Title of Invention

A NON-NATURALLY OCCURING POLYPEPTIDE HAVING IMPROVED GLUCOSE DEHYDROGENASE ACTIVITY

Abstract The present invention is directed to glucose dehydrogenase (GDH) polypeptides that have enhanced GDH activity and/or thermostability relative to the backbone wild-type glucose dehydrogenase polypeptide. In addition, the present invention is directed to a plynucleotide that encodes for the GDH polypeptides of the present invention, to nucleic acid sequences comrpising the polynucleotides, to expression vectors comprising the polynucleotides operatively linked to a promoter, to host cells transformed to express the GDH polypeptides, and to a method for producing the GDH polypeptides of the present invention.
Full Text IMPROVED GLUCOSE DEHYDROGENASE POLYPEPTIDES AND RELATED POLYNUCLEOTIDES
FIELD OF THE INVENTION
[01] The present invention is related to the field of enzymology, and particularly to the field of glucose dehydrogenase enzymology. More specifically, the present invention is directed to glucose dehydrogenase polypeptides having improved enzymatic activity (i e.fhigh substrate turnover) and stability, and to polynucleotides sequences encoding for the improved glucose dehydrogenase polypeptides. The present invention is useful because the glucose dehydrogenase polypeptides can be coupled to oxido- or reductase enzymes to produce synthetic organic chemicals or precursors in high yields.
BACKGROUND OF THE INVENTION
[02] Glucose dehydrogenase [EC 1.1.1.47] or "GDH" catalyzes the conversion of (3-glucose and nicotinamide adenine dinucleotide (NAD) to gluconolactone and reduced nicotinamide adenine dinucleotide (NADH). NAD serves as a co-factor in this reaction and may be phosphorylated in the form of NADP. GDH is an important enzyme for use in clinical tests and the food industry. GDH is also applied as a catalyst for chemical conversions where it serves a role in the regeneration of NADH and NADPH in enzymatic carbonyl reductions, such as aldehydes and ketones.
[03] Bacillus species have been an excellent source of GDH. The enzyme from B. megaterium M1286 was purified to homogeneity and found to be a homotetramer of 30,000 DA subunits with pH optimum of 8.0-9.0 depending on buffer conditions and uses either nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP) as cofactor (Pauly H.E. and Pfleiderer G., Hoppe Seylers Z. Physiol. Chem. 1975 356:1613-23). The enzyme from Cryptococcus uniguttulatus Y 0033 has a pH optimum of 6.0-8.0, an optimum temperature of 55° C and a molecular weight of 110 fcDa (U.S. Pat. 4,877,733). The enzyme from Pseudomonas sp. FH1227 has a pH optimum of

8.5-9.0, an optimum temperature of 55° C and a molecular weight of 101 kDa (U.S. Pat. 5,298,411).
[04] Commercially applied GDHs are primarily derived from microorganisms. Initially, GDH was produced by fermentation of the natural host organisms such as B. megaterium ATCC 39118 (U.S. Pat. 4,542,098), Bacillus cereus DSM 1644 (US4397952), Cryptococcus uniguttulatus Y 0033 (U.S. Pat. 4,877,733) and Pseudomonas sp. FH1227 (U.S. Pat. 5,298,411). Since then, GDH encoding genes have been identified, cloned and expressed in heterologous hosts such as Escherichia coli.
[05] The Bacillus subtilis 61297 GDH gene was expressed in E. coli and exhibited the same physicochemical properties as the enzyme produced in its native host (Vasantha et al. Proc. Natl. Acad.,Sci. USA 1983 80:785). The gene sequence of the B. subtilis GDH gene was reported by Lampel, K. A., Uratani, B., Chaudhry, R., Ramaley, R. F., and Rudikoff S., "Characterization of the developmentally regulated Bacillus subtilis glucose dehydrogenase gene," J. Bacteriol 166, 238-243 (1986) and Yamane,K., Kumano,M. and Kurita,K., "The 25 degrees-36 degrees region of the Bacillus subtilis chromosome: determination of the sequence of a 146 kb segment and identification of 113 genes," Microbiology 142 (Pt 11), 3047-3056 (1996), and is found in Genbank under Accession Nos. M12276 and D50453.
[06] Similarly, gene sequences were determined for GDH from 5. cereus ATCC14579 (Nature 2003 423:87-91; Genbank Ace. No. AE017013) and B. megaterium (Eur. J. Biochem. 1988 174:485-490, Genbank Ace. No. X12370; J. Ferment. Bioeng. 1990 70:363-369, Genbank Ace. No. D90044). The GDH enzymes from B. subtilis and B. megaterium are approximately 85% homologous (J. Theor. Biol. 1986 120:489-497).
[07] It has been well established that GDH enzymes suffer from limited stability. Ramaley and Vasantha reported that presence of glycerol in extraction and purification buffers is absolutely necessary to retain activity for GDH from B. subtilis (J. Biol. Chem. 1983 258:12558-12565). The enzyme instability can be largely attributed to the dissociation of the tetramer into its

