DESC:Priority Claim:
[0001] This application claims priority from the provisional application numbered 202321074743 filed with the Indian Patent Office, Mumbai on 2nd November 2023 post-dated to 2nd December 2023 entitled “A process of preparation of formaldehyde tolerant transaminases for the transamination of 4-hydroxy-2-butanone”, the entirety of which is expressly incorporated herein by reference.
PREAMBLE TO THE DESCRIPTION:
[0002] The following specification particularly describes the invention and the manner in which it is performed:
DESCRIPTION OF THE INVENTION
Technical field of the invention
[0003] The present invention discloses a process of preparation of formaldehyde tolerant transaminases for the transamination of 4-hydroxy-2-butanone (4H2B). More specifically, the process comprises in silico active site modification, distal mutation, in silico design to increase resistance to formaldehyde, stability improvement by evolutionary method. The process results in optimization of the formaldehyde tolerance. The process of preparation results in high percentage conversion by transaminases and is cost-effective and ecofriendly.
Background of the invention
[0004] Dolutegravir belongs to the class of human immunodeficiency virus (HIV) integrase inhibitors. Dolutegravir aids in the reduction of viral count in the blood and further mediates in increasing the immune cells.
[0005] The synthesis of dolutegravir comprises of (R)-3-amino-1-butanol (R3AB) as a vital intermediate. The biosynthesis of R3AB comprises transaminases as crucial biocatalysts. Site specific transaminases comprising R-selective and S-specific sites are of utmost importance.
[0006] In the bioprocess of synthesis of (R)-3-amino-1-butanol the formaldehyde content in substrate 4H2B is detrimental for transaminase enzyme. Formaldehyde is the raw material for synthesis of 4H2B.
[0007] The process of biosynthesis of (R)-3-amino-1-butanol by the addition of biocatalysts (transaminase) aids in high catalytic efficiency and reduced organic solvent dependence. The process is eco-friendly considering the mild reaction conditions.
[0008] The biocatalyst transaminase aids in the catalysis of transamination of 4 hydroxy-2-butanone with pyridoxal phosphate as cofactor. The transaminases are characterized by high regio-selectivity, chemo-selectivity, and stereoselectivity aiding in the synthesis of optically pure amines.
[0009] The optimization of enzyme structure by changing amino acids is essential in order to reduce the toxic effects of formaldehyde. The accumulation of formaldehyde is detrimental to the cell cycle.
[0010] The increased use of solvents in the synthesis of (R)-3-amino-1-butanol has been proven to be hazardous to the environment. The high amount of chemical oxygen demand (COD) is a challenge for the treatment of the generated waste.
[0011] In silico designing of the catalyst’s aids in the improvement of site specificity both qualitatively and quantitatively. In silico designing further aids in discovery of new targets and prediction of biological activity of the targets.
[0012] The Patent Application No. CN106754806A entitled “Improved transaminase and application thereof in preparation of (R)-3-aminobutanol” discloses a transaminase encoding gene in the preparation of (R)-3aminobutanol. The enzymatic activity of the improved transaminase in catalyzing ketone compound to chiral amine is higher than that of the wild transaminase. The invention discloses sequences of transaminase coding genes in Aspergillus terreus. The transaminase disclosed in the invention exhibits 95% sequence homology and comprises proline residue at the 215th position of the amino acid sequence. The improved transaminase aids in the catalysis of 4-hydroxy-2-butanone to (R)-3-aminobutanol. The improved transaminase is expressed in an expression vector comprising Escherichia coli BL21 host cell system.
[0013] The Patent Application No. CN104131048A entitled “Biological preparation method of R-3-aminobutanol” discloses a method for the preparation of R-3-aminobutanol. The method for the preparation of R-3-aminobutanol comprises steps of conducting whole gene synthesis, cloning into pET24a to obtain a recombinant expression vector, transferring the recombinant expression vector into E.coli to obtain a recombinant D-transaminase genetic engineering bacterium, culturing the bacterium to prepare recombinant D-transaminase, adding 3-carbonyl butanol with a final concentration of 10-300mmol/L, dimethyl sulfoxide or acetonitrile with a mass percentage concentration of 2-15%, pyridoxal phosphate with a final concentration of 0.1-1mmol/L, isopropylamine or D-alanine with a final concentration of 0.02-1mol/L, and recombinant D-transaminase with a mass percentage concentration of 0.01-1% are added into the reaction solution to compose a reaction system for reaction and, extracting R-3-aminobutanol. The method for the preparation of R-3-aminobutanol is cost-effective and exhibits a yield of 97% conversion rate. The method of preparation does not generate byproducts. The generated R-3-aminobutanol is analyzed and monitored by high performance liquid chromatography.
