Gluconobacter suboxydans sorbitol dehydrogenase, genes and methods of use thereof

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to the fields of molecular biology, bacteriology and industrial fermentation. More specifically, the invention relates to the identification and isolation of nucleic acid sequences and proteins for subunits of a novel, membrane bound sorbitol dehydrogenase in

Gluconobacter suboxydans

. The invention further relates to the fermentative production of L-sorbose from D-sorbitol and the subsequent production of 2-keto-L-gulonic acid.

2. Related Art

Sorbitol dehydrogenase (SDH) is an enzyme responsible for the efficient conversion of D-sorbitol into L-sorbose during sorbose fermentation in the process of the manufacturing of 2-keto-L-gulonic acid (2-KLG), an important precursor for vitamin C synthesis. Gluconobacter possesses several SDHs, which may be categorized according to their cofactor requirement: (1) NAD-dependent, (2) NADP-dependent and (3) NAD(P)-independent types. Among them, NAD(P)-independent enzyme is believed to be directly involved in efficient production of sorbose during industrial sorbose fermentation (Cummins. J. T. et al.,

J Biol. Chem

., 224, 323; 226, 3 01 (1957)).

The process of manufacturing L-sorbose from D-sorbitol is typically performed by fermentation with an acetic acid bacterium such as

Gluconobacter suboxydans

and

Acetobacter xylinum

. At room temperature, 96-99% of conversion is made in less than 24 hours (Liebster, J. et al.,

Chem. List

., 50:395 (1956)).

L-sorbose produced by the action of SDH is a substrate in the production of 2-keto-L-gulonic acid (2-KLG). A variety of processes for the production of 2KLG are known. For example, the fermentative production of 2-KLG via oxidation of L-sorbose to 2-KLG via a sorbosone intermediate is described for processes utilizing a wide range of bacteria:

Gluconobacter oxydans

(U.S. Pat. Nos. 4,935,359; 4,960,695; 5,312,741; and 5,541,108);

Pseudogluconobacter saccharoketogenes

(U.S. Pat. No. 4,877,735; European Pat. No. 221 707);

Pseudomonas sorbosoxidans

(U.S. Pat. Nos. 4,933,289 and 4,892,823); mixtures of microorganisms from these and other genera, such as Acetobacter, Bacillus, Serratia, Mycobacterium, and Streptomyces (U.S. Pat. Nos. 3,912,592; 3,907,639; and 3,234,105); and novel bacterial strains (U.S. Pat. No. 5,834,231).

A number of enzymes involved in the fermentative oxidation of sorbitol, sorbose and sorbosone are identified in the literature. U.S. Pat. Nos. 5,888,786; 5,861,292; 5,834,263 and 5,753,481 disclose nucleic acid molecules encoding and/or isolated proteins for L-sorbose dehydrogenase and L-sorbosone dehydrogenase; and U.S. Pat. No. 5,747,301 discloses an enzyme with specificity for sorbitol dehydrogenase.

In addition to distinguishing Gluconobacter SDH's on the basis of cofactor requirements, other physical characteristics may be found in the literature that distinguish these different enzymes. For example, the sorbitol dehydrogenase identified in U.S. Pat. No. 5,747,301 is distinguished on the basis of subcellular location (membrane-bound) and a haloenzyme molecular weight of 800±50 kDa (10 homologous subunits of 79±5 kDa). The membrane-bound D-sorbitol dehydrogenase isolated by Shinagawa et al.(E. Shinagawa, K. Matsushita, 0. Adachi and M. Ameyama (Agric. Biol. Chem., 46, 135-141, 1982)) consisted of three kinds of subunits with molecular weights of 63,000, 51,000 and 17,000.

In an effort to improve the productivity of commercial fermentation in the production of 2 KLG, the inventors have identified a novel, membrane-bound sorbital dehydrogenase in a strain of G. suboxydans that is distinct from others described in the literature (Choi, E. S. et al.,

FEMS Microbiol

. Lett., 125:45 (1995)).