monomers, which is an equilibrium process that is controlled by environmental factors such as pH and ionic strength (Maurer and Pfleiderer, Z. Naturforsch. 1987 42: 907-915). This has lead to the isolation and studies of GDH from other Bacillus sp. such as B. megaterium. For instance, U.S. Pats. 5,114,853 and 5,126,256 and Baik et al. Appl. Microbiol. Biotechnol. 2003 61:329-335 describe GDH encoding genes from B. megaterium and mutants thereof that exhibit increased thermostability and that can be produced in recombinant E. coli hosts. However, there remains an industrial need for GDH enzymes that not only have increased theimostability but that also have enhanced enzymatic activity. The above referenced publications and patents, and all other publications and patents referenced herein, are hereby incorporated by reference herein in their entirety.
BRIEF SUMMARY OF THE INVENTION
[08] The present invention has multiple aspects. In one aspect, the present invention is directed to a polypeptide having at least 1.5 times, typically 1.5 to about 25 times, more typically from 1.5 to about 11 times, the GDH activity of the wild-type GDH of SEQ ID NO: 2 (such as determined by the method of Example 4) and being selected from the group consisting of:
(a) a polypeptide having an amino acid sequence which has at least 91%
homology, preferably at least 95% homology, and more preferably at least 98%
homology with the amino acid sequence of SEQ ID NO: 54, 74, 84,160,164 or
168 (hereinafter "homologous polypeptides");
(b) a polypeptide encoded by a nucleic acid sequence which hybridizes under
medium stringency conditions with either (i) the nucleotide sequence of SEQ ID
NO: 53, 73, 83, 159, 163 or 167; (ii) a subsequence of (i) of at least 100
nucleotides, or (iii) a complementary strand of (i) or (ii) (J. Sambrook, E. F.
Fritsch, and T. Maniatis, 1989, Molecular Cloning, A Laboratory Manual, 2d
edition, Cold Spring Harbor, N.Y.);
(c) a variant of the polypeptide of SEQ ID NO: 54, 74, 84, 160, 164 or 168
comprising a substitution, deletion, and/or insertion of one to six amino acids;