[0014] Although multiple transaminases exist for the transamination reaction, the transaminases with formaldehyde tolerance along with higher percentage conversion is to be identified. The optimization of active site modification and formaldehyde tolerance is essential for the development of transaminases, for this substrate (4H2B).
Summary of the invention:
[0015] The present invention overcomes the drawbacks of existing state of art. The present invention discloses a process of preparation of formaldehyde tolerant transaminases for the transamination of 4-hydroxy-2-butanone. The process yields high catalytic efficiency for transamination.
[0016] The process of preparation of formaldehyde tolerant transaminases comprises steps of retrieval of R-selective transaminases from databases, crystallographic analysis and homology modelling of the enzymes, conformational exploration and stability analysis of the enzymes, golden gate cloning of the enzyme in E. coli host system, induction of gene expression in the cloned E. coli cells, purification of expressed protein by affinity chromatography and evaluation of enzyme activity and evaluation of yield.
[0017] The present invention discloses amino acid sequences of R-selective transaminases. The in-silico design evaluation is performed by active site mutation, distal mutation, mutation for formaldehyde tolerance, mutation for improvement of enzyme stability, mutation for improvement of chiral purity.
[0018] The process yields enhanced formaldehyde tolerance, high catalytic efficiency, improved chemoselectivity, stereoselectivity of transaminases for the transamination of 4-hydroxy-2-butanone to R-3-aminobutanol. The process aids in obtaining optically pure amines with site specific binding with enhanced intracellular R-selective transaminase enzyme. The process yields high percentage conversion by transaminases and is cost-effective and ecofriendly.
Brief description of the drawings:
[0019] The foregoing and other features of embodiments will become more apparent from the following detailed description of embodiments when read in conjunction with the accompanying drawings. In the drawings, like reference numerals refer to like elements.
[0020] FIG 1 illustrates a flowchart for the process of preparation of formaldehyde tolerant R-selective transaminases.
[0021] FIG 2 tabulates the percentage of biotransformation and ee% in formaldehyde tolerant cultures.

Detailed description of the invention:
[0022] In order to more clearly and concisely describe and point out the subject matter of the claimed invention, the following definitions are provided for specific terms, which are used in the following written description.
[0023] The term “in-silico design” refers to computer aided molecular designing of biomolecules.
[0024] The present invention discloses a process of preparation of formaldehyde tolerant transaminases for the transamination of 4-hydroxy-2-butanone. The present invention discloses the process of preparation of transaminases for the catalytic conversion of 4-hydroxy-2-butanone (4H2B) to R-3-aminobutanol (R3AB).
[0025] FIG 1 illustrates the flowchart for the process of preparation of formaldehyde tolerant R-selective transaminases. The process of preparation (100) begins with step (101) by retrieval of R-selective transaminases from bioinformatics databases. The transaminases are retrieved from different organisms such as bacteria, yeast and fungi. The amino acid sequences of the retrieved transaminases are aligned to identify and hence refine the filtered sequences. The sequences of the retrieved transaminases comprising R-selective motifs and specific binding sites are identified.
[0026] At step (102), the retrieved R-selective transaminases are subjected to crystallographic analysis and homology modelling. The crystallographic analysis is evaluated to identify and verify the redundancy of the R-selective transaminases. The R-selective transaminases are further subjected to homology modelling to convert the sequences of R-selective transaminases to 3-dimensional conformation. The obtained 3D conformation of the R-selective transaminases is evaluated for structural quality by in silico docking studies, thermostability studies and pyridoxal phosphate binding.
