DESC:FORM 2

THE PATENTS ACT, 1970

(39 of 1970)
&
The Patents Rules, 2003

COMPLETE SPECIFICATION

(Section 10 and Rule 13)

A PROCESS FOR THE ENANTIOSELECTIVE SYNTHESIS OF (R)-ß-AMINO BUTYRIC ACID

AUROBINDO PHARMA LTD HAVING CORPORATE OFFICE AT
GALAXY, FLOORS: 22-24,
PLOT No.1, SURVEY No.83/1,
HYDERABAD KNOWLEDGE CITY,
RAIDURG PANMAKTHA,
RANGA REDDY DISTRICT,
HYDERABAD – 500 032,
TELANGANA, INDIA
AN INDIAN ORGANIZATION

The following specification particularly describes and ascertains the nature of this invention and the manner in which the same is to be performed:

FIELD OF THE INVENTION

The present invention relates to a newly identified aspartate ammonia lyase (AAL) from Ureibacillus microorganism and its variants thereof. The invention also relates to nucleotides related to the AAL and it’s variant. The invention further relates to the corresponding protein and the use of the protein as a catalyst in the preparation of (R)-ß-amino butyric acid of formula – I from crotonic acid of formula – III.
; .

The compound of formula – I is a key precursor in the preparation of integrase inhibitor, Dolutegravir or pharmaceutically acceptable salts thereof of formula – II.

.

BACKGROUND OF THE INVENTION

Dolutegravir (Formula – II) is chemically known as (4R,12aS)-N-[(2,4-difluorophenyl)methyl]-3,4,6,8,12,12a-hexahydro-7-hydroxy-4-methyl-6,8-dioxo-2H-pyrido[1',2':4,5]pyrazino[2,1-b][1,3]oxazine-9-carboxamide. Dolutegravir is an investigational integrase inhibitor in development for the treatment of HIV infection.

(R)-3-Amino-1-butanol (VI) is a key precursor used in the preparation of Dolutegravir of Formula II.

US 8288575 discloses a process for the preparation of (R)-3-amino-1-butanol (VI) by hydrogenation of methyl (R)-3-aminobutanoate (VII) using a ruthenium complex. The process is depicted below as scheme-I:

Scheme-I

The major disadvantage associated with the above process is that sensitivity and the use of more expensive catalyst such as ruthenium complex, which is not easier to handle, and this process is not suitable in the commercial scale production of Dolutegravir.

ChemCatChem 2014, 6, 965 – 968, discloses mutant enzyme BSASP-C6, the substrate binding pocket of Aspartase B from Bacillus sp.YM55-1. The journal also discloses the use of BSASP-C6 as a catalyst in the preparation of (R)-ß-amino butyric acid of formula – I from crotonic acid of formula – III. The process is depicted below as scheme-II:

Scheme – II

Nature Chemical Biology, 2018, 14, pages 664-670 discloses the use of redesigned enzymes; wherein the design B19 (in Escherichia coli), contains four mutations (T187C, M321I, K324L and N326A) is used as a catalyst in the preparation of (R)-ß-amino butyric acid of formula – I. The process is depicted below as scheme-III:

Scheme – III

WO 2021058691 discloses the recombinant aspartase-like protein capable of catalyzing the reaction of acrylic acid of formula –IV with ammonia to ß-alanine of formula – V in an aqueous medium. The process is depicted below as scheme-IV:

Scheme – IV

By considering the need of Dolutegravir in the healthcare industry, there is a need to develop an improved process for the preparation of (R)-ß-amino butyric acid of formula – I, which is the key precursor in the preparation of Dolutegravir of formula – II.

Hence, the inventors of the present invention have identified a new AAL from microorganism of genus Ureibacillus. The nucleic acid sequence (synthetically or naturally) was altered to provide new DNA sequence, which on expression produced a protein that is capable of catalyzing the reaction of crotonic acid of formula – III with ammonia to obtain (R)-ß-amino butyric acid of formula – I, selectively. The present invention directed towards a process for the preparation of (R)-ß-amino butyric acid of formula – I with enhanced purity and yield.

OBJECTIVE OF INVENTION

The objective of the present invention is to provide a novel series of synthetic genes, SEQ ID NO: 1, 2, 3, 4, 5, 6 and 7.

Another objective of the present invention is the use of the novel series of synthetic genes, to express to corresponding proteins and use them as catalysts in the preparation of (R)-ß-amino butyric acid of formula – I from crotonic acid of formula – III.

Yet another objective of the present invention is the preparation of Dolutegravir or pharmaceutically acceptable salts thereof of formula – II by converting (R)-ß-amino butyric acid of formula – I, which is obtained from crotonic acid by using the novel series of proteins resulted from synthetic gene SEQ ID NO: 1, 2, 3, 4, 5, 6 and 7 as a catalyst.

SUMMARY OF THE INVENTION

In an embodiment, the present invention provides a novel series of synthetic gene comprising of nucleotide SEQ ID NO: 1, 2, 3, 4, 5, 6 and 7.

In another embodiment, the present invention provides a process for the preparation of (R)-ß-amino butyric acid of formula – I,
,
which comprises:
(i) reacting crotonic acid of formula – III with ammonia in the presence of a catalyst,

(ii) isolating (R)-ß-amino butyric acid of formula – I.

Yet, in another embodiment of the present invention provides a process for the preparation of Dolutegravir or pharmaceutically acceptable salts thereof of formula – II;
,
which comprises:
(i) reacting crotonic acid of formula – III with ammonia in the presence of a catalyst,
,
(ii) isolating (R)-ß-amino butyric acid of formula – I.
,
(iii) converting (R)-ß-amino butyric acid (I) to Dolutegravir (II) or pharmaceutically acceptable salts thereof.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a novel series of synthetic genes having nucleotide sequence comprising of SEQ ID NO: 1, 2, 3, 4, 5, 6 and 7 and their corresponding proteins are used as catalysts in the preparation of (R)-ß-amino butyric acid of formula – I from crotonic acid of formula – III.

