia, V.; Ruohonen, Laura; Penttilä, Merja.

3rd European Federation of Biotechnology Conference: Physiology of Yeasts and Filamentous Fungi PYFF3. Espoo : VTT Technical Research Centre of Finland, 2007. p. 165 P107 (VTT Symposium; No. 245).

Research output: Chapter in Book/Report/Conference proceedingConference abstract in proceedingsScientific

TY - CHAP

T1 - New molecular genetic tools for the non-conventional yeasts Candida sonorensis and Candida methanosorbosa

AU - Ilmen, Marja

AU - Koivuranta, Kari

AU - Suominen, Pirkko

AU - Rajgarhia, V.

AU - Ruohonen, Laura

AU - Penttilä, Merja

PY - 2007

Y1 - 2007

N2 - New molecular genetic tools were developed for Candida sonorensis and Candida methanosorbosa enabling their genetic engineering for the first time. Transformation was based on antibiotic markers and, alternatively, a dominant non-antibiotic marker, the MEL5 gene from Saccharomyces cerevisiae, enabling growth on melibiose as sole carbon source. Endogenous promoters were cloned from C. sonorensis and used to control the expression of the heterologous genes in both hosts. Introduction of the lactate dehydrogenase gene (ldhL) of Lactobacillus helveticus into C. sonorensis and C. methanosorbosa resulted in moderate (~10 g/l, ~20% yield) production of lactic acid with co-production of ethanol. Targeted gene deletions were accomplished in C. sonorensis. Two genes encoding pyruvate decarboxylase (PDC) were isolated. Strains deleted of PDC1, PDC2, or both PDC genes were constructed generating C. sonorensis strains producing reduced amounts or no ethanol. The PDC2 encoded enzyme was identified as the major activity contributing to ethanol production. Integration of the PDC1 and PDC2 replacement constructs into both homologous and non-homologous sites occurred in C. sonorensis. This work clearly demonstrates the feasibility of genetically engineering novel, non-conventional yeast species, and illustrates the usefulness of the molecular tools for analysing gene function.

AB - New molecular genetic tools were developed for Candida sonorensis and Candida methanosorbosa enabling their genetic engineering for the first time. Transformation was based on antibiotic markers and, alternatively, a dominant non-antibiotic marker, the MEL5 gene from Saccharomyces cerevisiae, enabling growth on melibiose as sole carbon source. Endogenous promoters were cloned from C. sonorensis and used to control the expression of the heterologous genes in both hosts. Introduction of the lactate dehydrogenase gene (ldhL) of Lactobacillus helveticus into C. sonorensis and C. methanosorbosa resulted in moderate (~10 g/l, ~20% yield) production of lactic acid with co-production of ethanol. Targeted gene deletions were accomplished in C. sonorensis. Two genes encoding pyruvate decarboxylase (PDC) were isolated. Strains deleted of PDC1, PDC2, or both PDC genes were constructed generating C. sonorensis strains producing reduced amounts or no ethanol. The PDC2 encoded enzyme was identified as the major activity contributing to ethanol production. Integration of the PDC1 and PDC2 replacement constructs into both homologous and non-homologous sites occurred in C. sonorensis. This work clearly demonstrates the feasibility of genetically engineering novel, non-conventional yeast species, and illustrates the usefulness of the molecular tools for analysing gene function.

M3 - Conference abstract in proceedings

SN - 978-951-38-6313-5

T3 - VTT Symposium

SP - 165

BT - 3rd European Federation of Biotechnology Conference

PB - VTT Technical Research Centre of Finland

CY - Espoo

ER -

Ilmen M, Koivuranta K, Suominen P, Rajgarhia V, Ruohonen L, Penttilä M. New molecular genetic tools for the non-conventional yeasts Candida sonorensis and Candida methanosorbosa. In 3rd European Federation of Biotechnology Conference: Physiology of Yeasts and Filamentous Fungi PYFF3. Espoo: VTT Technical Research Centre of Finland. 2007. p. 165. P107. (VTT Symposium; No. 245).