Efficient Synthesis Of ω-hydroxy Fatty Acids By Metabolic Engineering In Candida Tropicalis

ω-hydroxy fatty acid（ω-HFAs）is a green,safe,non-toxic,ideal biodegradable material with good biocompatibility.It is the precursor of high-performance polyester and polyamide,and is widely used in chemical industry,food,pharmacy.The production ofω-hydroxy fatty acids by microbial fermentation has important research significance and application prospects.This study constructs a metabolically engineered strain that efficiently synthesizesω-hydroxy fatty acids,and continuously knocks out diploid tropics through homologous recombination technology with reusable marker genes.The acyl-Co A oxidase（POX）inβ-oxidation in Candida,and the fatty alcohol oxidase（FAO）and fatty alcohol dehydrogenase（ADH）inω-oxidation were investigated.The effect of the ability to synthesizeω-hydroxy fatty acids.The main findings are as follows:According to the search of ADH gene in NCBI,6 ADH2 genes in C.tropicalis ATCC20336 genome were obtained,namely ADH2-A1,ADH2-A2,ADH2-A3,ADH2-A4,ADH2-B1and ADH2-B1.Using the URA3 marker gene to design a knockout box for ADH2 gene,and design a knockout box for the POX and FAO genes at the same time,six recombinant Candida tropicalis were constructed respectively:FT（△FAO）,AT（△ADH）,PT（△POX）,FTPT（△FAO△POX）,ATPT（△ADH△POX）,ATPTFT（△ADH△POX△FAO）.The growth plate dilution culture and liquid growth kinetic observations of six strains were carried out.It was found that the deletion of the POX gene affected the utilization of alkanes and fatty acids by the recombinant strain.The deletion of the ADH gene obviously affected the growth of the strain more than the deletion of the FAO gene.ADH,POX,and FAO can blockω-oxidation andβ-oxidation to a large extent,and the recombinant strains have certain differences in adaptation to substrates with different carbon chain lengths.Optimization of the fermentation ofω-hydroxy fatty acid produced by recombinant yeast is at the shake flask level.Through fermentation optimization,the optimal fermentation parameters are obtained:the seed liquid adopts 2×YPDU,the baffle shake flask fermentation.The output ofω-hydroxy fatty acid of FTPT,ATPT,and ATPTFT are different.The output of12-hydroxydodecanoic acid is 2.99 g·L^-1,1.76 g·L^-1,and 2.73 g·L^-1and the output of14-hydroxytetradecanoic acid were 8.5 g·L^-1,1.52 g·L^-1,and 16.48 g·L^-1,respectively.The highest conversion rate of 14-hydroxytetradecanoic acid was 63.35%.The fed-batch cultivation of the recombinant strain ATPTFT in the fermenter producesω-hydroxy fatty acids in the 5 L fermenter.After culturing for 144 hours,the cell concentration is stable at OD₆₀₀=100～114.When fermenting for 144 hours,the output of14-hydroxytetradecanoic acid reaches 20.14 g·L^-1,and the conversion rate reaches 29.21%.The output of dodecanedicarboxylic acid reached 5.39 g·L^-1,and the conversion rate was3.69%.