Multi-enzymatic Synthesis Of Chiral β-hydroxynitriles With Oxidoreductase And Halohydrin Dehalogenase

Multi-enzymatic biosynthesis is called the third wave of biocatalysis, either ex-vivo or in-vivo. Multi-enzymatic processes have been wildly used for the asymmetric synthesis of chiral chemicals, included cascade reaction and coupling reaction, such as coenzyme regeneration. Chiral β-hydroxynitriles are important building blocks in the pharmaceutical and chemical industry. At present, two major biocatalysis methods for prepration of chiral β-hydroxynitriles are kinetic resolution and asymmetric synthesis. Thereinto, cascade reaction by oxidoreductase and halohydrin dehalogenase, which catalyzed cascade directly from cheap prochiral a-halo ketones to chiral β-hydroxynitriles in 100% yield, have obvious advantages. However, these relative studies were all performed ex-vivo, and different optimal reaction conditions of enzymes resulted in the low conversion efficiency of these processes. Furthermore, the ex-vivo multi-enzymatic biosynthesis needed artificial addition of expensive co-factors, NAD(P)+, increasing the cost of reaction. And also, respective expression and islation of enzymes leaded to complicated process.As no need to respectively express and isolate the enzyme catalysts, and easily achieved coenzyme regeneration in site, the operation process can be further facilitated, and the cost of reaction can be further reduced. Moreover, internal environment and homeostasis of cells can stabilize enzymes, leading to that different enzymes can perform their functions in the same cell. Therefore, the in-vivo multi-enzymatic biosynthesis can make up for the shortcoming of the ex-vivo multi-enzymatic biosynthesis, further improve the efficiency of biocatalytic cascade reaction and coupling reaction. In this work, one bacterial multi-enzymatic process for asymmetric synthesis of chiral β-hydroxynitriles from α-halo ketones were developed, through co-expression of oxidoreductase and halohydrin dehalogenase in Escherichia coli. These processes have easy operation, cheap substrate,100% conversion rate and >99% ee value. Coupling reaction for coenzyme regeneration was introduced to enhance the carbonyl reduction reaction, and optimization of coexpression strategies was used to blance the enzyme activities. Meanwhile, the inhibition mechanism of HHDH by COBE was clarified. The results showed that COBE bound to the active center of HHDH via the formation of hydrogen bonds with the OH groups of S132 and Y145. This inhibition by COBE was found to be competitive reversible inhibition. Based on the inhibition mechanism, mutagenesis and screening were performed and the mutant F136V/W249F was successfully found to relieve the activity inhibition of HHDH by COBE. The half inhibiting concentration of mutant F136V/W249F was found to be 20-fold higher than wild-type HHDH. In addition, a new multi-enzymatic biosynthesis route, via three consecutive biocatalytic steps, was established for preparation of the key intermediate of atorvastatin, tert-butyl (3R,5R) 6-cyano-3,5-dihydroxyhexanoate.First, two double-enzymatic systems in-vivo (AdhR-HheC, AdhS-HheC) were established for the one-pot preparation of ethyl (R)-4-cyano-3-hydroxybutyrate ((R)-HN), ethyl 4-chloroacetoacetate (COBE) as model substrate, which combining opposite stereospecificity of alcohol dehydrogenases (AdhR and AdhS) from Lactobacillus kefiri DSM 20587 with halohydrin dehalogenase HheC. The two double-enzymatic systems have opposite stereospecificity. On that basis, AdhR, GdhBS and HheC were coupled to establish multi-enzymatic system in-vivo. Meanwhile, AdhS, FdhCB and HheC were coupled to establish another multi-enzymatic system in-vivo. Different co-expression strategies, such as poly cistron on single vector and single cistron on two vectors in E. coli were studied. Multi-enzymatic equilibrium expression was achieved. Compared with double-enzymatic systems, multi-enzymatic systems enhanced the carbonyl reduction reaction. And then, halohydrin dehalogenase HHDH(F136V/W249F) was used to replace HheC in two multi-enzymatic systems, leading to improvement of the dehalogenation cyanide reaction. Engineering bacteria Eco_RBH and Eco_SFH were established. The catalysis effiency of first step