| Resveratrol,a non-flavonoid polyphenol organic compound,has the effects of preventing and treating tumors,neuroprotection,autoimmune regulation,anti-asthma,anti-diabetic,etc.,and has broad market prospects.At present,the main source of resveratrol is extracted from plants such as Polygonum cuspidatum and grapes.However,the resveratrol extracted from natural plants has problems such as low content and difficulty in extraction,which can no longer meet people’s needs for it.Look for its replacement the source is imminent.At present,bioengineering technology is an ideal way to solve the problem of resveratrol deficiency.Model organisms such as Escherichia coli and Saccharomyces cerevisiae are used as the chassis to biosynthesize resveratrol,but there is still a problem of low yield.The synthesis pathway of resveratrol requires the participation of malonyl-Co A.The modification of its supply metabolic pathway has always been an important direction to improve resveratrol,and oil-producing microorganisms can accumulate a large amount of lipids,and cells can do Lipid synthesis provides a large amount of malonyl-Co A,which may be an ideal model organism for resveratrol synthesis.Yarrowia lipolytica yeast is currently the oil-producing microorganism with the clearest genetic background and the most complete genetic manipulation tools.In this study,Y.lipolytica yeast was selected as the chassis organism,and the resveratrol synthesis gene was introduced into yeast cells to explore.In order to explore the feasibility of Y.lipolytica yeast cells to synthesize resveratrol,this thesis made the following research:The 4-coumaric acid coenzyme A ligase(At4CL)gene from Arabidopsis thaliana and the stilbene synthase(Vv STS)gene from grapes were integrated into the Y.lipolytica genome by homologous recombination technology to make it successful Express and synthesize resveratrol.First,the sequences of the full-gene synthetic promoter p TEFin and terminator t LIP2 were digested and linked to the single-gene expression vector p INA1312 to transform it into a dualgene expression vector p CRCylm;then,the full-gene synthetic codon optimized At4 CL and Vv STS genes were connected to the T vector,and then respectively passed through the expression vector p CRCylm to obtain the recombinant expression vector p CRCyl-At4CL-Vv STS.Then,the expression vector was successfully transferred into the genome of Y.lipolytica competent cells using the lithium acetate chemical transformation method to obtain a recombinant yeast strain producing synthetic resveratrol.The recombinant strains and control strains verified by PCR were cultured in YEPD medium supplemented with a small amount of p-coumaric acid in shake flasks to explore the effects of At4 CL and Vv STS gene expression on the growth of Y.lipolytica yeast and the production of resveratrol.The experimental results showed that the expression of At4 CL and Vv STS genes in Y.lipolytica reduced the biomass from 12.5 g/L to 10.4 g/L,and the resveratrol production reached 674 μg/L,indicating that At4 CL and Vv STS genes were successfully transferred into Y.lipolytica yeast,the obtained recombinant strain can synthesize resveratrol.In order to further explore the potential of recombinant Y.lipolytica yeast cells to produce resveratrol,the culture conditions were optimized.A single factor experiment was designed to explore the effects of the concentration of p-coumaric acid,the time of p-coumaric acid addition,the culture time,and the culture temperature on the resveratrol production of recombinant Y.lipolytica yeast cells.The results of the study showed that the effect of the added concentration of p-coumaric acid on the yield of resveratrol showed a trend of first increasing and then decreasing,with the highest yield at 16 mg/L;when the p-coumaric acid was added for 12 h,the yield of resveratrol was the highest;as the cultivation time increased,the yield of resveratrol first increased and then decreased,and the yield was the highest at 72 h;the cultivation temperature has a great influence on the production of resveratrol.When the cultivation temperature is 28℃,the production of the recombinant yeast resveratrol is the highest.In order to further optimize the culture conditions,three factors with relatively large influencing factors were selected for response surface optimization experiments.The results of the study showed that by comparing the magnitude of the F value,the order of significance of the three factors on the yield of resveratrol was: culture temperature > addition of p-coumaric acid > addition time of p-coumaric acid.It was finally determined that the optimal culture parameters for the synthesis of resveratrol by recombinant Y.lipolytica yeast were the addition amount of p-coumaric acid 19.407 mg/L,the addition time of p-coumaric acid 11.437 h,and the culture temperature 26.2℃.The theoretical value of resveratrol synthesized by Y.lipolytica yeast is 1630.798 μg/L.Fermentation was carried out under this culture condition,and the resveratrol production was verified.The average value was 1604.7 μg/L after repeated three times,which was only 1.59% difference from the predicted value.Through single factor and response surface optimization,the production of recombinant Y.lipolytica yeast has been further improved.In short,the oleaginous microorganism Y.lipolytica can synthesize the functional factor resveratrol through genetic modification,which provides a theoretical and practical basis for studying the production of functional substances by this yeast. |