In another embodiment, the present invention provides a process for the preparation of (R)-ß-amino butyric acid of formula – I, which comprises, reacting crotonic acid of formula – III with ammonia in the presence of a catalyst and isolating (R)-ß-amino butyric acid of formula – I.

The catalyst used in above reaction comprises novel series of synthetic genes having nucleotide sequence comprising SEQ ID NO: 1, 2, 3, 4, 5, 6 and 7.

The gene with SEQ ID 1 cloned into pET28a expression vector at XhoI and NcoI restriction sites was used for protein engineering. Four mutations on the SEQ ID 1 was performed by site directed mutagenesis to produce the new DNA sequence with SEQ ID 3. The pET28a plasmid carrying the tetra mutant nucleotide sequence has SEQ ID: 7. The mutated plasmid with SEQ ID 7 was transformed in E. coli BL21(DE3) cells and used to produce the protein catalyst SEQ ID: 6.

SEQ ID NO: 6 herein referred as a AL1 tetra mutant (AL1 T188C-M322I-K325L-N327A), was synthesized by inducing the E. coli BL21(DE3)-pET28a- AL1-T188C-M322I-K325L-N327A plasmid, cultured with 0.5 mM IPTG at 30°C for 6 hours. The cell pellet obtained was lysed and purified by heat treatment at 60°C for 30 min followed by centrifugation at 12,000 g for 90 min to remove precipitates. SDS-PAGE of the purified protein confirms the presence of 51 kDa AL1 tetra-mutant, while the protein purity was found to be ~90%. Concentration of the purified protein determined by Bradford’s assay was found to be 7.92 mg/mL.
SEQ ID NO: 3 herein referred as AL1 tetra mutant (AL1 T188C-M322I-K325L-N327A) which is synthesized by inducing the E. coli BL21(DE3)-pET28a- AL1-T188C-M322I-K325L-N327A cultured with 0.5 mM IPTG at 30°C for 6 hours. The cell pellet obtained was lysed and purified by heat treatment.

Purified AL1 tetra mutant is employed in the enantioselective hydroamination reaction that involved ammonia addition to crotonic acid. The biotransformation was carried out in the presence of the pure enzyme. Analysis of the 6 h reaction mixture has shown the formation of ß-selective addition product, (R)-3-aminobutyric acid having 100% enantioselectivity.

In yet another preferred embodiment, purification of AL1 tetra mutant is carried out by growing of inoculum of an overnight grown E. coli Bl21(DE3) harboring AL1 tetra mutant in LB media with kanamycin is transferred to LB media with the same antibiotic and further grown until the OD600 reached. Protein expression of the AL1 tetra mutant is done by inducing the culture with IPTG at 30°C. The cells are then harvested by centrifugation and lysed by sonication in a buffer containing Tris-HCl (pH 7.5) and MgCl2 at 30 KHz amplitude with ÓN’ and ‘ÓFF’ cycle.

In another embodiment, the AL1 tetra mutant was purified by heat treatment at 60°C for 30 min followed by centrifugation at 12,000 g for 90 min to remove precipitates. The purity of the protein obtained by this method was generally >90%. The protein concentration was determined by Bradford assay taking BSA as standard and measuring the absorbance at 595 nm. A 12% SDS- PAGE was run by loading 17 µg of pure enzyme to confirm the presence of 51 kDa AL1 tetra mutant protein.

The AL1 tetra mutant is purified by heat treatment followed by centrifugation to remove precipitates. The purity of the protein obtained by this method is generally >90%. The protein concentration is determined by Bradford assay taking BSA as standard.

In another embodiment of the present invention provides a process for the preparation of Dolutegravir or pharmaceutically acceptable salts thereof of formula – II; which comprises: reacting crotonic acid of formula – III with ammonia in the presence of a catalyst, isolating (R)-ß-amino butyric acid of formula – I, converting (R)-ß-amino butyric acid (I) to Dolutegravir (II) or pharmaceutically acceptable salts thereof.

The catalysts used in above reaction comprises of proteins prepared from novel series of synthetic genes having nucleotide sequences comprising SEQ ID NO: 1, 2, 3, 4, 5, 6 and 7.

The (R)-ß-amino butyric acid of formula (I) prepared according to the present invention is converted to Dolutegravir or its salts as reported in the literature.

The invention is illustrated with the following examples, which are provided by way of illustration only and should not be construed to limit the scope of the invention in any manner whatsoever.

EXAMPLE 1: Synthesis of (R)-3-aminobutyric Acid:
A reaction mixture containing 300 mM ammonia solution, 100 mM Na2HPO4, and 100 mM crotonic acid was prepared and the pH was adjusted to 8 by adding 5 N HCl. To 482 µL of the above reaction mixture, 18 µL of purified enzyme having sequence selected from Sequences 1, 2, 3, 4, 5, 6 and 7, was added and the reaction was performed at 37°C with gentle shaking (450 rpm). After 6 hours the reaction was stopped by heating at 80°C for 15 min. The reaction mixture was derivatized as described above and analyzed by HPLC to determine the enantiomeric excess.

HPLC chromatogram of the reaction mixture shown 100.0% enantiomeric excess of (R)-3-aminobutyric acid.

