Cloning Of Marker Gene And Integrated Site Gene From Candida Glycerinogenes

Candida glycerinogenes has been used for commercial production of glycerol. To increase its glycerol productivity via genetic engineering, it is necessary to develop a transformation system for this industrial yeast. Isolating and cloning genetic marker gene and integration site gene is the basic job for constructing an integrative transformation system for the yeast Candida glycerinogenes.The URA3 gene and TRP1 gene of Candida glycerinogenes were isolated by complementation cloning in Saccharomyces cerevisiae. DNA sequence analysis of URA3 gene and TRP1 gene revealed the presence of an open reading frame (ORF) of 786 bp and 669 bp, encoding a 262 and 223 amino acid protein, which shares 76.60 % and 35 % amino acid sequence similarity to the S. cerevisiae Ura3 proteins and Trp1 proteins. Furthermore, the cloned ORF of URA3 gene and TRP1 gene fully complemented the S. cerevisiae Ura3~- and Trp1~- strain.A partial DNA fragment of ribosomal protein L41 from Candida glycerinogenes was amplified using degenerate primers. Partial genomic bank was used to isolate the complete nucleotide sequence of the coding region of the gene. DNA sequence analysis of L41 revealed the presence of an open reading frame (ORF) of 318 bp, encoding a 106 amino acid protein with a high level of homology to the L41 proteins from other yeast. The ORF is interrupted by a 386 bp putative intron just after the initiation codon, a characteristic reported in the other genes encoding the L41 proteins. After removing the intron from the ribosomal protein L41 from Candida glycerinogenes, modification of its 56th amino acid was done.The small-subunit (18S) rDNA gene and large-subunit (26S) rDNA gene D1/D2 region acquired by PCR amplification were used not only as a target for multicopy gene integration into the chromosome but also for phylogenetical analysis . Analyses of the sequences of 18S rDNA gene and 26S rDNA gene D1/D2 region revealed that Candida glycerinogenes was closely related to Issatchenkia orientalis (anamorph: Candida krusei).