SUMMARY OF THE INVENTION

This invention pertains to a novel, membrane-bound sorbitol dehydrogenase of

Gluconobacter suboxydans

. The isolated sorbitol dehydrogenase enzyme comprises three subunits: a first subunit of 75 kDa containing pyrroloquinoline quinone (PQQ) as cofactor; a second subunit of 50 kDa being a cytochrome c; and a third subunit of 29 kDa playing a very important role in the stability and the catalytic activity of the enzyme.

The present invention provides nucleic acid molecules for the 3 protein subunits of the Gluconobacter sorbitol dehydrogenase described herein. In a first specific embodiment, the invention provides an isolated nucleic acid molecule drawn to the first SDH subunit (75 kDA) identified by SEQ ID NO:1. In a second specific embodiment, the invention provides an isolated nucleic acid molecule drawn to the second SDH subunit (50 kDA) identified by SEQ ID NO:2. In a third specific embodiment the invention provides an isolated nucleic acid molecule drawn to the third SDH subunit (29 kDA) identified by SEQ ID NO:3. Other related embodiments are drawn to vectors, processes for producing the same and host cells carrying said vectors.

The invention also provides isolated nucleic acid molecules encoding the three subunits of the SDH of the invention. In one specific embodiment, the invention provides a cloned nucleic acid molecule encoding the 75 kDa and 50 kDa subunits. The structural genes for the first and the second subunit of sorbitol dehydrogenase are 2,265 bp and 1,437 bp, respectively, in size and are clustered in the cloned nucleic acid molecule which is a 5.7 kb Pstl DNA fragment that defines the operon. In another specific embodiment, the invention provides a cloned nucleic acid molecule encoding the third, 29 kDa, SDH subunit protein. The structural gene coding for the third subunit is 921 bp in size and found in a 4.5 kb Clal DNA fragment. Other related embodiments are drawn to vectors, processes to make the same and host cells containing said vectors.

The invention is also drawn to isolated polypeptides for the three subunits of the SDH described herein.

The invention also provides a method for the production of D-sorbose comprising: (a) transforming a host cell with at least one isolated nucleotide sequence selected from the group consisting of a polynucleotide comprising the polynucleotide sequence of SEQ ID NO:1; a polynucleotide comprising the polynucleotide sequence of SEQ ID NO:2; and a polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 3; and (b) selecting and propagating said transformed host cell.

Another aspect of the invention is drawn to a method for production of 2-KLG comprising: (a) transforming a host cell with at least one isolated nucleotide sequence selected from the group consisting of a polynucleotide comprising the polynucleotide sequence of SEQ ID NO:1; a polynucleotide comprising the polynucleotide sequence of SEQ ID NO:2; and a polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 3; and (b) selecting and propagating said transformed host cell.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1

presents DEAE-TSK column chromatography using a sodium acetate buffer of pH 5.0 (A) and pH 6.0 (B).

FIG. 2

presents SDS-PAGE analysis of peak I (A) and peak II fractions (B) separated by DEAE-TSK column chromatography.

FIG. 3

presents DEAE-TSK column chromatography using a sodium phosphate buffer of pH 6.5.

FIG. 4

presents SDS-PAGE analysis of column fractions of peak I (lane 1), peak II (lane 3) and peak III (lane 2) separated by DEAE-TSK column chromatography at pH 6.5. Lane M denotes molecular weight standard markers.

FIG. 5

presents an HPLC chromatogram of a tryptic digest of peak II protein from DEAE-TSK column chromatography at pH 6.5. The dotted line indicates the concentration gradient of acetonitrile in the mobile phase.

FIG. 6

presents a restriction enzyme map of the Lambda GEM 5- 1. The 1.53 kb DNA fragment (#SDH 2-1) used as probe is shown as solid bar.

FIG. 7

presents the locations of SI, S2 and S3 DNA fragments generated with different sets of restriction enzymes from 5.7 kb Pstl fragment of Lambda GEM 5-1.

