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Construction of new Pichia pastoris X-33 strains for production of lycopene and β-carotene

  • Applied genetics and molecular biotechnology
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Abstract

In this study, we used the non-carotenogenic yeast Pichia pastoris X33 as a receptor for β-carotene-encoding genes, in order to obtain new recombinant strains capable of producing different carotenoidic compounds. We designed and constructed two plasmids, pGAPZA-EBI* and pGAPZA-EBI*L*, containing the genes encoding lycopene and β-carotene, respectively. Plasmid pGAPZA-EBI*, expresses three genes, crtE, crtB, and crtI*, that encode three carotenogenic enzymes, geranylgeranyl diphosphate synthase, phytoene synthase, and phytoene desaturase, respectively. The other plasmid, pGAPZA-EBI*L*, carried not only the three genes above mentioned, but also the crtL* gene, that encodes lycopene β-cyclase. The genes crtE, crtB, and crtI were obtained from Erwinia uredovora, whereas crtL* was cloned from Ficus carica (JF279547). The plasmids were integrated into P. pastoris genomic DNA, and the resulting clones Pp-EBI and Pp-EBIL were selected for either lycopene or β-carotene production and purification, respectively. Cells of these strains were investigated for their carotenoid contents in YPD media. These carotenoids produced by the recombinant P. pastoris clones were qualitatively and quantitatively analyzed by high-resolution liquid chromatography, coupled to photodiode array detector. These analyses confirmed that the recombinant P. pastoris clones indeed produced either lycopene or β-carotene, according to the integrated vector, and productions of 1.141 μg of lycopene and 339 μg of β-carotene per gram of cells (dry weight) were achieved. To the best of our knowledge, this is the first time that P. pastoris has been genetically manipulated to produce β-carotene, thus providing an alternative source for large-scale biosynthesis of carotenoids.

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Acknowledgments

J. M. A-G. is the recipient of an AECID scholarship from the Spanish Foreign Affairs Ministry. The authors thank Dr. Norihiko Misawa (Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University) for the gift of plasmid pACCRT-EIB and both the Faculty of Pharmacy and School of Biotechnology for their support throughout this project. The authors also wish to thank Dr. Angeles Sánchez-Pérez, the University of Sydney, for critically reading and editing the English manuscript.

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Authors and Affiliations

  1. Department of Microbiology, University of Santiago de Compostela, Santiago de Compostela, Spain

    J. M. Araya-Garay, L. Feijoo-Siota, P. Veiga-Crespo & T. G. Villa

  2. School of Biotechnology, University of Santiago de Compostela, 15782, Santiago de Compostela, Spain

    P. Veiga-Crespo & T. G. Villa

  3. Department of Pharmacy and Pharmaceutical Technology, University of Santiago de Compostela, Santiago de Compostela, Spain

    F. Rosa-dos-Santos

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  1. J. M. Araya-Garay
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  2. L. Feijoo-Siota
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  3. F. Rosa-dos-Santos
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  4. P. Veiga-Crespo
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  5. T. G. Villa
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Correspondence to T. G. Villa.

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Araya-Garay, J.M., Feijoo-Siota, L., Rosa-dos-Santos, F. et al. Construction of new Pichia pastoris X-33 strains for production of lycopene and β-carotene. Appl Microbiol Biotechnol 93, 2483–2492 (2012). https://doi.org/10.1007/s00253-011-3764-7

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