WO2019123166A1

WO2019123166A1 - Nucleotide sequences encoding 3-quinuclidinone reductase and glucose dehydrogenase and soluble expression thereof

Info

Publication number: WO2019123166A1
Application number: PCT/IB2018/060096
Authority: WO
Inventors: Sachin Prabhakar BACHATE; Amol Arvind KANERI; Purshottam Sudin Sinai KUNCOLIENKAR; Rahul Subhash CHOUGULE; Nilabh ANAND; Conchita D’SOUZA; Mamata KATDARE; Sudeep Kumar; Dhananjay Sathe
Original assignee: Unichem Laboratories Ltd
Priority date: 2017-12-18
Filing date: 2018-12-14
Publication date: 2019-06-27

Abstract

The present invention relates to nucleotide sequence of SEQ ID 1 and SEQ ID 2 encoding recombinant 3-Quinuclidinone reductase and Glucose Dehydrogenase respectively. The invention further relates to the clone comprising the said nucleotide sequences and process to prepare 3-Quinuclidinone reductase and Glucose Dehydrogenase having amino acid sequence of SEQ ID 3 and SEQ ID 4, respectively.

Description

NUCLEOTIDE SEQUENCES ENCODING 3-QUINUCLIDINONE REDUCTASE AND GLUCOSE DEHYDROGENASE AND SOLUBLE

EXPRESSION THEREOF

TECHNICAL FIELD OF THE INVENTION:

The present invention relates to the nucleotide sequences encoding recombinant 3-quinuclidinone reductase and recombinant glucose dehydrogenase and their expression of soluble form in Escherichia coli.

BACKGROUND OF THE INVENTION:

(R)-3-Quinuclidinol of Formula I is an important building block for the production of anti-muscarinic drugs such as Solifenacin succinate, T alsaclidinefumarate, CevimelineHCl.

Formula I

Chemically (R)-3-quinuclidinol is prepared by either reduction of 3- quinuclidinone of Formula II, followed by separation of (R)-3-quinuclidinol or by asymmetric reduction of 3-quinuclidinone (Scheme I).

Scheme I

The two step process of reduction of 3-quinuclidinone of Formula II, followed by separation of (R)-3-quinuclidinol is tedious, time consuming, lacks reproducibility while separating the particular enantiomer and is low yielding. The single step conversion using asymmetric chemical catalyst is costly and less reproducible. Enzymatic reduction of 3-Quinuclidinone to (R)-3-Quinuclidinol is found to be easy, economical and highly reproducible process to prepare (R)-3-Quinuclidinol as compared to the chemical reduction. There are various methods of bio-catalytic conversion of 3-Quinuclidinone to (R)-3-Quinuclidinol using a co-factor dependent ketoreductase derived from different sources. Due to substrate-enzyme specificity bio-catalytic reaction is reproducible, high yielding without any trace of unwanted enantiomer.

The references disclose use of recombinant 3-quinuclidinone reductase, wherein 3-quinuclidinone specific reductase enzyme is derived from different microorganisms. Uzura et al (Appl. Microbiol Biotech, volume 83, pages 617- 626, year 2009)discloses ketoreductase isolated from Rhodotorula rubra JCM3782. EP2796548 Al and Isotani et al (Int. J. Mol Sci. volume 13, pages 13542-13553, year 2012) described 3-Quinuclidinone reductase derived from Microbacterium luteolum. In US20140147896, the enzyme to prepare (R)-3- Quinuclidinol from 3-Quinuclidinone is derived from Saccharomyces cerevisiae. EP2423320 uses polypeptide derived from the bacteria Burkholderia or a recombinant organism capable of producing the polypeptide. US5888804 discloses a process for the production of optically active (R)-3-Quinuclidinol from 3-Quinuclidinone using enzyme derived from microorganisms from the genus Nakazawaea, Candida and Proteus.

Though the specificity of the enzymes/recombinant enzymes derived from different sources is high towards 3-quinuclidinone, the enzyme capacity and the reaction time considerably differs.

Uzura et al. discloses a method for the production of (R)-3-quinuclidinol with 99% enantiomeric excess (e.e.). The process describes, E. coli strain co expressing the Nicotinamide Adenine Dinucleotide (NAD) or Nicotinamide Adenine Dinucleotide Phosphate (NADP) dependent ketoreductase and a co factor regenerating enzyme, glucose dehydrogenase (GDH) cloned in two different plasmids, to produce optically active (R)-3-quinuclidinol. The enzyme takes 2lh to convert 3-Quinuclidinone to (R)-3-Quinuclidinol with a substrate loading capacity of this enzyme is 63mg/mL. This recombinant enzyme takes longer time and produces less quantity of (R)-3-Quinuclidinol. The process by virtue of the said enzyme is less efficient, time consuming and less economical.

In the process of preparation of (R)-3-quinuclidinol Isotani el al. describes the use of immobilized enzymes for conversion of 3-quinuclidinone to (R)-3- quinuclidinol and organic solvents in the reaction mixture. The process becomes time consuming and expensive due to additional cumbersome steps of separation of product from the organic solvents. Use of immobilized-enzymes increase the cost of the process.