FIGS. 8A-8G

presents the nucleotide sequence of 4,830 bases (SEQ ID NO:7) of the 5.7 kb PstI fragment. The deduced amino acid sequence for the first and the second subunit is shown below the nucleotide sequence. The N-terminal amino acid sequence obtained by the N-terminal amino acid sequencing of the purified sorbitol dehydrogenase is underlined. Signal sequence cleavage site is marked as a triangle. The heme-binding sequences are underlined with dotted lines. Potential ribosome-binding sequences (SD) are enclosed in boxes. The transcription termination stem-and-loop structure is indicated by arrows. The complete coding sequence for the first subunit gene is located at position 665-2,929 (SEQ ID NO:1), with the signal sequence located at position 665-766, and the coding sequence for the mature protein of the SDH first subunit located at position 767-2,929 (SEQ ID NO:22). The complete coding sequence for the second subunit gene is located at position 2,964-4,400 (SEQ ID NO:2), with the signal sequence located at position 2,964-3,071, and the coding sequence for the mature protein of the SDH second subunit located at position 3,072-4,400 (SEQ ID NO:23).

FIG. 9

presents a restriction enzyme map of ClaI-#69. The closed box represents the coding region of the third subunit gene of sorbitol dehydrogenase.

FIGS. 10A-10D

presents the nucleotide sequence (SEQ ID NO:8) of DNA fragment containing the third subunit gene of sorbitol dehydrogenase. The deduced amino acid sequence is shown below the nucleotide sequence. Signal sequence cleavage site is marked as a triangle. Potential ribosome-binding sequence (SD) is enclosed in box. The complete coding sequence for the third subunit gene is located at position 1,384-2,304 (SEQ ID NO:3), with the signal sequence located at position 1,384-1,461, and the coding sequence for the mature protein of the SDH third subunit located at position 1,462-2,304 (SEQ ID NO:24).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

1. Definitions

Cloning Vector: A plasmid or phage DNA or other DNA sequence which is able to replicate autonomously in a host cell, and which is characterized by one or a small number of restriction endonuclease recognition sites at which such DNA sequences may be cut in a determinable fashion without loss of an essential biological function of the vector, and into which a DNA fragment may be spliced in order to bring about its replication and cloning. The cloning vector may further contain a marker suitable for use in the identification of cells transformed with the cloning vector. Markers, for example, provide tetracycline resistance or ampicillin resistance.

Expression: Expression is the process by which a polypeptide is produced from a structural gene. The process involves transcription of the gene into mRNA and the translation of such mRNA into polypeptide(s).

Expression Vector: A vector similar to a cloning vector but which is capable of enhancing the expression of a gene which has been cloned into it, after transformation into a host. The cloned gene is usually placed under the control of (i.e., operably linked to) certain control sequences such as promoter sequences. Promoter sequences may be either constitutive or inducible.

Gene: A DNA sequence that contains information needed for expressing a polypeptide or protein.

Host: Any prokaryotic or eukaryotic cell that is the recipient of a replicable expression vector or cloning vector. A “host,” as the term is used herein, also includes prokaryotic or eukaryotic cells that can be genetically engineered by well known techniques to contain desired gene(s) on its chromosome or genome. For examples of such hosts, see Sambrook et al.,

Molecular Cloning: A Laboratory Manual

, Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989).

Homologous/Nonhomologous: Two nucleic acid molecules are considered to be “homologous” if their nucleotide sequences share a similarity of greater than 50%, as determined by HASH-coding algorithms (Wilber, W. J. and Lipman, D. J.,

Proc. Natl. Acad. Sci

. 80:726-730 (1983)). Two nucleic acid molecules are considered to be “nonhomologous” if their nucleotide sequences share a similarity of less than 50%.

Mutation: As used herein, the term refers to a single base pair change, insertion or deletion in the nucleotide sequence of interest.

Mutagenesis: As used herein, the term refers to a process whereby a mutation is generated in DNA. With “random” mutatgenesis, the exact site of mutation is not predictable, occurring anywhere in the chromosome of the microorganism, and the mutation is brought about as a result of physical damage caused by agents such as radiation or chemical treatment.

