Metabolic Engineering Of Escherichia Coli For Efficient Production Of Salidroside

Salidroside is the main active ingredient of Rhodiola rosea,which has important and wide application value due to its rich pharmacological effects.At present,the main ways to obtain salidroside are direct extraction from the plant and chemical synthesis.However,these two preparation methods can hardly meet the market demand because of the yield or quality.In contrast,microbial synthesis has become an important production method for obtaining natural products because of its large-scale cultivation,high product purity,and compliance with sustainable development requirements.In recent years,more and more researches have been conducted on the biosynthesis of salidroside.In our laboratory,we have conducted research on the microbial synthesis of tyrosol and obtained a high-yielding strain YMG5A*R that can synthesize tyrosol from glucose without induction or addition of antibiotics through metabolic modification,so all strains in this study were constructed on this strain.The study was carried out in order to obtain a strain with high yield of salidroside.The main findings of this study are as follows.(1)Identify key enzymes for salidroside synthesis and construct recombinant strains expressing glycosyltransferase by plasmid.Two different sources of glycosyltransferases,Rs UGT72B14 and At UGT85A1,were selected according to the literature and the catalytic activity of UGT85A1 was confirmed by the fermentation results.After expression vector optimization,the obtained recombinant strain SA01 could synthesize salidroside from glucose up to 2.5 g/L in shake flasks.(2)Construction of recombinant strains with genomic integration expressing glycosyltransferase UGT85A1.Firstly,the effects of several different integrative expression sites of the coding and spacer regions in the genome on gene expression were investigated and several effective sites for integrative expression were identified by m easuring the production of salidroside of single site integration strain.Finally,a recombinant strain 5AS with genomic integrated expression of UGT85A1 was successfully obtained and the production of salidroside reached 120 mg/L.(3)The effect of the copy number of glycosyltransferase gene UGT85A1 on the production of salidroside was investigated.Firstly,based on the single copy number of strain5 AS,recombinant strain with 2 to 8 UGT85A1 gene were obtained sequentially and the fluorescence quantitative PCR results showed that the transcription level of UGT85A1 gene increased gradually with the copy number.Finally,strain 5A8 S was able to synthesize 2.42g/L of rhodopsin in shake flasks without the addition of inducer and antibiotics.(4)Expanded cultivation of strain 5A8 S and SA01.In order to further confirm the applicability of the recombinant strains for large-scale production,the strains SA01 and 5A8 S were subjected to scale-up fermentation in a 5 L fermenter.When the final fermentation time reached 120 h,the yields of strains SA01 and 5A8 S reached 9.48 g/L and 9.34 g/L,respectively.