Title:
CYCLODEXTRIN GLUCANOTRANSFERASE
Kind Code:
A1


Abstract:
The present invention relates to a novel cyclodextrin glucanotransferase (CGTase) enzyme which is obtainable from Clostridium saccharoperbutylacetonicum N1-4, N1-4(HMT) or N1-504. The invention further relates to nucleic acids encoding the enzyme, vectors and host cells, and uses of the CGTase.



Inventors:
Jenkinson, Elizabeth (Abingdon, GB)
Krabben, Preben (Didcot, GB)
Harding, Amanda (Abingdon, GB)
Application Number:
15/293189
Publication Date:
04/06/2017
Filing Date:
10/13/2016
Assignee:
GREEN BIOLOGICS LTD (Abingdon, GB)
Primary Class:
International Classes:
C12N9/10
View Patent Images:



Attorney, Agent or Firm:
KNOBBE MARTENS OLSON & BEAR LLP (2040 MAIN STREET FOURTEENTH FLOOR IRVINE CA 92614)
Claims:
What is claimed is:

1. A polypeptide, wherein the amino acid sequence of the polypeptide: (a) comprises the amino acid sequence set forth in SEQ ID NO: 1 or 3; (b) comprises an amino acid sequence which has at least 70% sequence identity with SEQ ID NO: 1 or 3; (c) is encoded by the nucleotide sequence set forth in SEQ ID NO: 2 or 4; or (d) is encoded by a nucleotide sequence which has at least 70% sequence identity with the nucleotide sequence set forth in SEQ ID NO: 2 or 4.

Description:

INCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATIONS

The entire contents of each of the applications listed in the accompanying Application Data Sheet is incorporated by reference herein.

REFERENCE TO SEQUENCE LISTING

A Sequence Listing submitted as in ASCII text file via EFS-Web is hereby incorporated by reference in accordance with 35 U.S.C. §1.52(e). The name of the ASCII text file for the Sequence Listing is 23415553.TXT, the date of creation of the ASCII text file is May 26, 2016, and the size of the ASCII text file is 98.7 KB.

The present invention relates to a novel cyclodextrin glucanotransferase (CGTase) enzyme which is obtainable from Clostridium saccharoperbutylacetonicum N1-4, N1-4(HMT) or N1-504. The invention further relates to nucleic acids encoding the enzyme, vectors and host cells, and uses of the CGTase.

Cyclodextrins are cyclic glucose oligosaccharides which are generally composed of α-(1,4) linked glucopyranose subunits. Common cyclodextrins include α-cyclodextrin (6-membered sugar ring), β-cyclodextrin (7-membered sugar ring) and γ-cyclodextrin (8-membered sugar ring). Cyclodextrins have many uses in industry, including in separation and extraction processes, as drug-delivery agents and as stabilisers in the food industry. Cyclodextrins have also been used as intermediates in the production of ethanol (e.g. WO 89/03421).

Cyclodextrins are generally produced by the enzymatic conversion of starch using enzymes such as cyclodextrin glucanotransferases. Cyclodextrin glucanotransferases (CGTases) are also known as cyclodextrin glycosyl transferases and cyclodextrin glucosyltransferases. These enzymes are generally only found in bacteria, particularly bacteria of the genus Bacillus (e.g. B. circulans, B. macerans and B. stearothermophilus). It should be noted that wherein Clostridium thermohydrosulfuricus was previously classified as a Clostridial species, it has now been reclassified as Thermoanaerobacter thermohydrosulfuricus (Collins, M. D. et al. (1994). The phylogeny of the genus Clostridium: proposal of five new genera and eleven new species combinations. Int. J. Syst. Bacteriol., 44(4), 812-26). The genus Thermoanaerobacter has now clearly established by sequence analysis and shown that it forms a separate and distinct genus from Clostridium sensu stricto (Cluster I) (Stackebrandt et al. (1999) Phylogenetic basis for a taxonomic dissection of the genus Clostridium. FEMS Immunol. Med. Microbiol., 24(3), 253-8).

Whilst CGTases are generally capable of catalysing more than one reaction, the most important activity is the production of cyclic dextrins from substrates such as starch, amylose and other polysaccharides. In this process, the polysaccharide chain is cleaved and the ends are joined by the CGTase in order to produce a cyclic dextrin, i.e. a cyclodextrin. The size of the cyclodextrin (i.e. the number of sugar residues it incorporates) is dependent on the distance apart of the ends.

There remains a need, however, for novel CGTases, particularly those that are capable of producing novel cyclodextrins.

In one embodiment, therefore, the invention provides a polypeptide, wherein the amino acid sequence of the polypeptide:

    • (a) comprises the amino acid sequence set forth in SEQ ID NO: 1 or 3;
    • (b) comprises an amino acid sequence which has at least 70% sequence identity with SEQ ID NO: 1 or 3;
    • (c) is encoded by the nucleotide sequence set forth in SEQ ID NO: 2 or 4; or
    • (d) is encoded by a nucleotide sequence which has at least 70% sequence identity with the nucleotide sequence set forth in SEQ ID NO: 2 or 4.
      The invention also provides a composition comprising the polypeptide of the invention.

The invention further provides a nucleic acid molecule comprising:

    • (a) the nucleotide sequence set forth in SEQ ID NO: 2 or 4;
    • (b) a nucleotide sequence which has at least 70% sequence identity with the nucleotide sequence set forth in SEQ ID NO: 2 or 4; or
    • (c) the complement of (a) or (b),
      preferably operably associated with one or more regulatory elements.

The invention also provides a vector comprising a nucleic acid molecule of the invention. Also provided is a host cell comprising a vector of the invention.

The invention further provides a method of hydrolysing a polysaccharide, comprising contacting the polysaccharide with a polypeptide of the invention.

Also provided is a process for producing a cyclodextrin, the process comprising the steps:

    • (i) contacting a polypeptide of the invention with a polysaccharide substrate in a reaction vessel, and
    • (ii) isolating a cyclodextrin from the reaction vessel,

and optionally purifying and/or concentrating the obtained cyclodextrin.

The invention also provides a cyclodextrin which is obtainable or obtained by a process of the invention.

The polypeptide of the invention may be isolated and/or purified. In particular, the polypeptide of the invention may be in a form which is isolated from one or more of the following: bacteria, polysaccharide (e.g. potato, starch), yeast extract, tryptone, other enzymes.

The polypeptide may be purified, i.e. the polypeptide may be substantially pure. In particular, the polypeptide may be at least 90%, preferably at least 95% and more preferably at least 99% pure. Purity may be assessed using SDS-PAGE or any other appropriate method.

The invention also provides variants or derivatives of the polypeptide of SEQ ID NO: 1 or 3. The proteins of the invention may be altered in various ways including substitutions, deletions, truncations, and/or insertions of one or more (e.g. 2-5, 2-10) amino acids, preferably in a manner which does not substantially alter the biological activity of the polypeptide of the invention. Guidance as to appropriate amino acid changes that do not affect biological activity of the protein of interest may be found in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Nat'l. Biomed. Res. Found., Washington, D.C.), herein incorporated by reference. Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be also made.

In particular, substitution of one hydrophobic amino acid such as isoleucine, valine, leucine or methionine for another may be made; or the substitution of one polar amino acid residue for another, such as arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine, may be made.

One or more (e.g. 1-5, 1-10) amino acids in the polypeptides of the invention may be substituted by their corresponding D-amino acids, preferably at the N- and/or C-terminus.

In particular, the invention provides a variant of the polypeptide of SEQ ID NO: 1 or 3, wherein the amino acid sequence of the variant comprises or consists of an amino acid sequence having at least 70%, preferably at least 80%, 85%, 90%, 95% or 99% sequence identity with SEQ ID NO: 1 or 3, preferably using the blastp method of alignment.

The invention particularly relates to polypeptides of SEQ ID NO: 1 or 3 or to variants of the polypeptide of SEQ ID NO: 1 or 3 as defined herein, wherein the amino acid which corresponds to the amino acid at position 223 is a small amino acid, e.g. glycine, alanine, leucine, serine, threonine or valine, preferably glycine. The size of the amino acid residue at position 223 may be involved in determining the size of any cyclodextrin products or ratio of any cyclodextrin products.

The CGTases of the invention are capable of producing one or more cyclodextrins from polysaccharides, e.g. starch.

