Screening Of Key Enzymes In Cinnamyl Alcohol Biosynthesis And The Preparation Of Cinnamyl Alcohol

Cinnamyl alcohol is a kind of natural plant secondary metabolite and it has good market demand in industrial and medical fields.At present,the preparation of cinnamyl alcohol is mainly by extraction and chemical synthesis methods.Relatively,the extraction method is costly and the separation is difficult.Although the method of chemical synthesis of is mature,there are problems such as severe reaction conditions and large environmental burden.Biological method is green,environmental protection,low cost and high specificity,which is a promising way to prepare cinnamyl alcohol.Recently,there are few reports on the biosynthesis of cinnamyl alcohol,and the research is still in its infancy.This paper focused on the whole-cell catalytic transformation of L-phenylalanine to cinnamyl alcohol.Through gene screening,analysis of enzymatic properties and comparison of catalytic activity,the best genes were selected and applied to the construction of recombinant strains.By combining with the optimization of whole-cell catalytic conditions,analysis of limiting factors and two-phase catalytic strategies,the efficient preparation of cinnamyl alcohol from L-phenylalanine was achieved.The main research results are as follows:（1）According to the annotation information of the Populus trichocarpa genome,Ptrpal genes were excavated and screened.The enzymatic properties of different recombinant PtrPAL were studied and they were applied to the whole-cell catalytic synthesis of cinnamic acid.Through bioinformatics analysis,PtrPAL3 and PtrPAL4 were selected from the five PtrPAL for further study.PtrPAL3 and PtrPAL4 were similar in enzymatic properties.The optimum temperature was 55℃ and 60℃,respectively.The optimum pH was 8.5 and 8.0,respectively.PtrPAL3（3.36 U/mg）and PtrPAL4（3.38 U/mg）exhibited similar PAL activity and both have weak tyrosine ammonia lyase activity.Kinetic analysis showed that PtrPAL3 has a higher affinity for L-phenylalanine,and the inhibition of cinnamic acid is weaker.The E.coli BLP3overexpressing the Ptrpal3 was applied to produce cinnamic acid from L-phenylalanine and104.97 mM（15.55 g/L）cinnamic acid was synthesized by whole cell catalysis after 7 h..（2）Based on plant monolignol biosynthesis pathway,excavating and screening Ptr4CL and PtrCCR from P.trichocarpa,and using them to construct cinnamyl alcohol biosynthesis pathway in Escherichia coli.An efficient whole cell catalytic synthesis system for cinnamyl alcohol was established.First,through bioinformatics analysis,Ptr4CL4,Ptr4CL5 and Ptr4CL7were selected from 17 Ptr4CL for further study.Among them,only Ptr4CL4 and Ptr4CL5 can be heterologously expressed in E.coli.Enzymatic analysis showed that the optimum pH of Ptr4CL4 and Ptr4CL5 from P.trichocarpa was 7.0 and 7.5 respectively,and the optimum temperature was 30℃.The V_max of Ptr4CL5 to cinnamic acid is 2.38 times that of Ptr4CL4,and it is more suitable for the construction of cinnamyl alcohol biosynthesis pathway.Then,excavating and screening Ptrccr2 and Ptrccr17 as candidate genes,which were respectively co-expressed in E.coli with Ptr4cl5.The whole-cell catalysis results showed that only PtrCCR2has cinnamoyl-CoA catalytic activity.Finally,the reaction conditions of whole-cell catalytic reduction of cinnamic acid to cinnamyl alcohol were optimized.After reaction at 30℃,pH 7.5,OD₆₀₀ 30,glucose 17 mM for 3 h,4.70 mM cinnamyl alcohol was synthesized from 8.5 mM cinnamic acid,and the selectivity was 90.5%.（3）The restrictive factors of cinnamyl alcohol biosynthesis in the microbial reduction pathway were explored,and the efficient biosynthesis system of cinnamyl alcohol was established.E.coli BLCS-1,co-expressing Nicar and Bssfp,had higher cinnamyl alcohol biosynthesis capacity.The maximum titer of cinnamyl alcohol under the optimal conditions was7.43 mM with a yield of 92.0%.However,severe product inhibition is a key factor limiting the synthesis of cinnamyl alcohol by E.coli BLCS-1.The construction of water/dibutyl phthalate two-phase system effectively removed the product inhibition.After reacting at a phase volume ratio of 0.4 and an initial aqueous cinnamic acid concentration of 17.4 mM for 6 h,cinnamic acid was completely converted and the output increased by 106.4%compared with single-phase catalysis.The synthesis of cinnamic acid by E.coli BLP3 was coupled with the reduction of cinnamic acid in two-phase system.0.049 mmol cinnamic alcohol was successfully synthesized from 0.06 mmol L-phenylalanine,and the yield was 81.7%.In addition,this process could concentrate and separate cinnamyl alcohol in situ.At the end of the reaction,the concentration of cinnamyl alcohol in the organic phase reaches was 38.23 mM（5.13 g/L）and the purity was92.9%.