Efficient Gene Disruption in the High-Ploidy YeastCandida utilisUsing the Cre-loxPSystem

A Combination of Flow Cytometry and Traditional Screening Using Chemicals to Isolate High Glutathione-Producing Yeast Mutants

“…This assumption is based on recent evidence from two experiments on DNA content and gene disruption in C. utilis, which showed polyploidity from three to five DNA content. 22) In addition, HN001 had enhanced GSH1 expression, while HN003 had a met30 mutation in the GSH pathway. Cer and MG resistance was evaluated in the GSH pathway.…”

Genetic Engineering ofCandida utilisYeast for Efficient Production ofL-Lactic Acid

“…Transformations of C. utilis were carried out by electroporation as previously described. 28) In order to construct a null disruptant of the CuPDC1 gene, designated Cupdc1Á4, a fourth deletion was carried out using the Cre-loxP system essentially as described for constructing the CuUra3-null deletant. 28) Two different gene disruption cassettes consisting of the aforementioned LHL module flanked by the 5 0 -and 3 0 -regions adjacent to the CuPDC1 gene for targeted replacement were generated in two-step PCRs.…”

“…Initially, the fragments forming these cassettes were amplified separately. The LHL module was constructed using primers IM-1 and 2 and pCU563 28) as template. A fragment upstream of CuPDC1, the targeted region, was amplified with primers IM-277 and -278, with C. utilis genomic DNA as template.…”

“…One is co-transformation, 27) and the other is based on the CreloxP recombination system. 28) Using the latter method, a four-round deletion of the CuURA3 gene, encoding orotidine-5 0 -phosphate decarboxylase, yielded an uracil auxotroph. 28) In the present study, we describe a geneticallyengineered strain of C. utilis which to our knowledge produces L-lactic acid more efficiently than previously reported in yeasts.…”

Ethanol Production from Xylose by a RecombinantCandida utilisStrain Expressing Protein-Engineered Xylose Reductase and Xylitol Dehydrogenase

“…[20][21][22] Since the development of an efficient electroporation-based method for transforming C. utilis, 23) this yeast has been used for the heterologous production of monellin, 24) -amylase, 25) and carotenoids such as lycopene. [26][27][28] Recently, we developed efficient transformation systems for C. utilis, 29) and to our knowledge achieved the highest level of production of L-lactic acid by a yeast species using a geneticallyengineered strain of C. utilis.…”