Abstract
Candida glycerinogenes WL2002-5 is an osmotolerant yeast used for the commercial production of glycerol. The TRP1 gene of Candida glycerinogenes (CgTRP1), encoding phosphoribosylanthranilate isomerase (PRAI) was cloned by complementation of the trp1 mutation of Saccharomyces cerevisiae W303-1A. DNA sequence analysis revealed a 735 bp open reading frame (ORF) encoding a polypeptide of 244 amino acids, which shared 32.9 ~ 49.2% amino acid sequence similarity to PRAI proteins from other species of Saccharomycetales. A trp1 auxotrophic mutant of C. glycerinogenes was selected in medium containing 5-fluoroanthranilic acid, and confirmed by functional and sequence analysis. An integrative vector was constructed with the 18S rDNA gene as integration target and CgTRP1 gene as selectable marker. The trp1 mutant of C. glycerinogenes was transformed with integrative vector, transformants were screened by trp1 complementation. Diagnostic PCR show that the plasmid could be integrated in the site of the 18S rDNA gene of C. glycerinogenes.
References
Chen XZ, Fang HY, Rao ZM et al (2008) An efficient genetic transformation method for glycerol producer Candida glycerinogenes. Microbiol Res 163:531–537
Jayaram M, Li YY, Broach JR (1983) The yeast plasmid 2 μ encodes components required for its high copy propagation. Cell 34:95–104
Kitada K, Yamaguchi E, Arisawa M (1995) Cloning of the Candida glabrata TRP1 and HIS3 genes, and consruction of their disruption strains by sequential integrative transformation. Gene 165:203–206
Li YL, Shen W, Wang ZX et al (2005) Isolation and sequence analysis of the gene URA3 encoding the orotidine-5′-phosphate decarboxylase from Candida glycerinogenes WL2002–5, an industrial glycerol producer. Yeast 22:423–430
Rose MD, Winston F, Hieter P (1988) Laboratory course manual for methods in yeast genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor
Toyn JH, Gunyuzlu PL, White WH et al (2000) A counterselection for the tryptophan pathway in yeast: 5-fluoroanthranilic acid resistance. Yeast 16:553–560
Ugolini S, Tosato V, Bruschi VC (2002) Selective fitness of four episomal shuttle-vector carrying HIS3, LEU2, TRP1 and URA3 selectable markers in Saccharomyces cerevisiae. Plasmid 47:94–107
Wang ZX, Zhuge J, Fang HY et al (2001) Glycerol production by microbial fermentation: a review. Biotechnol Adv 19:201–223
Acknowledgments
This work was financially supported by the National High Technology Research and Development Program of China (863 Program): Project 2006AA10Z307. Prof B. A. Prior is thanked for assistance with revision of the manuscript.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Shen, W., Wang, ZX., Rao, ZM. et al. A genetic transformation system based on trp1 complementation in Candida glycerinogenes . World J Microbiol Biotechnol 27, 1005–1008 (2011). https://doi.org/10.1007/s11274-010-0524-2
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11274-010-0524-2