EXAMPLE 2: Whole Cell bio-catalysis in the synthesis of (R)-3-Aminobutyric Acid:
A 2 mL reaction mixture containing 300 mg/mL of crotonic acid solution was prepared, and the pH was adjusted to 9.0 by adding 25% (w/w) ammonia (final concentration = 4.4 M). To this solution, whole E. coli BL21(DE3) whole cells carrying AL1 tetra mutant were added until the final OD600 of the reaction mixture reached 60 (16 µL of reaction mix was taken and the volume was made to 1 mL by water to check the OD600 1 in the UV-visible spectrophotometer). This gave a final volume of 2.5 mL. The reaction was performed at 50°C for 8 hours. The cells were removed by centrifugation (11,000 rpm, 1 hour). The supernatant of the reaction mixture was further clarified using Amicon tube (10 kDa cutoff) and the flow through was decolorized by adding a pinch of activated carbon and heating the mixture at 60°C in a thermo shaker for 1 h. The mixture was filtered using Whatman filter paper and the collected supernatant was concentrated (75°C, 0.05 MPa) using rotary evaporator to get the viscous product. The product obtained was cooled to 4°C. The products were characterized by 1H and 13C NMR using D2O as solvent. Enantiomeric excess was determined by HPLC after NAC-OPA derivatization.

HPLC chromatogram of the reaction mixture shown 97.4% enantiomeric excess of (R)-3-aminobutyric acid.

Sequence listing:

<110> Applicant name: Aurobindo Pharma Limited
<120> Title of invention: A process for the enantioselective synthesis of (R)-ß-amino butyric acid
<160> Number of SEQ ID NOs: 7
<210> SEQ ID NO: 1
<211> Length: 1407
<212> Type: Artificial DNA
<213> Organism: – Ureibacillus thermophilus
<400> Sequence: 1

1 ATGGGTGAAA AGAACGTGCG TATCGAGAAA GACTTCCTGG GCGAGAAGGA AATCCCGATT
61 GATGCGTACT ATGGCGTTCA GACCATGCGT GCGACCGAAA ACTTTCCGAT CACCGGTTAT
121 CGTATTCACC CGGAGCTGAT CAAGAGCCTG GGCATTGTGA AGAAGGCGGC GGCGCTGGCG
181 AACATGGAAG TTGGTCTGCT GGACAAAACC ATCGGCGAGT ACATTGTGAA GGCGGCGGAT
241 GAGGTTATCG AGGGTAAATG GGACGATCAG TTCATCGTGG ACCCGATTCA AGGTGGCGCG
301 GGCACCAGCA TCAACATGAA CGCGAACGAA GTTATTGCGA ACCGTGCGCT GGAGCTGATG
361 GGTGCGGAAA AGGGCAACTA TAGCCTGATC AGCCCGAACA GCCACGTGAA CATGAGCCAG
421 AGCACCAACG ATGCGTTTCC GACCGCGACC CACATTGCGG TGCTGAGCCT GCTGAACCAA
481 CTGATTGACA CCACCAAAAC CATGCAGCAA GTTTTCCTGA ACAAGGCGGA TGAGTTTGCG
541 GGTATCATTA AAATGGGTCG TACCCACCTG CAGGATGCGG TTCCGATCCT GCTGGGTCAA
601 GAGTTCGAAG CGTACGCGCG TGTGATCGCG CGTGATGTTG AACGTATTAG CAACACCAAA
661 AACAACCTGT ATGAGGTGAA CATGGGTGCG ACCGCGGTTG GCACCGGTCT GAACGCGGAG
721 CCGGAATACA TCAAAATTGT GACCGAACAC CTGGTTAAGC TGAGCGGTCA CCCGCTGCGT
781 AGCGCGAAGC ACCTGGTGGA CGCGACCCAG AACACCGATT GCTATACCGA GGTTTCTGCG
841 GCGCTGAAAA TCTGCATGAT TAACATGAGC AAGATCGCGA ACGACCTGCG TCTGATGGCG
901 AGCGGTCCGC GTGCGGGTCT GAGCGAAATC ATTCTGCCGG CGCGTCAACC GGGTAGCAGC
961 ATTATGCCGG GCAAAGTGAA CCCGGTTATG CCGGAAGTGG TTAACCAGGT GGCGTTCCAA
1021 GTTATCGGTA ACGATCTGAC CATTAGCGCG GCGAGCGAGG CGGGCCAGTT TGAACTGAAC
1081 GTGATGGAGC CGGTTCTGTT CTTTAACCTG ATCCAAAGCA TCAGCATTAT GAACAACGTG
1141 TTCAAAACCT TTACCGAAAA CTGCCTGAAG GGTATTCAGG CGAACGAGGA ACGTATGAAA
1201 GAGTACGTGG AACGTAGCAT CGGTATCATT ACCGCGATTA ACCCGCACGT GGGTTATGAG
1261 ACCGCGGCGA AGCTGGCGCG TGAAGCGTAT CTGACCGGCG AGAGCATCCG TGACCTGTGC
1321 ATTAAATACG ATGTTCTGAC CGAGGAACAA CTGAACGAAA TCCTGAACCC GTATGAGATG
1381 ACCCACCCGG GTATTGCGGG CAAGCAC

