Study On Construction Of Candida Tropicalis Engineering Strain Producing Dodecyl Dicarboxylic Acid And Efficient Conversion Of Fat Oils

Long-chain dicarboxylic acid is just an extremely widely used aliphatic dicarboxylic acid.Candida tropicalis is a high-yield microorganism that can produce long-chain dicarboxylic acid(DCA)by fermenting fatty acids and alkanes.In this study,C.tropicalis 1798 was used as the target strain,construction of a suicide vector pPICPJm using the mazF gene from E.coli as a reverse selection marker,the gene of GK which promotes glycerol utilization in C.tropicalis,CRAT,which regulates the fatty acid?-oxidation process,and P450,the key enzyme in?-oxidation,the promoter genes of gk gene and cytochrome P450 enzymes and NADH-cytochrome P450reductase were replaced on the basis of the markerless genome editing system,and the carnitine acetyltransferase gene CRAT was single copy knockout by molecular biology techniques,The main results were as follows:(1)The toxic protein gene mazF from E.coli and the promoter gene pGAL was obtained from C.lipolytica 1457 were obtained,pGAL-mazF was constructed by overlapping PCR method and was inserted into the vector pPICz?A with the Kan~r gene by One Step Cloning Kit.The suicide plasmid pPICPJm was constructed as markerless genome edit vector in the molecular modification of C.tropicalis.After the suicide plasmid pPICPJm was electrotransformed into C.tropicalis and screened by G418 resistance and PCR,the cultures were streaked on plates containing galactose concentrations of 0 g/L,5 g/L and 10 g/L,respectively.It was shown that 10g/L of galactose can effectively inhibit the growth of C.tropicalis-pPICPJm,so the second single exchanged recombinant was screened with 10 g/L of galactose subsequently.(2)On the basis of the suicide plasmid pPICPJm,C.tropicalis 1798 was used as the starting strain to construct C.tropicalis gkPr by means of gene recombination.The promoter of the gk gene was confirmed by G418 resistance and PCR screening.C.tropicalis gkPr was successfully constructed.The fermentation results showed that the utilization rate of glycerol in the promoter replacement gene C.tropicalis gkPr increased by 56.1%,indicating that promoter replacement can promote the absorption and utilization of glycerin by C.tropicalis 1798 and provides energy for fermentation.When the oil was used as a substrate for fermentation,it was also found that the amount of dodecanedioic acid produced by the recombinant strain was increased by32.7%compared to the starting strain.(3)The C.tropicalis homologous arms CRATpF-CRATpR were obtained by overlapping PCR and connected to the suicide plasmid pPICPJm by One Step Cloning Kit.The C.tropicalis gkPc was successfully constructed on the basis of C.tropicalis gkPr through the screening of G418 resistance and PCR screening by a gene homologous recombination.Shake flask experiments showed that the production of dodecanedioic acid from C.tropicalis gkPc is higher than that of the original strain,and the yield was 7.13 g/L.This indicates that due to the single copy knockdown of the CRAT gene,the consumption of intracellular fatty acids is reduced,resulting in an increase in acid production levels.(4)Based on the above study,the promoter genes of cytochrome P450 and NADPH-cytochrome P450 reductase were replaced and the engineering strain C.tropicalis GCPP was constructed on the basis of C.tropicalis gkPc.Shake flask fermentation showed that the production of dodecanedioic acid of engineering bacteria reached 11.39 g/L of C.tropicalis GCPP,which was 2.8 times of the original strain.5L fermentor fermentation showed that the production of dodecanedioic acid of engineering bacteria reached 35.64g/L.