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Applied and Environmental Microbiology, April 2005, p. 1964-1970, Vol. 71, No. 4
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.4.1964-1970.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Efficient Production of L-Lactic Acid by Metabolically Engineered Saccharomyces cerevisiae with a Genome-Integrated L-Lactate Dehydrogenase Gene

Nobuhiro Ishida,^,1* Satoshi Saitoh,^2, Kenro Tokuhiro,¹ Eiji Nagamori,¹ Takashi Matsuyama,¹ Katsuhiko Kitamoto,³ and Haruo Takahashi¹

Biotechnology Laboratory, Toyota Central R&D Labs Inc.,¹ Toyota Biotechnology & Afforestation Laboratory, Toyota Motor Co., Aichi,² Department of Biotechnology, The University of Tokyo, Bunkyo-ku, Tokyo, Japan³

Received 26 July 2004/ Accepted 6 November 2004

We developed a metabolically engineered yeast which produces lactic acid efficiently. In this recombinant strain, the coding region for pyruvate decarboxylase 1 (PDC1) on chromosome XII is substituted for that of the L-lactate dehydrogenase gene (LDH) through homologous recombination. The expression of mRNA for the genome-integrated LDH is regulated under the control of the native PDC1 promoter, while PDC1 is completely disrupted. Using this method, we constructed a diploid yeast transformant, with each haploid genome having a single insertion of bovine LDH. Yeast cells expressing LDH were observed to convert glucose to both lactate (55.6 g/liter) and ethanol (16.9 g/liter), with up to 62.2% of the glucose being transformed into lactic acid under neutralizing conditions. This transgenic strain, which expresses bovine LDH under the control of the PDC1 promoter, also showed high lactic acid production (50.2 g/liter) under nonneutralizing conditions. The differences in lactic acid production were compared among four different recombinants expressing a heterologous LDH gene (i.e., either the bovine LDH gene or the Bifidobacterium longum LDH gene): two transgenic strains with 2µm plasmid-based vectors and two genome-integrated strains.

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* Corresponding author. Mailing address: Biotechnology Laboratory, Toyota Central R&D Labs Inc., Nagakute-cho, Aichi 480-1192, Japan. Phone: 81-561-63-5375. Fax: 81-561-63-6498. E-mail: e1168{at}mosk.tytlabs.co.jp.

N.I. and S.S. contributed equally to this work.

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Applied and Environmental Microbiology, April 2005, p. 1964-1970, Vol. 71, No. 4
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.4.1964-1970.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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