Operon: As used herein, the term refers to a unit of bacterial gene expression and regulation, including the structural genes and regulatory elements in DNA.

Parental Strain: As used herein, the term refers to a strain of microorganism subjected to some form of mutagenesis to yield the microorganism of the invention.

Phenotype: As used herein, the term refers to observable physical characteristics dependent upon the genetic constitution of a microorganism.

Promoter: A DNA sequence generally described as the 5′ region of a gene, located proximal to the start codon. The transcription of an adjacent gene(s) is initiated at the promoter region. If a promoter is an inducible promoter, then the rate of transcription increases in response to an inducing agent. In contrast, the rate of transcription is not regulated by an inducing agent if the promoter is a constitutive promoter.

Recombinant Host: According to the invention, a recombinant host may be any prokaryotic or eukaryotic cell which contains the desired cloned genes on an expression vector or cloning vector. This term is also meant to include those prokaryotic or eukaryotic cells that have been genetically engineered to contain the desired gene(s) in the chromosome or genome of that organism.

Recombinant Vector: Any cloning vector or expression vector which contains the desired cloned gene(s).

2. Isolation and Purification of Sorbitol Dehydrogenase

The present invention isolates and purifies SDH from the cytoplasmic membrane of

G. suboxydans

KCTC (Korea Culture Type Collection) 2111 (equivalent to ATCC 621) using a series of column chromatographic steps. Biochemical properties of the purified enzyme are provided, as well as the isolation of each subunit and a determination of the N-terminal amino acid sequence of each subunit using an amino acid sequence analyzer (Applied Biosystems, 477A).

The newly characterized enzyme is different from the reported FAD-dependent SDH from

G. suboxydans

IFO 3254 strain (Shinagawa, E. et al.,

Agric. Biol. Chem

., 46:135 (1982)), containing pyrroloquinoline quinone (PQQ) as a cofactor and comprising three subunits (Choi, E. S. etal.,

FEMS Microbiol. Lett

., 125:45 (1995)).

The SDH of the invention may be isolated using standard protein techniques. Briefly,

G. suboxydans

KCTC 2111 is cultured in SYP medium (5% D-sorbitol, 1% Bacto-Peptone, 0.5% yeast extract) and the cells are lysed in a 10 mM sodium acetate buffer solution (pH 5.0). After centrifuged at 12,000 g to remove cell debris, the supernatant is centrifuged in an ultracentrifuge to recover cytoplasmic membrane fraction. Purification is completed by solubilizing the cytoplasmic membrane fraction with 1.5% n-octylglucoside (Boehringer Mannheim) and passage over a series of chromatographic columns, including CM-TSK 650 (S) (Merck), DEAE-TSK 650 (S) (Merck), Mono-S (Pharmacia) and Superose 12 (Pharmacia).

The purified enzyme is active towards polyols such as D-sorbitol (100%), D-mannitol (68%) and D-ribitol (70%). Activity of the enzyme increases up to nine fold when pyrroloquinoline quinone (PQQ) is added, suggesting that PQQ is a cofactor for the enzyme; fluorescence spectrum analysis confirmed that the purified enzyme contains pyrroloquinoline quinone (PQQ). The absorption spectrum analysis of the purified enzyme demonstrates that this enzyme contains cytochrome c. When the purified enzyme is subjected to polyacrylamide gel electrophoresis (PAGE) at pH 4.3 (Reisfeld, R. A. et al.,

Nature

, 195:281 (1962)), it forms a single activity band after activity staining on the gel.

