Discovery And Engineering Of Key Enzyme In Chemoenzymatic Synthesis Of Chiral Epichlorohydrin From Glycerol

Enantiopure epichlorohydrin(ECH) is recognized as a versatile chiral C3 platform compounds and has practical applications in the synthesis of atorvastatin, β-adrenergic blocking agents, L-carnitine, and trehalostatin. Glycerol, as one of the by-products of biodiesel production, has become an inexpensive and renewable material. Researchers showed a surge of interest in using glycerol as renewable feedstock to produce functional chemicals. In fact, much effort had already been devoted to enantiopure ECH, which include chemical and biological routes. Biological methods for enantiopure ECH preparation have been paid much attention with respect to high enantioselectivity, extensive enzyme sources, low production costs and green environmental protection. Biocatalytic transformations include epoxide hydrolase(EH) mediated kinetic resolution and non-hydrolytic enantioselective ring opening by halohydrin dehalogenases(HHDHs). As an efficient and straightforward access to chiral ECH, biocatalytic asymmetric dehalogenation of prochiral 1,3-dichloro-2-propanol(DCP) is particularly attractive since 100% of the starting material can be converted to product, in contrast to a resolution approach where 50% of raw material is unused. This research was focused on the screening and molecular engineering of novel HHDHs and EHs for the synthesis of chiral ECH.Seven HHDHs were discovered by traditional screening, genome hunting and data mining. In the screening of seven E. coli strains overexpressing recombinant HHDHs, four HHDHs were identified with capability of higher catalytic activities and enantioselectivities for producing chiral ECH and purified to homogeneity. They showed an optimal temperature of 40-50 ?C, while all of them exhibited the highest activity at pH 10.0. All four enzymes were active with most of chlorinated and brominated C2 and C3 vicinal halohydrins tested. They could catalyze the asymmetric dehalogenation of prochiral halohydrin to chiral epoxides and the enantioselectivity that HHDH display when catalyzing ring-closure reactions makes them promising biocatalysts for the production of optically active epoxides. HHDHIs and HHDHTm showed high activity toward ethyl(S)-4-chloro-3-hydroxybutyrate(CHBE).HHDHSg exhibited highest enantioselectivity toward 1,3-DCP than other HHDHs. Asymmetric conversions of 20-100 mM 1,3-DCP with recombinant E. coli overexpressing HHDHSg afforded(S)-ECH in 83.4-84.9% enantiomeric excess(ee) and 56.2-85.9% yield.(R)-ECH was enantioselectively biotransformed by HHDHIs from the 20-100 mM 1,3-DCP with about 38.9-73.5% yield and 30.2-58.6% ee. Further, we explored the potential of HHDHTm to catalyze the synthesis of optically pure(R)-HN, which can be applied as an intermediate in the production of cholesterol-lowering drugs of the statin type. With 100 g/L(S)-CHBE and 50 g/L recombinant E. coli cells harboring the HHDHTm, the conversion and yield reached 92.8% and 89.2%. With 300 g/L of(S)-CHBE and 65 g/L recombinant E. coli cells harboring the HHDHIs, the reaction was formed(R)-HN in 94.5% conversion and 89.3% yield.This research was focused on systhesis of(S)-ECH by kinetic resolution of ECH using epoxide hydrolase. Two new strains were successfully isolated from soil samples, which were proven to be useful in enantioselective resolution of ECH to produce(S)-ECH. Three EHs from the two strains were discovered. AmEH showed the highest enantioselectivity and was purified to homogeneity by a Ni-NTA column. Its basic catalytic properties were investigated. AmEH showed its optimum pH and temperature at 8.0 and 35 °C, respectively. Moreover, this AmEH showed broad substrates specificity toward epoxides. Nevertheless, among these substrates there are significant differences in activity and enantioselectivity. The purified AmEH showed an enantioselective hydrolysis toward monosubstituted epoxides at C-1 position with bulky ring such as styrene oxide and benzyl glycidyl ether and with aliphatic chains such as ECH. There were marked differences in enantioselectivity with methyl position, a general trend can be found that the ee value increases as the methyl on the phenyl ring is shifted from the para- to the ortho-position. Enantiopure(S)-ECH could be obtained with enantiomeric excess(ee) of > 99% and yield of 21.5%(E value of 12.9) from 64 mM(R,S)-ECH.The site-saturation mutagenesis technique was employed to improve the enantioselectivity of HHDHs toward 1,3-DCP. The three-dimensional homology model of HHDHs were generated, and series key residues were suggested to play critical roles in enantioselectivity by modeling and docking. The saturation mutagenesis libraries were screened using the high-throughput colorimetric activity assay on a 96-well plate format and chiral GC. The best HheC mutant(P175S/W249P) displayed greatly improved the ee of(S)-ECH from 5.2% to 95.3% in the catalyzed dehalogenation of 1,3-DCP at pH 8.0. The best HHDHSg mutant(V137I) displayed greatly improved the ee of(S)-ECH from 84.6% to 91.8%. The HHDHTm mutant(N183L) displayed greatly improved the ee of(S)-ECH from 40.2% to 70.2%. Modeling and docking studies demonstrated that the enhanced enantioselectivity is caused by the decreasing steric hindrance of one of halogen-bearing carbon atom of 1,3-DCP, resulting in asymmetric dehalogenation. Reactions of 20-100 mM 1,3-DCP with HheC mutant(P175S/W249P) afforded(S)-ECH in 90.4-92.7% ee and 58.0-91.4% yield. Reactions of 20-100 mM 1,3-DCP with HHDHSg mutant(V137I) afforded(S)-ECH in 87.2-91.5% ee and 48.2-90.7% yield.Subsequently, to improve the enantioselectivity and activity of AmEH, we used a protein engineering approach that involves building a homology model of AmEH, library generation based on the model, screening for AmEH activity and higher enantioselectivity. The best mutant VDF(W182F/S207V/N240D) has 7-fold enhanced enantioselectivity toward racemic ECH, with the enantiomeric ratio value(E value) preferring(R)-ECH increased from 12.9 of wild-type to 90.0, as well as 1.7-fold improved activity. Modeling and docking studies demonstrated that the enhanced enantioselectivity is caused by increasing the through-space distance, d, between the attacking O-atom of Asp181 and the epoxide C-atom. In the mutant VDF, the △d value was increased from 0.3 ? to 1.1 ?. Furthermore, we successfully applied the created recombinant E. coli whole cells expressing variant VDF in the kinetic resolution of racemic ECH. Enantiopure(S)-ECH could be obtained with an enantiopurity of > 99% ee and a yield of 40.5-45.8% from 75- 450 mM racemic ECH, which is better than other reported EHs.To increase the optical purity of chiral ECH, Two-enzyme systems were constructed and applied for the efficient synthesis of chiral ECH. Firstly, HHDH was coexpressed with EH, respectively on two different styles. Higher ee of(S)-ECH was observed by using two-plasmids coexpression system, and higher ee of(R)-ECH was obtained by using E. coli harboring a coexpression plasmid. Secondly, a practical, two-pot, two-step catalytic method is successfully constructed for conversion of 1,3-DCP to chiral ECH in >99% ee. This suggests that the stepwise procedure is more effective than “one pot” conversion, since higher optical purity of chiral ECH was obtained. The study on immobilized bienzyme system has been carried out on the basis of immobilized HHDH and immobilized EH. Biosynthesis of chiral ECH with immobilized enzyme coupled HHDH and EH allowed us to re-use the enzyme for an extended period of time, enables easier separation of the catalyst from the product and achieve continuous operation. It also shows good potential for synthesis of other chiral epoxides and halohydrins.