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Applied and Environmental Microbiology, July 2007, p. 4342-4350, Vol. 73, No. 13
0099-2240/07/$08.00+0     doi:10.1128/AEM.02759-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

High-Level Production of Beta-Carotene in Saccharomyces cerevisiae by Successive Transformation with Carotenogenic Genes from Xanthophyllomyces dendrorhous{triangledown}

René Verwaal,1,{dagger} Jing Wang,1 Jean-Paul Meijnen,1 Hans Visser,1,{ddagger} Gerhard Sandmann,2 Johan A. van den Berg,1 and Albert J. J. van Ooyen1*

Fungal Genomics, Laboratory of Microbiology, Wageningen University, Dreijenlaan 2, 6703 HA Wageningen, The Netherlands,1 Biosynthesis Group, Molecular Biosciences 213, J. W. Goethe Universität, Siesmayerstrasse 70, P.O. Box 111932, D-60054 Frankfurt, Germany2

Received 26 November 2006/ Accepted 5 May 2007

To determine whether Saccharomyces cerevisiae can serve as a host for efficient carotenoid and especially ß-carotene production, carotenogenic genes from the carotenoid-producing yeast Xanthophyllomyces dendrorhous were introduced and overexpressed in S. cerevisiae. Because overexpression of these genes from an episomal expression vector resulted in unstable strains, the genes were integrated into genomic DNA to yield stable, carotenoid-producing S. cerevisiae cells. Furthermore, carotenoid production levels were higher in strains containing integrated carotenogenic genes. Overexpression of crtYB (which encodes a bifunctional phytoene synthase and lycopene cyclase) and crtI (phytoene desaturase) from X. dendrorhous was sufficient to enable carotenoid production. Carotenoid production levels were increased by additional overexpression of a homologous geranylgeranyl diphosphate (GGPP) synthase from S. cerevisiae that is encoded by BTS1. Combined overexpression of crtE (heterologous GGPP synthase) from X. dendrorhous with crtYB and crtI and introduction of an additional copy of a truncated 3-hydroxy-3-methylglutaryl-coenzyme A reductase gene (tHMG1) into carotenoid-producing cells resulted in a successive increase in carotenoid production levels. The strains mentioned produced high levels of intermediates of the carotenogenic pathway and comparable low levels of the preferred end product ß-carotene, as determined by high-performance liquid chromatography. We finally succeeded in constructing an S. cerevisiae strain capable of producing high levels of ß-carotene, up to 5.9 mg/g (dry weight), which was accomplished by the introduction of an additional copy of crtI and tHMG1 into carotenoid-producing yeast cells. This transformant is promising for further development toward the biotechnological production of ß-carotene by S. cerevisiae.

* Corresponding author. Mailing address: Fungal Genomics, Laboratory of Microbiology, Wageningen University, Dreijenlaan 2, 6703 HA Wageningen, The Netherlands. Phone: 31-6-42255321. Fax: 31-3-17484011. E-mail: Ab.vanOoyen{at}wur.nl

{triangledown} Published ahead of print on 11 May 2007.

{dagger} Present address: DSM Food Specialties, 2600 MA Delft, The Netherlands.

{ddagger} Present address: Dyadic Nederland BV, 3704 HE Zeist, The Netherlands.

Applied and Environmental Microbiology, July 2007, p. 4342-4350, Vol. 73, No. 13
0099-2240/07/$08.00+0     doi:10.1128/AEM.02759-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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