(d) a fragment of (a), (b) or (c) that has from 1.5 to about 11 times the GDH
activity of the wild-type GDH of SEQ ID NO: 2,; and
(e) a polypeptide of (a), (b) or (c) that retains more than 80% of the initial GDH
activity after 20 minutes of incubation at 50° C and pH 7. In one embodiment,
the present invention is also directed to a variant GDH polypeptide as described
herein in isolated and purified form. In another embodiment, the present
invention is directed to a variant GDH polypeptide as described herein in
lyophilized form. In yet another embodiment, the present invention is directed
to a composition comprising a variant GDH polypeptide as described herein and
a suitable carrier, typically a buffer solution, more typically a buffer solution
having a pH between 6.0 and 8.0.
[09] The novel GDH polypeptides of the present invention have enhanced GDH activity (>1.5 fold) relative to the backbone GDH polypeptide from 5. subtilis of SEQ ID NO: 2 and typically vary from SEQ ID NO: 2 by 1-7 amino acid residues, more typically by 1-6 amino acid residues, even more typically by 1-5 amino acid residues, and most typically by 1-4 amino acid residues. For purposes of the present invention, the degree of homology between two amino acid sequences was determined using the Needleman Wunsch global alignment algorithm, i.e., using dynamic programming algorithm for Global Alignment Scoring Matrix: PAM 120 matrix with gap penalties for introducing gap = -22.183 and extending gap = -1.396. The percent identity = number of identical residues between the first sequence and the second sequence divided by the length of first sequence in alignment (with gaps)(p) indicates partial match. See Needleman, S.B. & Wunsch, CD., "A general method applicable to the search for similarities in the amino acid sequence of two proteins," Journal of Molecular Biology, 48:443-453 (1970).
[10] The various residue positions of the 5, subtilis GDH polypeptide that have been substituted to yield enhanced GDH activity and/or thermostability are summarized in Table 1 herein. The amino acid sequences for a number of the inventive GDH polypeptides that have demonstrated enhanced GDH activity and/or thermostability at 50° C are disclosed herein as SEQ ID NOS: 6, 8, 10,



83, 159, 163, and 167 that encode for the novel glucose dehydrogenase polypeptides of SEQ ID NOS: 54,74, 84,160,164 and 168, respectively.
[14] In a third aspect, the present invention is directed to a nucleic acid construct, a vector, or a host cell comprising a polynucleotide sequence encoding a GDH polypeptide of the present invention operatively linked to a
promoter.
[15] In a fourth aspect, the present invention is directed to a method of making a GDH polypeptide of the present invention comprising (a) cultivating a host cell comprising a nucleic acid construct comprising a nucleic acid sequence encoding a GDH polypeptide of the present invention under conditions suitable for production of the polypeptide; and (b) recovering the polypeptide.
BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS
[16] FIG. 1 exemplifies an oxidation-reduction cycle wherein glucose is oxidized by GDH to gluconic acid in the presence of NAD+ (or NADP+) to produce the corresponding reduced form NADH (or NADPH), respectively, which in turn drives the reduction of a substrate to a reduced substrate while being oxidized back to NAD (or NADP) by a reductase. The gluconic acid formed in this reaction is neutralized by sodium hydroxide to sodium-gluconate.
[17] FIGS. 2A-2B in combination provide a table comparing the % amino acid identity of the GDH polypeptides of the present invention versus the GDH polypeptides of the indicated prior art references. In col. 4 of FIG. 2, the GDH polypeptide of B subtilis (S06-3) has the same amino acid sequence as disclosed in EP 955375 (col. 8). To generate FIGS 2A-2B, alignments were done using dynamic programming algorithm for Global Alignment Scoring Matrix: PAM 120 matrix with gap penalties for introducing gap = -22.183 and extending gap = -1.396. The percent identity = number of identical residues between the first sequence and the second sequence divided by the length of first sequence in alignment (with gaps)(p) indicates partial match. See Needleman, S.B. & Wunsch, CD., "A general method applicable to the search for similarities in the

