Isolation and structural analysis of efficient autonomously replicating sequences (ARSs) of the yeastCandida utilis

Abstract

The industrially important yeast Candida utilis is widely used in production of food and medical materials, but its host-vector system has not been well developed. We screened for compact and efficient ARSs to construct practically useful vectors. The C. utilis strain AHU3053 was found to be efficiently transformed by the conventional lithium acetate method and was used as the host. The C. utilis IAM4264 genomic library was constructed by inserting the partial Sau3AI digests in pRI51, which has a kanMX gene expressible in C. utilis. By examining 98 C. utilis G418-resistant transformants, five plasmids had the highest ARS activity. By trimming of the inserts, the 1490 and 552 bp fragments with transformation activity of over 10(3)/microg DNA were obtained from ARS3 and ARS4, respectively. Although several sequences identical to S. cerevisiae ARS consensus sequences (ACSs) were found in ARS3 and ARS4, our deletion analysis indicated that these were not essential for the activity. Because the minimal functional ARS fragment was also several-fold larger than that of S. cerevisiae, the C. utilis ARSs have some unique characteristics resembling the Sz. pombe ARSs. These ARSs were functional in other C. utilis strains tested and useful for constructing practical vectors.

Identification and Application of Novel Autonomously Replicating Sequences (ARSs) for Promoter-Cloning and Co-Transformation inCandida utilis

“…3, are comparable with those of ARS3 and ARS4, 11) although the values can depend on the methods and conditions of transformation, the strains used as hosts, and so on.…”

Isolation of theYAP1 homologue ofCandida utilis and its use as an efficient selection marker

“…Yeast transformation was performed by the conventional lithium acetate method (Gietz et al , 1995). The C. utilis strain AHU3053 was used as the host, because the transformation frequency was high using this method (Iwakiri et al , 2005). The cells were incubated in YPD medium after transformation for 1 h before being spread on the selective medium.…”

“…The overexpression plasmid vector pRI177 for C. utilis was constructed as follows. The Eco RI–S pe I fragment of C. utilis ARS4 (Iwakiri et al , 2005) was inserted in the Eco RV site of pBluescript II SK after blunt‐ending to generate an ARS vector pARS4‐2. An Eco RI– Pst I DNA fragment of the C. utilis GAP promoter region was prepared by PCR, using primers CuPgap‐F and CuPgap‐R and the template C. utilis IAM4264 DNA, and then was inserted into the Eco RI– Pst I site of pBluescript II SK, yielding pCuPgap.…”

Candida utilis and Cyberlindnera (Pichia) jadinii: yeast relatives with expanding applications

“…By FACS analyses, a ploidy of 3-5 was previously suggested for C. utilis strain ATCC9950 (Ikushima et al 2009a;Kondo et al 1995) and sequential disruption of the URA3 and PDC1 loci indicated tetraploidy (Ikushima et al 2009a(Ikushima et al , 2009b, while other transformation experiments suggested diploidy (Rodriguez et al 1998;Iwakiri et al 2005a). It should be noted, however, that ploidy determinations by sequential disruption are not necessarily reliable because chromosomal duplications may occur and become selected during transformation procedures (Enloe et al 2000).…”