EP2796548 Al discloses a process for the preparation of a polynucleotide sequence encoding a chimeric fusion protein comprising a glucose dehydrogenase and NADP dependent 3-quinuclidinone reductase and expressing the said proteins in the host E. coli cells. The process describes the use of whole cell and crude extract for the enzymatic conversion in the presence of phosphate buffer at a suitable pH, temperature and reaction time. The process requires 48-96h for conversion at 30°C to achieve more than 95% conversion and 99% e.e. Thus the process is lengthy and less efficient.

In all the cited references the enzyme/recombinant enzyme system of ketoreductase and glucose dehydrogenase has low capacity of substrate conversion, takes longer time for conversion, is low yielding and less efficient. Thus the process of production of (R)-3-quinuclidinol is expensive and difficult to carry out on an industrial scale. Hence there is an unmet need to develop an enzyme/recombinant enzyme system that has high conversion capacity, higher substrate loading capacity, high yielding, economical and could be prepared easily on larger scale. Also the enzyme or recombinant enzyme should be highly efficient towards conversion of 3-quinuclidinone to (R)-3-quinuclidinol, giving high yield of the product in shorter period.

OBJECT OF THE INVENTION:

The main object of the invention is to provide nucleotide sequence encoding highly efficient recombinant3-quinuclidinone reductase. Yet another object of the invention is to provide nucleotide sequence encoding highly efficient recombinant glucose dehydrogenase.

Another object of the invention is to devise a reproducible, economical, industrially feasible and efficient cloning system for the preparation of recombinant3 -quinuclidinone reductase .

Yet another object of the invention is to devise a reproducible, economical, industrially feasible and efficient cloning system for the preparation of recombinantgluco se dehydrogenase .

Another object of the invention is to develop a quick, high yielding, and economical process to prepare recombinant3 -quinuclidinone reductase in short period.

Yet another object of the invention is to develop a rapid, highly efficient, cost effective process to prepare recombinant glucose dehydrogenase in short period.

SUMMARY OF THE INVENTION:

The main embodiment of the present invention relates to the nucleotide sequence of SEQ ID 1 encoding 3-quinuclidinone reductase derived from Rhodotorula rubra (Rr).

Another embodiment of the present invention relates to the nucleotide sequence of SEQ ID 2 encoding Glucose Dehydrogenase derived from Bacillus megaterium (Bm).

Yet another embodiment of the present invention relates to the clone for the preparation of recombinant 3-quinuclidinone reductase in soluble form comprising bacterial host E. coll BL21 DE3 Gold cells, a vector pET28a harboring nucleotide sequence of SEQ ID 1 encoding 3-quinuclidinone reductase. The invention further relates to the process for the production of clone.

Further in yet another embodiment of the present invention clone for the preparation of recombinant glucose dehydrogenase in soluble form comprising bacterial host E. coll BL21 DE3 Gold cells, a vector pET28a harboring nucleotide sequence of SEQ ID 2 encoding glucose dehydrogenase is disclosed. Yet another embodiment of the present invention relates to the process for the production of said clone.

Another embodiment of present invention relates to the process to prepare recombinant 3-quinuclidinone reductase in soluble form, wherein the process comprises;

a. producing a clone comprising a bacterial host E. coli BL21 DE3 Gold cells, a vector pET28a harbouring nucleotide sequence of SEQ ID 1 ;

b. fed-batch fermentation of the clone obtained in step‘a’ in a fermentation medium;

c. separation of soluble form of 3-quinuclidinone reductase from the fermentation broth obtained in step‘b’.

Yet another embodiment of present invention relates to the process to prepare recombinant glucose dehydrogenase in soluble form, wherein the process comprises;

a. producing a clone comprising a bacterial host E. coli BL21 DE3 Gold cells, a vector pET28a harbouring nucleotide sequence of SEQ ID 2;

b. fed-batch fermentation of the clone obtained in step ‘a’ in a fermentation medium;

c. separation of soluble form of glucose dehydrogenase from the fermentation broth obtained in step‘b’.

The present invention further relates to the production of (R)-3-quinuclidinol using 3-quinuclidinone reductase encoded by nucleotide sequence of SEQ ID 1 and glucose dehydrogenase encoded by nucleotide sequence of SEQ ID 2.

BRIEF DESCRIPTION OF THE FIGURES:

Figure 1: Annotated diagram of pET28a Vector with SEQ ID NO: 1: Nucleotide Sequence Encoding variant of 3-Quinuclidonone reductase derived from Sequence of SEQ ID NO: 3. DETAILED DESCRIPTION OF THE INVENTION:

The terms‘ketoreductase enzyme’,‘3-quinuclidinone reductase enzyme’ are used interchangeably and all refer to an enzyme synthesized by encoding nucleotide sequence of SEQ ID 1 or an enzyme derived from the amino acid sequence from Rhodotorula rubra. The enzyme isused in the enzymatic reduction of 3- Quinuclidinone to (R)-3-Quinuclidinol.