amino acid sequence of two proteins," Journal of Molecular Biology, 48:443-453 (1970).
[18] FIG. 3 is a 4036 bp expression vector (pCKl 10900) of the present invention comprising a P15A origin of replication (P15A ori) a lacl repressor, a CAP binding site, a lac promoter (lac), a T7 ribosomal binding site (T7gl0 RBS), and a chloramphenicol resistance gene (camR),
[19] The foregoing summary, as well as the following detailed description of certain embodiments of the present invention, will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there is shown in the drawings, certain embodiments. It should be understood, however, that the present invention is not limited to the arrangements and instrumentality shown in the attached drawings.
DETAILED DESCRIPTION OF THE INVENTION
[20] The present invention has multiple aspects. In one aspect, the present invention is directed to a polypeptide having at least 1.5 times, typically 1.5 to about 25 times, more typically from 1.5 to about 11 times the GDH activity of the wild-type GDH of SEQ ID NO: 2 (such as determined by the method of Example 4) and being selected from the group consisting of;
(a) a polypeptide having an amino acid sequence which has at least 91%
homology, preferably at least 95% homology, and more preferably at least 98%
homology with the amino acid sequence of SEQ ID NO: 54, 74, 84, 160, 164 or
168 (hereinafter "homologous polypeptides");
(b) a polypeptide encoded by a nucleic acid sequence which hybridizes under
medium stringency conditions with either (i) the nucleotide sequence of SEQ ID
NO: 53, 73 or 83, (ii) a subsequence of (i) of at least 100 nucleotides, or (iii) a
complementary strand of (i) or (ii) (J. Sambrook, E. F. Fritsch, and T. Maniatis,
1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring
Harbor, N.Y.);
(c) a variant of the polypeptide of SEQ ID NO: 54, 74, 84, 160, 164 or 168
comprising a substitution, deletion, and/or insertion of one to six amino acids;

(d) a fragment of (a), (b) or (c) that has from 1.5 to about 11 times the GDH
activity of the wild-type GDH of SEQ ID NO: 2; and
(e) a polypeptide of (a), (b) or (c) that retains more than 80% of the initial GDH
activity after 20 minutes of incubation at 50° C and pH 7.
[21] Unless otherwise noted, as used throughout this specification, the terms "percent identity," "% identity," "percent identical," and "% identical" are used interchangeably herein to refer to the percent amino acid sequence identity that is determined using the Needleman Wunsch global alignment algorithm, i.e., using dynamic programming algorithm for Global Alignment Scoring Matrix: PAM 120 matrix with gap penalties for introducing gap = -22.183 and extending gap = -1.396. The percent identity = number of identical residues between the first sequence and the second sequence divided by the length of first sequence in alignment (with gaps)(p) indicates partial match. See Needleman, S.B. & Wunsch, CD., "A general method applicable to the search for similarities in the amino acid sequence of two proteins," Journal of Molecular Biology, 48:443-453 (1970).
[22] As used herein, the terms "glucose dehydrogenase" and "GDH" are used interchangeably herein to refer to a polypeptide that has the ability to catalyze the conversion of glucose and nicotinamide adenine dinucleotide (NAD) to gluconolactone and reduced nicotinamide adenine dinucleotide (NADH). Alternatively, the phosphorylated cofactors NADP and NADPH can replace NAD and NADH in the above reaction. In nature, GDH is made up of four subunits that are loosely held together in a homo-tetramer. Based upon the crystal structure of wild-type B. megaterium GDH polypeptide (SEQ ID NO: 4) (Yamamoto et al. J. Biochem. 2001 129:303-312), residue positions 188-217 of the polypeptide define a protein loop region that is involved in NAD+ and glucose binding.
[23] In use, the enhanced GDH polypeptides of the present invention are preferably coupled to a synthetic reaction as a cofactor regeneration system (See Figure 1) to provide a continuing source of reduced cofactor. As used herein, the term "cofactor" refers to a non-protein compound that operates in