[0027] At step (103), the R-selective transaminases are subjected to conformational exploration and stability analysis. Conformational exploration aids in the analysis of alternate bioactive conformations of the 3D conformations of R-selective transaminases. The conformational exploration of the R-selective transaminases is analyzed by evaluating the structural divergence at a threshold lower than 8Ao. The obtained 3D conformations of the R-selective transaminases are required to comprise an active site and average associated score of > -5kcal/mol. The average stability of the R-selective transaminases is evaluated to analyze the enzyme-substrate bond subjected to local minimization not to exceed 2.0Ao.
[0028] At step (104), the R-selective transaminases are subjected to golden gate cloning in Escherichia coli host system. The obtained sequences of R-selective transaminases are subjected to codon optimization and further subjected to golden gate cloning in E. coli host system in polyhistidine tag.
[0029] At step (105), the cloned E. coli cells comprising R-selective transaminases are induced for gene expression. The gene expression of cloned E. coli cells is induced at a temperature of 24 °C and for a duration of 20 hours in an enriched broth media comprising arabinose at a concentration of 0.02%. The induced cells are subjected to harvesting by centrifugation. Conversion of 4-hydroxy-2- butanone (4H2B) to R-3-aminobutanol (R3AB) occurs by whole cell or cell lysate/purified enzyme with equivalent efficiency. The selection of appropriate host system aids in obtaining enhanced intracellular enzyme concentration.
[0030] The cells are further resuspended in potassium phosphate (KPi) buffer at a concentration of 50 mM, at a pH of 7.5, and sodium chloride at a concentration of 150 mM. The cells were further subjected to sonication and centrifugation to obtain the cell free extract.
[0031] At step (106), the expressed R-selective transaminases are subjected to purification by affinity chromatography. The affinity chromatography set-up comprises Nickel-Sepharose resin and the cell free extract. The Ni-Sepharose resin is further subjected to washing with KPi buffer at a concentration of 50 mM, at a pH of 7.5, sodium chloride at a concentration of 150 mM and imidazole at a concentration of 10mM. The obtained enzymes are subjected to elution in KPi buffer at a concentration of 50 mM at a pH of 7.5, sodium chloride at a concentration of 150 mM and imidazole at a concentration of 500mM and desalted in Tris HCl at a concentration of 100 mM and at a pH of 8.0. The enzyme is further subjected to freeze drying. The suitable R-selective transaminase construct for soluble expression of the enzyme is evaluated by sodium dodecyl sulphate poly acrylamide gel electrophoresis (SDS-PAGE) analysis.
[0032] At step (107), the obtained R-selective transaminases are evaluated for enzyme stability analysis. The increase in temperature results in the unfolding of protein. Upon exposure to a fluorescent dye, the hydrophobic regions of the bound enzyme are exposed for analysis. At step (108), the R-selective transaminases are evaluated for yield of conversion by high throughput screening. At step (109) the substrate 4-hydroxy-2-butanone is subjected to recycling and R3AB is purified by adsorption to selective ion exchange resin. The removal of R3AB from reaction mass facilitates the forward reaction of transaminase recycling the starting material 4H2B.
[0033] The present invention further discloses sequences of the screened and retrieved R-selective transaminases from multiple organisms. The present invention discloses SEQ ID NO.: 1 comprising branched chain amino acid R-selective transaminases comprising 340 amino acids retrieved from Mycobacterium aurum and further related sequences.
[0034] The following examples are offered to illustrate various aspects of the invention. However, the examples are not intended to limit or define the scope of the invention in any manner.
Example 1: In silico design and analysis of R-selective transaminases
[0035] The in silico design of R-selective transaminases is evaluated to analyze the transaminase activity and the formaldehyde tolerance. The evaluation comprises detecting the mutations which stabilize the ligand pre-catalytic binding mode at the enzyme active site. The enzyme residues comprising ligands are subjected to mutation detected in sequence alignment. The resulting binding energies (?G) and the distance between the amino group nitrogen bound to pyridoxal 5 phosphate (PLP) and the receptor carbon atom of the substrate were measured and analyzed. The analysis yielded 23 single point mutations including K148H, K148Q, A294G, A294M, A294S, A167V, A167P, F132H, F132L, W202H, W202Y, T292S, T292G, T292A, T292N, G71Q, H72L, Y160F, Y200N, Y200H, Y77F, Y77W.
