The Effect Of Long Chain Fatty Acid Transport Protein On The Production Of Dicarboxylic Acid In Candida Tropicalis

Candida tropicalis can use alkanes and fatty acids as the sole carbon source to synthesize long chain dibasic acids and is an important long chain dibasic acid producing strain.Long-chain dicarboxylic acid is an important chemical raw materials and pharmaceutical intermediates which can not be obtained directly from nature,microbial fermentation method has the advantages of low cost,high safety and green environmental protection,and is widely regarded at home and abroad.At present,the use of Candida tropical fermentation of n-alkanes to produce long-chain dicarboxylic acid fermentation industry amplification technology has matured.With the depletion of oil resources,the production of long chain dicarboxylic acids with alkanes as raw materials is facing increasing cost pressures and raw material pressures.Therefore,the search for renewable,cheap alkane alternatives as raw materials is a major challenge for carbon chain dicarboxylic acid industryis.Experiments showed that ScFat1p in Saccharomyces cerevisiae has the function of transporting long chain fatty acids,which was reported in Candida tropicalis.In this study,C.tropicalis 1798 was used as the target strain,and the key gene ctfat1p of fatty acid transporter protein FATPs was used to target single-copy knockout and double-knock knockout of the fatty acid transporter gene?ctfat1p?.The copy number of ctfat1p gene was increased and the function of ctfat1p gene was identified.In addition,the fermentation medium of C.tropicalis 1798+ctfat1p was optimized by Plackett-Burman design and orthogonal test.The main results were as follows:?1?The ctfat1p gene in Candida tropicalis 1798 was used as the study object.The homologous single exchange was used to insert the resistance gene kanr into the ctfat1p gene to achieve single copy knockout of the target gene and analyze the expression of CtFat1p in Candida tropicalis The results showed that the OD₆₀₀ nm of the recombinant strain was only 47.9%of the wild type strain and the yield was decreased by 65.3%after 12 h of C.tropicalis 1798 with the oil as substrate.The results showed that the ctfat1p knockout.The results showed that the ctfat1p gene could inhibit the uptake of long-chain fatty acids by C.tropicalis 1798,and the ctfat1p gene could be used as a key gene for absorption and utilization of Candida tropicalis.Then,the ctfat1p gene double deletion strain was obtained by constructing pZERO-Blunt-ctfat1p2-hygroB knockout vector with hygroB knockout vector.However,the ctfat1p gene double deletion strain could not grow and metabolize normally when the oil was the sole carbon source,and further inferred that the Fat1p protein in yeast could promote the activity of long-chain fatty acid transport.?2?Based on the above study,we obtained the overexpression of ctfat1p gene containing double copy through the method of seamless cloning on the basis of obtaining the full length of ctfat1p gene C.tropicalis 1798+ctfat1p.Fermentation experiments showed that the DCA yield of the multi-copy recombinant Candida tropicalis obtained after overexpressing the ctfat1p gene was 30%higher than that of the Candida tropicalis,and the cells did not show the phenomenon of poor growth due to the increase in copy number.It can be seen that the ability of the yeast to absorb and use the oil after the ctfat1p gene copy number increased,indicating that the ctfat1p gene is the key gene for the absorption of Candida tropicalis.?3?A series of experiments were carried out to optimize the culture medium of DCA,which was produced by fermentation of recombinant strain C.tropicalis1798+ctfat1p.The effect of 4 kinds of important medium DCA yield components were determined by Plackett-Burman experimental design,respectively MgSO4·7H2O?NH₄?₂SO₄,Na₂HPO₄·12H₂O and KH₂PO₄;through the orthogonal test analysis of the optimal fermentation production of DCA culture medium?flask?:?NH₄?₂SO₄ 1 g/L,yeast extract powder 2 g/L VB₁ 0.2 g/L?KH₂PO₄ 6 g/L?Na₂HPO₄·12H₂O 10 g/L?MgSO₄·7H₂O 2 g/L?glucose 62.5 g/L glucose;the culture medium in shake flask fermentation the measured DCA yield was 8.78 g/L,28.6%higher than before optimization medium producing acid.