Construction Of Biosynthesis Of P-Coumaric Abcid In E. Coli Based On Phenylalnine Ammonia-Lyase Pathway

P-Coumaric acid as bioactive phenolic acid serves as a synthetic precursor for various phenylpropanoid compounds widely used in the nutritional,pharmaceutical,materials,and chemical industries.Microbial production of p-coumaric acid could utilize L-phenylalanine（PAL branch）or L-tyrosine（TAL branch）as precursors.Currently,the highest production of p-coumaric acid was achieved by the synergistic PAL and TAL pathways in engineered Saccharomyces cerevisiae.In Escherichia coli,the biosynthesis ofp-coumaric acid previously focused on the TAL branch using tyrosine as the precursor.In this study,an engineered strain capable of de novo production of p-coumaric acid via the PAL pathway was constructed in the L-phenylalanine-producing E.coli.The specific results were as follows.（1）Screen and evaluation of phenylalanine ammonia-lyase and trans-cinnamate 4hydroxylases in the PAL branch for p-coumaric acid biosynthesis.Among the nine phenylalanine ammonia-lyase（PAL）candidates selected,three candidates,including the PALs from Rhodosporidium glutinis（RgPAL）,Arabidopsis thaliana（AtPAL2）,and Anabaena variabilis（AvPAL）of 3.11,1.88,and 1.60 U·mg^-1,respectively.For this,we selected AtPAL2,as it was more soluble than other PALs in E.coli.In contrast,the C4H enzyme from Lycoris aurea（LauC4H）among five candidates trans-cinnamate 4-hydroxylases（C4H）was selected based on its best soluble expression and enzyme activity.Furthermore,"GSTSSGSG" fusion expression strategy was used to optimize the connection between the reductase partner（AtATR2）and LauC4H,resulting in 13.48 μM p-coumaric acid.In addition,we tested the protein expression conditions and catalytic capacity of AtPAL2 and LauC4H in vitro.The best soluble expression of AtPAL2 and LauC4H were determined as follows:0.4mM IPTG was added into the culture when the OD600 of cells reached 0.8,and enzymes were induced at 25℃or 16℃ for 15 h;the best conditions for testing the catalytic capacity of AtPAL2 and LauC4H were at 50℃,pH=9 and 25℃,pH=7.5,respectively.（2）Protein engineering of the rate-limiting enzyme in the PAL branch for p-coumaric acid biosynthesis.The L-phenylalanine was converted with equal amounts of purified AtPAL2 and LauC4H enzymes in vitro,and the p-CA was determined by the liquid mass combination（HPLC-MS）and high performance liquid chromatography（HPLC）,suggesting that the p-CA synthesis pathway was achieved.When the LauC4H concentration was doubled,the initial reaction rate of L-phenylalanine to p-coumaric acid was 1.9-fold higher than that of the control.Also the k_cat/K_m of AtPAL2 was 22-fold higher than that of LauC4H,which collectively indicated LauC4H as the rate-limiting enzyme in the p-CA synthesis pathway.11 candidate residues of LauC4H were screened by the semi-rational design,in which LauC4H^V91A,LauC4H^V278S and LauC4H^S187T resulted inp-coumaric acid of 12.13 μM,11.65 μM and 15.37μM,respectively,which were 42.1%,36.4%and 80%higher than LauC4Hwt.In contrast,the double and triple mutants with random combinations of three mutants showed poor effect.Finally,LauC4H^S187T was subjected to six N-terminal modifications.It can improve in the folding and localization of LauC4H under the ’KKK’ modification,that the yield of pcoumaric acid was increased by 2.88-fold compared to the original strain.（3）Construction and optimization of the de novo biosynthetic pathway for p-coumaric acid in E.coli.Plasmid pEMAL7 was constructed and transformed into E.coli JNYPQ-5.The obtained strain JNYPQ-12 synthesized p-coumaric acid using glucose.The expression levels of AtPAL2 and LauC4H were adjusted by promoters with different strength.When AtPAL2 and LauC4H were combined with PT5 and Ptac,respectively,and fermented at 25℃,the yield ofp-coumaric acid was 15.7 μM.It was found that NADPH plays a key role in the production of p-CA.To enhance the supply of the cofactor NADPH,the genes zwf and gnd were overexpressed in the engineered strain E.coli JNYPQ-16.The NADP+/NADPH ratio was 83.9%higher than JNYPQ-12,and the titer of p-coumaric acid was 38.4 μM in shake flask.（4）Fermentation capacities of the engineered strains were evaluated in a 5 L fermenter.Improving the ability of producing L-phenylalanine in chassis bacteria by optimizing the temperature and the concentration of yeast powder in the culture medium.When the yeast powder concentration was increased to 5 g/L,the bacteria first grew at 33.5℃ to an OD₆₁₀ of about 20 and then fermented at 38.5℃,the production,a yield on glucose and the intensity of L-phenylalanine production increased to 64.9 g/L,0.259 g·g^-1 and 1.47 g/L/h,respectively.The effect of IPTG addition timing on p-coumaric acid production of E.coli JNYPQ-16 was initially evaluated.It was showed that p-CA production reached 378.6 μM with an average production intensity of 1.29 mg/L/h when IPTG was added at a OD₆₁₀ of 10.Overall,the seleted AtPAL2 and LauC4H from candidate enzymes had good solube expression and catalytic activities in E.coli by protein engineering.Additionally,the PAL branch was inserted in the L-phenylalanine producing strain to achieve the de novo accumulation ofp-coumaric acid,which could be used for improving the microbial synthesis of p-coumaric acid and related phenylpropanes.Protein engineering of C4H enzyme may also provide insight into enhancing hydroxylation reactions of p450 on aromatic hydrocarbons.