The terms Glucose Dehydrogenase and GDH are interchangeably used and refer to the enzyme derived from the amino acid sequence from Bacillus megaterium (Bm), wherein GDH has the ability to regenerate co-factors like NADH⁺ or NADPH⁺ using glucose as the substrate.

v/v or V/V means volume/volume; w/v or W/V means weight/volume and w/w or W/W means weight/weight.

The terms“identity” or“identical” as used herein refer to two or more referenced entities that share at least partial similarities over a given region or portion. Substantial identity refers to a molecule that is structurally or functionally conserved such that it has or is predicted to have at least partial structure or function of one or more of the structures or functions (e.g., a biological function or activity) of the reference molecule, or a relevant/corresponding region or portion of the reference molecule to which it shares identity.

Unless otherwise indicated, the term“homology” is used interchangeably with the term“identity” in the present specification.

The term“soluble form” as used herein with respect to protein and refers to the modified protein being expressed in a soluble or in partially soluble form which means the modified protein is not completely forming inclusion bodies. In one embodiment, solubility of the modified protein is determined by cell lysis of a host cell that expresses the modified protein and subsequent SDS-PAGE analysis of the lysis supernatant and pellet. The presence of the modified protein in the lysis supernatant indicates that it is soluble. The presence of the modified protein in the lysis supernatant and the pellet indicates that it is partially soluble and presence of the modified protein in the lysis pellet indicating that the modified protein is expressed as inclusion bodies. The term“nucleotide sequence” refers to the sequence comprising nucleotides wherein “nucleotide” may be naturally occurring nucleotides or a synthetic nucleotide analogues that are recognized by cellular enzymes. The DNA of the present invention means a genomic sequence containing regulatory sequences such as a promoter and a terminator, which are involved in the expression of the gene of interest.

The present invention relates to the nucleotide sequence of SEQ ID 1 encoding 3- quinuclidinone reductase derived from Rhodotorula rubra.

According to the aspect of the invention 3-quinuclidinone reductase derived from Rhodotorula rubra has amino acid sequence of SEQ ID 3.

According to the aspect of the invention nucleotide sequence of SEQ ID 1 is derived from the amino acid of SEQ ID 3, obtained from Rhodotorula rubra. The sequence ofSEQ ID 1 is nucleotide derivative of SEQ ID 3 with bio-informatics analysis such as codon optimization, mRNA (messenger Ribonucleic Acid) stability and GC (Guanine-Cytosine) content.

The preferred embodiment of the present invention relates to nucleotide sequence, which is at least 70% identical to nucleotide sequence of SEQ ID 1.

The more preferred embodiment of the present invention relates to nucleotide sequence, which is at least 80% identical to nucleotide sequence of SEQ ID 1.

The most preferred embodiment of the present invention relates to nucleotide sequence, which is at least 90% identical to nucleotide sequence of SEQ ID 1.

The present invention further relates to the nucleotide sequence of SEQ ID 2 encoding glucose dehydrogenase derived from Bacillus megaterium.

According to the aspect of the invention glucose dehydrogenase derived from Bacillus megateriumhas, amino acid sequence of SEQ ID 4.

According to the aspect of the invention nucleotide sequence of SEQ ID 2 is derived from the amino acid sequence of SEQ ID 4, obtained from B. megaterium. The nucleotide sequence ofSEQ ID 2 is nucleotide derivative of SEQ ID 4 with bio-informatics analysis such as codon optimization, mRNA stability and GC content. The preferred embodiment of the present invention comprises nucleotide sequence, which is at least 70% identical to nucleotide sequence of SEQ ID 2.

The preferred embodiment of the present invention relates to nucleotide sequence, which is at least 80% identical to nucleotide sequence of SEQ ID 2.

A most preferred embodiment of the present invention comprises nucleotide sequence, which is at least 90% identical to nucleotide sequence of SEQ ID 2.

The present invention further relates to the clone for the preparation of recombinant 3-quinuclidinone reductase in soluble form comprising a bacterial host, a vector harboring nucleotide sequence encoding 3-quinuclidinone reductase.

Accordingly, the host strain for the expression of 3-quinuclidinone reductase is chosen such that it expresses the protein of interest. The preferred bacterial host is E. coli. More preferred host strain is E. coli BL 21 DE3 Gold strain. The vector having gene of interest is transformed into E. coli BL 21 DE3 Gold strain. The gene is switched on when culture is induced by the addition of an inducer like Isopropyl-P-D- l-thiogalactopyranoside (IPTG).

According to another aspect of the invention, the vector used for expression of recombinant 3-quinuclidinone reductase is pET28a. The vector pET28a is cloned with the variant nucleotide sequence of SEQ ID 1, transformed and expressed in the host E. coli BL21 DE3 Gold cells.

According to the aspect of the invention nucleotide sequence of SEQ ID 1 is derived from the amino acid of SEQ ID 3, obtained from Rhodotorula rubra. The sequence ofSEQ ID 1 is nucleotide derivative of SEQ ID 3 with bio-informatics analysis such as codon optimization, mRNA stability and GC content.