The CGTases fall within the general EC classification 2.4.1. (hexosyltransferases). In some embodiments, the CGTase of the invention falls within classification EC 2.4.1.248 (cycloisomaltooligosaccharide glucanotransferase). In other embodiments of the invention, the CGTase falls within classification EC 2.4.1.19 (cyclomaltodextrin glucanotransferase).

The invention also provides a composition comprising or consisting essentially of a polypeptide of the invention. The polypeptide may be present in the composition in the absence of one or more of the following: bacteria, polysaccharide (e.g. potato, starch), yeast extract, tryptone.

The polypeptide of the invention may be provided in any suitable form, e.g. in lyophilised form or in a buffer.

As used herein, the term “nucleic acid molecule” refers to a DNA or RNA molecule, which might be single- or double-stranded. Preferably, the nucleic acid molecule is a DNA molecule, most preferably a double-stranded DNA molecule. The nucleic acid molecule is preferably one which contains no introns. The nucleic acid molecule may, for example, be intron-less genomic DNA or cDNA.

The nucleic acid molecule of the invention is preferably isolated or purified. As used herein, the term “isolated nucleic acid” means that the nucleic acid molecule is not contiguous with other genes with which it is normally associated in the natural source of the polypeptide-encoding nucleic acid. For example, an isolated nucleic acid of the invention will not be contiguous with a nucleic acid encoding a maltose binding protein periplasmic precursor; or it will not be contiguous with a nucleic acid encoding a neopullananse/cyclomaltodextrinase.

As used herein, the term “purified nucleic acid” means a nucleic acid molecule which is free or substantially free from other non-contiguous nucleic acids and/or is free or substantially free from one or more of the following: bacteria, polysaccharide (e.g. potato, starch), yeast extract, tryptone.

As used herein, the term “polysaccharide” or “polysaccharide substrate” refers preferably to a glucose-based polysaccharide, e.g. a starch or a starch-based material. Most preferably, the polysaccharide is starch or a starch-based material, e.g. corn, corn starch, corn mash, potato, potato starch, potato mash, potato peeling, potato chips, cassava, cassava starch, cassava chips, sago, sago starch or ‘soluble starch’. e.g. as sold by Fisher/Sigma. In some embodiments of the invention, the nucleic acid molecule is a recombinant nucleic acid.

The nucleic acid of the invention is preferably operably associated with one or more regulatory elements, e.g. a promoter and/or a terminator element. As used herein the term “operably associated” or “operably linked” with a promoter means that the polypeptide-encoding region is transcribable from that promoter. The polypeptide-encoding region may, for example, be immediately 3′ to the promoter, in which case the promoter will direct the transcription of the coding sequence. Alternatively, the polypeptide-encoding region may be part of an operon in which case the associated or linked promoter will direct the transcription of all of the polypeptide-encoding regions within that operon.

The promoter or promoters are preferably ones which are operable in bacterial cells. More preferably, the promoters are bacterial promoters. Suitable promoters include inducible promoters, such as those that are inducible with specific sugars or sugar analogues, e.g. arabinose (e.g. lac, ara), those inducible with antibiotics (e.g. tetracycline, tet), those inducible with IPTG (e.g. trp, tac, Pspac), those inducible with heat (e.g. hsp70), those inducible with anaerobic induction (e.g. nisA, pfl, trc, IPL, IPR, T7), P11, ldh, sec (secDF), SV40 promoter, those inducible with xylose (e.g. Pxyl promoter), those inducible with osmotic shock, cell density (quorum sensing), anaerobicity, antibiotics, or growth phase. In some embodiments, the promoter is a constitutive promoter, e.g. the promoters for the thiolase gene (thl) or the permease operon (hfuC). In other embodiments, the promoter is one from Clostridia, e.g. a promoter from the pta/ptb genes. In yet other embodiments, the promoter is one from a butanol and/or butyrate biosynthetic pathway gene.

In other embodiments, the promoter is an early onset promoter, i.e. a promoter from a gene which is upregulated during early exponential phase and reduced during transition phase and stationary phase. Examples of such promoters include promoters from glcK, hydA genes, or vitamin B12 synthesis, pta, ptb promoters.

In other embodiments, the promoter is a promoter from a gene which is normally active in the exponential phase of solventogenic bacteria. Examples include promoters from genes that are expressed constitutively throughout exponential phase, e.g. from glycolysis genes and those in the pathway to produce butyryl-CoA (pfk, gap, pgk, bcd).

Other examples of suitable promoters include the P2 (pta-ack, CAC1742, promoter), P6 (luxS, CAC2942, promoter) and P7 (CAC2941) promoters (Alsaker and Papoutsakis, 2005, J. Bacteriol. 187:7103-7118).

The P2 promoter is the promoter region from the operon encoding the phosphotransferase and the acetate kinase involved in acetate production from acetyl-CoA. The P6 promoter is the promoter region from a single chromosomal open reading frame encoding a LuxS homolog (CAC 2942), predicted to be involved in quorum sensing. The P7 promoter is the promoter region from a chromosomal operon (CAC 2938-2941) encoded downstream and in the reverse orientation to CAC 2942 and putatively involved in quorum sensing. The operon encodes a hydrolase (CAC 2941), a histidine kinase (CAC 2940), a response regulator (CAC 2939) and a hypothetical protein (CAC 2938).

In a further embodiment, the invention provides a variant of the nucleic acid molecule of SEQ ID NO: 2 or 4, wherein the nucleotide sequence of the variant comprises or consists of an nucleotide sequence having at least 70%, preferably at least 80%, 85%, 90%, 95% or 99% sequence identity with SEQ ID NO: 2 or 4, preferably using the BLASTN method of alignment.

Percentage amino acid sequence identities and nucleotide sequence identities may be obtained using the BLAST methods of alignment (Altschul et al. (1997), “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs”, Nucleic Acids Res. 25:3389-3402. Preferably the standard or default alignment parameters are used.

Standard protein-protein BLAST (blastp) may be used for finding similar sequences in protein databases. Like other BLAST programs, blastp is designed to find local regions of similarity. When sequence similarity spans the whole sequence, blastp will also report a global alignment, which is the preferred result for protein identification purposes. Preferably the standard or default alignment parameters are used. In some instances, the “low complexity filter” may be taken off.

BLAST protein searches may also be performed with the BLASTX program, score=50, wordlength=3. To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25: 3389. Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform an iterated search that detects distant relationships between molecules. (See Altschul et al. (1997) supra). When utilizing BLAST, Gapped BLAST, PSI-BLAST, the default parameters of the respective programs may be used.

With regard to nucleotide sequence comparisons, MEGABLAST, discontiguous-megablast, and blastn may be used to accomplish this goal. Preferably the standard or default alignment parameters are used. MEGABLAST is specifically designed to efficiently find long alignments between very similar sequences. Discontiguous MEGABLAST may be used to find nucleotide sequences which are similar, but not identical, to the nucleic acids of the invention. The BLAST nucleotide algorithm finds similar sequences by breaking the query into short subsequences called words. The program identifies the exact matches to the query words first (word hits). The BLAST program then extends these word hits in multiple steps to generate the final gapped alignments. In some embodiments, the BLAST nucleotide searches can be performed with the BLASTN program, score=100, wordlength=12.

One of the important parameters governing the sensitivity of BLAST searches is the word size. The most important reason that blastn is more sensitive than MEGABLAST is that it uses a shorter default word size (11). Because of this, blastn is better than MEGABLAST at finding alignments to related nucleotide sequences from other organisms. The word size is adjustable in blastn and can be reduced from the default value to a minimum of 7 to increase search sensitivity.

A more sensitive search can be achieved by using the newly-introduced discontiguous megablast page that is available through the website of the National Center for Biotechnology Information. This page uses an algorithm which is similar to that reported by Ma et al. (Bioinformatics. 2002 March; 18(3): 440-5). Rather than requiring exact word matches as seeds for alignment extension, discontiguous megablast uses non-contiguous word within a longer window of template. In coding mode, the third base wobbling is taken into consideration by focusing on finding matches at the first and second codon positions while ignoring the mismatches in the third position. Searching in discontiguous MEGABLAST using the same word size is more sensitive and efficient than standard blastn using the same word size. Parameters unique for discontiguous megablast are: word size: 11 or 12; template: 16, 18, or 21; template type: coding (0), non-coding (1), or both (2).