The initial SDS-PAGE analysis of the purified enzyme showed that the enzyme comprised three subunits of 75 kDa, 50 kDa and 14 kDa which were named the first subunit, the second subunit, and the third subunit, respectively (Choi, E. S. et al.,

FEMS Microbiol. Lett

., 125:45 (1995)). In a further study of the enzyme, however, it was discovered that another subunit of 29 kDa played a very important role in the stability and the catalytic activity of SDH. That is, while investigating a variety of pH conditions in an effort to increase protein separation capability during purification on DEAE-TSK column, it was discovered that the partially resolved activity peaks eluting at pH 5.0 were completely resolved into two separate activity peaks when eluted at pH 6.0. The early eluting activity peak quickly lost enzyme activity and the late eluting activity peak remained stable. By SDS-PAGE analysis of these two peaks, it was found that the peak with stable enzyme activity contained an additional subunit of 29 kDa in addition to the 75 and 50 kDa subunits, whereas the one that quickly lost enzyme activity contained only 75 and 50 kDa subunits. This additional subunit of 29 kDa was renamed the third subunit: it is uncertain whether the 14 kDa subunit previously assigned the third subunit is a true subunit of the enzyme when comparing the relative amount with other subunits on the acrylamide gel. It was also found that a further increase of the pH ofthe elution buffer to pH 6.5 resulted in a complete separation of three subunits into individual subunits.

When different combinations of two or three subunits were tested for the restoration of enzyme activity by the Ferric-Dupanol assay method (Wood, W. A. et al.,

Meth. Enzymol

, 5:287 (1962)), it was found that enzyme activity was fully restored only in the presence of the third subunit of 29 kDa. Therefore, it was concluded that the third subunit of 29 kDa plays important roles in the stability and the catalytic activity of SDH.

The Michaelis-Menten constants, when using D-sorbitol as substrate, was determined to be Km=120 mM and Vmax=3.9×10

−5

M/min. Dichlorophenol indophenol (DCIP) or ferricyanide worked effectively as electron acceptor of the enzyme. When phenazine methosulfate (PMS) was added as an electron mediator, the enzyme activity increased. Calcium or magnesium ion addition significantly increased purified enzyme activity, whereas copper ion addition seriously inhibited activity.

Further details ofthe purification and characterization ofthe SDH enzyme of the invention are provided in Example 1.

3. Nucleic Acid Molecules of the Invention

The invention provides isolated nucleic acid molecules encoding one or more of the three subunits of the SDH enzyme described herein. Methods and techniques designed for the manipulation of isolated nucleic acid molecules are well known in the art. For example, methods for the isolation, purification and cloning of nucleic acid molecules, as well as methods and techniques describing the use of eukaryotic and prokaryotic host cells and nucleic acid and protein expression therein, are described by Sambrook, et al.,

Molecular Cloning: A Laboratory Manual

, Second Edition, Cold Spring Harbor, N.Y., 1989, and Current Protocols in Molecular Biology, Frederick M. Ausubel et al. Eds., John Wiley & Sons, Inc., 1987, the disclosure of which is hereby incorporated by reference.

More particularly, the invention provides several isolated nucleic acid molecules encoding the individual 75 kDa, 50 kDa, and 29 kDa subunit proteins of SDH enzyme of the invention. Additionally, the invention provides several isolated nucleic acid molecules encoding one or more of subunit proteins of the SDH enzyme of the invention. For the purposes of clarity, the particular isolated nucleic molecules ofthe invention are described. Thereafter, specific properties and characteristics of these isolated nucleic acid molecules are described in more detail.

Unless otherwise indicated, all nucleotide sequences determined by sequencing a DNA molecule herein were determined using an automated DNA sequencer (such as the Model 373A from Applied Biosystems, Inc.), and all amino acid sequences of polypeptides encoded by DNA molecules determined herein were predicted by translation of a DNA sequence determined as above. Therefore, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors. Nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule. The actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well known in the art. As is also known in the art, a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the predicted amino acid sequence encoded by a determined nucleotide sequence will be completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, beginning at the point of such an insertion or deletion.

In one embodiment, the invention provides an isolated nucleic acid molecule for the first (75 kDa) subunit of the SDH enzyme of the invention comprising a polynucleotide sequence selected from the group consisting of (a) the polynucleotide of SEQ ID NO:1; (b) a polynucleotide fragment at least about 20 nucleotides in length of the polynucleotide of SEQ ID NO:1; (c) a polynucleotide encoding the amino acid sequence of SEQ ID NO:4; and (d) a polynucleotide encoding a fragment at least about 10 amino acids in length of the amino acid sequence of SEQ ID NO:4.

