A Genetic Transformation System Based On A Reusable Selection Marker For Candida Tropicalis And Its Application

Candida tropicalis has important potential in industrial biotehnological application.However, the complicated genetic structure with diploid and the low genetic transformationefficiency limits its capability for strain improvement through metabolic engineering.Althoghvarious genetic transformation and screening methods were developed for Saccharomycescerevisiae, their feasibility were very low when applied to C. tropicalis directly. Therefore,development of reliable transformation methods of C. tropicalis yeast for academic researchand industrial applications is important. This paper developed an efficient genetictransformation system for C. tropicalis in which the selective marker can be reutilized, andusing this method, two pyruvate decarboxylasegene (PDC) alleles genes were disruptedsuccessfully and mutant strain C. tropicalis (pdc/pdc) was obtained, which paved a solidfoundation for further genetic engineering of C. tropicalis. After it, we used the similar strategyto disrupt the heat shock protein70-2allel genes (HSP) of C. tropicalis. The main researchcontent is as follows:Firstly, using C. tropicalis ATCC20336as parent strain,13auxotrophic mutant strainswhich is defective in orotidine monophosphate decarboxylase (URA3) was isolated by chemicalmutagenesis and a double-selection procedure that combined nystatin enrichment selection and5-fluoro-orotic acid (5-FOA) resistance selection. Three of them were chosen, designated C.tropicalis XZW, XZX and XZB, to be analysed their URA3gene mutants after PCRamplification and DNA sequencing. Found that they had the common mutant which the G608converted to A, indicating that the change of its amino acid sequence caused by the mutant ofthis site was the main reason for the deficiency of URA3gene’s function.Secondly,1.6kb URA3gene and1.7kb PDC gene from ATCC20336, and1.1kb hisGfragment from Salmonella were cloned by PCR, respectively for construction of the PDCdisruption cassette. URA3gene was flanked by400bp PDC homologous arms and generatedPDC-URA3-PDC cassette. Then two hisG fragments were inserted into the ends of URA3geneof the PDC-URA3-PDC cassette with the same direction and the the PDC disruption cassettePDC-hisG-URA3-hisG-PDC(PHUHP) was constructed. Using the auxotrophic host C.tropicalis XZX as host, the PHUHP disruption cassette was transformed into the competentcells according to lithium chloride procedure and heterozygous transformants C. tropicalisXZX02was obtained and identified by PCR and DNA sequencing, in which one of two PDCallele genes was replaced by the disruption cassette PHUHP.For isolating mutants with URA3pop-out from PDC-hisG-URA3-hisG-PDC, C. tropicalisXZX02cells were sprayed onto the5-FOA selective medium plate and incubated for three daysat30℃. The colonies formed were isolated and characterized. Molecular identification showedthat the mutant the strain restoring the uracil requirement because of poping-out the URA3genefrom the location of PDC-hisG-URA3-hisG-PDC of C. tropicalis XZX02chromosome.Therefore, the mutant isolated was designated C. tropicalis XZX03(pdc::hisG/PDC) and usedfor disruption of the second PDC allele gene. Furthermore, the second PDC gene disruptioncassette were constructed. PDCm fragment which was a600bp inner section of PDC gene was cloned by PCR. Using the similar strategy, the second PDC disruption cassette PDCm-URA3-PDCm (MUM) consisting of a URA3gene inserted into a pair of PDC homologous arms wasconstructed. After introducing the MUM cassette into the C. tropicalis XZX03according to thelithium chloride procedure, the transformants in which both copies of PDC alleles have beendeleted were obtained and subsequently identified by PCR amplification and DNA sequencing.The resulting transformants were named C. tropicalis XZX04.Finally, the effects of PDC gene disruption on the PDC activity in the various mutantstrains were evaluated. The crude cell extractions of the strains of C. tropicalis XZX, C.tropicalis XZX02, C. tropicalis XZX03and C. tropicalis XZX04were used to enzyme activitydetection. Results demenstrated that the PDC activity of C. tropicalis XZX02and C. tropicalisXZX03shwoed the similar PDC activity, however their activity decreased by20.56%and20.00%, respectively compared to the strain C. tropicalis XZX. Furthermore, the strain C.tropicalis XZX04showed the lowest activity, which decreased by43.60%compared to thestrain C. tropicalis XZXMoreover, the heat shock protein70-2allel genes (HSP) of C. tropicalis were deleted usingthe similar strategy, which demenstrates the method developed in this study is useful andefficient in the genetic manipulation of the C. tropicalis.In this study, an efficient genetic transformation method was developed for diploid yeastC. tropicalis and using this method, two allele genes encoding pyruvate decarboxylasegene andanother two allele genes encoding the heat shock protein70-2were successfully deleted. Thisstudy will provide a powerful tool for metabolic engineering of C. tropicalis in future.