reaction was 195 U/L, and the catalysis effiency of second step reaction was 184 U/L by Eco_RBH. The catalysis effiency of first step reaction was 18.1 U/L, and the catalysis effiency of second step reaction was 107 U/L by Eco_SFH. The multi-enzymatic processes in-vivo provide the higher stability of the biocatalysts in the cell environment, compare with the extracellular environment, and can be reusable. The substrate spectra of HheC, HHDH, AdhR and AdhS were investigated. Furthermore, two chiral β-hydroxynitriles were achieved by Eco_RBH, (R)-HN and (S)-3-hydroxy phenyl propionitrile with 99% ee value. Two chiral β-hydroxynitriles were achieved by Eco_SFH, (S)-HN with 60.2% ee value and (S)-3-hydroxy phenyl propionitrile with 93.2% ee value.Second, halohydrin dehalogenase HHDH was cloned and over-expressed. The specific activity for (S)-CHBE was 3408 U/g, and for (R)-CHBE was 3712 U/g. However the activity of HHDH can be serious inhibited by COBE. Half inhibiting concentration of COBE to HHDH is 10 μM. Substrate inhibition kinetics analysis was performed, illustrating that the inhibition of HHDH by COBE is competitive reversible inhibition. Molecule simulation analysis elucidated that the inhibitor COBE can binding to the enzymatic active site by formation of halogen bonds with the OH groups of S132, Y145. Moreover, binding energy calculation was performed to further support the results. Site saturation mutagenesis on the residues around the active site and the entrance of access tunnel was used to identify two key residues, F136 and W249, which can affect the binding of COBE for the enzyme. Furthermore, small focused mutagenesis analysis on the two key sites were performed, leading to achieving one outstanding variant mutant F136V/W249F, which successful relieving the activity inhibition of HHDH by COBE. Half inhibiting concentration of COBE to mutant F136V/W249F is 200 μM,20 times more than HHDH (wild-type). Third, optimization of fermentation medium for Eco_RBH was performed in flasks, obtaining the industrial grade medium to replace LB medium. And expression conditions were optimized in the industrial grade medium. The optimized catalysis effiency of first step reaction was 857.9 U/L, and the optimized catalysis effiency of second step reaction was 932.3 U/L, about 4 times more than catalysis effiency in LB medium. Furthermore, high-density fermentation of Eco_RBH was studied in 15 L bioreactor. By means of fed-batch fermentation, dry cell weight reached 20.44 g/L. And the catalysis effiency of first step reaction reached 8620 U/L, and the catalysis effiency of second step reaction reached 8587 U/L, about 10 times more than best effiency in flasks. And then, the influences of temperature and pH on catalysis reaction by Eco_RBH were investigated. The results showed that 30 ℃ and pH 7.0 was most appropriately. The investigation of catalysis reaction by Eco_RBH with higher substrate concentration showed that 100 mM COBE can be completely transformed within 2 h,100 mM 2-chloroacetophenone can be completely transformed within 1 h, by Eco_RBH cells achieved by high-density fermentation (load cells:20 g dry cells/L).Fourth, engineering bacteria BL21_pACYCDuet-AdhR was used to prepare tert-butyl (S) 6-chloro-5-hydroxy-3-oxohexanoate, with tert-butyl 6-chloro-3,5-dioxohexanoate as substrate. The ee value of product was >99%. The catalysis effiency was about 12 U/L. Carbonyl reductase from Candidia magnoliae and GdhBS were co-expressed to establish engineering bacteria BL21_pACYCDuet-CB, which was successfully used to prepare tert-butyl (3R,5S) 6-chloro-3,5-dihydroxyhexanoate, with tert-butyl (S) 6-chloro-5-hydroxy-3-oxohexanoate as substrate. The de value of product was about 98.2%. This catalysis effiency was about 35 U/L. Engineering bacteria Eco_RCB was established by co-expression of AdhR, Cr and GdhBS, and successful used for preparation of tert-butyl (3R,5S) 6-chloro-3,5-dihydroxyhexanoate, the catalysis effiency reached about 11 U/L. Meanwhile, engineering bacteria Eco_H was established with HHDH, and successfully used for preparation of the key intermediate of atorvastatin, tert-butyl (3R,5R) 6-cyano-3,5-dihydroxyhexanoate, with tert-butyl (3R,5S) 6-chloro-3,5-dihydroxyhexanoate as substrate. This catalysis effiency was about 21.4 U/L.