Effect Of Pxa1 Gene Deletion In Candida Tropicalis On Long Chain Dicarboxylic Acid Yield

Long chain dicarboxylic acid was a kind of important fine chemical intermediates.C.tropicalis could use alkanes and fatty acid product of long chain dicarboxylic acid by microbial fermentation.But the metabolic pathway of excessive?-oxidation would cause the yield decline in long chain dicarboxylic acid.Pxa1p was a membrane protein of Saccharomyces cerevisiae,which was able to transport long chain fatty acids into peroxisomes in a specific way.In this study,through the comparative analysis Candida tropicalis transporter Pxa1p by means of genetic engineering,and knocking out the pxa1 gene,explore the hinder part of the long chain fatty acids into peroxisomes,a new method for reducing the consumption of long chain fatty acids.?1?C.tropicalis 1798 was used as the starting strain,using single exchange method,by overlapping PCR method directly constructed knockout fragment pxa1p-kanr,to homologous recombination by electroporation,pxa1 gene single deletion C.tropicalis1798-pxa1 was constructed.And through antibiotic G418 resistance,PCR screening and other experiments.It was confirmed that the C.tropicalis 1798-pxa1 was successfully constructed which pxa1 gene was single deleted.After fermentation test,single deletion strains C.tropicalis 1798-pxa1 than the original strain C.tropicalis 1798 outputing dodecanedioic acid increased by 56.1%,the output concentration was 6.4 g/L.The results indicated that pxa1 gene knockout,blocking part of long chain fatty acids into peroxisomes,reduced?-oxidation and long chain dicarboxylic acid consumption,improved the production of long chain dicarboxylic acid.?2?The plasmid pxa1p2 of Candida tropicalis was amplified by PCR,it was ligated to the plasmid pFA6a-hphMX-tetO-Pcyc1-3xFLAG with the hygromycin resistance gene hph by degestion.to homologous recombination by electroporation,pxa1gene double deletion strain C.tropicalis 1798-pxa1p2 was constructed,through antibiotic hygromycin B resistance,PCR screening and other experiments.It was confirmed that the C.tropicalis 1798-pxa1p2 was successfully constructed which pxa1gene was double deleted.After fermentation test,double deletion strains C.tropicalis1798-pxa1p2 was lower than the original strain C.tropicalis 1798 outputing dodecanedioic acid,the output concentration was 2.3 g/L.It was speculated that the complete knockout of the pxa1 gene affected the normal metabolism of the bacteria,resulting in a low level of acid production.?3?The fermentation conditions and the culture components of C.tropicalis1798-pxa1 were optimized.The optimum temperature for the fermentation of dodecanedioic acid was 30?,the optimum growth pH was 5.0,and the optimum pH of the dodecanedioic acid was 7.5.The four factors influencing the yield of dodecanedioic acid in fermentation medium were determined by Plackett-Burman?PB?method:glucose,urea,yeast extract,KH₂PO₄.The effects of the fermentation medium on the yield of dodecanedioic acid were studied by single factor experiment and orthogonal experiment.The optimum medium was determined as follows:glucose 30 g/L,yeast extract powder 1 g/L,?NH₄?₂SO₄ 1 g/L,VB1 0.2 g/L,NaCl 2 g/L,KH₂PO₄ 8 g/L,Na₂HPO₄·12H₂O 10.08 g/L,urea 1.5 g/L,Mg₂SO₄·7H₂O 6.15 g/L.In this paper,the genetic transformation of Candida tropicalis metabolic pathway was carried out.The relationship between the long chain fatty acid transporter Pxa1p and the long chain dibasic acid in Candida tropicalis was studied.The effects of long-chain fatty acid transporter Pxa1p on Candida tropicalis were studied.Pxa1 gene single deletion C.tropicalis 1798-pxa1 and double deletion C.tropicalis 1798-pxa1p2were constructed.The fermentation conditions and media components of C.tropicalis1798-pxa1 were optimized to increase the yield of dodecanedioic acid.