| Synthetic biology is based on systems biology,applying the standardized,modularized,and systematized design concepts of engineering to design and create parts,devices and motifs,and use these designed and created "Synthetic biology parts"to transform and optimize existing biological systems or artificially resynthesize biological systems with preset specific functions to achieve large-scale production and application in the fields of medicine,agriculture and environment.Resveratrol is a kind of natural antitoxin that exists in nature and secreted when plants are under stress.It has antibacterial,antiviral and cardiovascular protection effects.At present,a large amount of resveratrol is industrially obtained by extracting and separating from natural plant tissues.The traditional method of obtaining resveratrol has low eff-iciency and the methods are complicated which cause damage to environment seriously and it also consumes natural plant resources.Based on the concepts of synthetic biology,we select Saccharomyces cerevisiae as the chassis cell,resveratrol as the target product.Saccharomyces cerevisiae W303-1A is constructed as a Cell Factory”of resveratrol heterologous synthesis.The research of this paper is mainly divided into the following five parts:(1)Based on the detection technology of high performance liquid chromatography,the detection method of resveratrol and galactose in fermentation process were established.This method has high detection accuracy and good detection repeatability,which lays the foundation for the rapid and efficient detection of key fermentation parameters during the experiment;(2)According to the preference of S.cerevisiae codons,five key enzyme genes from different sources in resveratrol biosynthesis pathway were optimized:the phenylalanine ammonia lyase gene pal from Rhodotorula sphaeroides,the tyrosine ammonia lyase gene tal from Rhodobacter sphaeroides,the cinnamic acid 4-hydroxycarboxylase gene c4h and the 4-coumarinyl-CoAligase gene 4cll derived from Arabidopsis thaliana and the resveratrol synthase gene sts derived from peanut.And then the five key enzyme genes are fully synthesized.Two heterologous synthetic pathways:PAL-C4H-4CL1-STS and TAL-4CL1-STS were designed and constructed in Saccharomyces cerevisiae W303-1A to obtain Saccharomyces cerevisiae engineering bacteria with three deficiences and Saccharomyces cerevisiae engineering bacteria with two deficiences.After cultured by shaking flask,the yield of resveratrol reached 0.94 mg/L and 4.72 mg/L respectively.Resveratrol heterogeneous biosynthesis in S.cerevisiae was successfully achieved and the efficiency of two artificial preset ways for producing resveratrol was verified respectively.(3)The fermentation conditions of yeast engineering bacteria were initially explored and optimized.The content of galactose in the fermentation broth was optimized by solid feeding,respectively,to make the resveratrol production of S.cerevisiae engineered bacteria with three deficiences and S.cerevisiae engineered bacteria with two deficiences increase to 1.64 times and 1.31 times at the shake flask level.The feeding method was used to optimize the content of galactose in the fermentation broth,so that the content of galactose during the cultivation process was maintained at a relatively stable level,and the resveratrol production of S.cerevisiae engineering bacteria is further increased to 2.09 times.At the same time,three batches of Saccharomyces cerevisiae engineering bacteria were continuously cultivated in three batches of fermenters,and finally 4.84 mg/L of resveratrol was harvested,which was 5.14 times higher than that of shake flask culture.The conclusion indicates that the optimization of fermentation conditions of Saccharomyces cerevisiae engineering bacteria can indeed increase the production of resveratrol to a certain extent.(4)To investigate the effect of partial knock-out of the citrate synthase CIT,a key enzyme in the tricarboxylic acid cycle on yeast,on heterologous synthesis pathways,and use the CRISPR-Cas9 technology to perform the citl gene on the yeast chromosome Knockout to construct the three deficient yeast engineering bacteria Acitl and the two deficient yeast engineering bacteria Acitl.The results showed that the yield of resveratrol after fermentation of the three deficient yeast engineering bacteria Acitl reached 1.47 mg/L,which was an increase of 1.56 times compared with the initial yield;the yield of resveratrol after the fermentation of the two deficient yeast engineering bacteria Acitl reached 6.09 mg/L,an increase of 1.29 times compared to the initial production.It is shown that the deletion of the citl gene will weaken the tricarboxylic acid cycle of yeast and slow the growth and reproduction rate of bacteria.However,at the same time,the prerequisite substance malonyl-CoA will flow into the heterologous synthesis pathway and increase the production of resveratrol.(5)The Gibson assembly method was used to construct a tool yeast fluorescent strain for library screening.The green fluorescent protein-expressing egfp gene and the rate-limiting enzyme resveratrol synthase gene sts in the heterologous synthetic pathway were constructed using a flexible linker phase.Ligation Design Construction Tools Yeast Fluorescent Strains.Using the fluorescence intensity of green fluorescent protein to characterize the expression of resveratrol synthase,a yeast strain with high resveratrol synthase activity can be quickly and conveniently screened.At the same time,a three-deficient fluorescent yeast strain and a positive control fluorescent yeast strain were constructed as experimental controls.By measuring the fluorescence expression levels of the three yeast fluorescent strains,the fluorescence reference value at the time of screening and the induction time required for the screening were determined for the subsequent library screening.This work provides convenient and effective detection methodsIn this study,we use the ideas and concepts of synthetic biology,the natural resveratrol synthesis pathway in the plant was designed and constructed into two lines respectively introduced into Saccharomyces cerevisiae,and the three-deficient yeast engineering bacteria and the two-deficient yeast producing resveratrol were successfully obtained Engineering bacteria.At the same time,detection methods for resveratrol and galactose were established based on high-performance liquid chromatography technology.The ability of two yeast engineering bacteria in different heterologous synthetic pathways to produce resveratrol was evaluated,laying a foundation for industrial biosynthesis of resveratrol.The foundation.On this basis,preliminary exploration and optimization of fermentation conditions were carried out The yield of resveratrol was further increased by adding galactose and phenylalanine and culturing in a fermenter.Then use CRISPR-Cas9 technology to knock out the citrate synthase gene citl in the tricarboxylic acid cycle,and explore the partial knock-out of the citrate synthase CIT,a key enzyme in the tricarboxylic acid cycle in yeast influences.Finally,we constructed a yeast tool strain for library screening,which provided a convenient and effective detection method for subsequent library screening The research in this thesis has universal reference significance for the biosynthesis of resveratrol and similar phenolic compounds. |
PDF Full Text Request