[0036] The in silico designing for functional hotspots yielded 70 single point mutations including K62T, F66L, F66M, Y141V, Y141S, T206Q, T206S, Q201N, Q201S, L106M, L106F, D74T,T192W, T192S, V193L, V193I, A188N, A188H, I159V, I159A, L135P, L135I, W50Y, P57A,P57N, F44L, F44Y, S131A, S131G, S131C, L128I, L128M, I162W, I162F, I162V, Y158V,Y158I, M328V, M328I, W325F, T260S, F262I, F262M, I239V, L288A, L288V, V237I, V237A,I221V, A290V, A179G, A179V, P233S, P233T, S249P, A252S, A252I, L282V, A246V, P195C, P195T, N251G, N251S, K244E, D204N, G295A, P182V, W166L, L222M, F168L. The variants indicate the impact of conformational dynamics on enzyme activity.
[0037] The in-silico design for formaldehyde tolerance was analyzed by evaluating the arginine and lysine residues impacting the enzyme stability upon methylation. The design model comprising of 14 lysine residues and 19 arginine residues of the system were independently di-methylated and tri-methylated. The methylation of the lysine and arginine residues does not indicate significant enhancement in the flexibility of the enzyme binding sites. The residues were further analyzed for mutagenesis. The percentage yield conversion of R-selective transaminases for the variant A167V is 24.62%. The A167V variant was found to exhibit higher yield of conversion and higher tolerance to formaldehyde. The present invention discloses SEQ ID NO.: 2 comprising 340 amino acids expressed in Escherichia coli. The present invention discloses related mutant sequences of transaminases.
[0038] The enzyme variants were further subjected to in silico design analysis by proprietary in silico design software. The analysis comprises evaluating a single mutant target in turn affecting the activity and stability of the enzyme system. The enzyme variants were selected on the basis of sequence-based virtual library construction, computational design of mutants and computational screening of mutants with multiple filters. The selected enzyme variants were analyzed for the rate of conversion by R-selective transaminases.
[0039] The present invention further discloses related single point mutant sequences of transaminases after subjecting the sequence to another round of point mutation. Single mutants suggested in the second round of mutagenesis are A167I, A167L, A167M, A167Y, A167T, A294T, A294S, K148M, V79T, L135V, L135M, L135A, T119V, T119I, N134D, S131V, E60A, L222Q, L222N, V193T, Y141Q, A246S, A80I, A80V, A80L, V137L, V137I, A108I, A108L, V133I, A188S, A188V, T192V, T192I, T192M, G146A, G146V, G146L, T68L, N191D, R129L, R129M, E60A, R186I, R186L, E312R, E52N, M56V, H72Y, H72F, S73G, L75G, T76V, T78E, H81R, W83Y, G100S, S101A, G109P, S126N, F132Y, N134R, H155P, G203L, A207L, S209K, T219E, N227G, C238F, S247T, A272V, L274E, M289F, V291T, G296E. A167I was shortlisted as the best of the above listed mutants. The present invention discloses SEQ ID NO.: 3 comprising 340 amino acids expressed in Escherichia coli.
[0040] Structural methods were applied to combine these mutations aiming for additive effects. The best mutant found, A167I was selected as a template and double, triple and quadruple mutants were predicted over it generating a list of 20 combined mutations. Mutant combinations suggested as A167I_A188S, A167I_A272V, A167I_A246S, A167I_A188S_A272V, A167I_A188S_A272V_E52N, A167I_A188S_A246S, 167I_E52N_M56V, A167I_E52N_E60A, A167I_E52N, A167I_M56V, A167I_E60A, A167I_A188S_E52N, A167I_A188S_M56V, A167I_A188S_E60A, A167I_A188S_L135M, A167I_A246S_E52N, A167I_A246S_M56V, A167I_A246S_E60A, A167I_A246S_L135M, A167I_A272V_E52N. A167I_A188S was shortlisted as the best mutant depicted by SEQ ID NO.:4.
