Construction Of Engineering Strain For De Novo Synthesis Of Phenylethanol Based On Escherichia Coli

2-Phenylethanol(2-PE)is an aromatic alcohol substance with a lasting and elegant rose aroma,which is widely used in the food,cosmetic and tobacco industries.Natural 2-PE mainly exists in the flowers and fruits of flowering plants and the essential oils extracted from them.Due to its low concentration extracted from plants,it is difficult to extract.Therefore,2-PE is mainly synthesized by chemical methods at present.Although the cost of chemical synthesis is convenient,there are disadvantages such as serious environmental pollution and toxic by-products,which do not in line with the green consumption concept of modern consumers advocating"natural,safe and environmentally friendly".The fragrance prepared by microbial transformation,known as natural equivalent fragrance,meets the safety standards of"green production",and its configuration and chirality are single,which contributes to the improvement of fragrance quality.The synthesis of 2-PE by microorganisms is an important development direction in the preparation of natural flavors in recent years.In this paper,L-phenylalanine(L-Phe)high-producing Escherichia coli M4 was used as the host to construct the 2-PE biosynthesis pathway.The main research contents are as follows:(1)Study on the introduction of styrene-derived pathway in E.coliThe novel alternative pathway for 2-PE synthesis is a styrene-derived pathway,which was introduced into the E.coli by CRISPR/Cas9.This pathway starts from intracellular L-Phe and involves four heterologous enzymes,encoded by pal2,fdc1,sty AB and sty C.Four key genes were integrated into the E.coli M4 genome,and plasmid-free engineering strains E.coli BYC2were successfully obtained.However,only a trace amount of 2-PE was detected in the shaking flask fermentation.It is indicating that the styrene-derived pathway did not work well and not suitable for constructing 2-PE biosynthesis engineered strain.(2)Study on the introduction of Ehrlich pathway in E.coliThe Ehrlich pathway also starts from intracellular L-Phe,involving phenylpyruvate decarboxylase encoded by ARO10 gene and endogenous alcohol dehydrogenase,was introduced into E.coli M4.Different recombinant plasmids were successfully constructed based on the principle of POE-PCR and further transformed into E.coli M4,and the corresponding transformant named as E.coli BYC3(E.coli M4;p Trc99a-ARO10),E.coli BYC4(E.coli M4;p STV28-ARO10),E.coli BYC5(E.coli M4;p WSK29-ARO10)and ARO10gene codon-optimized engineered strains E.coli BYC6(E.coli M4;p Trc99a-ARO10*),respectively.The results of shake flask fermentation showed that E.coli BYC3 containing the high-copy plasmid exhibited the highest yield.After 72 h of fermentation,the production of 2-PE achieved 761.20±20.03 mg/L and the yield reached 0.031 g/g glucose.Surprisingly,E.coli BYC6 carried codon-optimization AR010 gene exhibited the lowest 2-PE production,indicating that the codon-optimization might decrease the catalytic activity or structural stability of phenylpyruvate decarboxylase which suggested that it was not suitable for 2-PE biosynthesis.(3)Enhancement of the Ehrlich pathwayThe engineering strain E.coli BYC7(E.coli M4;?fea B;p Trc99a-ARO10)was constructed by knocking out the fea B gene in the competitive metabolic pathway.The concentration of 2-PE reached 1041.03±34.31 mg/L,which was increase by 35%.In addition,it showed that the engineering strain E.coli BYC7 had the best performance to biosynthesize 2-PE(2.15±0.021g/L)by adding 0.5 mmol/L IPTG.Furthermore,the crr gene was selected and knocked out in the genome to improve the supply of the precursor PEP and engineered strain E.coli BYC8(E.coli M4;?fea B;?crr;p Trc99a-ARO10)was obtained.Compared with start strain E.coli BYC3,the production of 2-PE increased by 20%(reached 2.12±0.018 g/L)under the 0.5 mmol/L IPTG induction.To reduce the usage of antibiotics and inducers in the process of strain incubation,a plasmid-free engineering strain E.coli BYC9(E.coli M4;?fea B;?crr;yee P::P_trc-ARO10)was reconstructed based on the Ehrlich pathway.The production of 2-PE reached 1.61±0.104 g/L and the glucose conversion efficiency reached 0.071 g/g,which was the highest level of de novo synthesis of 2-PE by E.coli so far.(4)Study on 2-PE fermentation in 5 L fermenterThe preliminary exploration of the biosynthesis of 2-phenylethanol in the 5 L fermenter confirmed that the engineering strain E.coli BYC9 has the preconditions for high-yield 2-PE under the condition of continuous sugar supplementation;the final production of 2-PE is about1.8 g/L under the batch sugar supplementation strategy,which laid a theoretical foundation for subsequent industrial production and needed further research.