In yet other embodiments, the nucleic acid of the invention is present in an operon, preferably with one or more genes which are involved in starch metabolism. Preferably, the nucleic acid of the invention is in an operon, wherein the nucleic acid of the invention is contiguous with one or more nucleic acid molecules which encode one or more of the following: a maltose binding protein periplasmic precursor, a neopullanase/cyclomaltodextrinase, one or more maltose/maltodextrin ABC transporter permease proteins, an alpha amylase catalytic domain protein and a glycogen debranching protein.

As used herein, the term “operon” refers to a segment of a nucleic acid molecule which comprises a linear sequence of two, three, four or more polypeptide-encoding regions which are all in the same 5′-3′ orientation and which are transcribable as a single (polycistronic) unit from an associated promoter. The promoter will in general be at the 5′ end of the operon.

In this case, transcription will be initiated from the first promoter and a single polycistronic mRNA transcript will be produced from the said two, three, four or more of the polypeptide-encoding regions.

The remaining polypeptide-encoding regions of the nucleic acid molecule may independently be operably linked together with a second promoter in a second operon, wherein the second operon is transcribable from the second promoter; or they may each be operably linked to and transcribable from further promoters.

Preferably, the operon has the sequence as given in SEQ ID NO: 5, or a variant thereof having at least 70%, preferably at least 80%, 90%, 95% or 99% sequence identity with SEQ ID NO: 5 using the BLASTN method of alignment.

In some embodiments, the operon encodes the following polypeptides in the order 5′-3′ (and each coding sequence being in the 5′-3′ direction): a maltose binding protein periplasmic precursor, an isocyclomaltooligosaccharide glucanotransferase (of the invention), a neopullanase/cyclomaltodextrinase, two maltose/maltodextrin ABC transporter permease proteins, an alpha amylase catalytic domain protein and a glycogen debranching protein.

The nucleic acid molecule of the invention or operon will preferably be in the form of a vector, particularly an expression vector, or a plasmid. The vector or plasmid may comprise one or more selectable markers and/or other genetic elements. Preferably, the vector or plasmid is less than 100 Kb, more preferably less than 90, 80, 70, 60, 50, 40, 30 or 20 Kb. Preferably, the vector or plasmid additionally comprises one or more antibiotic resistance genes. Examples of such genes include genes conferring resistance to ampicillin, erythromycin, neomycin/kanamycin, tetracycline, chloramphenicol, spectinomycin, bleomycin and puromycin. In some embodiments, the vector or plasmid also comprises one or more genes conferring tolerance to one or more heavy metals, e.g. mercury. Other selectable markers include auxotrophy genes, e.g. genes for essential amino acids.

The vector or plasmid may also comprise an origin of replication, for example a Gram positive and/or a Gram negative origin of replication. The vector or plasmid may also comprise one or more insertion sequences, e.g. Tn10, Tn5, Tn1545, Tn916 and/or ISCb.

The nucleic acid molecule of the invention or operon or the plasmid or vector, may be introduced into a host cell, e.g. a micro-organism, preferably a bacterial cell.

The bacterial cell may, for example, be a Gram-positive or Gram-negative bacterium. In some embodiments, the micro-organism is a spore-forming bacterium. In other embodiments, the micro-organism is a saccharolytic bacterium.

The bacterium may be an aerobic or an anaerobic bacteria. Preferably it is an anaerobic bacteria. The bacteria may be a thermophilic bacterium. In yet other embodiments, the bacterium is a biphasic bacterium. As used herein, the term “biphasic” refers to a bacterium which has an acidogenic growth phase and a solventogenic growth phase. The term “acidogenic growth phase” refers to the ability of the bacterium to convert a substrate into R-COOH, for example, into acetate and/or butyrate. In this context, wherein R is an aliphatic C1-C5, preferably C1-3, alkyl or alkenyl group. The term “solventogenic growth phase” refers to the ability of the bacterium to convert the RCOOH into a solvent, preferably into one or more of acetone, ethanol and/or butanol.

In other embodiments, the bacterium is a solvent-producing bacterium. As used herein, the term “solvent-producing” means that the bacterium is one which is capable of producing a solvent, preferably a solvent such as acetone, ethanol, propanol and/or butanol. In certain particularly preferred embodiments, the bacterium is capable of producing ethanol, acetone and butanol. Preferably, the bacteria is a butanol-producing bacteria or a butanol-tolerant bacterium.

In some preferred embodiments, the bacterium is of the genus Clostridium. Preferred Clostridium species include C. acetobutylicum, C. aurantibutyricum, C. beijerinckii, C. thermocellum, C. thermobutyricum, C. pasteurianum, C. kluyveri, C. saccharobutylicum, C. thermosaccharolyticum, C. saccharolyticum, C. tyrobutyricum, C. butyricum, C. puniceum, C. diolis and C. roseum.

In some embodiments, the bacteria is a Cluster I Clostridia. Preferred examples of Cluster I Clostridia include C. acetobutylicum, C. arbusti, C. argentinense, C. beijerinckii, C. butyricum, C. cellulovorans, C. diolis, C. kluyveri, C. novyi, C. pasteurianum, C. puniceum, C. roseum, C. saccharobutylicum, C. saccharoperbutylacetonicum and C. tyrobutyricum.

In some embodiments of the invention, the host cell is not C. saccharoperbutylacetonicum N1-4. In other embodiments of the invention, the host cell is not C. saccharoperbutylacetonicum N1-4(HMT). In yet other embodiments of the invention, the host cell is not C. saccharoperbutylacetonicum N1-504.

In other preferred embodiments, the bacterium is of the genus Bacillus or Geobacillus.

The invention further provides a process for making a recombinant bacterial host cell, comprising introducing a nucleic acid molecule of the invention, or an operon or a vector or plasmid of the invention, into a bacterial host. Methods of introducing nucleic acid molecules, operons, plasmids and vectors into bacterial hosts are well known in the art. These include transformation, transfection and electroporation techniques.

The invention also provides a recombinant bacterial host comprising a nucleic acid molecule of the invention, or an operon or a vector or plasmid of the invention.

The nucleic acid molecule or operon may be present in the cytoplasm of the host, e.g. as a plasmid or a vector, or it may be integrated in the host genome.

The invention therefore provides a bacterial cell comprising a nucleic acid molecule, an operon, a vector or plasmid of the invention, wherein the nucleic acid molecule, operon, vector or plasmid is present in the cytoplasm of the cell.

The invention also provides a bacterial cell comprising a nucleic acid molecule of the invention or an operon or vector or plasmid of the invention, wherein the nucleic acid molecule, operon, vector or plasmid is stably integrated into the genome of the cell.

In a further embodiment, the invention provides a method of hydrolysing a polysaccharide, comprising contacting the polysaccharide with a polypeptide of the invention. Preferably, the polypeptide is in isolated or purified form.

The invention also provides the use of a polypeptide of the invention in the hydrolysis of a polysaccharide.

The invention also provides a method of hydrolysing a polysaccharide, comprising contacting the polysaccharide with a host cell of the invention, preferably a recombinant bacterial host cell of the invention.

The invention particularly provides a method of hydrolysing a polysaccharide, comprising contacting the polysaccharide with a host cell of the invention which has been stably transformed with a nucleic acid or operon or vector or plasmid of the invention, such that the host cell expresses a CGTase and optionally one or more other polypeptides which are involved in starch metabolism.

As used herein, the term “polypeptides which are involved in starch metabolism” includes maltose binding protein periplasmic precursors, isocyclomaltooligosaccharide glucanotransferases, neopullanase/cyclomaltodextrinases, maltose/maltodextrin ABC transporter permease proteins, alpha amylase catalytic domain proteins and glycogen debranching proteins.

Preferably, the host cell is also capable of converting the hydrolysed polysaccharide to an acid such as R-COOH, for example into acetate and/or butyrate. Optionally, the host cell is also capable of converting the RCOOH into a solvent, preferably into one or more of acetone, ethanol and/or butanol.

The invention also provides a method of producing a solvent comprising the steps:

    • (i) incubating a host cell of the invention, preferably a recombinant bacterial host cell of the invention, with a polysaccharide substrate;
      wherein the host cell is also capable of converting hydrolysed polysaccharide to an acid such as R-COOH, for example into acetate and/or butyrate. Optionally, the host cell is also capable of converting the RCOOH into a solvent, preferably into one or more of acetone, ethanol and/or butanol.