The present invention further relates to the clone for the preparation of recombinant glucose dehydrogenase in soluble form comprising a bacterial host, a vector harboring a nucleotide sequence encoding glucose dehydrogenase.

Accordingly the host strain for the expression of glucose dehydrogenase is chosen such that it expresses the protein of interest. The preferred bacterial host is E. coli. More preferred host strain is E. coli BL 21 DE3 Gold strain. The vector having gene of interest is transformed into E. coli BL 21 DE3 Gold strain. The gene is switched on when culture is induced by the addition of an inducer like Isopropyl- b-D- l-thiogalactopyranoside (IPTG).

According to another aspect of the invention vector used for expression of recombinant glucose dehydrogenase is pET28a. The vector pET28a is cloned with the nucleotide sequence of SEQ ID 2, transformed and expressed in host strain E. coli BL21 DE3 Gold cells.

According to the aspect of the invention nucleotide sequence of SEQ ID 2 is derived from the amino acid sequence of SEQ ID 4, obtained from Bacillus megaterium. The nucleotide sequence ofSEQ ID 2 is nucleotide derivative of SEQ ID 4 with bio -informatics analysis such as codon optimization, mRNA stability and GC content.

The present invention relates to the process to prepare recombinant 3- quinuclidinone reductase in soluble form, wherein the process comprises;

a. preparing a clone comprising a bacterial host E. coli BL21 DE3 Gold cells, a vector pET28a harboring a nucleotide sequence of SEQ ID 1 ;

b. fed-batch fermentation of the clone obtained in step‘a’ in a fermentation broth; c. separation of soluble form of 3-quinuclidinone reductase from the fermentation broth obtained in step‘b’.

According to the aspect of the invention the clone is prepared by transforming E. coli BL21 DE3 Gold cells with pET28a vector that carries the nucleotide sequence of SEQ ID 1.

According to another aspect of the invention, the clone is cultured at 30°C-40°C preferably 35°C - 39°C in inoculum medium containing lOg/L - 30g/L of Luria Broth, 3g/L - 7g/L of dextrose, 6g/L - 9g/L disodium hydrogen phosphate, 0.5g - 2.0g Magnesium Sulphate. A three hundred milliliter of culture is used as inoculum for 3L fermenter. Fermentation is carried out at 30°C- 40°C preferably between 35°C - 39°C for l0h-l4h under fed-batch mode using a glycerol and yeast extract. The rate of feeding of glycerol and yeast extract in fed-batch stage was 2g/L/h - 6g/L/h from lh - 5h, 4g/L/h - lOg/L/h from 5h - 8h, 2g/L/h - 6g/L/h from 8h - l4h. The Carbon to Nitrogen (C : N) ratio is 3:1 to 5:1. At the end of fermentation, the broth was centrifuged at 7,000-12,000 rpm for 20 min - 40 min at l2°C - l8°C. Supernatant was carefully decanted to get the wet cell pellet. An output of 200g - 260g of wet cell pellet per liter of the culture broth was obtained.

Another aspect of the invention is isolation of 3-quinuclidinone reductase, wherein the wet cell pellet is suspended in 0.08M - 0.12M potassium phosphate buffer pH 7.0 - 8.0 in a ratio of 1:6 to 1:15 and stirred to form a uniform suspension. The suspended cells were then lysed by high pressure homogenization at -15,000 psi - 20,000 psi (pounds per square inch). The lysed cells were centrifuged at 7,000 - 12,000 rpm for 20-40 min at l2°C - l8°C to pellet down the cell debris. The supernatant is the source of soluble 3-Quinuclidinone reductase enzyme.

The present invention relates to the process to prepare recombinant glucose dehydrogenase in soluble form, wherein the process comprises;

a. preparing a clone comprising a bacterial host E. coli BL21 DE3 Gold cells, a vector pET28a harboring a variant nucleotide sequence of SEQ ID 2;

b. fed-batch fermentation of the clone obtained in step‘a’ in a fermentation broth; c. separation of soluble form of glucose dehydrogenase from the fermentation broth obtained in step‘b’.

According to the aspect of the invention the clone is prepared by transforming E coli BL21 DE3 Gold cells with pET28a vector carrying the variant nucleotide sequence of SEQ ID 2.

According to another aspect of the invention the clone is cultured at 30°C-40°C preferably 35°C - 39°C in inoculum medium containing lOg/L - 30g/L of Luria Broth, 3g/L - 7g/L of dextrose, 6g/L - 9g/L of disodium hydrogen phosphate, 0.5g - 2.0g Magnesium Sulphate. A three hundred milliliter of culture is used as inoculum for 3L fermenter. Fermentation run is carried out at l8°C- 30°C preferably between 20°C - 27°C for 22h - 28h under fed-batch mode using a glycerol and yeast extract. The rate of feeding of glycerol-yeast extract in fed- batch stage is l.5g/L/h - 4.0g/L/h from 2h - 4h, 4g/L/h - 8g/L/h for 5h - 22h, and l.5g/L/h - 4.0g/L/h after 22h. The Carbon to Nitrogen (C:N) ratio is 3:1 to 5:1. The fermentation broth was centrifuged at 7,000-12,000 rpm for 20-40 min at l2°C -l8°C. Supernatant was carefully decanted to get the wet cell pellet. An output of l80g - 240g of wet cell pellet per liter of the culture broth was obtained.