In another embodiment, the invention provides an isolated nucleic acid molecule for the first (75 kDa) subunit of the SDH enzyme of the invention comprising a polynucleotide at least about 95% identical to the isolated nucleic acid sequence for the first (75 kDa) subunit of the SDH enzyme of the invention described above.

Another embodiment of the invention provides an isolated nucleic acid molecule for the second (50 kDa) subunit of the SDH enzyme of the invention comprising a polynucleotide sequence selected from the group consisting of: (a) the polynucleotide, or fragment thereof, of SEQ ID NO:2; (b) a polynucleotide fragment at least about 20 nucleotides in length of the polynucleotide of SEQ ID NO:2; (c) a polynucleotide encoding the amino acid sequence of SEQ ID NO:5; and (d) a polynucleotide encoding a fragment at least about 10 amino acids in length of the amino acid sequence of SEQ ID NO:5.

In another embodiment, the invention provides an isolated nucleic acid molecule for the second (50 kDa) subunit of the SDH enzyme of the invention comprising a polynucleotide at least about 95% identical to the isolated nucleic acid sequence for the second (50 kDa) subunit of the SDH enzyme of the invention described above.

Another embodiment of the invention provides an isolated nucleic acid molecule for the third (29 kDa) subunit of the SDH enzyme of the invention comprising a polynucleotide sequence selected from the group consisting of: (a) the polynucleotide of SEQ ID NO:3; (b)a polynucleotide fragment at least about 20 nucleotides in length of the polynucleotide of SEQ ID NO:3; (c) a polynucleotide encoding the amino acid sequence of SEQ ID NO:6; and (d) a polynucleotide encoding a fragment at least about 10 amino acids in length of the amino acid sequence of SEQ ID NO:6.

In another embodiment, the invention provides an isolated nucleic acid molecule for the third (29 kDa) subunit of the SDH enzyme of the invention comprising a polynucleotide at least about 95% identical to the isolated nucleic acid sequence for the third (29 kDa) subunit of the SDH enzyme of the invention described above.

Another embodiment the invention provides an isolated nucleic acid molecule encoding both the first (75 kDa) and second (50 kDa) subunit proteins of the SDH enzyme of the invention comprising a polynucleotide sequence selected from the group consisting of: (a) the polynucleotide of SEQ ID NO:7; and (b) a polynucleotide fragment at least about 20 nucleotides in length of the polynucleotide of SEQ ID NO:7.

In another embodiment, the invention provides an isolated nucleic acid molecule for the first (75 kDa) and second (50 kDa) subunit proteins of the SDH enzyme ofthe invention comprising a polynucleotide at least about 95% identical to the isolated nucleic acid sequence for the first (75 kDa) and second (50 kDa) subunit proteins of the SDH enzyme of the invention.

Another embodiment of the invention provides an isolated nucleic acid molecule for the third (29 kDa) subunit of the SDH enzyme of the invention comprising a polynucleotide sequence selected from the group consisting of: (a) the polynucleotide of SEQ ID NO:8; and (b) a polynucleotide fragment at least about 20 nucleotides in length of the polynucleotide of SEQ ID NO:8.

In another embodiment, the invention provides an isolated nucleic acid molecule for the third (29 kDa) subunit of the SDH enzyme of the invention comprising an isolated nucleic acid molecule at least about 95% identical to the isolated nucleic acid molecule for the third (29 kDa) subunit of the SDH enzyme of the invention described above.

By “isolated” nucleic acid molecule is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment. For example, recombinant DNA molecules contained in a vector are considered isolated for the purposes of the present invention. Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules of the present invention. Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.

