Research Of Producing 3-hydroxypropionic Acid From Yarrowia Lipolytica Via Malonyl-CoA Pathway

3-hydroxypropionic acid(3-HP)is not only an important platform compound,but also an important chemical precursor,which has the potential to be further converted into useful chemicals and has been widely used in industry,agriculture and medical fields.Therefore,3-HP is listed as one of the 12 most potential renewable chemical products by DOE.With the lack of non renewable resources such as oil,the demand of 3-HP is increasing.Yarrowia lipolytica is a kind of unconventional yeast,because Y.lipolytica is widely found in ecosystems(such as soil,marine waters,mycorrhiza and oil polluted environment)and various foods(especially meat and dairy products,including cheese),and it has significant activity of decomposing fat and can secrete fat and produce organic acids.With the development of many molecular and genetic tools,Y.lipolytica has become a recognized system for expressing foreign genes and secreting proteins,and has successfully produced more than 130 kinds of proteins from more than 80 species.In this study,we choose to construct malonyl-Co A pathway to produce 3-HP.Because the genome of yeasts contains one of the two genes involved in the pathway,acc.Therefore,it is necessary to transfer another gene mcr into Y.lipolytica.In order to compare the expression effect of the original mcr gene and the optimized synthesis of the codon mcr gene,as well as the heterologous gene expression effect of the single copy and multi copy plasmids,and to improve the production of 3-HP as much as possible,the following studies were carried out:The original mcr gene(named mcr1)and the password free synthesis mcr gene(named mcr2)were obtained by PCR.Four recombinant expression vectors p1312-mcr1,p1312-mcr2,p1269-mcr1 and p1269-mcr2 were constructed by seamless connection with multiple copy plasmids p INA1312 and single copy plasmids p INA1269,respectively.Four recombinant yeasts,YL(p1312-mcr1),YL(p1312-mcr2),YL(p1269-mcr1)and YL(p1269-mcr2),which can produce 3-HP,were constructed and screened.The results of liquid chromatography showed that YL(p1312-mcr2)was the best production strain.The most significant effect of fermentation temperature on 3-HP synthesis was found by orthogonal test,followed by p H,fermentation time and inoculation amount.At the same time,the optimal fermentation conditions were fermentation temperature 30 ?,p H 5.0,fermentation time 84 h,inoculation amount 2.5%.The yield of 3-HP was 1.397 g/L,which was49.2% higher than before.