| In order to reduce the dependence on non-renewable petroleum resources,the production of bio-based materials and chemicals using fatty acids,a renewable resource abundant in nature,as green raw materials has attracted widespread interest in recent years.ω-Amino fatty acids（ω-AmFAs）are excellent monomers for the synthesis of nylon.In this study,we used Candida tropicalis and Saccharomyces cerevisiae as the chassis cells to try to achieve efficient synthesis ofω-AmFAs through two strategies:biotransformation and de novo synthesis.We successfully achieved the efficient conversion of fatty acid methyl esters intoω-AmFAs in C.tropicalis and the first de novo synthesis of long-chainω-AmFAs in S.cerevisiae.The main contents are as follows:1.Screening and identification ofω-transaminases.Based on the codon optimization and integrated expression strategy,we evaluated the heterologous expression and functional analysis ofω-transaminase gene Cv TA from Chromobacterium violaceum andω-transaminase gene Sp TA from Silicibacter pomeroyi in C.tropicalis cells,respectively.Expression and functional analysis ofω-transaminase Cv TA andω-transaminase Sp TA from Silicibacter pomeroyi in C.tropicalis cells showed thatω-transaminase Cv TA has higher activity and can synthesizeω-AmFAs more efficiently,with a yield of 30 mg·L-1.2.A high-yielding strain ofω-AmFAs was constructed by modifying the metabolic pathway of the engineered strain.Firstly,the CAT gene was knocked out and the engineered strain Ct03 was constructed,theβ-oxidation activity of the engineered strain was blocked and the yield of 12-aminododecanoic acid（12-ADA）was increased by 4.2-fold to 157.8 mg·L-1;further knocking out the fatty aldehyde dehydrogenase genes Ct ALD1 and Ct ALD2 of the engineered strain Ct03,the by-product dodecanedioic acid（12-DCA）decreased by 26.3%and the yield of 12-ADA reached 486.6 mg·L-1;overexpression of CYP52A17,Ct NCP,FAO1,and Cv TA,key genes in the synthesis pathway ofω-AmFAs,further increased the yield of 12-ADA by 94.6%;enhancement of the endogenous long-chain The enhanced endogenous long-chain fatty acid transport protein gene Ct FAT1 was able to further enhance the yield of 12-ADA and the yield of 12-ADA reached 1104.6 mg·L-1;finally,the ability of the engineered strain to produce 14-aminotetradecanoic acid and 16-aminohexadecanoic acid was evaluated.Finally,the ability of the engineered strain to produce 14-aminotetradecanoic acid and 16-aminohexadecanoic acid was evaluated,and their yields reached 433.7 mg·L-1 and 43 mg·L-1,respectively.3.Rational modification of Saccharomyces cerevisiae chassis cells to enhance the synthesis of free fatty acids（FFAs）.Using S.cerevisiae BY4741 as the chassis cell,the engineered strain FFAs reached 384.4 mg·L-1 by deleting the genes encoding acyl-Co A synthases FAA1,FAA4,and FAT1.Further,the level of FFAs synthesis was increased to 394.9mg·L-1 by knocking down POX1,FAA2,and PXA2 genes.By knocking down the genes encoding phosphatidic acid phosphatase DPP1,LPP1,and PAH1 to reduce the dephosphorylation level of phosphatidic acid（PA）and up-regulate the expression of key genes related to fatty acid synthesis,the obtained yeast engineered strain BY9（Δfaa1Δfaa4Δfat1Δpox1Δfaa2Δpxa2Δdpp1Δlpp1Δpah1）was able to produce 497.3 mg·L-1 of extracellular free fatty acids and 1332.2 mg·L-1 of total fatty acids.4.Theω-hydroxylation ability of two different sources of P450 enzymes in S.cerevisiae was explored in the fatty acid producing strain BY9,and the P450 enzyme CYP52A17 has high efficiency in catalyzing the terminal hydroxylation of fatty acids.On this basis,heterologousω-transaminase were expressed,and an engineered strain of S.cerevisiae yeast with ab initio synthesis of long-chainω-AmFAs was constructed.And the yields ofω-AmFAs for C12,C14and C16 were 2.3 mg·L-1,9.7 mg·L-1 and 14.6 mg·L-1,respectively. |