Preferably, step (i) is carried out under conditions wherein the host cell expresses the CGTase and wherein the CGTase hydrolyses some or all of the polysaccharide substrate.

The host cell may be one which is naturally capable of converting the hydrolysed polysaccharide to an acid such as R-COOH and/or which is naturally capable of converting the RCOOH into a solvent. Alternatively, the host cell is one which has been transformed with one or more nucleic acid molecules encoding polypeptides which are capable of converting the hydrolysed polysaccharide to an acid such as R-COOH and/or which are capable of converting the RCOOH into a solvent.

The invention also provides a process for producing a cyclodextrin, the process comprising the steps:

    • (i) contacting a polypeptide of the invention with a polysaccharide substrate in a reaction vessel, and
    • (ii) isolating a cyclodextrin from the reaction vessel,
      and optionally purifying and/or concentrating the obtained cyclodextrin.

Preferably, the polysaccharide substrate a glucose-based polysaccharide. More preferably, the polysaccharide substrate is starch or a starch-based material, e.g. corn mash, potato mash, potato peeling,

The invention also provides a cyclodextrin which is obtainable or obtained by a process of the invention.

The invention further provides a cyclodextrin with an elution profile as shown in FIG. 7 herein. Preferably, the cyclodextrin is resistant to hydrolysis by amylases.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows starch plate onto which supernatant samples were spotted. Supernatant from C. saccharoperbutylacetonicum grown on glucose does not show any hydrolytic activity whereas supernatant from starch and corn does.

FIG. 2 shows C. saccharoperbutylacetonicum grown on glucose, starch or corn mash. Supernatants were concentrated and proteins analysed by 12.5% SDS-PAGE. The highlighted protein shows clear induction in starch and corn mash with no polypeptide in the glucose-grown sample.

FIG. 3. The CGTase enzyme is located as the second gene in an operon in which the rest of the genes also appear to have a role in starch metabolism, from conversion to cyclodextrin through substrate uptake to internal conversion of the substrate, probably to glucose. Gene annotations are based on BLAST searches.

FIG. 4. The protein sequence of the C. saccharoperbutylacetonicum CGTase enzyme was aligned with various previously reported CGTases. It does not cluster with the well characterised α-, β-, γ-CGTases, instead being more closely related to enzymes that cyclise starch using various other mechanisms.

FIG. 5. Amino acid alignment of highly conserved residues required for cyclisation. α-, β-, γ-CGTases that use α1-4 linkages have a Tyr or Phe at this position (highlighted with arrow). The CGTases that use a different cyclisation mechanism, all have small residues, e.g. Gly. C. saccharoperbutylacetonicum (lines 31 and 32) sequences both have a Gly at this position. The sequences identified in FIG. 5 as numbers 1-41 correspond to SEQ ID NOs: 6-46.

FIG. 6. The 75% fraction from ammonium sulphate cuts was found to have the most starch degradation activity so this was added to a starch solution to identify conversion products. Soluble starch already contains some linear dextrins (labelled LD). This peak increased in size and a new peak was also observed when the starch was treated with the 75% ‘cut’ (labelled CD).

FIG. 7. Crude supernatant extracts were added to 10 g/L starch solution and incubated overnight to compare starch degradation products by HPLC. C. acetobutylicum has a well studied α-amylase, glucoamylase, method of hydrolysing starch to glucose. C. saccharoperbutylacetonicum, on the other hand, does not convert starch to glucose, instead processing it only as far as the cyclic compound.

FIGS. 8A-8L. Starch metabolism operon from C. saccharoperbutylacetonicum, illustrating the location of the CGTase-encoding gene. The sense and anti-sense strand genomic sequences are SEQ ID NOs: 5 and 55, respectively. The SEQ ID NOs of the amino acid sequences are as follows: transcriptional regulator—SEQ ID NO: 47; maltose binding protein—SEQ ID NO: 48; isocyclomaltooligosaccharide—SEQ ID NO: 1; neopullanase—SEQ ID NO: 49; ABC transporters—SEQ ID NOs: 50 and 51; protein of unknown function—SEQ ID NO: 52; alpha amylase catalytic region—SEQ ID NO: 53; and glycogen debranching protein—SEQ ID NO: 54.

EXAMPLES

The present invention is further illustrated by the following Examples, in which parts and percentages are by weight and degrees are Celsius, unless otherwise stated. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. The disclosure of each reference set forth herein is incorporated herein by reference in its entirety.

Example 1

Identification of Starch Hydrolytic Activity

C. saccharoperbutylacetonicum was grown on various substrates. Supernatant samples were taken after 72 hours, concentrated and then spotted onto a starch plate. Supernatant from C. saccharoperbutylacetonicum grown on glucose does not show any hydrolytic activity, whereas supernatant from starch and corn does (FIG. 1).

These supernatant samples were also analysed by SDS-PAGE and an induced enzyme was identified (FIG. 2).

Example 2

Sequencing of the Novel Polypeptide

The induced peptide was excised from the SDS-PAGE gel and identified by mass spectrometry as being an isocyclomaltooligosaccharide glucanotransferase (CGTase).

The portion of the C. saccharoperbutylacetonicum genome coding for the CGTase was sequenced. It was found that the CGTase is located within a starch metabolism operon (FIG. 3 and SEQ ID NO: 5).

The amino acid sequence of the CGTase from C. saccharoperbutylacetonicum N1-4(HMT) is given in SEQ ID NO: 1. The corresponding nucleic acid sequence is given in SEQ ID NO: 2.

The amino acid sequence of the CGTase from C. saccharoperbutylacetonicum N1-504 is given in SEQ ID NO: 3. The corresponding nucleic acid sequence is given in SEQ ID NO: 4.

Based on sequence alignments and homology searches, the CGTase from C. saccharoperbutylacetonicum appears to be a novel enzyme. A number of features make it different from the well-characterised α-, β-, and γ-CGTases for example, protein alignments show it clusters with CGTases from B. circulans and Arthrobacter which have been characterised and do not form the standard α-, β- or γ-cyclodextrins containing 6, 7 or 8 glucose units with α1-4 linkages (FIG. 4). Instead this class of CGTase enzymes appears to be much less conserved and converts starch to cyclodextrins containing 4, 5 or 6 glucose units with both α1-4 and α1-6 linkages. A key feature of these enzymes is a highly conserved residue required for efficient cyclisation. The α-, β-, and γ-CGTases all have tyr or phe at this position. α-amylases have a small residue at this equivalent position, as do the CGTases from B. circulans, Arthrobacter and C. saccharoperbutylacetonicum (FIG. 5).

Based on these sequence comparisons, it is inferred that the CGTase from C. saccharoperbutylacetonicum does not convert starch through the well characterised α-, β-, γ-cyclodextrin route. Instead it appears to cyclise starch using a different mechanism.

Example 3

Initial Identification of Cyclodextrins

Proteins secreted into the supernatant during a C. saccharoperbutylacetonicum fermentation on a starch-based substrate were fractionated using ammonium sulphate cuts. The starch degradation activity was followed by spotting each fraction onto a starch plate and staining with iodine to detect zones of clearing. The fraction containing starch hydrolysis activity was added to a flask containing 10 g/L starch solution and incubated overnight at 35° C. in a shaking incubator. The starch solution was known to contain some linear dextrins.

In the morning, a mixture of starch and various starch hydrolysis products were detected in the flask, including linear- and cyclo-dextrins.

Example 4

Purification of Cyclodextrin

The hydrolysis products from Example 3 were detected by HPLC. As shown in FIG. 6, various starch hydrolysis products were detected, including linear- and cyclo-dextrins.

The 75% ammonium sulphate ‘cut’ was also separated on an SDS-PAGE gel and the bands were isolated. Mass spectrometry was used to confirm the CGTase was still present in this fraction (data not shown).

The CGTase enzyme from C. saccharoperbutylacetonicum produced a cyclic dextrin with an elution profile which was different from known α-, β- and γ-cyclodextrins (FIG. 7). The elution profile was also clearly different to the elution profile one would expect to see if the strain was converting starch to dextrins and glucose using α-amylase and glucoamylase.

Furthermore, the cyclic compound was found to be relatively resistant to further hydrolysis by other amylases.