combination with an enzyme that catalyzes a reaction of interest. Suitable cofactors employed with the GDH polypeptides of the present invention include NADP (nicotinamide-adenine dinucleotide phosphate) and NAD (nicotinamide adenine dinucleotide).
[24] The term "cofactor regeneration system" refers herein to a set of reactants that participate in a reaction that regenerates a utilized cofactor back to its pre-reaction state. An example is the regeneration of oxidized cofactor regeneration back to reduced cofactor, e.g., NADP to NADPH. The reduced (regenerated) cofactor is then capable of participating in a reaction with a substrate and an enzyme, such as a reducing enzyme, to produce the reduced substrate and the oxidized (utilized) cofactor, which can again be regenerated by the cofactor regeneration system. The above-described operation of the glucose/glucose dehydrogenase cofactor regeneration system is exemplified in Figure 1.
[25] In FIG. 1, the reaction catalyzed by the reducing enzyme is shown as being coupled to the glucose dehydrogenase cofactor regeneration system. The term "coupled" is used herein to refer to the use of the reduced form of cofactor in the reduction of a substrate, and the concomitant use of the oxidized form of the same cofactor, generated in the aforementioned reaction, in the oxidation of a component (e.g., glucose) of the cofactor regeneration system, which generates the reduced form of the same cofactor. One possible limiting factor in the overall reaction speed in a coupled system is the speed (activity) of the GDH polypeptide in regenerating cofactor.
[26] The GDH polypeptides of the present invention have enhanced GDH activity (such as measured by the method of Example 4) that is 1.5 fold to about 11 fold greater than the GDH activity of the backbone GDH polypeptide from B. subtilis of SEQ ID NO; 2, and typically vary from SEQ ID NO: 2 by 1-7 amino acid residues, more typically by 1-6 amino acid residues, even more typically by 1-5 amino acid residues, and most typically by 1-4 amino acid residues. Preferably, the GDH polypeptides of the present invention have enhanced GDH activity that is 2.5 fold to about 11 fold greater than the GDH













screened to find the polypeptides having the highest GDH activity. Then, a second mutagenic or evolutionary technique is applied to polynucleotides encoding the most active polypeptides to create a second library, which in turn is screened for GDH activity by the same technique. The process of mutating and screening can be repeated as many times as needed, including the insertion of point mutations, to arrive at a polynucleotide that encodes a polypeptide with the desired activity, thermostability, and cofactor preference.
[44] Alternatively, polynucleotides and oligonucleotides of the invention can be prepared by standard solid-phase methods, according to known synthetic methods. Typically, fragments of up to about 100 bases are individually synthesized, then joined (e.g., by enzymatic or chemical litigation methods, or polymerase mediated methods) to form essentially any desired continuous sequence. For example, polynucleotides and oligonucleotides of the invention can be prepared by chemical synthesis using, e.g., the classical phosphoramidite method described by Beaucage et al (1981) Tetrahedron Letters 22:1859-69, or the method described by Matthes et al (1984) EMBO J. 3:801-05, e.g., as it is typically practiced in automated synthetic methods. According to the phosphoramidite method, oligonucleotides are synthesized, e.g., in an automatic DNA synthesizer, purified, annealed, ligated and cloned in appropriate vectors.
[45] In addition, essentially any nucleic acid can be custom ordered from any of a variety of commercial sources, such as The Midland Certified Reagent Company, Midland, TX, The Great American Gene Company (Ramona, CA), ExpressGen Inc., Chicago, EL, Operon Technologies Inc. (Alameda, CA), and many others. Similarly, peptides and antibodies can be custom ordered from any of a variety of sources, such as PeptidoGenic (pkim@ccnet.com), HTI Bio-products, Inc. (http://www.htibio.com), BMA Biomedicals Ltd. (U.K.), Bio.Synthesis, Inc., and many others.
[46] Polynucleotides may also be synthesized by well-known techniques as described in the technical literature. See, e.g., Carruthers et al, Cold Spring Harbor Symp. Quant. Biol 47:411-418 (1982), and Adams et al, /. Ant Chem. Soc. 105:661 (1983). Double stranded DNA fragments may then be obtained