<210> SEQ ID NO: 2
<211> Length: 1407
<212> Type: Artificial DNA
<213> Organism: – Ureibacillus terrenus
<400> Sequence: 2
1 ATGGGTAACA AGAACATCCG TATTGAAAAA GACTTCCTGG GCGAGAAGGA AATCCCGGCG
61 GAGGCGTACT ATGGCGTGCA GACCGCGCGT GCGACCGAAA ACTTTCCGAT CACCGGTTAT
121 CGTATTCACC CGGAGCTGAT CAAAGCGCTG GGCATTGTGA AGAAGGCGGC GGCGCTGGCG
181 AACATGGAAG TTGGTCTGCT GGATAAAGAG ATCGGCCAAT ACATTGTGAA GGCGGCGGAG
241 GAAGTTATCG AGGGTAAGTG GAACGACCAG TTCGTGGTTG ATCCGATCCA AGGTGGCGCG
301 GGCACCAGCA TTAACATGAA CGCGAACGAA GTGATCGCGA ACCGTGCGCT GGAGCTGATG
361 GGCGAGGAAA AAGGCAACTA TAGCGTGATT AGCCCGAACA GCCACGTTAA CATGAGCCAG
421 AGCACCAACG ACGCGTTTCC GACCGCGACC CGTATTGCGG TTCTGAGCCT GCTGGATCAA
481 CTGATCGAAA CCACCACCAC CATGCAGCAA GCGTTCCTGA AGAAAGCGGA CGAGTTTGCG
541 GGTGTGATTA AGATGGGCCG TACCCACCTG CAGGATGCGG TTCCGATCCT GCTGGGTCAA
601 GAGTTCGAAG CGTACGCGAA CGTGATTGCG CGTGACATCG AACGTATTAG CAACACCAAG
661 AAACACCTGT ATGACATCAA CATGGGTGCG ACCGCGGTTG GCACCGGTCT GAACGCGGAC
721 CCGAAATACA TCGAAATTGT GACCAAGCAT CTGGTTGAGC TGAGCGGTCA CCCGCTGCGT
781 ACCGCGAAGC ACCTGGTGGA CGCGACCCAG AACACCGATT GCTACACCGA GGTGAGCAGC
841 GCGCTGAAAG TTTGCATGCT GAACATGAGC AAGATCGCGA ACGACCTGCG TCTGATGGCG
901 AGCGGTCCGC GTGCGGGCCT GGCGGAAATT GTTCTGCCGG CGCGTCAACC GGGTAGCAGC
961 ATCATGCCGG GCAAAGTGAA CCCGGTTATG CCGGAAGTGG TTAACCAGGT GGCGTTCCAA
1021 GTTTTTGGTA ACGATGTTAC CATCAGCAGC GCGAGCGAGG CGGGCCAGTT TGAACTGAAC
1081 GTGATGGAGC CGGTTCTGTT CTTTAACCTG ATCCAAAGCA TCAGCATTAT GAACAACGTT
1141 TTCAAAGCGT TTACCGAATA CTGCCTGAAG GGTATTCAGG CGAACGAGAA GCGTATGAAA
1201 GAGTATGTGG AAAAGAGCAT CGGTATCATT ACCGCGATTA ACCCGCACGT TGGCTACGAG
1261 ACCGCGGCGA AACTGGCGCG TGAAGCGTAT CTGACCGGCG AGAGCATCCG TGATCTGTGC
1321 ATTAAGTACG GCGTTCTGAC CGAGGAACAG CTGAACGAAA TTCTGAACCC GTATGAGATG
1381 ACCCACCCGG GTATCGCGGG CAAACAC

<210> SEQ ID NO: 3
<211> Length: 1407
<212> Type: Artificial DNA
<213> Organism: – Ureibacillus terrenus
<400> Sequence: 3

1 ATGGGTAACA AGAACATCCG TATTGAAAAA GACTTCCTGG GCGAGAAGGA AATCCCGGCG
61 GAGGCGTACT ATGGCGTGCA GACCGCGCGT GCGACCGAAA ACTTTCCGAT CACCGGTTAT
121 CGTATTCACC CGGAGCTGAT CAAAGCGCTG GGCATTGTGA AGAAGGCGGC GGCGCTGGCG
181 AACATGGAAG TTGGTCTGCT GGATAAAGAG ATCGGCCAAT ACATTGTGAA GGCGGCGGAG
241 GAAGTTATCG AGGGTAAGTG GAACGACCAG TTCGTGGTTG ATCCGATCCA AGGTGGCGCG
301 GGCACCAGCA TTAACATGAA CGCGAACGAA GTGATCGCGA ACCGTGCGCT GGAGCTGATG
361 GGCGAGGAAA AAGGCAACTA TAGCGTGATT AGCCCGAACA GCCACGTTAA CATGAGCCAG
421 AGCACCAACG ACGCGTTTCC GACCGCGACC CGTATTGCGG TTCTGAGCCT GCTGGATCAA
481 CTGATCGAAA CCACCACCAC CATGCAGCAA GCGTTCCTGA AGAAAGCGGA CGAGTTTGCG
541 GGTATCATTA AAATGGGTCG TTGCCACCTG CAGGATGCGG TTCCGATCCT GCTGGGTCAA
601 GAGTTCGAAG CGTACGCGAA CGTGATTGCG CGTGACATCG AACGTATTAG CAACACCAAG
661 AAACACCTGT ATGACATCAA CATGGGTGCG ACCGCGGTTG GCACCGGTCT GAACGCGGAC
721 CCGAAATACA TCGAAATTGT GACCAAGCAT CTGGTTGAGC TGAGCGGTCA CCCGCTGCGT
781 ACCGCGAAGC ACCTGGTGGA CGCGACCCAG AACACCGATT GCTACACCGA GGTGAGCAGC
841 GCGCTGAAAG TTTGCATGCT GAACATGAGC AAGATCGCGA ACGACCTGCG TCTGATGGCG
901 AGCGGTCCGC GTGCGGGCCT GGCGGAAATT GTTCTGCCGG CGCGTCAACC GGGTAGCAGC
961 ATTATTCCGG GCCTGGTGGC CCCGGTTATG CCGGAAGTGG TTAACCAGGT GGCGTTCCAA
1021 GTTTTTGGTA ACGATGTTAC CATCAGCAGC GCGAGCGAGG CGGGCCAGTT TGAACTGAAC
1081 GTGATGGAGC CGGTTCTGTT CTTTAACCTG ATCCAAAGCA TCAGCATTAT GAACAACGTT
1141 TTCAAAGCGT TTACCGAATA CTGCCTGAAG GGTATTCAGG CGAACGAGAA GCGTATGAAA
1201 GAGTATGTGG AAAAGAGCAT CGGTATCATT ACCGCGATTA ACCCGCACGT TGGCTACGAG
1261 ACCGCGGCGA AACTGGCGCG TGAAGCGTAT CTGACCGGCG AGAGCATCCG TGATCTGTGC
1321 ATTAAGTACG GCGTTCTGAC CGAGGAACAG CTGAACGAAA TTCTGAACCC GTATGAGATG
1381 ACCCACCCGG GTATCGCGGG CAAACAC