SEQUENCES
SEQ ID NO: 1 C. saccharoperbutylacetonicum strain N1-4(HMT)
MFRRKFNKVILSILVATIVSSTNMFMSGSKAQAAIGNLSENDTIYQIMVDRFYDGDKTNN
ATGDAFRNTENLEDDFRYMHGGDWQGVIDKLDYIKGMGYSAIWISPVAEPQMWSRADGTG
KVWPTAYHGYNVKDPNKANPYFGTKEKLKELVDKAHEKGIKVIIDIVPNHVGDYMLGKQA
YYDIKGFEPAAPFNNPNWYHHNGDIDWSREHSDPQMLDDHDLGGLDDLNQDNSDAKAAMN
NAIKSWFDYTGADAARVDAAKCMKPSYINELQKYIGVNTFGENFDMNVDFVKKWVGSDAE
WGMLDFPLYQAINNDFASGQSFDDMSSSGTCSIKNILAQDNKYNGYANHMVTFIDNHDRN
RFLTVANGNVKKLQNALVFMFTVRGVPTVFQGTEQNKGNANGASINGIADTWNRWSMVKK
DYNGNVITDYFNENTDTYKLINKLNSFRQKYEALREGTQREMWSSPHLYAFSRRMDSGEN
VGQEVVNVFNNSDGDQSATIPIRAESTIKVGDKFVNLFDVNDSITVQQGGVTGKQISVNL
GENSGKIYVVNNETPNPDQKNVQYKVSYKNTNAQKVTLHYGTNGWKNIQDVNMTKNSNGE
FEATITVNNNDILNYCIHIISPTDYWDNNGGQNWNVKVTKAEDYINDGVKSNLKSVNTTT
SAAIDSGIDSTVNR
The predicted N-terminal signal sequence is highlighted (predicted using signalP).
SEQ ID NO: 2 C. saccharoperbutylacetonicum strain N1-4(HMT)
ATGTTTAGAAGAAAATTTAACAAGGTAATATTATCTATCTTAGTTGCAACAATTGTTTCA
AGCACTAACATGTTTATGAGTGGAAGCAAGGCACAAGCGGCAATTGGAAATCTAAGTGAA
AACGATACTATTTATCAAATTATGGTAGACAGATTTTATGATGGAGATAAAACAAATAAT
GCTACAGGAGATGCATTTCGTAATACAGAAAATCTTGAAGATGATTTTAGATATATGCAC
GGCGGAGATTGGCAAGGTGTTATTGATAAGTTAGATTATATTAAGGGCATGGGATACTCA
GCCATTTGGATATCACCGGTTGCGGAACCACAAATGTGGTCTAGAGCTGATGGCACAGGA
AAAGTATGGCCTACAGCTTATCATGGATATAATGTGAAAGATCCCAATAAGGCAAATCCT
TATTTTGGAACAAAAGAAAAGCTAAAGGAGTTAGTAGATAAAGCTCACGAAAAGGGGATT
AAAGTAATAATAGATATAGTTCCAAATCATGTTGGGGATTATATGTTAGGAAAACAAGCT
TATTATGACATCAAGGGGTTTGAGCCGGCAGCACCTTTTAATAATCCAAATTGGTATCAT
CATAATGGCGATATTGATTGGTCAAGAGAACACTCTGATCCCCAAATGTTAGATGATCAT
GATTTGGGCGGTTTAGATGATTTAAATCAAGATAATTCTGATGCTAAGGCAGCTATGAAT
AATGCTATTAAGTCATGGTTTGATTATACTGGAGCTGATGCAGCAAGGGTTGACGCAGCA
AAATGTATGAAACCATCTTATATTAACGAGTTACAAAAGTATATAGGAGTTAATACTTTT
GGAGAAAATTTTGATATGAATGTAGATTTTGTGAAGAAGTGGGTTGGATCCGATGCAGAA
TGGGGAATGCTAGATTTTCCATTATATCAAGCAATAAATAATGATTTTGCATCAGGACAA
TCTTTTGATGACATGTCATCATCAGGTACTTGCTCTATTAAAAATATTTTAGCACAAGAC
AATAAATATAATGGTTATGCAAATCATATGGTGACTTTTATAGATAATCATGATCGTAAT
AGATTTTTAACAGTAGCAAATGGTAATGTAAAAAAACTTCAAAATGCACTTGTTTTCATG
TTTACTGTAAGAGGGGTACCAACAGTATTTCAAGGTACAGAACAAAACAAAGGTAATGCA
AATGGAGCAAGTATAAATGGTATTGCAGATACATGGAATCGTTGGTCAATGGTTAAAAAG
GATTACAATGGAAATGTAATTACAGATTATTTTAATGAGAATACAGATACTTATAAACTA
ATTAACAAATTGAATTCATTTAGGCAAAAATATGAAGCCTTAAGAGAAGGTACTCAAAGA
GAAATGTGGTCTTCACCACATTTATATGCATTCTCAAGAAGGATGGATTCAGGAGAAAAT
GTTGGACAAGAAGTTGTAAATGTATTTAATAATTCAGATGGAGATCAAAGTGCGACCATT
CCAATTAGAGCTGAAAGTACTATAAAAGTTGGAGATAAATTTGTAAATCTTTTTGATGTA
AATGATTCGATCACAGTTCAACAAGGAGGTGTTACAGGAAAACAAATATCAGTGAATTTA
GGAGAAAATAGTGGGAAGATTTATGTTGTTAATAATGAAACACCAAATCCAGATCAAAAG
AACGTACAATATAAAGTTTCATATAAGAATACTAATGCACAAAAAGTAACACTTCATTAT
GGAACTAATGGATGGAAAAACATTCAAGATGTAAATATGACTAAGAATTCCAATGGAGAA
TTTGAAGCAACTATTACAGTAAATAATAATGATATTCTAAATTACTGTATTCATATTATT
TCACCAACAGACTATTGGGATAATAATGGTGGACAGAATTGGAATGTAAAAGTGACTAAG
GCAGAAGATTATATAAATGATGGTGTAAAGAGTAATTTGAAGAGCGTTAATACAACTACA
TCAGCAGCTATAGACTCTGGGATTGATAGTACTGTAAATCGTTAA
SEQ ID NO: 3 C. saccharoperbutylacetonicum strain N1-504
MFRRKFNKVILSILVATIVSSTNMFMSGSKAQAAIGNLSENDTIYQIMVDRFYDGDKTNNATGDAFRNTENLE
DDFRYMHGGDWQ
GVIDKLDYIKGMGYSAIWISPVAEPQMWSRADGTGKVWPTAYHGYNVKDPNKANPYFGTK
EKLKELVDKAHEKGIKVIIDIVPNHVGDYMLGKQAYYDIKGFEPAAPFNNPNWYHHNGDI
DWSREHSDPQMLDDHDLGGLDDLNQDNSDAKAAMNNAIKSWFDYTGADAARVDAAKCMKP
SYINELQKYIGVNTFGENFDMNVDFVKKWVGSDAEWGMLDFPLYQAINNDFASGQSFDDM
SSSGTCSIKNILAQDNKYNGYANHMVTFIDNHDRNRFLTVANGNVKKLQNALVFMFTVRG
VPTVFQGTEQNKGNGNGAILNGIADTWNRWSMVKKDYNGNIITDYFNENTDTYKLISKLN
SFRQKYEALREGTQREMWSSPHLYAFSRRMDSGENVGQEVVNVFNNSDGDQSATIPIRAE
STIKVGDKLVNLFDVNDSITVQQGGVTGKQISVNLGENSGKIYVVNNETPNPDQKNVQYK
VSYKNTNAQKVTLHYGTNGWKNIQDVNMTKNSNGEFEATITVNNNDILNYCIHIISPTDY
WDNNGGQNWNVKVTKAEDYINDGVKSNLKSVNTTTSAAIESGIDSTVNR
The predicted N-terminal signal sequence is highlighted (predicted using signalP).
SEQ ID NO: 4 C. saccharoperbutylacetonicum strain N1-504
atgtttagaagaaaatttaacaaggtaatattatctattttagttgcaacaattgtttca
agcactaacatgttt
ATGAGTGGAAGCAAGGCACAAGCGGCAATTGGAAATTTAAGTGAAAACGATACTATTTAT
CAAATTATGGTAGACAGATTTTATGATGGAGATAAAACAAATAATGCTACAGGAGATGCA
TTTCGTAATACAGAAAATCTTGAAGATGATTTTAGATATATGCACGGCGGAGATTGGCAA
GGTGTTATTGATAAGTTAGATTATATTAAGGGCATGGGATACTCAGCCATTTGGATATCA
CCGGTTGCGGAACCACAAATGTGGTCTAGAGCTGATGGCACAGGAAAAGTATGGCCTACA
GCTTACCATGGATATAATGTGAAAGATCCCAATAAGGCAAATCCTTATTTTGGAACAAAA
GAAAAGCTAAAGGAGTTAGTAGATAAAGCTCACGAAAAGGGGATTAAAGTAATAATAGAT
ATAGTTCCAAATCATGTTGGGGATTATATGTTAGGAAAACAAGCTTATTATGACATCAAG
GGGTTTGAGCCGGCAGCACCTTTTAATAATCCAAATTGGTATCATCATAATGGCGATATT
GATTGGTCAAGAGAACACTCTGATCCCCAAATGTTAGATGATCATGATTTGGGCGGTTTA
GATGATTTAAATCAAGATAATTCTGATGCTAAGGCAGCTATGAATAATGCTATTAAGTCA
TGGTTTGATTATACTGGAGCTGATGCAGCAAGGGTTGACGCAGCAAAATGTATGAAACCA
TCTTATATTAACGAGTTACAAAAGTATATAGGAGTTAATACTTTTGGAGAAAATTTTGAT
ATGAATGTAGATTTTGTGAAGAAGTGGGTTGGATCCGATGCAGAATGGGGAATGCTAGAT
TTTCCATTATATCAAGCAATAAATAATGATTTTGCATCAGGACAATCTTTTGATGACATG
TCATCATCAGGTACTTGCTCTATTAAAAATATTTTAGCACAAGACAATAAATATAATGGT
