Caffeic acid phenethyl ester is a natural compound mainly existing in propolis, which has obvious pharmacological effects such as anti-cancer and anti-inflammation, and is widely used in the medical field. The production of caffeic acid phenylethyl ester usually adopts chemical synthesis method, but in the process of synthesis, environmental pollution problems will inevitably be involved, such as the use of acylating agent in acyl chloride method; moreover, some raw materials needed for the reaction are difficult to prepare and expensive, such as triphenylphosphine, which is the raw material of Witting reaction. Therefore, the chemical synthesis method does not meet the requirements of green chemical industry today. In vitro enzyme catalysis is another method to synthesize caffeic acid phenylethyl ester, but its synthesis efficiency is not high due to the specificity of enzyme and the limitation of temperature. In recent years, the rapid development of metabolic engineering and synthetic biology has enabled the secondary metabolic pathway to be rebuilt in rapidly dividing microorganisms such as Escherichia coli and Saccharomyces cerevisiae, providing an important platform for the synthesis of secondary metabolites. Therefore, this paper attempts to construct a system for synthesizing caffeic acid phenylethyl ester by microorganisms. The main results are as follows:
（1）Through database mining and literature analysis, phenylalanine lyase (PAL), cinnamic acid -4- hydroxylase (C4H), 4- coumaroyl -CoA ligase (4CL) and hydroxycinnoyltransferase (HCT) were screened for the synthesis of caffeoyl-CoA from phenylalanine. After codon optimization, they were integrated into the site of Invsc1 YPRC δ15 in Saccharomyces cerevisiae. Under the catalysis of yeast's own acyltransferase, In order to improve the synthesis efficiency, the expression of acyltransferase (Eht1 coding) was strengthened, and the supply of 2- phenylethanol was optimized by expressing kdc/Aro10 (encoding phenylpyruvate decarboxylase) and Adh6 (encoding alcohol dehydrogenase), so that the yield of caffeic acid phenylethyl ester was increased to 56.44 μg/L.
As phenylpyruvic acid is the common precursor of caffeoyl-CoA and 2- phenylethanol, in order to improve the supply of phenylpyruvic acid, the yield of caffeic acid phenylethyl ester was further increased to 98.56 μ g/L by overexpression of phenylpyruvic acid (S4). The accumulation of intermediate products p-coumaric acid and 2- phenylethanol reached 700.36 μg/L and 188.92 mg/L, and the accumulation of low concentration of p-coumaric acid showed that caffeoyl-CoA was still the limit of the synthesis of caffeic acid phenylethyl ester.
（2）Considering that phenylpyruvic acid is the common precursor of caffeoyl-CoA and 2- phenylethanol, in order to balance the two substrates of caffeoyl-CoA and 2- phenylethanol in the fermentation system, a mixed fermentation system was designed. Firstly, tyrosine was introduced into Escherichia coli to synthesize caffeoyl-CoA, and p-coumaric acid synthesis was enhanced by expressing pal and c4h, and an engineering strain M2 for caffeoyl-CoA synthesis based on Escherichia coli MG1655 was established. Then it was coupled with the constructed Saccharomyces cerevisiae engineering strain S4 to form a mixed-strain fermentation system, and the yield of caffeic acid phenylethyl ester was increased to 272.37 μg/L, which was 2.76 times that of single-strain (S4) fermentation.
（3）In order to further improve the fermentation yield of caffeic acid phenylethyl ester, the fermentation conditions of mixed bacteria were optimized to balance the different needs of Escherichia coli and Saccharomyces cerevisiae for culture environment and nutrition. The results showed that the highest yield of caffeic acid phenylethyl ester reached 667.64 μg/L after 120h of fermentation, when Saccharomyces cerevisiae was cultured for 60h and then inoculated with Escherichia coli according to the inoculation ratio of 1∶1, the fermentation temperature was controlled at 35℃, the rotation speed was 240 rpm, and 1.0 g/L Mg2SO4 and 40 mg/L K2HPO4·3H2O were added. Compared with the initial fermentation, the yield of caffeic acid phenylethyl ester increased by 41.
In this paper, through the optimization of synthetic biotechnology and metabolic pathway, the biosynthesis system of phenylethyl caffeic acid mixed with Saccharomyces cerevisiae INVSc1 and Escherichia coli MG1655 was constructed, which provided a reference for the synthesis of caffeic acid phenylethyl ester by microorganisms.
谷歌
火狐