<210> SEQ ID NO: 4
<211> Length: 480
<212> Type: Artificial DNA
<213> Organism: – Escherichia coli BL21 (DE3)
<400> Sequence: 4

1 ACAACCTGTA TGAGGTGAAC ATGGGTGCGA CCGCGGTTGG CACCGGTCTG AACGCGGAGC
61 CGGAATACAT CAAAATTGTG ACCGAACACC TGGTTAAGCT GAGCGGTCAC CCGCTGCGTA
121 GCGCGAAGCA CCTGGTGGAC GCGACCCAGA ACACCGATTG CTATACCGAG GTTTCTGCGG
181 CGCTGAAAAT CTGCATGATT AACATGAGCA AGATCGCGAA CGACCTGCGT CTGATGGCGA
241 GCGGTCCGCG TGCGGGTCTG AGCGAAATCA TTCTGCCGGC GCGTCAACCG GGTAGCAGCA
301 TTATTCCGGG CCTGGTGGCC CCGGTTATGC CGGAAGTGGT TAACCAGGTG GCGTTCCAAG
361 TTATCGGTAA CGATCTGACC ATTAGCGCGG CGAGCGAGGC GGGCCAGTTT GAACTGAACG
421 TGATGGAGCC GGTTCTGTTC TTTAACCTGA TCCAAAGCAT CAGCATTATG AACAACGTGT

<210> SEQ ID NO: 5
<211> Length: 469
<212> Type: Polypeptide
<213> Organism: – Ureibacillus thermophiles
<400> Sequence: 5

1 MGEKNVRIEK DFLGEKEIPI DAYYGVQTMR ATENFPITGY RIHPELIKSL GIVKKAAALA
61 NMEVGLLDKT IGEYIVKAAD EVIEGKWDDQ FIVDPIQGGA GTSINMNANE VIANRALELM
121 GAEKGNYSLI SPNSHVNMSQ STNDAFPTAT HIAVLSLLNQ LIDTTKTMQQ VFLNKADEFA
181 GIIKMGRTHL QDAVPILLGQ EFEAYARVIA RDVERISNTK NNLYEVNMGA TAVGTGLNAE
241 PEYIKIVTEH LVKLSGHPLR SAKHLVDATQ NTDCYTEVSA ALKICMINMS KIANDLRLMA
301 SGPRAGLSEI ILPARQPGSS IMPGKVNPVM PEVVNQVAFQ VIGNDLTISA ASEAGQFELN
361 VMEPVLFFNL IQSISIMNNV FKTFTENCLK GIQANEERMK EYVERSIGII TAINPHVGYE
421 TAAKLAREAY LTGESIRDLC IKYDVLTEEQ LNEILNPYEM THPGIAGKH

<210> SEQ ID NO: 6
<211> Length: 469
<212> Type: Polypeptide sequence of ALA 1 tetra mutant
<213> Organism: – Ureibacillus thermophiles
<400> Sequence: 6

1 MGEKNVRIEK DFLGEKEIPI DAYYGVQTMR ATENFPITGY RIHPELIKSL GIVKKAAALA
61 NMEVGLLDKT IGEYIVKAAD EVIEGKWDDQ FIVDPIQGGA GTSINMNANE VIANRALELM
121 GAEKGNYSLI SPNSHVNMSQ STNDAFPTAT HIAVLSLLNQ LIDTTKTMQQ VFLNKADEFA
181 GIIKMGRCHL QDAVPILLGQ EFEAYARVIA RDVERISNTK NNLYEVNMGA TAVGTGLNAE
241 PEYIKIVTEH LVKLSGHPLR SAKHLVDATQ NTDCYTEVSA ALKICMINMS KIANDLRLMA
301 SGPRAGLSEI ILPARQPGSS IIPGLVAPVM PEVVNQVAFQ VIGNDLTISA ASEAGQFELN
361 VMEPVLFFNL IQSISIMNNV FKTFTENCLK GIQANEERMK EYVERSIGII TAINPHVGYE
421 TAAKLAREAY LTGESIRDLC IKYDVLTEEQ LNEILNPYEM THPGIAGKH

<210> SEQ ID NO: 7
<211> Length: 6,640
<212> Type: Nucleotide sequence of pET28a-AL1-T188C-M322I-K325L-N327A
<400> Sequence: 7