RNA vectors may also be utilized with the SDH nucleic acid molecules disclosed in the invention. These vectors are based on positive or negative strand RNA viruses that naturally replicate in a wide variety of eukaryotic cells (Bredenbeek, P. J. and Rice, C. M.,

Virology

3:297-310 (1992)). Unlike retroviruses, these viruses lack an intermediate DNA life-cycle phase, existing entirely in RNA form. For example, alpha viruses are used as expression vectors for foreign proteins because they can be utilized in a broad range of host cells and provide a high level of expression; examples of viruses of this type include the Sindbis virus and Semliki Forest virus (Schlesinger, S.,

TIBTECH

11:18-22 (1993); Frolov, I., et al.,

Proc. Natl. Acad. Sci

. (USA) 93:11371-11377 (1996)). As exemplified by Invitrogen's Sinbis expression system, the investigator may conveniently maintain the recombinant molecule in DNA form (pSinrep5 plasmid) in the laboratory, but propagation in RNA form is feasible as well. In the host cell used for expression, the vector containing the gene of interest exists completely in RNA form and may be continuously propagated in that state if desired.

In another embodiment, the invention further provides variant nucleic acid molecules that encode portions, analogs or derivatives ofthe isolated nucleic acid molecules described herein. Variants include those produced by nucleotide substitutions, deletions or additions, which may involve one or more nucleotides. The variants may be altered in coding regions, non-coding regions, or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions.

Variants of the isolated nucleic acid molecules of the invention may occur naturally, such as a natural allelic variant. By an “allelic variant” is intended one of several alternate forms of a gene occupying a given locus on a chromosome of an organism.

Genes II

, Lewin, B., ed., John Wiley & Sons, New York (1985). Non-naturally occurring variants may be produced using art-known mutagenesis techniques.

Isolated nucleic acid molecules of the invention also include polynucleotide sequences that are 95%, 96%, 97%, 98% and 99% identical to the isolated nucleic acid molecules described herein. Computer programs such as the BestFit program (Wisconsin Sequence Analysis Package, Version 10 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711) may be used to determine whether any particular nucleic acid molecule is at least 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequences disclosed herein or the the nucleotides sequences of the deposited clones described herein. BestFit uses the local homology algorithm of Smith and Waterman,

Advances in Applied Mathematics

2: 482-489 (1981), to find the best segment of homology between two sequences.

By way of example, when a computer alignment program such as BestFit is utilized to determine 95% identity to a reference nucleotide sequence, the percentage of identity is calculated over the full length of the reference nucleotide sequence and gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed. Thus, per 100 base pairs analyzed, 95% identity indicates that as many as 5 of 100 nucleotides in the subject sequence may vary from the reference nucleotide sequence.

The invention also encompasses fragments ofthe nucleotide sequences and isolated nucleic acid molecules described herein. In a preferred embodiment the invention provides for fragments that are at least 20 bases in length. The length of such fragments may be easily defined algebraically. For example, SEQ ID NO:1 provides for an isolated nucleotide molecule that is 2, 265 bases in length. A fragment (F1) of SEQ ID NO:1 at least 20 bases in length may be defined as F1=20+X, wherein X is defined to be zero or any whole integer from 1 to 2,245. Similarly, fragments for other isolated nucleic acid molecules described herein may be defined as follows: for SEQ ID NO:2 which is 1,437 bases in length, a fragment (F2) of SEQ ID NO:2 at least 20 bases in length may be defined as F2=20+X, wherein X is defined to be zero or any whole integer from 1 to 1,417; for SEQ ID NO:3 which is 921 bases in length, a fragment (F3) of SEQ ID NO:3 at least 20 bases in length may be defined as F3=20+X, wherein X is defined to be zero or any whole integer from 1 to 901; for SEQ ID NO:7 which is 4,830 bases in length, a fragment (F7) of SEQ ID NO:7 at least 20 bases in length may be defined as F7=20+X, wherein X is defined to be zero or any whole integer from 1 to 4,810; and for SEQ ID NO:8 which is 2,700 bases in length, a fragment (F8) of SEQ ID NO:8 at least 20 bases in length may be defined as F8=20+X, wherein X is defined to be zero or any whole integer from 1 to 2,680. As will be understood by those skilled in the art, the isolated nucleic acid sequence fragments of the invention may single stranded or double stranded molecules.