reverse transcriptase and a polymerase. See, Ausubel, Sambrook and Berger,
all supra.
[48] It will be appreciated by those skilled in the art due to the degeneracy of the genetic code, a multitude of nucleotide sequences encoding GDH polypeptides of the invention may be produced, some of which bear substantial identity to the nucleic acid sequences explicitly disclosed herein.
[49] In the present case, several round No. 1 libraries were created by applying a variety of mutagenic techniques to the coding region of the B. subtilis gdh gene (SEQ ID NO: 1) or to the coding region of the B. megaterium gdh gene (SEQ ID NO: 3), as obtained by PCR.
[50] To obtain expression of the variant gene encoding a GDH, the variant gene was first operatively linked to one or more heterologous regulatory sequences that control gene expression to create a nucleic acid construct, such as an expression vector or expression cassette. Thereafter, the resulting nucleic acid construct, such as an expression vector or expression cassette, was inserted into an appropriate host cell for ultimate expression of the GDH polypeptide encoded by the shuffled gene. A "nucleic acid construct" is defined herein as a nucleic acid molecule, either single-or double-stranded, which is isolated from a naturally occurring gene or which has been modified to contain segments of nucleic acid combined and juxtaposed in a manner that would not otherwise exist in nature. Thus, in one aspect, the present invention is directed to a nucleic acid construct comprising a polynucleotide encoding a GDH polypeptide of the present invention.
[51] The term "nucleic acid construct" is synonymous with the term "expression cassette" when the nucleic acid construct contains all the control sequences required for expression of a coding sequence of the present invention. The term "coding sequence" is defined herein as a nucleic acid sequence, which directly specifies the amino acid sequence of its protein product. The boundaries of a genomic coding sequence are generally determined by a ribosome binding site (prokaryotes) or by the ATG start codon (eukaryotes) located just upstream of the open reading frame at the 5' end of the mRNA and a transcription

terminator sequence located just downstream of the open reading frame at the 3' end of the mRNA. A coding sequence can include, but is not limited to, DNA, cDNA, and recombinant nucleic acid sequences.
[52] An isolated polynucleotide encoding a GDH polypeptide of the present invention may be manipulated in a variety of ways to provide for expression of the polypeptide. Manipulation of the isolated polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotides and nucleic acid sequences utilizing recombinant DNA methods are well known in the art.
[53] The term "control sequence" is defined herein to include all components, which are necessary or advantageous for the expression of a polypeptide of the present invention. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleic acid sequence encoding a polypeptide.
[54] The term "operably linked" is defined herein as a configuration in which a control sequence is appropriately placed at a position relative to the coding sequence of the DNA sequence such that the control sequence directs the expression of a polypeptide.
[55] The control sequence may be an appropriate promoter sequence. The "promoter sequence" is a relatively short nucleic acid sequence that is recognized by a host cell for expression of the longer coding region that follows. The promoter sequence contains transcriptional control sequences, which mediate the expression of the polypeptide. The promoter may be any nucleic acid sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained



Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are described by Romanos et al., 1992, Yeast 8:423-488.
[59] The control sequence may also be a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3' terminus of the nucleic acid sequence encoding the polypeptide. Any terminator, which is functional in the host cell of choice, may be used in the present invention.
[60] Preferred terminators for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.
[61] Preferred terminators for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanos et al., 1992, supra.
[62] The control sequence may also be a suitable leader sequence, a nontranslated region of an mRNA which is important for translation by the host cell. The leader sequence is operably linked to the 5' terminus of the nucleic acid sequence encoding the polypeptide. Any leader sequence that is functional in the host cell of choice may be used in the present invention. Preferred leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase. Suitable leaders for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).
[63] The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3' terminus of the nucleic acid sequence and

which, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence that is functional in the host cell of choice may be used in the present invention. Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger alpha-glucosidase. Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Molecular Cellular Biology 15: 5983-5990.
[64] The control sequence may also be a signal peptide coding region that codes for an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded polypeptide into the cell's secretory pathway. The 5' end of the coding sequence of the nucleic acid sequence may inherently contain a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region that encodes the secreted polypeptide. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding region that is foreign to the coding sequence. The foreign signal peptide coding region may be required where the coding sequence does not naturally contain a signal peptide coding region.
[65] Alternatively, the foreign signal peptide coding region may simply replace the natural signal peptide coding region in order to enhance secretion of the polypeptide. However, any signal peptide coding region that directs the expressed polypeptide into the secretory pathway of a host cell of choice may be used in the present invention.
[66] Effective signal peptide coding regions for bacterial host cells are the signal peptide coding regions obtained from the genes for Bacillus NC1B 11837 maltogenic amylase, Bacillus stearothennophilus alpha-amylase, Bacillus lichenifonnis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothennophilus neutral proteases (nprT, nprS, nprM), and Bacillus subtilis prsA. Further signal peptides are described by Simonen and Palva, 1993, Microbiological Reviews 57: 109-137.





cel5] The expression vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell, or a transposon may be used.
[76] The expression vector of the present invention preferably contains one or more selectable markers, which permit easy selection of transformed cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. Examples of bacterial selectable markers are the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers, which confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol (Example 1) or tetracycline resistance. Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3.
[77] Selectable markers for use in a filamentous fungal host cell include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof. Preferred for use in an Aspergillus cell are the amdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzae and the bar gene of Streptomyces hygroscopicus.
[78] The vectors of the present invention preferably contain an element(s) that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome. For integration into the host cell genome, the vector may rely on the nucleic acid sequence

encoding the polypeptide or any other element of the vector for integration of the vector into the genome by homologous or nonhomologous recombination.
[79] Alternatively, the vector may contain additional nucleic acid sequences for directing integration by homologous recombination into the genome of the host cell. The additional nucleic acid sequences enable the vector to be integrated into the host cell genome at a precise location(s) in the chromosome(s). To increase the likelihood of integration at a precise location, the integrational elements should preferably contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000 base pairs, which are highly homologous with the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding nucleic acid sequences. On the other hand, the vector may be integrated into the genome of the host cell by non-homologous recombination.
[80] For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. Examples of bacterial origins of replication are P15A, the origins of replication of plasmids pBR322, pUC19, pACYC177, which has the P15A origin of replication), or pACYC184 which permit replication in E. coli; and pUBHO, pE194, pTA1060, or pAM.beta.l which permit replication in Bacillus. Examples of origins of replication for use in a yeast host cell are the 2 micron origin of replication, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6. The origin of replication may be one having a mutation which makes it's functioning temperature-sensitive in the host cell (see, e.g., Ehrlich, 1978, Proceedings of the National Academy of Sciences USA 75: 1433).
[81] More than one copy of a nucleic acid sequence of the present invention may be inserted into the host cell to increase production of the gene product. An increase in the copy number of the nucleic acid sequence can be obtained by







[91] More preferred GDH polypeptides of the present invention are those polypeptides having 95% homology, more preferably 97% homology, and even more preferred 100% homology with the polypeptides of SEQ ID NOs: 160, 164 and 168. As shown above, each of these polypeptides has from 5-8 mutations but has the following five mutations in common: I165M, P194T, A197K, K204E and K206R. Thus, stated in other terms, one embodiment of the present invention is directed to a GDH polypeptide of SEQ ID NO: 2 having from 5-8 residue substitutions wherein five of the residue substitution are I165M, P194T, A197K, K204E, and K206R.
[92] Only a very few (≤ 0.5%) of the mutations to the wild-type B. subtilis GDH (SEQ ID NO: 2) backbone were found to be beneficial. Specifically, for every 1000 clones screened, there occurred only 3-5 single point or double point mutations that were beneficial. In fact, many of the mutations were found to be detrimental. For example, Y253C rendered the GDH polypeptide inactive, whereas Q252L slightly reduced the initial GDH activity wild-type B. subtilis GDH (SEQ ID NO: 2). Interestingly, the beneficial effects of one mutation were not found to be additive with the beneficial effects of another mutation. Thus, for example, it was discovered that the combination of a first mutation that increased GDH activity 2 fold compared to the wild-type activity, with a second mutation at a second residue position that increased GDH activity 3 fold compared to the wild-type activity most often did not result in a GDH polypeptide that had a 5 or 6 fold increase in GDH activity.
[93] The GDH polypeptides of the present invention have the activities described herein, as well as other desirable properties, e.g., altered temperature and/or pH optimums, solvent resistance (e g., butyl acetate), and the like. Moreover, the GDH polynucleotide may be mutated or evolved to generate libraries that can be screened to identify those modified GDH polypeptides having the ability to preferentially accept other compounds as cofactors, such as, for example, NADP (also referred to as NADP+).
[94] The polynucleotides encoding the GDH polypeptides of the present invention may be codon optimized for optimal production from the host











