Synthesis Of Statin Side Chain Precursor By Using Enzymatic Cascade Reaction

Statins are groups of cholesterol lowering drugs,which are clinically used to reduce the risk of coronary heart diseases.All these statin drugs,either natural products or synthetic compounds,share a common pharmacophore of syn-3,5-dihydroxy carboxylate side chain,which is essential for statin's biological activity.Generally,there are three chemo-enzymatic routes to synthesize the key 5-dihydroxy carboxylate side chain precursors.Among these routes,route III starting from asymmetric aldol condensation by a 2-deoxyribose-5-phosphate aldolase(DERA)has attracted enormous attention recently due to simpler and cheaper starting chemicals,fewer synthesis steps and higher atom economy.In this study,a novel aldolase LbDERA was discovered from Lactobacillus brevis,which showed outstanding activity and substrate tolerance for catalyzing the condensation of acetaldehyde and chloroacetaldehyde into(3R,5S)-6-chloro-2,4,6-trideoxyhexapyranoside[CTeHP];a new NADPH-dependent dehydrogenase LeADH was discovered from Lodderomyces elongisporus with an obvious activity for CTeHP oxidation;an NADPH oxidase SmNOXv193R/V194H was used for effective cofactor recycling.The catalytic efficiency of LbDERA and LeADH were further improved by protein engineering.Finally,a high efficiency and green bioprocess was constructed for the preparation of statin side chain precursor.Firstly,a screening for new microbial DERAs with the ability to catalyze the sequential aldol condensation of acetaldehyde and chloroacetaldehyde was carried out among various bacterial strains deposited in our laboratory.Four potential aldolases were cloned and heterologously expressed in E.coli.Among them,LbDERA from Lactobacillus brevis was selected for further study due to its high activity and outstanding tolerance against aldehyde substrates.The LbDERA showed the highest activity at pH 6.0-7.0 and 40?,and 75%of the maximum activity was maintained at 30?.The LbDERA showed very good thermostability,with half-lives of 24 days,12 days and 5.5 hours at 30?,40? and 50?,respectively.Substrates tolerability is a key factor for DERA application,and the half-lives of LbDERA in 300 mM chloroacetaldehyde or acetaldehyde were 198 and 37.3 min,respectively,which are 7.4-fold and 2-fold higher than those of EcDERA from Escherichia coli.Secondly,the crystal structure of LbDERA was determined at 1.95 A resolution in the space group P41212 by a molecular replacement method.The resultant structure contains two subunits and each subunit folds into an(?/?)8-barrel similar to the other class I aldolases.The dimeric structure of LbDERA is quite similar to that of other DERAs,but different from that of EcDERA.However,the interface area of LbDERA subunits is comparable to those of hyperthermophilic DERAs,but much larger than that of EcDERA.The hydrophobic clusters between two subunits composed of hydrophobic residues Phe68,Pro69,Leu70 and Phe163 are considered as important factors for larger inter-subunit hydrophobic interactions and mutations at these sites led to degressive acetaldehyde tolerability of the protein.In the third part,the aldehyde tolerability of LbDERA was enhanced through improving the global stability of LbDERA by consensus sequence approach.Among 13 mutants out of 229 positions,LbDERAE78K showed a significantly improved stability.Its melting temperature(Tm)increases from 56.0?(LbDERA)to 61.9? as determined by circular dichroism spectroscopy,and the half-life in the presence of 300 mM acetaldehyde was also remarkably enhanced from 37.3 min(wild type)to 85.7 min.The crystal structure of LbDERAE78K was determined at 2.17 A resolution by a molecular replacement method.It was found that the substitution E78K gives two additional hydrogen bonds and one salt bridge with adjacent residues:the side chain of Lys78 forms hydrogen bonds with Gly71 and Val96 at distances of 2.8 and 2.9 A,respectively,as well as one salt bridge with Asp113 at a distance of 2.6 A.The newly introduced salt bridge and hydrogen bonds should contribute to the overall stability of LbDERAE78K.In some negative mutants with obviously poor aldehyde tolerability,the hydrogen bonds or salt bridges were destroyed by the newly introduced mutations,implying that the intramolecular hydrogen bond and salt bridge play an important role for maintenance of the aldehyde tolerability.With the purified LbDERAE78K as catalyst,the substrate concentrations could be further increased up to 0.7/1.4 M,resulting in 84%yield of CTeHP at 3.0 h.Using E.coli cells harboring LbDERAE78K as catalyst without changing any of the other conditions,the catalyst loading was lowered to 7.5 g/L?In the fourth part,we performed a screening using dehydrogenase toolboxs developed in our laboratory to identify a novel NAD(P)+-dependent CTeHP oxidation enzyme.Among the 30 dehydrogenases in one of the toolboxes,an enzyme marked as LeADH isolated from Lodderomyces longisporus,showed the highest CTeHP oxidation activity and was therefore selected for further study.Though the activity of LeADH was the highest among the tested dehydrogenases,the specific activity of wild-type LeADH was only 2.1 U/mgprot,which is still relatively low since CTeHP is an artificial substrate and does not exist in the nature.A semi-rational strategy was applied to further improve the catalytic activity of LeADH.The simulated structure of LeADH was constructed and the substrate CTeHP was then docked into the catalytic center of LeADH.A triple mutant LeADH187F/N235H/P236H increased the activity sharply up to 26.5 U/mgprot,which is an 11.7-fold improvement compared with the wild-type LeADH.Finally,an NAD(P)H oxidase SmNOXv193R/V194H from Streptococcus mutans was introduced for the oxidation of reductive cofactor NADPH using molecular oxygen as a cosubstrate.Then,two E.coli transformant strains(pETDuet-nox-adh and pETDuet-adh-nox)were constructed based on different co-expression orders.A higher productivity of CTeHL was observed with the E.coli cells possessing pETDuet-nox-adh plasmid.When the substrate concentration was 300 mM,a conversion of 95%was achieved within 10 h.In contrast to chemical oxidation of CTeHP with excessive phosphoric acid and calcium hypochlorite,the aforementioned bio-oxidation process uses oxygen as a cosubstrate and produces water as a by-product,which representa a green approach for oxidation of statin side-chain intermediates.