Epoxy-norbornane Production From Norbornene Using Whole-cell Catalysts

Epoxy-norbornane（EPO-NBE）is a synthetic intermediate of new heterocyclic compounds,which has been employed widespreadly in the construction of various heterocyclic systems.EPO-NBE is mainly prepared from norbornene（NBE）with the complicated transition metal complexes as catalysts.The industrial production of EPO-NBE still lacks an efficient and economic biosynthesis method.In this study,an engineered H₂O₂-dependent cytochrome P450peroxygenase（PpCytP）was developed to realize EPO-NBE bio-preparation;Then,the crystal structure of PpCytP（PDB code:7X53）was resolved for the first time,and the epoxidation activity of PpCytP was improved by protein engineering;In addition,the optimization of transformation conditions and fed-batch of H₂O₂ were employed to further increase the catalytic efficiency.The main study results are described as follows:（1）Design and verification of the two-phase system for EPO-NBE bio-preparation.Cytochrome P450 enzyme（PpCytP）from Pseudomonas putida was selected as the parent enzyme;Then,the expression and transformation conditions were optimized;Furthermore,the formation of EPO-NBE was detected by MS and NMR,which confirmed the feasibility of this transformation system.（2）Protein crystallization and mechanism analysis.The crystal structure of PpCytP was obtained for the first time（PDB code:7X53）.On the one hand,MD simulations of PpCytP^WT-NBE docking model suggested that the incompatible substrate-binding pocket led to the overlong catalytic distance;On the other hand,the mechanism analysis of PpCytP-catalyzed epoxidation with QM/MM calculations,indicated that the key to improving the epoxidation activity of PpCytP was how to decrease the energy barrier related to the iron-alkoxy radical complex formation,and which was mainly affected by the catalytic distance.（3）Improving epoxidation activity of PpCytP by protein engineering.The strategy was designed to reconstruct the substrate-binding pocket compatible with NBE,and the optimal mutant PpCytP^NPV(PpCytP^{A250V/T255E/S296V/A297P/P298N})and the corresponding recombinant strain E.coli 05 were obtained;Compared to PpCytP^WT,the enzyme activity and k_cat/K_M value of PpCytP^NPV variant increased by 7.54-fold and 23.33-fold,respectively.Besides,the whole-cell transformation with strain E.coli 05 led to the EPO-NBE titer of 57.83 g·L^-1 with 49.43%conversion,which exhibited a 6.40-fold increase compared to that of starting strain;MD and QM/MM calculations of PpCytP^NPV-NBE docking model indicated that:（i）The local hydrophobic“fence”increased the stability of the whole structure of PpCytP,shortened the catalytic distance,and improved the affinity for NBE;（ii）The energy barrier related to the iron-alkoxy radical complex formation decreased by 16.11 kcal·mol^-1,suggesting that the Cpd I intermediate was more apt to attack NBE to form EPO-NBE;（iii）The energy barrier related to the protonation of Cpd 0 decreased by 6.13 kcal·mol^-1,confirming the deduction that Glu255participated in proton transfer and thus constructing the efficient acid-alcohol pairs system.（4）Production of EPO-NBE at fermenter-scale.Firstly,fed-batch of H₂O₂ was adopted to weaken the damage of H₂O₂ on PpCytP;Then,the transformation system was amplified to fermenter level in order to confirm the industrial application potential of strain E.coli 05.In 30L fermenter,the titer of EPO-NBE reached to 77.59 g·L^-1 with 66.32%conversion after 72 h.