Another aspect of the invention is the process for the preparation of the cell lysate of GDH enzyme. The wet cell pellet was suspended in 0.08M - 0.12M potassium phosphate buffer pH 7.0 - 8.0 in a ratio of 1:6 to 1:15 and stirred to form a uniform suspension which was then subjected to lysis by high pressure homogenization at ~l5,000psi - 20,000 psi. The lysed cells were centrifuged at 7,000-12,000 rpm for 20-40 min at l2°C - l8°C to pellet down the cell debris. The clear supernatant free from the cell debris is the source of soluble Glucose Dehydrogenase enzyme.

The present invention also relates to the (R)-3-quinuclidinol prepared using 3- quinuclidinone reductase encoded by nucleotide sequence of SEQ ID 1 and glucose dehydrogenase encoded by nucleotide sequence of SEQ ID 2.

According to this embodiment (R)-3-quinuclidinol is prepared by reacting 3- quinuclidinone with 3-quinuclidinone reductase encoded by nucleotide sequence of SEQ ID l,in the presence of a suitable co-factor regenerating system comprising of glucose dehydrogenase encoded by nucleotide sequence of SEQ ID 2 and co-factors NAD or NADP, wherein both the enzymes are in the cell lysate.

According to the aspect of the invention the enzymatic reduction of 3- quinuclidinone to (R)-3-quinuclidinol is carried out using the process comprising steps of:

a. preparation of a clone comprising a bacterial expression host viz. E. coli BL 21 DE3 Gold, a vector pET28a and a variant nucleotide sequence of SEQ ID 1 encoding 3-quinuclidinone reductase derived from Rhodotorula rubra;

b. preparation of a clone comprising a bacterial expression host viz. E. coli BL 21 DE3 Gold, a vector pET28a and a variant nucleotide sequence of SEQ ID 2 encoding glucose dehydrogenase derived from Bacillus megaterium;

c. preparation of 3-quinuclidinone reductase in soluble form by fed-batch fermentation of clone obtained in step‘a’; d. separation of soluble form of 3-quinuclidinone reductase as cell lysate from fermentation broth of step‘c’;

e. preparation of glucose dehydrogenase in soluble form by fed-batch fermentation of clone obtained in step‘b’;

f. separation of soluble form of glucose dehydrogenase as cell lysate from fermentation broth of step‘e’;

g. reacting 3-quinuclidinone with the cell lysate containing 3-quinuclidinone reductase obtained in step‘d’, in the presence of cell lysate containing glucose dehydrogenase obtained in step‘f , to produce (R)-3-quinuclidinol;

h. extracting and purifying (R)-3-quinuclidinol obtained in step‘g’.

According to the aspect of the invention the nucleotide sequence of having SEQ ID 1 obtained from Rhodotorularubraby subjecting amino acid sequence of SEQ ID 3 to bio-informatics analysis like codon optimization, mRNA stability and GC content of the sequence.

According to the aspect of the invention the nucleotide sequencehaving SEQ ID 2 of glucose dehydrogenase obtained from Bacillus megateriumby subjecting SEQ ID 4 to bio-informatics analysis like codon optimization, mRNA stability and GC content of the sequence.

Accordingly the host strain for the expression of 3-quinuclidinone reductase and glucose dehydrogenase was chosen such that it expresses the proteins of interest. This vector having gene of interest is transformed into E. coll BL 21 DE3 Gold strain. This cloned vector has T7 promoter and optimized ribosome binding site for overexpression of cloned gene. The gene is switched on when culture is induced by addition of an inducer like IPTG.

According to another aspect of the invention, 3-quinuclidinone reductase is produced by fed-batch fermentation of clone E. coll BL 21 DE3 Gold, a vector pET28a and a nucleotide sequence of SEQ ID 1, wherein the clone is cultured at 30°C-40°C preferably 35°C - 39°C in inoculum medium containing lOg/L - 30g/L of Luria Broth, 3g/L - 7g/L of dextrose, 6g/L - 9g/L disodium hydrogen phosphate, 0.5g/L - 2.0g/L magnesium sulphate. A three hundred milliliter of culture is used as inoculum for 3L fermenter. Fermentation was carried out at 30°C -40°Cpreferably between 35°C - 39°C for lOh - l4h under fed-batch mode using a glycerol and yeast extract. The rate of feeding of glycerol-yeast extract in fed-batch stage is 2g/L/h - 6g/L/h from lh - 5h, 4g/L/h - lOg/L/h for 5h - 8h, 2g/L/h - 6g/L/h for 8h - l4h. The Carbon to Nitrogen (C:N) ratio is 3:1 to 5:1. The fermentation harvest broth was centrifuged at 7,000-12,000 rpm for 20-40 min at l2°C -l8°C. The culture supernatant was carefully decanted to get bacterial wet cell mass. An output of 200g - 260g of wet cell mass per liter of the culture broth was obtained.