1 TGGCGAATGG GACGCGCCCT GTAGCGGCGC ATTAAGCGCG GCGGGTGTGG TGGTTACGCG
61 CAGCGTGACC GCTACACTTG CCAGCGCCCT AGCGCCCGCT CCTTTCGCTT TCTTCCCTTC
121 CTTTCTCGCC ACGTTCGCCG GCTTTCCCCG TCAAGCTCTA AATCGGGGGC TCCCTTTAGG
181 GTTCCGATTT AGTGCTTTAC GGCACCTCGA CCCCAAAAAA CTTGATTAGG GTGATGGTTC
241 ACGTAGTGGG CCATCGCCCT GATAGACGGT TTTTCGCCCT TTGACGTTGG AGTCCACGTT
301 CTTTAATAGT GGACTCTTGT TCCAAACTGG AACAACACTC AACCCTATCT CGGTCTATTC
361 TTTTGATTTA TAAGGGATTT TGCCGATTTC GGCCTATTGG TTAAAAAATG AGCTGATTTA
421 ACAAAAATTT AACGCGAATT TTAACAAAAT ATTAACGTTT ACAATTTCAG GTGGCACTTT
481 TCGGGGAAAT GTGCGCGGAA CCCCTATTTG TTTATTTTTC TAAATACATT CAAATATGTA
541 TCCGCTCATG AATTAATTCT TAGAAAAACT CATCGAGCAT CAAATGAAAC TGCAATTTAT
601 TCATATCAGG ATTATCAATA CCATATTTTT GAAAAAGCCG TTTCTGTAAT GAAGGAGAAA
661 ACTCACCGAG GCAGTTCCAT AGGATGGCAA GATCCTGGTA TCGGTCTGCG ATTCCGACTC
721 GTCCAACATC AATACAACCT ATTAATTTCC CCTCGTCAAA AATAAGGTTA TCAAGTGAGA
781 AATCACCATG AGTGACGACT GAATCCGGTG AGAATGGCAA AAGTTTATGC ATTTCTTTCC
841 AGACTTGTTC AACAGGCCAG CCATTACGCT CGTCATCAAA ATCACTCGCA TCAACCAAAC
901 CGTTATTCAT TCGTGATTGC GCCTGAGCGA GACGAAATAC GCGATCGCTG TTAAAAGGAC
961 AATTACAAAC AGGAATCGAA TGCAACCGGC GCAGGAACAC TGCCAGCGCA TCAACAATAT
1021 TTTCACCTGA ATCAGGATAT TCTTCTAATA CCTGGAATGC TGTTTTCCCG GGGATCGCAG
1081 TGGTGAGTAA CCATGCATCA TCAGGAGTAC GGATAAAATG CTTGATGGTC GGAAGAGGCA
1141 TAAATTCCGT CAGCCAGTTT AGTCTGACCA TCTCATCTGT AACATCATTG GCAACGCTAC
1201 CTTTGCCATG TTTCAGAAAC AACTCTGGCG CATCGGGCTT CCCATACAAT CGATAGATTG
1261 TCGCACCTGA TTGCCCGACA TTATCGCGAG CCCATTTATA CCCATATAAA TCAGCATCCA
1321 TGTTGGAATT TAATCGCGGC CTAGAGCAAG ACGTTTCCCG TTGAATATGG CTCATAACAC
1381 CCCTTGTATT ACTGTTTATG TAAGCAGACA GTTTTATTGT TCATGACCAA AATCCCTTAA
1441 CGTGAGTTTT CGTTCCACTG AGCGTCAGAC CCCGTAGAAA AGATCAAAGG ATCTTCTTGA
1501 GATCCTTTTT TTCTGCGCGT AATCTGCTGC TTGCAAACAA AAAAACCACC GCTACCAGCG
1561 GTGGTTTGTT TGCCGGATCA AGAGCTACCA ACTCTTTTTC CGAAGGTAAC TGGCTTCAGC
1621 AGAGCGCAGA TACCAAATAC TGTCCTTCTA GTGTAGCCGT AGTTAGGCCA CCACTTCAAG
1681 AACTCTGTAG CACCGCCTAC ATACCTCGCT CTGCTAATCC TGTTACCAGT GGCTGCTGCC
1741 AGTGGCGATA AGTCGTGTCT TACCGGGTTG GACTCAAGAC GATAGTTACC GGATAAGGCG
1801 CAGCGGTCGG GCTGAACGGG GGGTTCGTGC ACACAGCCCA GCTTGGAGCG AACGACCTAC
1861 ACCGAACTGA GATACCTACA GCGTGAGCTA TGAGAAAGCG CCACGCTTCC CGAAGGGAGA
1921 AAGGCGGACA GGTATCCGGT AAGCGGCAGG GTCGGAACAG GAGAGCGCAC GAGGGAGCTT
1981 CCAGGGGGAA ACGCCTGGTA TCTTTATAGT CCTGTCGGGT TTCGCCACCT CTGACTTGAG
2041 CGTCGATTTT TGTGATGCTC GTCAGGGGGG CGGAGCCTAT GGAAAAACGC CAGCAACGCG
2101 GCCTTTTTAC GGTTCCTGGC CTTTTGCTGG CCTTTTGCTC ACATGTTCTT TCCTGCGTTA
2161 TCCCCTGATT CTGTGGATAA CCGTATTACC GCCTTTGAGT GAGCTGATAC CGCTCGCCGC
2221 AGCCGAACGA CCGAGCGCAG CGAGTCAGTG AGCGAGGAAG CGGAAGAGCG CCTGATGCGG
2281 TATTTTCTCC TTACGCATCT GTGCGGTATT TCACACCGCA TATATGGTGC ACTCTCAGTA
2341 CAATCTGCTC TGATGCCGCA TAGTTAAGCC AGTATACACT CCGCTATCGC TACGTGACTG
2401 GGTCATGGCT GCGCCCCGAC ACCCGCCAAC ACCCGCTGAC GCGCCCTGAC GGGCTTGTCT