TATGCAAATCATATGGTGACTTTTATAGATAATCATGATCGTAATAGATTTTTAACAGTA
GCAAATGGTAATGTTAAAAAACTTCAAAATGCACTTGTTTTCATGTTTACTGTAAGAGGG
GTACCAACAGTATTTCAAGGTACAGAACAAAACAAAGGTAATGGAAATGGAGCAATTCTA
AATGGTATTGCAGATACATGGAATCGTTGGTCAATGGTTAAAAAGGACTATAATGGAAAT
ATAATTACAGATTATTTTAATGAGAATACAGATACTTATAAACTAATTAGCAAATTGAAT
TCATTTAGGCAAAAATATGAAGCCTTAAGAGAAGGTACTCAAAGAGAAATGTGGTCTTCA
CCACATTTATATGCATTCTCAAGAAGGATGGATTCAGGAGAAAATGTTGGACAAGAAGTT
GTAAATGTATTTAATAATTCAGATGGAGATCAAAGTGCGACCATTCCAATTAGAGCTGAA
AGTACTATAAAAGTTGGAGATAAACTTGTAAATCTTTTTGATGTAAATGATTCGATCACA
GTTCAACAAGGAGGTGTTACAGGAAAACAAATATCAGTGAATTTAGGAGAAAATAGTGGG
AAGATTTATGTTGTTAATAATGAAACACCAAATCCAGATCAAAAGAACGTACAATATAAA
GTTTCATATAAGAATACTAATGCACAAAAAGTAACACTTCATTATGGAACTAATGGATGG
AAAAACATTCAAGATGTAAATATGACTAAGAATTCCAATGGAGAATTTGAAGCAACTATT
ACAGTAAATAATAATGATATTCTAAATTACTGTATTCATATTATTTCACCAACAGACTAT
TGGGATAATAATGGTGGACAGAATTGGAATGTAAAAGTGACTAAGGCAGAAGATTATATA
AATGATGGTGTAAAGAGTAATTTGAAGAGCGTTAATACAACTACATCAGCAGCGATAGAA
TCTGGTATTGATAGTACTGTAAATCGTTAA
SEQ ID NO: 5 C. saccharoperbutylacetonicum strain N1-4(HMT)
gttcatggttttttcaataggctagattcacgtataagtaatttaggtgatattagggttatttca
ttctgagaatttggattttgaatttttttcattaaaatttcagctgccacttcaccaagtttaaat
gtatctacatctagacaagttaaggatggagtggtgtaagcagaaaatggttcattgtcaaaagtc
acaattccaatatctgtggggctatttaagcccttttctttaagtgctttaagtacaccaaatgca
acataattatttatgcatagtatggcatctatttcaggaaaatctgataataattgaagtgtaagt
tcataaccactttccttgtctgagttgccttcttttatgtataaattatttttagtaaaatttaat
ttagaaagtatgtttttataaccaagaagccgattgaaagatattatttcatttgatttgccacca
ataaaggctatatttttataaccttgttctattaaatgacatgtggccagttctcctcctacagta
ttattaacatcaacccagctggtactattcttaaattcctttggctgaccaataaggacataagga
aagtttaaaccgttgagcttactaataatttccgaattgactattgaggtaggtattatgattcca
tctacttttttactatatatcaatcgatttagaaattctgctttgcattctagtgaatttatgttt
gatagggttaactcatagttattgattccaactatactttcaactccaccaataatattatagaaa
aagaaatcaagaaaatattcctttctgctaatatctactagaagacctatattaaaactattctgg
cgagctagctgccttgccgaattatttggaatatagttaagctctttcataatgtttcttactttg
agctttgtactttcagaaatagatttatgattatttataactttggataccgttgatttagataca
tttgctgcgtgagctatatcatttatagtaactttcatttttaactccttatgtgtgaaattgatt
atttaaatattataaaacattatttgatttttttcgatatgtacattgttattaataatattacta
tttattgtaaagtattttagaaattttttatacttctataagtttagcataataatctaaaaatac
atatatagttgcacatttcagtgctaaagattaaatttatttatatcatctaaatcctcttaaact
catttaaagttccttctctaaattgaaaaatgaatttccaattgtactcattgcataatttataga
tttgttcacaagcataaagactattacctaaaatgtaggtattaagtgttataaacttatatttta
attttttcataagtcttttagcatatgccttaataaaacaatatatctggtggaagttaatacaat
atattgaaattgattcaatattgcagtatactaaaatgagtaaaccggtttcttaaattttcaatg
tatttaaagaatttgtaagacagaagaatataattttcaactttataatatgcttatttgaatgaa
ttaataaaaagatgatttttagtattttgtttataagcctagtagttacaagaagtaaatattttc
tgtaaaagattataaattaggagggagaattgaaaaatggtaaaaaaaaataaagtattagcatca
atcgtggcagcaactttagttgcaggaacatttgtaggatgtggaggaacaacagctacaagtaat
aatgctaaagaaattacagtttggtcacatttgaaagaaaaagagattacagagcttactaaagta
gcggaaaaatggggaagtgaaaagggagttaaggttaatgttgtagatgataaaggggagatgcaa
gcatatatacaagccgctaatagttctaaaggtccagatatactttttggtgtacctaatgataac
ttaggaacatttcaaaaagctggtttactttcagaagtgccaagtggttttatagatgagagtaaa
tatacatctaaacaagtaatagattcagtgactatagaaggaaaaaaatatgcagttccattagca
gctgaaactagtgctctattttataataaagataaagtttcagaagtaccaaaaactatggaagaa
gttgttgaattaggcaaaaaagtaggatttgaatacgatgtaactgatttatacagaagttatgga
tttttagcatcgcaaggtagctatatttttaaaaataataatggaactgttgattcaaacgatatt
ggattaggcaatgaaggtgcgataaaaggatatcaattcattcaagatttaattgttaaagacaaa
ttaatgtctcaagatattactgatgatatagctaaagcagatttccaatcaggtaaatcagcattt
tatatttcaggaccatgggatatagaagcatttaaagattcaggaattaattttggtatagctcca
atgccaacattaggtgggaaaactgtttcaacattgatgggagttcaaactgcatttgtaagttca
aagtcacctaatcaagacttatcatgggagttaatgaagtatcttatggaaaatagtgatgaccta
atgattaagcaaggaaatagaattccagtttcaaaagcaggtatagaaagtgatgcgtttaaagcg
gccggaaacatggatgtatttgctaaacaattagaagttgctacagcaatgcctaatattccagaa
attcaaactacttggactccggtaaaaaataatataatatctttaataagcggatcaatggattcg
aaagaaactgcaaaacaaatagtagatcaaattaaagaaggtataaagcaacaaaaataaaaaagt
aaattaagaaaataacatgtagtgctaaagtgattgaaaacaattaactttagcatctttttaata
ttaatattttcaaaatacacaagccaaaagaagttctctagctatatagtggagtttagtgtacat
tctatgtaattatattggtaaatgttttcagaaatgtattgaaatccatttaagatggtagtatac
taaatagggaaaccggtttacctaatgtaatacatttaaagaaataaaggaatgcgaagacgacga
tatttttgatttttatgtgcttattttgatgaaattatgacaaggtaattttataaaatcatttat
tagtttttcataattatgtgaatgactaataaaaatatataatagtgggggaaagttatgtttaga
agaaaatttaacaaggtaatattatctatcttagttgcaacaattgtttcaagcactaacatgttt
atgagtggaagcaaggcacaagcggcaattggaaatctaagtgaaaacgatactatttatcaaatt
atggtagacagattttatgatggagataaaacaaataatgctacaggagatgcatttcgtaataca
gaaaatcttgaagatgattttagatatatgcacggcggagattggcaaggtgttattgataagtta
gattatattaagggcatgggatactcagccatttggatatcaccggttgcggaaccacaaatgtgg
tctagagctgatggcacaggaaaagtatggcctacagcttatcatggatataatgtgaaagatccc
aataaggcaaatccttattttggaacaaaagaaaagctaaaggagttagtagataaagctcacgaa
aaggggattaaagtaataatagatatagttccaaatcatgttggggattatatgttaggaaaacaa