According to another aspect of the invention, glucose dehydrogenase is produced by fed -batch fermentation of clone E. coli BL 21 DE3 Gold harboring a vector pET 28a cloned with a variant nucleotide sequence of SEQ ID 2, wherein the clone is cultured at 30-40°C preferably between 35°C -39°C in inoculum medium containing lOg/L - 30g/L of Luria Broth, 3g/L - 7g/L of dextrose, 6g/L - 9g/L of disodium hydrogen phosphate and 0.5g - 2.0g of magnesium sulphate. A three hundred milliliter of culture was used as inoculum for 3L fermentation medium. Fermentation was carried out at 20°C - 28°C preferably at 22°C - 28°C for 20h - 28h under fed-batch mode using a glycerol and yeast extract. The rate of feeding of glycerol- yeast extract in fed-batch stage is l.5g/L/h - 4g/L/h from 2h - 4h, 4g/L/h - 8g/L/h for 5h - 22h and l.5g/L/h - 4.0g/L/hfor 23h - 28h. The Carbon to Nitrogen (C: N) ratio is 3:1 to 5:1

Cells were separated by centrifugation of fermentation broth at 7,000-12,000 rpm for 20-40 min at l2°C-l8°C. The clear supernatant was carefully decanted and the wet cell pellet was recovered. An output of l80g - 240g of wet cell pellet per liter of the culture broth was obtained.

Another aspect of the invention is isolation of 3-quinuclidinone reductase, wherein the cell pellet was suspended in 0.08M - 0.12M potassium phosphate buffer pH 7.0 - 8.0 in a ratio of 1:6 to 1:15 and stirred to form a uniform suspension which is then subjected to lysis on a homogenizer at ~l5,000psi - 20,000psi. The lysed cells were centrifuged at 7,000 - 12,000 rpm for 20-40 min at l2°C - l8°C to remove the cell debris. The resultant clear supernatant was the source of enzyme 3-quinuclidinone reductase Another aspect of the invention is isolation of glucose dehydrogenase, wherein the cell pellet is suspended in 0.08M - 1.12M potassium phosphate buffer with pH 7.0 to 8.0 in a ratio of 1:6 to 1:15 and stirred to form a uniform suspension which is then subjected to lysis on a homogenizer at -15,000 psi - 20,000 psi. The lysate is centrifuged at 7,000-12,000 rpm for 20-40 min at l2°C - l8°C. The cell debris settle at the bottom giving clear supernatant. This clear supernatant is the source of glucose dehydrogenase enzyme

Yet another aspect of present invention is reacting 3-quinuclidinone with cell lysate containing 3-quinuclidinone reductase in the presence of cell lysate containing glucose dehydrogenase to obtain (R)-3-quinuclidinol, wherein the reaction mixture comprises:

a) 3-quinuclidinone as substrate

b) cofactor NAD or NADP;

c) glucose, 1 to 2 times of the substrate concentration;

d) cell lysate containing the enzyme 3-quinuclidinone reductase;

e) cell lysate containing the enzyme glucose dehydrogenase;

f) phosphate buffer medium;

g) pH of the reaction is maintained at 6.8 - 7.7

The completion of the reaction is achieved in 3h - lOh

Another aspect of the invention is a process for extracting and purifying (R)-3- quinuclidinol. The process comprises the steps of:

a) basifying/acidifying the reaction mixture to pH 11.0 - 13.5 or pH 1.5 - 3.0, respectively, to obtain basified/acidified reaction mixture;

b) adding 1 - 3 volumes (v/v) of acetone to the basified/acidified reaction mixture in Step‘a’, precipitating the impurities, filtering of precipitate formed and removing acetone by evaporation to obtain aqueous solution of product;

c) alternatively, adding celite to the basified/acidified reaction mixture in step‘a’, stirring at room temperature for 10 min to 2h and filtering to obtain aqueous filtrate containing the product;

d) extracting the product from the aqueous solution obtained in step‘b’ or‘c’ using n-butanol and concentrating the extract to dryness to obtain the product; e) solubilizing the product obtained in step‘d’ in hot toluene at 80°C - l05°C to obtain solution containing insoluble impurities;

f) filtering the insoluble impurities from the solution obtained in step‘e’, cooling the filtrate gradually to room temperature to obtain pure crystals of (R)-3- quinuclidinol and recovering the crystals by filtration.

According to this aspect of the invention, the reaction mixture is basified using 20% sodium hydroxide (NaOH) or acidified using hydrochloric acid (HC1). The substrate input to celite ratio is 1:0.2 to 1:2.0 (w/w). Filtration of precipitate is carried out using muslin cloth or filter of 4 m - 5m. For solubilization the product to hot toluene ratio is from 1:10 to 1:50 (w/v).

Examples:

Following examples of the present invention demonstrate the best mode of carrying out the present invention. These examples do not limit the scope of invention in any manner and should be considered as purely illustrative.

