Study On The Biological Production Of Farnesene And R-3-Aminobutyric Acid By Recombinant Escherichia Coli

This paper explores a route for preparing high value-added products（farnesene,fatty acid methyl ester and R-3-aminobutyric acid）,which helps greatly reduce production costs and effectively suppress environmental problems caused by the accumulation of waste resources.The specific research contents are as follows:（1）Studys on the efficient production of farnesene and fatty acid methyl ester（FAME）by waste cooking oil（WCO）through a two-step biological method:In this study,a technique for preparing high-yield FAME and high-concentration by-product crude glycerol by immobilized lipase Novozym 435,and a technique for producing high-yield farnesene by recombinant E.coli F4 fermentation using the by-product crude glycerol as a substrate were developed.Recombinant E.coli F4 consumes crude glycerol to produce farnesene by fermentation,the yield is very low,the reason is that the methanol content in crude glycerol is too high.WCO pretreatment and enzymatic transesterification process optimization were carried out.FAME with 96%yield and1200 g/L by-product glycerol with zero methanol content were obtained,which solved the problem of methanol suppression.At the same time,the composition comparison of crude glycerol obtained by the enzymatic and chemical transesterification reactions and the performance comparison of the preparation of farnesene with glycerol from different sources as fermentation substrates were explored.The crude glycerol obtained by enzymatic fermentation was used as a substrate to produce farnesene with a yield of5.29 g/L,which is 100%higher than the yield（2.62 g/L）of chemically prepared crude glycerol to produce farnesene.Therefore,this green bio-conversion method has achieved two-step production of high-yield FAME（96%）and high-yield farnesene（5.29 g/L）.（2）Studies on high yield of R-3-aminobutyric acid by recombinant E.coli biological method:In this study,based on the analysis of enzyme catalytic mechanism and PDB library screening,the resulting aspartase mutant can specifically catalyze the conversion of crotonic acid to R-3-aminobutyric acid,and RBS was optimized to obtain an engineered strain BL21-22b-P_T7-Asp-H that can efficiently express high enzyme activity（1516 U/mg）aspartase.After optimization of the conversion system process and substitution of industrial raw materials（fermentation raw materials and biological conversion raw materials）,after 24 hours of conversion,the conversion rate of crotonic acid and the yield of R-3-aminobutyric acid reached 97%and 96%,and the output reached 287.6 g/L.At the same time,in the scale-up system,through the performance analysis and process optimization of the fermentation,permeation and biotransformation operation units,after conversion for 24 h,the output of R-3-aminobutyric acid reached 284 g/L and the yield reached 95%with a selectivity of 98%.According to the raw material cost calculation of R-3-aminobutyric acid（1 kg）,the cost of all industrial raw materials（（?）116.21/kg）is about 1/4 of the laboratory raw material cost（（?）479.76/kg）.In summary,this research route has the advantages of mild conditions,environmental friendliness,short process route,high yield,preparation,which can provide reliable technical support for the industrial production of the important raw material R-3-aminobutyric acid of dulutaway（anti-AIDS drug）.