2461 GCTCCCGGCA TCCGCTTACA GACAAGCTGT GACCGTCTCC GGGAGCTGCA TGTGTCAGAG
2521 GTTTTCACCG TCATCACCGA AACGCGCGAG GCAGCTGCGG TAAAGCTCAT CAGCGTGGTC
2581 GTGAAGCGAT TCACAGATGT CTGCCTGTTC ATCCGCGTCC AGCTCGTTGA GTTTCTCCAG
2641 AAGCGTTAAT GTCTGGCTTC TGATAAAGCG GGCCATGTTA AGGGCGGTTT TTTCCTGTTT
2701 GGTCACTGAT GCCTCCGTGT AAGGGGGATT TCTGTTCATG GGGGTAATGA TACCGATGAA
2761 ACGAGAGAGG ATGCTCACGA TACGGGTTAC TGATGATGAA CATGCCCGGT TACTGGAACG
2821 TTGTGAGGGT AAACAACTGG CGGTATGGAT GCGGCGGGAC CAGAGAAAAA TCACTCAGGG
2881 TCAATGCCAG CGCTTCGTTA ATACAGATGT AGGTGTTCCA CAGGGTAGCC AGCAGCATCC
2941 TGCGATGCAG ATCCGGAACA TAATGGTGCA GGGCGCTGAC TTCCGCGTTT CCAGACTTTA
3001 CGAAACACGG AAACCGAAGA CCATTCATGT TGTTGCTCAG GTCGCAGACG TTTTGCAGCA
3061 GCAGTCGCTT CACGTTCGCT CGCGTATCGG TGATTCATTC TGCTAACCAG TAAGGCAACC
3121 CCGCCAGCCT AGCCGGGTCC TCAACGACAG GAGCACGATC ATGCGCACCC GTGGGGCCGC
3181 CATGCCGGCG ATAATGGCCT GCTTCTCGCC GAAACGTTTG GTGGCGGGAC CAGTGACGAA
3241 GGCTTGAGCG AGGGCGTGCA AGATTCCGAA TACCGCAAGC GACAGGCCGA TCATCGTCGC
3301 GCTCCAGCGA AAGCGGTCCT CGCCGAAAAT GACCCAGAGC GCTGCCGGCA CCTGTCCTAC
3361 GAGTTGCATG ATAAAGAAGA CAGTCATAAG TGCGGCGACG ATAGTCATGC CCCGCGCCCA
3421 CCGGAAGGAG CTGACTGGGT TGAAGGCTCT CAAGGGCATC GGTCGAGATC CCGGTGCCTA
3481 ATGAGTGAGC TAACTTACAT TAATTGCGTT GCGCTCACTG CCCGCTTTCC AGTCGGGAAA
3541 CCTGTCGTGC CAGCTGCATT AATGAATCGG CCAACGCGCG GGGAGAGGCG GTTTGCGTAT
3601 TGGGCGCCAG GGTGGTTTTT CTTTTCACCA GTGAGACGGG CAACAGCTGA TTGCCCTTCA
3661 CCGCCTGGCC CTGAGAGAGT TGCAGCAAGC GGTCCACGCT GGTTTGCCCC AGCAGGCGAA
3721 AATCCTGTTT GATGGTGGTT AACGGCGGGA TATAACATGA GCTGTCTTCG GTATCGTCGT
3781 ATCCCACTAC CGAGATATCC GCACCAACGC GCAGCCCGGA CTCGGTAATG GCGCGCATTG
3841 CGCCCAGCGC CATCTGATCG TTGGCAACCA GCATCGCAGT GGGAACGATG CCCTCATTCA
3901 GCATTTGCAT GGTTTGTTGA AAACCGGACA TGGCACTCCA GTCGCCTTCC CGTTCCGCTA
3961 TCGGCTGAAT TTGATTGCGA GTGAGATATT TATGCCAGCC AGCCAGACGC AGACGCGCCG
4021 AGACAGAACT TAATGGGCCC GCTAACAGCG CGATTTGCTG GTGACCCAAT GCGACCAGAT
4081 GCTCCACGCC CAGTCGCGTA CCGTCTTCAT GGGAGAAAAT AATACTGTTG ATGGGTGTCT
4141 GGTCAGAGAC ATCAAGAAAT AACGCCGGAA CATTAGTGCA GGCAGCTTCC ACAGCAATGG
4201 CATCCTGGTC ATCCAGCGGA TAGTTAATGA TCAGCCCACT GACGCGTTGC GCGAGAAGAT
4261 TGTGCACCGC CGCTTTACAG GCTTCGACGC CGCTTCGTTC TACCATCGAC ACCACCACGC
4321 TGGCACCCAG TTGATCGGCG CGAGATTTAA TCGCCGCGAC AATTTGCGAC GGCGCGTGCA
4381 GGGCCAGACT GGAGGTGGCA ACGCCAATCA GCAACGACTG TTTGCCCGCC AGTTGTTGTG
4441 CCACGCGGTT GGGAATGTAA TTCAGCTCCG CCATCGCCGC TTCCACTTTT TCCCGCGTTT
4501 TCGCAGAAAC GTGGCTGGCC TGGTTCACCA CGCGGGAAAC GGTCTGATAA GAGACACCGG
4561 CATACTCTGC GACATCGTAT AACGTTACTG GTTTCACATT CACCACCCTG AATTGACTCT
4621 CTTCCGGGCG CTATCATGCC ATACCGCGAA AGGTTTTGCG CCATTCGATG GTGTCCGGGA
4681 TCTCGACGCT CTCCCTTATG CGACTCCTGC ATTAGGAAGC AGCCCAGTAG TAGGTTGAGG
4741 CCGTTGAGCA CCGCCGCCGC AAGGAATGGT GCATGCAAGG AGATGGCGCC CAACAGTCCC
4801 CCGGCCACGG GGCCTGCCAC CATACCCACG CCGAAACAAG CGCTCATGAG CCCGAAGTGG
4861 CGAGCCCGAT CTTCCCCATC GGTGATGTCG GCGATATAGG CGCCAGCAAC CGCACCTGTG