gcttattatgacatcaaggggtttgagccggcagcaccttttaataatccaaattggtatcatcat
aatggcgatattgattggtcaagagaacactctgatccccaaatgttagatgatcatgatttgggc
ggtttagatgatttaaatcaagataattctgatgctaaggcagctatgaataatgctattaagtca
tggtttgattatactggagctgatgcagcaagggttgacgcagcaaaatgtatgaaaccatcttat
attaacgagttacaaaagtatataggagttaatacttttggagaaaattttgatatgaatgtagat
tttgtgaagaagtgggttggatccgatgcagaatggggaatgctagattttccattatatcaagca
ataaataatgattttgcatcaggacaatcttttgatgacatgtcatcatcaggtacttgctctatt
aaaaatattttagcacaagacaataaatataatggttatgcaaatcatatggtgacttttatagat
aatcatgatcgtaatagatttttaacagtagcaaatggtaatgtaaaaaaacttcaaaatgcactt
gttttcatgtttactgtaagaggggtaccaacagtatttcaaggtacagaacaaaacaaaggtaat
gcaaatggagcaagtataaatggtattgcagatacatggaatcgttggtcaatggttaaaaaggat
tacaatggaaatgtaattacagattattttaatgagaatacagatacttataaactaattaacaaa
ttgaattcatttaggcaaaaatatgaagccttaagagaaggtactcaaagagaaatgtggtcttca
ccacatttatatgcattctcaagaaggatggattcaggagaaaatgttggacaagaagttgtaaat
gtatttaataattcagatggagatcaaagtgcgaccattccaattagagctgaaagtactataaaa
gttggagataaatttgtaaatctttttgatgtaaatgattcgatcacagttcaacaaggaggtgtt
acaggaaaacaaatatcagtgaatttaggagaaaatagtgggaagatttatgttgttaataatgaa
acaccaaatccagatcaaaagaacgtacaatataaagtttcatataagaatactaatgcacaaaaa
gtaacacttcattatggaactaatggatggaaaaacattcaagatgtaaatatgactaagaattcc
aatggagaatttgaagcaactattacagtaaataataatgatattctaaattactgtattcatatt
atttcaccaacagactattgggataataatggtggacagaattggaatgtaaaagtgactaaggca
gaagattatataaatgatggtgtaaagagtaatttgaagagcgttaatacaactacatcagcagct
atagactctgggattgatagtactgtaaatcgttaaatataaatgttaatttaaagaaaaatttca
tcatgcatattatatttggcacacaaaaatattaaatatctacttttcgcttctaaatggaaaaac
cgcatggttagatcctaaagccttataaaatccatgtttccataattgaagcgaaatataggtaga
taataatgtataaattaggaggaataattgatgaaaggtgaaataatatatcaaatttttccagac
agatttaataaatcaagacaaaataataatgttgaaggtttaaaagaatgggaaagtgaagttgat
ggacaatgtgttatgggaggtgatttaattggaattaaagagaaacttgattatctatcaaaactc
ggtgttagtgcaatttatttaaatccaatttttcaggcaaattctaatcataagtatgatactgtt
aactattataatatagatagttcttttggaactttagatgattttagagaattagtagattcatgt
cataaaaaaaatataaaagttattattgatggagtttttaaccatactagcccagatttttttgct
ttcaaagatatattagaaaatcaagaaagatcaaaatataaggattggtatactatttttagttat
ccagttaaagtggaaagtccacctaattatagaaattttggaggatgtatagatatgccgcgtctt
aatactgaaaatgttgaagttcaaaagtatatagttgatgttattaagtattgggaagggatgaaa
atagatggattaagactagatgtaccatattatattgaagactctatgttagaaaaaataagaaaa
tctactagcttatatatagtaggtgaaatatgggggtgtggcaagaaatttgtgcctcaatatttt
gatggagtaatgaattattcatttagagatttagtgcaaaaagcagttataagacaaagcattgat
gcatcaatattcatagatgaatggaatttcatagaagaaacatacgggcagaatatacattgctgc
tttaatatgtctggaagtcatgatacagaaaggatttttaatttctgcagaggagatataaagaga
gaaaaattattctatgcatttttatttttattcccaggaatgcctcttgtatattatggagatgaa
ataggtatgaaaggagaaaatgacccttattgtagaggaactatggaatggaatgaaagtaaatgg
aattatgatatatataatcatgtaaaaggtttaatagaacttagaaatagtagtgaagcattgcaa
aaagggactatacaatttgttggacataaagaaatgatgtttgcatttgaaagagtgtatgcagaa
aaaagagttaaagtatttatgaattttggacatagcaaacagtctattgatggatttgaactagat
ggtcttagttataaagttatagtttagcattcaaggataactttgcaagttataaaatagcaactt
taaacaatcaatgttctttaattggacattggttatcacaatatgtttatctgtttggataaatat
atgaataaatttcattaattttatttttttccatgataaaaatcatagagaaaaaggcatatattt
aaatttggctttattagtaaattcaattagtataatatattttagtgatattgacataagagatta
aataaattatttatataaaaaaagtaagattaagaaatactagatttaaatttttttatatcaaag
aggtgggcaattatgaaacaagccaaaacaaaaaaaataacacatactttgaaatcagtgccgtat
ttattaccagccattatttcaataattatattttcaatattaccaatacttaatacaatatatttg
gcatttacagactatactatgtattcacaaggaaaaattaattttgtaggaattgcaaattttaaa
gaagtatttgctggtccatttaaagaagtattttttccggtatttatatggacatgtgtctttgct
acattggcaactgcaggaacatttttgttaggactaattatggcaattcttgtaaataatgaaaat
ataaaagaacgagggctttataaagcaattttaattattccatgggcattaccagctactgttgca
atactttcatggcaaggtttattaaatggaagttatggggcaattaataatttacttataagtgta
catgctatttcagcgcctattccatggttaactaatccattatgggcaagaattgcaataatcata
gtaactatatggctaggatttccatatgccatgaatatttgtttgggttcacttcaatcgatacct
aaaacatattatgaagcagctgacgttgatggagccagcaagtttgtaaaatttattaaaataact
ttaccttcgcttgcacaaacagcatatccattagttatttcatcctttgcatttaactttaataat
tttggtcaagcatatttaattactaatggtaatccggcaagacctggaacacaatttgcaggtttc
acagatatattggcttcagtaaattataaattgtcaataacatttggaagatatgaaattgcttcc
actataagcattattatatttataattttagctacaatttcatacatacaaatgaaagcatcagga
caatttgaggaggttgattaaaatgacatcaaatgcagggaatttgaaattaaataatacagaagg
acaaagtgaagaaatacaaaacataaaattaaaatatgtaaaaaaattaagaccagcagaaataag
aactgcatggatttcaaggatagtactttggattatgattgtaatagttcttattccaatcatggc
agttgtttcagcatctatggctaaaggtaattcatttacgcaaacctctatttttcctaaatcatt
tactttagagaattatgtaaaagtaataactcaaactaagtttttaatatgggcaagaaattcatt
agttgtttgttttagcgttgctatgatgcagctaatcatgacaattccagcagcttttgcgttttc
taagcttaggtttaaaggtagaaaatttggacttatgacacttttgatattacagatgtttccaaa
tacaatggcattaccagcaattttaagtgttgcatataatattcggggtggaatggataatttatt
accattaatattaattatatcagtaggtagtgcatataacatctggcttatgaagggatacatgga
tggaattccaaaagaattaactgaaactgcatatatagatggagcaacaacttttcaagctttcat