Example 1: Construction of pET28a Rr-KRED Clone and Expression of recombinant enzymes:

DNA sequence encoding a polypeptide of 3-Quinuclidinone reductase, derived from Rhodotorula rubra, was codon optimized, chemically synthesized and cloned into expression vector pET28a. The nucleotide coding for 3- Quinuclidinone reductase has short additional amino acid residues at the N- terminal end. Vector carrying the sequence was then transformed into a propagation host, E. coli DH5a. The bacteria carrying the cloned vector were selected on the basis of colony PCR. From these selected bacterial cells, the plasmids were isolated and purified. The purified pET28a vector carrying the gene of interest was then transformed into E. coli BL21 DE3 Gold cells. The transformed colonies were grown in LB broth medium at 35°C - 40°C preferably 37°C. Once the culture attained the desired level as measured by absorption at 600nm, it was induced with 0.5mM - l.OmM IPTG. Incubation was continued for 2h - 4h preferably 4h. The culture was monitored for the expression of the desired polypeptide. Cells were harvested by centrifugation and lysed on a cell disruptor. The expression was analyzed by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and it was found that 20% - 40% of the desired polypeptide was produced in soluble form.

Example 2: Construction of pET28a Bm-GDH Clone and Expression:

DNA sequence encoding a polypeptide of Glucose Dehydrogenase derived from Bacillus megaterium, was codon optimized, chemically synthesized and cloned into expression vector pET28a. The nucleotide coding for Glucose Dehydrogenase has a short additional amino acid residues sequence at the N- terminal end. Vector carrying the sequence was then transformed into a propagation host E. coll DH5a. The bacteria carrying the cloned vector were selected on the basis of colony PCR. From these selected bacterial cells, the plasmids were isolated and purified. The purified pET28a vector carrying the gene of interest was then transformed into E. coll BL21 DE3 Gold cells. The transformed colonies were grown in LB broth medium at 35°C - 40°C preferably 37°C. Once the culture attained the desired level as measured by absorption at 600nm, it was induced with 0.5mM - 1.0 mM IPTG. Incubation was continued for 2h - 4h preferably 4h. The culture was monitored for the expression of Glucose Dehydrogenase. The cells were harvested by centrifugation and lysed on a cell disruptor. The expression was analyzed by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and it was found that 20% - 40% of the desired polypeptide was produced in soluble form.

Example 3: Reduction of 3-Quinuclidinone to (R)-3-Quinuclidinol Using whole Cell and Lysates:

Cells harvested from Example 1 and 2, were washed with phosphate buffer (0.1M, pH 7.4). The first set of cells (E. coli pET28a KRED) served as the catalysts for the conversion of 3-Quinuclidinone to (R)-3-Quinuclidinol. The second set of cells (E. coli pET28a GDH) served as a source of the enzyme Glucose dehydrogenase, which is required for the regeneration of cofactor NAD/NADP.

Alternatively, cells harvested from Example 1 and 2, were washed with phosphate buffer (0.1M and pH 7.4) and lysed on Vibra Cell™ sonicator at cold condition. The lysed cells were centrifuged and the clear supernatants free from cell debris were used as source of enzyme ketoreductase and glucose dehydrogenase.

Example 4: Production of 3-Quinuclidinone Reductase at 3L Fermenter:

Clone obtained from nucleotide of SEQ ID 1 was cultured at 37°C in Luria Broth and 300 ml culture used as inoculum for 3L fermentation medium. Fermentation medium comprised of Yeast extract lOg/L, Glucose lOg/L, KH₂P0₄ 3g/L, Na₂HP0₄ 7g/L, (NH₄)₂S0₄ 2 g/L, NaCl 0.33g/L, MgS0₄.7H₂0 l.Og/L, Thiamine O.Olg/L, Trace metal solution lml/L and Kanamycin 0.02g/L. Fermentation was carried out at 37°C for l2h under fed batch mode using a glycerol-yeast extract based feed. The pH was maintained at 6.8 throughout the fermentation using 20% NaOH solution. Dissolved oxygen was maintained at 50-60% with aeration (1-2 vvm). When the OD₆oo_nm of the culture reached -140.0 the culture was induced with IPTG (lmM) and continued the incubation for another 4h. At the end of the fermentation, the final OD₆oo_nm reached 190 - 200. The culture broth was harvested by centrifugation at 9000 rpm at l5°C for l5min. Clear supernatant was carefully decanted to obtain the wet cell pellet. An output of 300g/F of wet cell mass was obtained.

Example 5: Production of Glucose Dehydrogenase at 3L Fermentor:

Clone obtained from SEQ ID No. 2 was cultured at 37°C in Furia Broth and 300 ml culture was used as inoculum for 3F fermentation medium. The Fermentation medium comprised of Yeast extract lO.Og/F, Glucose l2.0g/F, KH₂P0₄ 3.0g/F, K₂HP0₄ l2.5g/F, (NH₄)₂S0₄ 5.0g/F, NaCl 0.5g/F, MgS0₄.7H₂0 l.Og/F, Trace metal solution lml/F and Kanamycin 0.02g/F. Fermentation was carried out at 22°C for 24h under fed batch mode using a glycerol-yeast extract based feed. The pH was maintained at 6.8 throughout the fermentation using 20% NaOH solution. Dissolved oxygen was maintained at 50-60% with aeration of 1 - 2vvm. When the OD₆oo_nm of the culture reached -60.0 the culture was induced with O.lmM IPTG and the incubation continued for four more hours. At the end of the fermentation, the final OD₆oo_nm of the culture reached 190 - 200. The culture broth was harvested at 9000 rpm at l5°C for l5min. The clear supernatant was carefully decanted to separate the wet cell mass. A 200g/F of wet cell mass was obtained. Example 6: Preparation of Crude Cell Lysate as Source of Enzyme:

