| Tyrosol is a natural polyphenol compound that has many physiological and pharmacological effects,such as anti-oxidation,anti-aging,reducing altitude sickness,protecting the nervous system,and improving immunity.Due to its great potential in the food industry,the demand for tyrosol is increasing.At present,the main preparation method is extracting from plants;however,the lower tyrosol content and the harsh growth requirements of plants greatly reduce the extraction efficiency,restricting the yield of tyrosol.With the development of metabolic engineering,the tyrosol synthesis pathway has become clear,and it is possible to produce tyrosol via fermentations.In addition,researchers have found that tyrosol is also a direct precursor of some other natural products,like hydroxytyrosol.Therefore,it is important to establish a strain with high tyrosol titer.This article constructed a synthesis pathway of tyrosol in Escherichia coli.The main contents are as follows:(1)Overexpressing the aromatic aldehyde synthase from Petroselinum crispum obtained tyrosol105.97 mg·L-1,and the titer of tyrosol increased to 1055.36 mg·L-1 by blocking its main branched metabolic pathways.In this study,T7 promoter in the plasmid p RSFDuet-1 was replaced by a constitutive promoter Pcsp A,introducing the heterologous synthesis pathway of tyrosol successfully,as well as avoiding the addition of inducers during the fermentation process.Then,the gene fea B in the branch pathway was knocked out by CRISPR/Cas9,and the above recombinant plasmid was overexpressed in the strain that lacked the 6 genes(tyr R,pts G,crr,pyk F,phe A and fea B)of 5 branch pathways.As a result,the titer of tyrosol increased by 895.90%to 1055.36 mg·L-1.(2)Screening the alcohol dehydrogenases increased the tyrosol titer to 1516.86 mg·L-1,and the strategy of combination regulations was conducted to balance the flux,increasing the tyrosol titer to1810.46 mg·L-1.In this study,the alcohol dehydrogenases(Adh E,Adh P,Yah K and Frm A derived from E.coli and ADH6 derived from Saccharomyces cerevisiae)were detected,in order to enhance the metabolic flux.The results showed that the alcohol dehydrogenase from S.cerevisiae showed the most suitable for tyrosol production,producing 1516.86 mg·L-1 tyrosol.It was 43.73%higher than that of non-expression of that alcohol dehydrogenase.High-strength,medium-strength and low-strength promoters were applied to overexpress the key genes.It was found that using a medium-strength promoter to overexpress Pc AAS and a low-strength promoter to overexpress Sc ADH6 was the best combination,and the titer of tyrosol reached 1810.46 mg·L-1,increasing by 19.40%.(3)Conducting the optimizations of fermentation process increased tyrosol titer to 2120.58mg·L-1,which was an increase of 17.13%.In this study,the culture method(the inoculation ratio and culture temperature)and fermentation medium(carbon source,nitrogen source,Ca CO3,Mg2+,Ca2+and citric acid)were taken into consideration.It was discovered that Ca CO3 is beneficial to the production of tyrosol,as well as the optimal concentrations of carbon source.The optimal medium ingredients are as follows:glucose 25 g·L-1,glycerol 20 g·L-1,(NH4)2SO4 7.5 g·L-1,K2HPO4·3H2O3 g·L-1,KH2PO4 2 g·L-1,Mg SO4·7H2O 2.0 g·L-1,sodium citrate 1.0 g·L-1,vitamin B1 0.1 g·L-1,yeast extract 7 g·L-1,Ca CO3 5 g·L-1.Trace element nutrient solution 1 m L·L-1.This studys lay a good foundation for the production of tyrosol and related downstream products by fermentation. |
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