Engineering Of Lipase Lip From Thermomyces Lanuginosus For Efficient Biosynthesis Of Chiral Intermediate Of Pregabalin

Pregabalin[(S)-3-(aminomethyl)-5-methylhexanoic acid]is a lipophilic derivative of the inhibitory neurotransmitter ?-aminobutyric acid(GABA).Due to its good curative effect on neuropathic pain and epilepsy,Pregabalin has been one of the fastest growing and block-buster drugs.Since(S)-Pregabalin is significantly more potent than the(R)-enantiomer and racemic-Pregabalin,more interest have been focused on the asymmetric synthesis of optical pure(S)-Pregabalin domestically as well as globally.The most successful scalable chemoenzymatic strategy for(S)-Pregabalin involved lipase-catalyzed resolution of 2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester(CNDE)to(3S)-2-carboxyethyl-3-cyano-5-methylhexanoic acid,which was converted to Pregabalin after decarboxylation,hydrolysis and hydrogenation.The biocatalyst involved in Pfizer's chemoenzymatic manufacturing process is Lipolase(?),a commercial available Thermomyces lanuginosus lipase(TLL)supplied by Novozymes,there's no other robust biocatalyst used in industrial production of Pregabalin.Accordingly,development of novel biocatalyst with high process efficiency will be of great significance and value for Pregabalin production.In this thesis,we focused on the development a novel robust biocatalyst for kinetic resolution of CNDE.The gene mining and cloning,heterologous expression,molecular engineering of the biocatalyst,and its application in efficient biosynthesis of chiral intermediate of Pregabalin were investigated in details.The amino-acid sequence of known TLL,the robust biocatalyst used in industrial production of Pregabalin,was used to search for homologues in GenBank.Lipase LN,the amino acid sequence with 78%identity to that of TLL,was discovered by gene mining for lipase.The lip gene(GenBank accession no.KC479729),with 99%identity to that of In,was cloned from T.lanuginosus DSM 1065 by RT-PCR using the primers that designed from the available In sequence.The lip gene was cloned into the expression vector pET-28b,and overexpressed in biologically active in Escherichia coli BL21(DE3).The kinetic resolution of CNDE by recombinant E.coli cell overexpressing Lip suggested that the enzyme showed excellent enantioselectivity(98%eep,E>200)for CNDE.However,the catalytic activity of the recombinant Lip was too low to fulfill the industrial application.Meanwhile,a novel esterase encoding gene,tle,was successfully cloned from T.lanuginosus DSM10635 with degenerate RT-PCR and RACE-PCR methods.The tle(GenBank accession no.KF305767)had an open reading frame of 945 bp encoding TLE of 314 amino acids.TLE was heterologous expressed in E.coli and Pichia pastoris in biologically active,respectively.Recombinant TLE was purified to homogeneity by single-step affinity chromatography on a Ni-NTA column.Several biochemical properties of TLE were studied.Among the reported esterases,TLE showed the highest enantioselectivity(E=95)in the kinetic resolution of CNDE.The unique properties of the esterase indicate that TLE is a potential candidate for industrial application.The site-saturation mutagenesis technique was used to improve the hydrolytic activity of Lip towards CNDE.The three-dimensional homology modelof Lip was generated by modeling,and series key residues were revealed by modeling and docking studies.The site-saturation mutagenesis libraries were created at oxyanion hole and the lid hinge region.The saturation mutagenesis libraries were screened using the high-throughput colorimetric activity assay on a 96-well plate format.The most active variant,S88T/A99N/V116D(2.35 U/mg),demonstrated a 60.3-fold improvement over the wild-type Lip(0.039 U/mg)in specific activity for CNDE,which almost matches TLL's performance(2.40 U/mg).Modeling and docking studies demonstrated that the mutant could more effectively stabilize oxygen anions in transition states and the lid of Lip in the open conformation.The increased activity for S88T might be explained by the increase in steric bulk introduced in oxyanion hole,result in more effectively stabilized oxygen anions in transition states in the hydrolytic reaction.The substitution of A99N and V116D exhibited higher hydrolytic activity towards CNDE due to three new hydrogen bonds were formed to stabilize the lid in the maximally open position,one hydrogen bond was between Asp101 and Asp 116,and the other two was between Asn99 and Asp 116.In order to develop a novel biocatalyst with high process efficiency compatible with the industrial prerequisites,we tried to further enhance its activity toward CNDE of Lip-T(S88T/A99N/V116D)by complementary protein engineering strategy,including error-prone PCR,site-directed mutagenesis and site-saturation mutagenesis.By screening approximately 2,500 clones from random-mutant libraries created by epPCR,the mutant S63L/D232A with 2.0-fold improved activity toward CNDE was obtained.To identify whether both substitutions in S63L/D232A are necessary for activity improvement,site-directed mutagenesis was performed to construct the single-point mutants S63L and D232A,respectively.Interestingly,the mutant S63L and D232A showed opposite effect on activity,S63L exhibited a significant improvement on activity,whereas D232A exerted a slight inhibitory effect.To enhance the effectiveness of directed evolution,the saturation mutagenesis libraries at site S63 and D232 were created and screened for mutants with improved activity,respectively.The mutant S63M/S88T/A99N/V116D with the highest activity(8.68 U/mg)was obtained from the site-saturation mutagenesis library at the site S63.It is worth noting that the specific activity of mutant S63M/S88T/A99N/V116D was about 3.7-fold,3.6-fold and 222.6-fold that of the S88T/A99N/V116D,TLL and wild-type Lip,respectively.Structural changes resulting from the mutations were analyzed and the mechanism responsible for the enhanced activity was discussed.Moreover,the engineered lipase was used as a robust biocatalyst for enantioselective hydrolysis of CNDE at a very high substrate loading(3 M,765 g/L).As only 5%(w/v)resting cells were used,the bioprocess is much more cost-effective than Pfizer's process using 8%(w/v)commercially available lipase Lipolase(?).During the kinetic resolution of CNDE by whole cells,2-carboxyethyl-3-cyano-5-methylhexanoic acid was found to inhibit its own production.To enhance catalytic efficiency,a new biocatalytic process of in situ product adsorption(ISPA)has been developed utilizing anion-exchange resin.To optimize the bioconversion of 2-carboxyethyl-3-cyano-5-methylhexanoic acid from CNDE,seven anion-exchange resins were examined,and the resin 201×7 was selected.In fed-batch biotransformation with ISPA with 20%resin 201×7 and 3 M CNDE was performed,and>45%conversion was achieved after 12 h.As compared to the conventional fed-batch mode,this approach reduced the reaction times by about 50 percent(from 24 h to 12 h),and allowed(3S)-2-carboxyethyl-3-cyano-5-methylhexanoic acid space-time yield and biocatalyst productivity to be increased from 56.25 mmol/L/h and 1.125 mmol/h/g catalyst to 112.50 mmol/L/h and 2.250 mmol/h/g catalyst,respectively.The batch biotransformation with ISPA demonstrated 2.0-fold and 3.2-fold improvement over Pfizer's process in space-time yield and biocatalyst productivity,respectively.The ISPA method by addition of resin 201×7 was proven to be an effective way to eliminate the product inhibition and enhance process productivity.After phase separation of the aqueous solution from the enzymatic reaction,a rapid chemical decarboxylation of(3S)-2-carboxyethyl-3-cyano-5-methyl-hexanoic acid into(S)-3-cyano-5-methylhexanoic acid ethyl ester took place in 105? reflux(40 min).(S)-3-cyano-5-methylhexanoic acid ethyl ester with 99%purity and 99%ee was obtained.