Cell mass obtained in example 4 or 5 was suspended in phosphate buffer (0.1M pH 7.4) in a ratio of 1:10 (w/v) and stirred for at least lh - 2h on an overhead stirrer to form a homogenous suspension. The process was carried out on ice throughout the work. The suspension was lysed by high pressure homogenization at ~l8,000psi. At least two passes were carried out to achieve maximum lysis of the cells. The lysate was centrifuged at 9,000 rpm for 30min at l5°C and the resultant clear supernatant was used as the source of enzyme for the bioconversion reaction.

Example 7: Production of (R)-3-Quinuclidinol Using Whole Cells:

For the bioconversion of lg of 3-Quinuclidinone HC1, 4g of cell mass was used in a reaction mix comprising of lOmg of NADP, 6g of Glucose, lOmg of glucose dehydrogenase in a final volume of 40ml. Reaction mass was mixed at l50rpm on a rotary shaker at 25°C for 3h - 4h. pH was adjusted intermittently to ~ 6.5 - 7.5 using 20% NaOH solution. The reaction was monitored for completion by Silica gel Thin Layer Chromatography (TLC).

Example 8: Production of (R)-3-Quinuclidinol Using Cell Lysate:

A 10 ml supernatant containing ketoreductase enzyme and 5ml supernatant containing glucose dehydrogenase enzyme (from Example 6) were used in the reaction comprising 3mg of NADP and l.4g of Glucose, lg 3-Quinuclidinone. The final volume of the reaction mixture was l5ml. Reaction mass was stirred at l50rpm on a rotary shaker at 25°C for 3h - 4h. pH was constantly adjusted to ~ 6.8 - 7.5 using 20% NaOH solution. At end of reaction time, the mixture was sampled to analyze conversion and optical purity.

Example 9: Purification of (R)-3-Quinuclidinol from Reaction Mixture:

A lOOml reaction mixture was alkalified with 20% NaOH solution to pH 12 - 13 followed by addition of 200ml of acetone. The precipitate thus formed was separated by filtration. Acetone was evaporated and 2 volumes of n-butanol was added to the aqueous filtrate and mixed vigorously. Aqueous and organic layers were separated and aqueous phase is re-extracted with 200ml of n-butanol. The n- butanol extract was then concentrated to dryness. The dried product was solubilized in toluene at 90°C - l00°C. Hot toluene was filtered and the filtrate was allowed to gradually cool to room temperature. (R)-3-Quinuclidinol crystals were filtered and dried. A white to off-white crystals of (R)-3-Quinuclidinol were obtained which had more than 99% purity and more than 99.9% e.e.

Claims

We Claim:

1. A recombinant DNA comprising a nucleotide sequence, wherein said nucleotide sequence is selected from:

(i) SEQ ID 1;

(ii) SEQ ID 2;

(iii) a nucleotide sequence encoding amino acid sequence of SEQ ID 3 or SEQ ID 4;

(iv) a nucleotide sequence which is at least 70% identical to the nucleotide sequence of (i), (ii) or (iii).

2. A vector comprising the recombinant DNA as claimed in claim 1.

3. A host cell transformed by the vector as claimed in claim 2.

4. The host cell claimed in claim 3, wherein the preferred host cell is E. coli cells.

5. A clone comprising the recombinant DNA as claimed in claim 1.

6. The clone as claimed in claim5, further comprises the vector as claimed in claim 2 and the host cell as claimed in claim 4.

7. A process for soluble expression of amino acid sequence selected from SEQ ID 3 and SEQ ID 4, wherein the process comprises:

a. preparing a clone comprising a host, a vector containing a variant nucleotide sequence encoding targeted amino acid sequence;

b. fed-batch fermentation of clone obtained in step‘a’ in fermentation broth;

c. separation of soluble form of encoded amino acid from the fermentation broth in step‘b’.

8. The process as claimed as claim 7, wherein the variant nucleotide sequence in step‘a’ is SEQ ID 1 for amino acid sequence of SEQ ID 3 and SEQ ID 2 for amino acid sequence of SEQ ID 4 and wherein the host is as claimed in claim 4.

9. The process as claimed in claim 8, wherein the fermentation broth in step ‘b’ comprises lOg - 30g/L of Luria Broth, 3g - 7g/L of dextrose, 6g - 9g/L disodium hydrogen phosphate, 0.5g -2.0g magnesium sulphate.

10. (R)-3-quinuclidinol prepared by reduction of 3-quinuclidinone using one or more enzymes encoded by recombinant DNA as claimed in claim 1.