taaggtagtattgccactaataaagaatatgataatagtaatatttatatttgcttttgttggagc
ttatagcgaatttttatttacatcagctcttataaaagatcaatatacagaaactctagcaacagg
tatgcaaggattcattaaagatcatttttcagctaactggactcaatattcagcggctgcaataat
ggcatcattaccagttgttttgatatcagtattttcacaaaaattctttgcaaaaggattaactgc
tggatcagtaaaaggctaaagtggggacgtgagtatataatgaagccaactaaggattagaaaata
agtagcaattttaataaaattaaaaagtttttctataaaaatctttcaactaaagaaaattcaaaa
aacaatgatgtaactattaaaaaatctaaaagtttgcattttaaattattggaaagtatgttctgt
atttcaattattccaataataattatttgcagcgttacttttattaaaattaattttattaagtaa
tgttaattcataagctgaagatgtgaattcagaatcattgattttaaatgataaaagtaaggaatt
atcgttagctatatctaatgtaaatgagactttaggcaagattgatcttggaactatagattctac
aaataatttggaatctttagtgttaaatatggaggaggtatcaaatccaatgatcaaagtagcgta
atagttatatataagtgaaaaggggaaggaattcttatgagattcgaagctgtatatcatagagcc
tcagataacttatgttattcaattgataaagacaatttaattgtaaatataaagactggttatgat
gtagaaaaagtgtttatatattatggtgatccatttgatggaggaattttaggtggcgaatggaaa
tggaaaggaaaaagagaagaaattccatttaaaaagagattaaagcaccaaatatggtggacaact
actttgaaactgaagtataaaagatgtaaatactattttgaattaacggggaatgaagaaacctgg
ttttattttgaagattgttttttaagtgaaaaacaaatgcaattggacggaaaaatgttgcaatgt
tttacatttccatggatgaatgaagctgatataaataaaacaccagcatgggtaaatgatatggtt
tggtatcagatattcccagagcgtttttgtaatggaaatccttcaattaatcccaaaggggtccag
ccttggcataaaggaggcgttacaaatgaagagttttatggtggggatttgcaggggataataaat
aaattaaattatttaaaagaaataggaattacaggcatatatttaaatccaatattcgaatctccg
tcagcacataaatatgatacaactgattatatgaaaatagatcctaattttggagatgaaaatgta
tttagaaagcttgtaaataaagcacatgaaaaagggattaggattatgcttgatggagtgtttaat
cattgtggagctaagtttggaccatggttagatgtacttgaaaatggtcctagttccaaatattat
agttggtttatggtaaataagtggccttttgatgataataatcacgatacaaaggatggacgattt
tattcttttgcctttaatcagaaaatgccaaaattaaatacaaataatccagaagtaattgattat
ttaattaaggtatgtgagtattgggtaaaaaattataagattgatggattaagattagatgttgca
aatgaaatttcgcataagttctgtaagaagcttagagaaaaaatgaaatcgttaaatccagacttc
tatattttaggtgaaatatggcatgattctattccttggcttagaggtgatgaatttgatgctatt
atgaattattcactaactagtagtatatcagacttctggatagataagagtttaactaaggatgat
tttgagtacacaataaatagatgctatacaatatatatgcagcaaaataatgatgtgttatttaat
ttgttggattctcatgatacagaacgcttaatttcaagagtaaaagacattaacgtattttatcaa
cagctagctgtactatttacgatgccaggaagtccatgcatattttatggtacagaagttgcactt
gaaggaaagtatgatcccgattgccgaagatgtatgccatgggatgaaataaaaagtggaatttat
gatgataagattaatataatgaaggcgttgattaatttaagaaaagagcaaaaattatttagaagc
cgtaattttcattttccaaatacaattaaaaatagcagggtaatagaatatattaaaatagatgaa
aatgggaataggttagaaattttactaaattgttcaaatatagatgttttaatagagaataatggt
agtgttttgtttagtaatttatattctaataatagactgcttaaaaaaggtgtattaattaggaag
gttgattctatataaggttcaatcaaataaataacaagtccgtttacatcatgggctgataaaaaa
tatccattttgcgatttgattttatttataaatgaaaaagatatttaattaaatcagcaatatgta
cttattaattataaatgaaaaaataattttgaagaggagtagtcatggaattaacatataggttcg
gaagaggatactggagaaatataaaggaaggaaacgagagagaatggatgataggcaatggtattg
gcgggtatagcagtcaaactatcattaatagtggatttagatgtcataatgggtatttaatagcag
caatgaatccgccagtagaacgttattcaatattatatagaactcaggaaaaaatcgtcacagatg
gaagaacatatgatttgacttgtcaggaatataaggattatacgaagaatggttatgagtatctca
aaagttttatatttgattcagtgcctcaatatatttatcaaatagaagatataaatgtaaaaaaaa
ctatagctatggaatatggatataatactgtagctatatgttatgagattgaaaatggaagttcta
aggctaaaattgatattacaccactgtttaattttaaggaagctggtacatttaaggcttctgagc
agctggattttaaaactgaattacaagacgatatattaaaattgtatcctaatgaagatgataaga
agataataagttttatgtcatcagcaggcatatttaaggacagaagtcttataaaagtacagaatg
attttaattataatccattaattgaagagaatcattactatgaatttgaaaatagaaatggattta
ttgggttaaataatcattatacgccatatgatattgaaattgaattagagccttttgaaactaaaa
agttttatttaaaatgtacagtagaagagttaggtgataaagacggatttgatattgttaaagaat
ataaggaaagaacaaatgaattattgaatagatcaggctataaagatttttttgcattaaatttag
taaaagcagctgaccattttattgtagatagaaaaagtactggattgaaaacaatacttgcaggat
ttccttggtttgttgattgggggagagacactatgatagcttttgaaggtttaacgctgtgtacaa
agagatttgaggatgcaagagaaatattaaagtcttttgcagaatatataaaagatggacttgttc
caaatgtttttgcggataaaggaacacaagcgttttacaatactgcagatgcatcattatggtata
tacaagctgtatataagtatttaaaatatactggaaagaaaagtgattttaagtttgttaatgata
aattattcgacaagttaattgaaattattgatgcttattcaaatggcacacatttttcaataggta
tggatgatgattgtcttattcatgctggcagcggattggatcaagtaacgtggatggatgtaagag
tagacgaaatggttgttactccaaggcatggtaaaccagtagaaataaatgctctttggtataatg
ccctttgcataatggattggttatgtagaaagtatgaaatgaatggatcaaaatatgaaagtttag
cgagaaaagttaaaaactcctttaacaaaaaattctggaatgaaaaagaacagtgtttatttgatg
ttgttgatgattatgatgggaaagttaggccaaatcaaatatgggcagtatcattgccatttacta
tgttagaaaaagaaaaggaagcgaaagttgtgaataaagtatataaagaattatattcgacttatg
gattgagatcgctgtcatacttagataaagattttaagagcgaatatataggaccacttatgaaaa
gggatttagcatatcatatggggacaacatgggcattcttaatagggagctttatatcagcatatt
gtaaggtaaataatcactctaaagaagcagtaagtagagcaaaagaaatgtgtgaagtatttcagg
atcatatgaaagatggatgcataaatggaatagctgaagtatttgatggaaaattttcagctacag
gcaggggatgctatagtcaagcctggagtgtaggcgaagttttaagagcatatactaacgatgtac
tgccatttatttgatctactttgca

In the above sequence, the coding regions have been underlined and the start and stop codons have been highlighted.