Development Of The Genetic Manipulation System For Candida Tropicalis And Its Application In Dicarboxylic Acid Production

The n-alkane utilizing yeast Candida tropicalis is an important strain for long-chain dicarboxylic(DCA) production. In this study, deletion of carnitine acetyltransferase genes(CAT) and/or acyl-CoA oxidase genes(POX4 and POX5) of C. tropicalis XZX were employed using a novel gene deletion strategy. Then, the growth pattern and 1, 12-dodecanedioic acid(DCA12) production were characterized. In addition, heterologous gene expression in C. tropicalis was developed using the GFP gene as reporter. Lastly, cytochrome P450 gene(YlALK1) and NADPH-cytochrome P450 reductase gene(Yl CPR1) from Yarrowia lipolytica were expressed in C. tropicalis, respectively. The recombinant strains were evaluated for their gene expression and DCA12 produciton. Main results in this research were as follows:1. An efficient multi-gene disruption strategy was developed based on gda as gene disruption auxiliary(gda) sequence for C. tropicalis. The disruption cassette, which consists of a functional yeast URA3 gene flanked by a 0.3 kb gda sequence direct repeat derived from 5’ or 3’ of the URA3 gene, and homologous arms of the target gene, was constructed and introduced into the yeast genome by integrative transformation. The recombination efficiency can be increased by almost one order of magnitude when compared to the traditional URA3 blaster, and the frequency of URA3 gene pop-out is similar.2. The mutant strains of 02(cat::gda324/CAT) and 04(cat::gda324/cat::gda324) were constructed, respectively. The growth rate and final biomass of C. tropicalis 02 and XZX were similar when cultured in medium containing glucose or dodecane as the sole carbon source. C. tropicalis 04 could not grow on dodecane and the pathway of glucose metabolism was affected by the disruption of the CAT alleles. Moreover, the CAT activity of strain C. tropicalis 02 decreased by 80% compared to XZX, however no higher DCA12 concentration was observed. Furthermore, no CAT activity was detected in C. tropicalis 04, while the capability of conversion of alkane to DCA12 increased significantly and higher DCA12 concentration was obtained.3. The mutant strains of C. tropicalis 22(ura3/ura3 CAT/CAT pox4::gda324/POX4 POX5/POX5), 24(ura3/ura3 CAT/CAT pox4::gda324/pox4::gda324 POX5/POX5), 26(ura3/ura3 CAT/CAT pox4::gda324/pox4::gda324 pox5::gda324/POX5) and 28(ura3/ura3 CAT/CAT pox4::gda324/pox4::gda324 pox5::gda324/pox5::gda324) were constructed, and the DCA12 concentration of C. tropicalis 28 was increased compared to XZX.4. The GFP was functionally expressed in C. tropicalis. The expression cassette CAT2-gda324-URA3-P-GFP-T-CAT2 was constructed and transformed into C. tropicalis 02. Thus, both copies of the CAT genes were destroyed and the GFP expression cassette was specifically integrated into CAT locus. In this way, wtGFP(wild type GFP), GFPCTC(CUG codons were optimized as CTC) and yeGFP3(all codons were optimized in GFP) were expressed in C. tropicalis, respectively. High level of fluorescence was observed in C. tropicalis with yeGFP3. However only a slight amount of fluorescence was observed in C. tropicalis with GFPCTC, It is not surprising that no fluorescence was observed in C. tropicalis expressing wtGFP gene.5. In order to increase DCA12 production, YlALK1 and YlCPR1 from Y. lipolytica were expressed in C. tropicalis 28, respectively. The gene expression was investigated for different yeast transformants using qRT-PCR. In addition, fermentation performances of recombinant strains were evaluated and the results showed that heterologous expression in C. tropicalis 28 could improve DCA12 production compared to strains of C. tropicalis 04 and 28.