Regulating And Optimizing The Process Of Prenylation Of (2S)-Naringenin In Saccharomyces Cerevisiae

8-Prenylnaringenin（8-PN）is a valuable medicinal phytoestrogen,and also the precursor of many prenylated flavonoids.Currently,the production of 8-PN relies on plant extraction.However,the content of 8-PN in plants is relatively low,and the existence of isomers causes difficulties in the downstream separation and purification.The use of synthetic biology technology to transform microorganisms to synthesize 8-PN is an important method to ensure its production.Microbial synthesis of 8-PN mainly faces three problems:the interference of signal peptides on the structure and function of prenyltransferases（PTs）,the insufficient supply of precursors,and the low activity of PTs.In order to solve the above problems,this experiment used a Saccharomyces cerevisiae strain E32 as the chassis strain that can produce a high titer of（2S）-naringenin,and combined the methods of metabolic engineering and protein engineering to realize the efficient heterologous biosynthesis of 8-PN.The research results provide a certain reference value for the biosynthesis of prenylated flavonoids.The main results of the thesis are as follows:（1）Construction and expression optimization of the 8-PN synthetic pathway.In this experiment,PTs from six different species were screened by sequence alignment,and the signal peptides were predicted and truncated,respectively.It was determined thatSfN8DT-1derived from Sophora flavescens and truncated cognate tSfN8DT-1 that truncated at E81 site,had the ability to synthesize 8-PN.At the same time,it was found that the truncation significantly improved the synthesis ability of SfN8DT-1,and the yield of 8-PN increased by108.37%.Subsequently,the expression of tSfN8DT-1 was optimized by picking over 14endogenous promoters with different expression strengths from S.cerevisiae.After being verified by fermentation,GAL7p was shown to be the most suitable promoter for the expression of tSfN8DT-1.Finally,after 120 h of fermentation,the production of 8-PN in a 250mL shaking flask reached 4.98 mg×L^-1.（2）N-terminal signal peptide truncation of SfN8DT-1 enhancing the synthesis of 8-PN.The structure prediction of wild-type SfN8DT-1 showed that it consists of a main structure,containing multipleα-helices and a side chain of 120 amino acids.According to the structure of the side chain,17 different amino acid positions in the side chain were selected for truncation.The results showed that the truncation of different amino acid residue positions in the side chain had different effects in the synthesis of 8-PN,and the truncation to K62 showed the most obvious improvement on the synthesis of the 8-PN.Subsequently,molecular docking,alanine scanning mutation and saturation mutagenesis were performed to identify K185 as a conserved key amino acid residue,which may be involved in the binding process of（2S）-naringenin and DMAPP.Compared the ability of SfN8DT-1 and tSfN8DT-1 to synthesize8-PN,result showed that tSfN8DT-1 has better synthetic ability and potential.（3）Enhancing precursor supply and utilizing protein redesign methods to improve the synthesis of 8-PN.First,12 low-conserved sites were selected by sequence alignment,and site-directed mutagenesis was performed on these sites.6 positive mutants were obtained,among them,the mutant tSfN8DT-1^Q12E increased the titer of 8-PN by 114.51%.Then,by overexpressing tHMGR and IDI1,the key genes of mevalonate pathway,with different copy numbers,the production of 8-PN was significantly increased when two copies of tHMGR and IDI1 were simultaneously overexpressed.Through protein 3D structure prediction and virtual saturation mutagenesis,two key amino acid residues were selected:P229 and N305.The mutant tSfN8DT-1^{Q12E N305M} was increased the titer of 8-PN to 49.35 mg×L^-1 by saturation mutagenesis.Finally,the 8-PN production reached 101.40 mg×L^-1 after cultivating in a 5-L bioreactor.