[0041] SEQ ID NO.: 4 comprising 340 amino acids is expressed in Escherichia coli. The sequences disclosed in the present invention subjected to rounds of point mutation exhibit enhanced throughput and enantioselectivity.
[0042] FIG 2 tabulates the percentage of biotransformation and ee% in formaldehyde tolerant cultures. The percentage of biotransformation at a concentration of 50 mM of 4H2B and 0.02% HCHO was found to be 4.94% by Phase 1 shortlisted culture. The percentage of biotransformation at a concentration of 50 mM 4H2B and 0.02% HCHO was found to be 24.62% by shortlisted Phase 2 culture, Phase 3 and Phase 4 cultures are tolerant for 0.1% formaldehyde at 300mM and 500mM 4H2B respectively. The percentage of biotransformation at a concentration of 300mM is 21% with 89.28 % chiral purity by Phase 3 shortlisted culture. The percentage of biotransformation at a concentration of 500mM is 40% with 94.40 % chiral purity by Phase 4 shortlisted culture. The values indicate, the impact of subsequent rounds of in-silico design to generate mutants with improved conversion, formaldehyde tolerance, chiral purity and throughput with increased input.
Example 2: Use of whole cell bacterial biomass containing over-expressed transaminase for conversion of 4-hydroy-2-butanone (4H2B) to R-3-aminobutanol (R3AB)
[0043] The use of wet biomass for the conversion of 4-hydroy-2-butanone to R-3-aminobutanol was evaluated by obtaining culture from glycerol stock inoculated in terrific broth at a volume of 25ml with 100 PPM ampicillin. The culture conditions were incubated at a temperature ranging from 28oC to 32°C at 150 RPM till optical density range of 2.5 to 3.0 was achieved. Optical density was achieved at a value of 2.0, Arabinose was added at a concentration of 0.02% as an inducer. Biomass was further harvested 20 hours after induction by subjecting to centrifugation at a speed of 10,000 rpm for 40 minutes. Wet biomass was stored at a temperature of -20°C till use. Stored wet biomass was thawed at room temperature. Wet biomass of weight 10g was added to 250 ml capacity round bottom flask 50 ml distilled water and mixed using magnetic stirrer. PLP at a concentration of 1mM was incorporated into the biomass solution and mixed on magnetic stirrer for a duration of 7 to 10 minutes. To the biomass solution isopropyl amine HCl solution to reach a concentration of 750mM and 4H2B to reach a concentration of 500mM and mixed well. pH was adjusted to 9.0 by the addition of potassium hydroxide solution of 6M concentration. Biotransformation reaction was continued at a temperature range of 28-30°C.for a duration of 24 hours on overhead stirrer. After a duration of 6 hours upon obtaining>100 mM 4H2B conversion to R3AB the biotransformation mass is processed through a microfiltration/centrifugation setup to recover broth and separate biomass rich phase which goes back to reaction. The broth is taken to ion exchange column for R3AB absorption and unreacted 4H2B (along with top-up) is recycled back to biotransformation reaction mass. This thermodynamic equilibrium is effectively shifted to give improved conversion and increased throughput.
[0044] The samples after biotransformation were analyzed by HPLC for quantification of R3AB. The glass column filled with ion exchange resin was regenerated by the addition of ammonia solution at a concentration of 5%. The supernatant of biotransformation reaction was passed through resin column to achieve binding of product. The obtained eluate was collected and analyzed for remaining R3AB, 4H2B by HPLC. Resin bound R3AB was eluted by using ammonia solution at a concentration of 5%. Eluent was subjected to concentration by the process of distillation. Assay of 98.64% was achieved with ~1% water content.
Example 3: Use of immobilized R-selective transaminases for the conversion of 4-hydroy-2-butanone (4H2B) to R-3-aminobutanol (R3AB) for recovery of product and recycling of the substrate
[0045] The use of immobilized R-selective transaminases for the conversion of 4-hydroy-2-butanone (4H2B) to R-3-aminobutanol (R3AB). The crude cell lysate was passed through nickel and cobalt resins for binding of histidine tag of enzyme. The unbound enzyme was eluted by passing of wash buffer through resin column. The resin column with bound enzyme was further passed with the solution of PLP at a concentration of 1mM, substrate 4H2B at a concentration of 500mM and IPA-HCl solution at a concentration of 750mM.