4921 GCGCCGGTGA TGCCGGCCAC GATGCGTCCG GCGTAGAGGA TCGAGATCTC GATCCCGCGA
4981 AATTAATACG ACTCACTATA GGGGAATTGT GAGCGGATAA CAATTCCCCT CTAGAAATAA
5041 TTTTGTTTAA CTTTAAGAAG GAGATATACC ATGGGTGAAA AGAACGTGCG TATCGAGAAA
5101 GACTTCCTGG GCGAGAAGGA AATCCCGATT GATGCGTACT ATGGCGTTCA GACCATGCGT
5161 GCGACCGAAA ACTTTCCGAT CACCGGTTAT CGTATTCACC CGGAGCTGAT CAAGAGCCTG
5221 GGCATTGTGA AGAAGGCGGC GGCGCTGGCG AACATGGAAG TTGGTCTGCT GGACAAAACC
5281 ATCGGCGAGT ACATTGTGAA GGCGGCGGAT GAGGTTATCG AGGGTAAATG GGACGATCAG
5341 TTCATCGTGG ACCCGATTCA AGGTGGCGCG GGCACCAGCA TCAACATGAA CGCGAACGAA
5401 GTTATTGCGA ACCGTGCGCT GGAGCTGATG GGTGCGGAAA AGGGCAACTA TAGCCTGATC
5461 AGCCCGAACA GCCACGTGAA CATGAGCCAG AGCACCAACG ATGCGTTTCC GACCGCGACC
5521 CACATTGCGG TGCTGAGCCT GCTGAACCAA CTGATTGACA CCACCAAAAC CATGCAGCAA
5581 GTTTTCCTGA ACAAGGCGGA TGAGTTTGCG GGTATCATTA AAATGGGTCG TTGCCACCTG
5641 CAGGATGCGG TTCCGATCCT GCTGGGTCAA GAGTTCGAAG CGTACGCGCG TGTGATCGCG
5701 CGTGATGTTG AACGTATTAG CAACACCAAA AACAACCTGT ATGAGGTGAA CATGGGTGCG
5761 ACCGCGGTTG GCACCGGTCT GAACGCGGAG CCGGAATACA TCAAAATTGT GACCGAACAC
5821 CTGGTTAAGC TGAGCGGTCA CCCGCTGCGT AGCGCGAAGC ACCTGGTGGA CGCGACCCAG
5881 AACACCGATT GCTATACCGA GGTTTCTGCG GCGCTGAAAA TCTGCATGAT TAACATGAGC
5941 AAGATCGCGA ACGACCTGCG TCTGATGGCG AGCGGTCCGC GTGCGGGTCT GAGCGAAATC
6001 ATTCTGCCGG CGCGTCAACC GGGTAGCAGC ATTATTCCGG GCCTGGTGGC CCCGGTTATG
6061 CCGGAAGTGG TTAACCAGGT GGCGTTCCAA GTTATCGGTA ACGATCTGAC CATTAGCGCG
6121 GCGAGCGAGG CGGGCCAGTT TGAACTGAAC GTGATGGAGC CGGTTCTGTT CTTTAACCTG
6181 ATCCAAAGCA TCAGCATTAT GAACAACGTG TTCAAAACCT TTACCGAAAA CTGCCTGAAG
6241 GGTATTCAGG CGAACGAGGA ACGTATGAAA GAGTACGTGG AACGTAGCAT CGGTATCATT
6301 ACCGCGATTA ACCCGCACGT GGGTTATGAG ACCGCGGCGA AGCTGGCGCG TGAAGCGTAT
6361 CTGACCGGCG AGAGCATCCG TGACCTGTGC ATTAAATACG ATGTTCTGAC CGAGGAACAA
6421 CTGAACGAAA TCCTGAACCC GTATGAGATG ACCCACCCGG GTATTGCGGG CAAGCACCTC
6481 GAGCACCACC ACCACCACCA CTGAGATCCG GCTGCTAACA AAGCCCGAAA GGAAGCTGAG
6541 TTGGCTGCTG CCACCGCTGA GCAATAACTA GCATAACCCC TTGGGGCCTC TAAACGGGTC
6601 TTGAGGGGTT TTTTGCTGAA AGGAGGAACT ATATCCGGAT ,CLAIMS:WE CLAIM:
1. A novel series of synthetic gene comprising of nucleotide SEQ ID NO: 1, 2, 3, 4, 5, 6 and 7.

2. A process for the preparation of (R)-ß-amino butyric acid of formula – I,
,
which comprises:
(i) reacting crotonic acid of formula – III with ammonia in the presence of a
catalyst selected from any of the SEQ ID NO as claimed in claim 1,
,
(ii) isolating (R)-ß-amino butyric acid of formula – I.

3. A process for the preparation of Dolutegravir or pharmaceutically acceptable salts thereof of formula – II;
,
or its salts which comprises:
(i) reacting crotonic acid of formula – III
,

with ammonia in the presence of a catalyst selected from any of the SEQ ID NO as claimed in claim 1,
(ii) isolating (R)-ß-amino butyric acid of formula – I.
,
(iii) converting (R)- ß-amino butyric acid (I) to Dolutegravir (II) or pharmaceutically acceptable salts thereof.