dropwise on demand by the automatic titrator (a pH of 6.85 was set as a lower limit) to constantly adjust the pH to 7.0. The reaction was complete when no more caustic was needed. The reaction rates were determined by measuring the amount of base added per unit time or by taking samples of the reaction mixture, extracting the sample 3 times with an equal volume of ethyl acetate, and analyzing the combined organic layers by gas chromatography to determine the amount of ethyl-S- 4-chloro-3-hydroxybutyrate produced per unit time.
[104] While the invention has been described with reference to certain embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from its scope. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed, but that the invention will include all embodiments falling within the scope of the appended claims.

Documents:

521-CHENP-2006 AMANDED CLAIMS 26-11-2009.pdf

521-CHENP-2006 CORRESPONDENCE OTHERS 26-11-2009.pdf

521-CHENP-2006 CORRESPONDENCE OTHERS.pdf

521-CHENP-2006 CORRESPONDENCE PO.pdf

521-CHENP-2006 FORM-13.pdf

521-CHENP-2006 FORM-18.pdf

521-CHENP-2006 FORM-2 26-11-2009.pdf

521-CHENP-2006 FORM-2.pdf

521-CHENP-2006 FORM-3 26-11-2009.pdf

521-CHENP-2006 FORM-3.pdf

521-CHENP-2006 OTHER DOCUMENT 26-11-2009.pdf

521-CHENP-2006 OTHER PATENT DOCUMENT 26-11-2009.pdf

521-CHENP-2006 POWER OF ATTORNEY 26-11-2009.pdf

521-chenp-2006 correspondance others.pdf

521-CHENP-2006 DRAWINGS 26-11-2009.pdf

521-chenp-2006 pct.pdf

521-chenp-2006-abstract.pdf

521-chenp-2006-assignement.pdf

521-chenp-2006-claims.pdf

521-chenp-2006-correspondnece-others.pdf

521-chenp-2006-description(complete).pdf

521-chenp-2006-drawings.pdf

521-chenp-2006-form 1.pdf

521-chenp-2006-form 3.pdf

521-chenp-2006-form 5.pdf


Patent Number 239922
Indian Patent Application Number 521/CHENP/2006
PG Journal Number 16/2010
Publication Date 16-Apr-2010
Grant Date 09-Apr-2010
Date of Filing 10-Feb-2006
Name of Patentee CODEXIS, INC.
Applicant Address 200 PENOBSCOT DRIVE, REDWOOD CITY, CA 94063, USA;
Inventors:
# Inventor's Name Inventor's Address
1 SHRI. KREBBER, ANKE 3500 LOUIS ROAD, PALO ALTO, CA 94303, USA
2 NEWMAN, LISA, MARIE, 1002 KING STREET, REDWOOD CITY, CA 94061, USA
3 DAVIS S., CHRISTOPHER 104 WINFIELD STREET, SAN FRANCISCO, CA 94001, USA
4 JENNE, STEPHANE, J., 821 E1 CAMINO REAL, #202, BURLINGAME, CA 94010, USA
PCT International Classification Number C12N 9/00
PCT International Application Number PCT/US04/26194
PCT International Filing date 2004-08-11
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/494,300 2003-08-11 U.S.A.
2 60/545,657 2004-02-10 U.S.A.