[0046] The eluent was analyzed for R3AB concentration by HPLC. The eluent was passed further through an ion exchange resin for binding of R3AB. R3AB was further recovered by ammonia solution (5%). The recovered substrate 4H2B was further utilized for biotransformation reaction. Being a continuous process, the thermodynamic equilibrium is partly alleviated by inline product R3AB removal.
[0047] The present invention discloses a process of preparation of formaldehyde tolerant transaminases for the transamination of 4-hydroxy-2-butanone. The in-silico designing of formaldehyde tolerant transaminases for the conversion of 4-hydroxy-2-butanone to R-3-aminobutanol. The in silico active site modification aids in optimization of formaldehyde tolerance. The process yields enhanced formaldehyde tolerance, high catalytic efficiency, improved region-selectivity, chemo-selectivity and stereo-selectivity. The process aids in obtaining optically pure amines with site specific binding with enhanced intracellular R-selective transaminase concentration. The process is efficient and commercially viable. The process yields high percentage conversion by transaminases and is cost-effective and ecofriendly. ,CLAIMS:We Claim:
1. A process of preparation of formaldehyde tolerant transaminase for the transamination of 4-hydroxy-2-butanone, the process comprising steps of:
a) retrieving one or more R-selective transaminases from bioinformatics database (101);
b) subjecting retrieved R-selective transaminases to in-silico crystallographic analysis to identify the redundancy of transaminases, homology modelling for 3-dimensional conformation of transaminases, docking studies, thermostability studies (102);
c) subjecting R-selective transaminases to conformational exploration by active site in-silico design, distal in-silico design, evaluation of formaldehyde tolerance based on surface reactive groups of enzyme and stability analysis (103);
d) subjecting R-selective transaminases to golden gate cloning in Escherichia coli host system (104);
e) inducing gene expression in the R-selective transaminase cloned Escherichia. coli cells (105);
f) subjecting the expressed R-selective transaminases to purification using affinity chromatography and whole cell biotransformation (106);
g) evaluating thermostability of enzyme by fluorescent dye binding to hydrophobic regions (107);
h) evaluating the conversion of R-selective transaminases using high throughput screening (108); and
i) isolating the product R-3-aminobutanol (R-3AB) from the reaction mass and facilitating the forward reaction by adsorption on selective ion exchange resin (109).
wherein the R-selective transaminases obtained by the process facilitates the transamination of 4-hydroxy-2-butanone to R-3-aminobutanol with enhanced catalytic efficiency, chemo-selectivity and stereo-selectivity.
2. The process as claimed in claim 1, wherein the successive rounds of in-silico design of R-selective transaminases involving one or more-point mutations exhibits improved transaminase activity, formaldehyde tolerance with enhanced throughput and enantioselectivity.

3. The process as claimed in Claim 1, wherein the successive phases of in-silico design/cloning and expression obtained R-selective transaminases with increased formaldehyde tolerance from a percentage of 0.02% w/v to a percentage of 0.1% w/v.

4. The process as claimed in claim 1, wherein R-selective transaminase is depicted by SEQ ID NO.: 1 of 340 amino acids expressed in E. coli host system.

5. The process as claimed in claim 1, wherein one or more single-point mutation variants of R-selective transaminases are depicted by SEQ ID NO.: 2, SEQ ID NO.:3 and SEQ ID NO.:4 comprising 340 amino acids expressed in E. coli host system.

6. The process as claimed in claim 1, wherein the retrieved one or more R-selective transaminases comprises one or more R-selective motifs and one or more binding sites.

7. The process as claimed in claim 1, wherein 3D conformations of the R-selective transaminases comprise structural divergence at a threshold at a value lesser than 8Ao, average associated score of value greater than -5kcal/mol and local minimization at a value of 2.0Ao.

8. The process as claimed in claim 1, wherein the gene expression for R-selective transaminases is induced at a temperature of 240 C for a duration of 24 hours in in an enriched broth media comprising arabinose at a concentration of 0.02%.