The invention discloses isolated nucleic acid sequences encoding the three subunit proteins of the SDH enzyme ofthe invention. Computer analysis provides information regarding the open reading frames, putative signal sequence and mature protein forms of each subunit. Genes encoding the first (75 kDa) and second (50 kDa) subunits are completely contained in a 5.7 kb Pst I fragment of the lambda GEM 5-1 clone, which was deposited in bacteria under the accession number KCTC 0593BP on Mar. 25, 1999 and as DNA under accession number KCTC 0597BP on Apr. 2, 1999 with the Korean Collection for Type Cultures (KCTC), Korea esearch Institute of Bioscience and Biotechnology (KRIBB), 52, Oun-Dong, Yusong-Ku, Taejon 305-33, Republic of Korea. The third (29 kDa) subunit gene is contained in a sequence 4.5 kb in length, referred to as Cla I-#69, which was deposited in bacteria under the accession number KCTC 0594BP on Mar. 25, 1999 and as DNA under accession number KCTC 0598BP on Apr. 2, 1999 with the Korean Collection for Type Cultures (KCTC), Korea Research Institute of Bioscience and Biotechnology (KRIBB), 52, Oun-Dong, Yusong-Ku, Taejon 305-33, Republic of Korea.

Thus, the invention provides an isolated nucleic acid molecule (KCTC 0597BP) carried in the novel strain KCTC 0593BP, and the invention also provides an isolated nucleic acid molecule (KCTC 0598BP) carried in the novel strain KCTC 0594BP.

Sequence obtained from the lambda GEM 5-1, 5.7 kb Pst I fragment is presented in FIG. 8 and assigned SEQ IDNO:7. The complete coding sequence for the first subunit gene is located at position 665-2,929,(SEQ ID NO:1), with the signal sequence located at position 665-766, and the coding sequence for the mature protein of the SDH first subunit located at position 767-2,929 (SEQ ID NO:22). The complete coding sequence for the second subunit gene is located at position 2,964-4,400 (SEQ ID NO:2), withthe signal sequence located at position 2,964-3,071, and the coding sequence for the mature protein of the SDH second subunit located at position 3,072-4,400 (SEQ ID NO:23).

Thus, another embodiment ofthe invention provides isolated nucleic acid molecules derived from sequence obtained from the lambda GEM 5-1, 5.7 kb Pst I fragment that is presented in FIG.

8

and identified as SEQ ID NO:7. Such isolated nucleic acid molecules include the following: (1) nucleotides 1-664 of SEQ ID NO:7 identified as SEQ ID NO:28; (2) nucleotides 50-664 of SEQ ID NO:7 identified as SEQ ID NO:29; (3) nucleotides 100-664 of SEQ ID NO:7 identified as SEQ ID NO:30; (4) nucleotides 150-664 of SEQ ID NO:7 identified as SEQ ID NO:31; (5) nucleotides 200-664 of SEQ ID NO:7 identified as SEQ ID NO:32; (6) nucleotides 250-664 ofSEQ ID NO:7 dentified as SEQ ID NO:33; (7) nucleotides 300-664 of SEQ ID NO:7 identified as SEQ ID NO:34; (8) nucleotides 350-664 of SEQ ID NO:7 identified as SEQ ID NO:35; (9) nucleotides 400-664 of SEQ ID NO:7 identified as SEQ ID NO:36; (10) nucleotides 450-664 of SEQ ID NO:7 identified as SEQ ID NO:37; (11) nucleotides 500-664 of SEQ ID NO:7 identified as SEQ ID NO:38; (12) nucleotides 550-664 of SEQ ID NO:7 identified as SEQ ID NO:39; (13) nucleotides 600-664 of SEQ ID NO:7 identified as SEQ ID NO:40; (14) the nucleotide sequence encoding the full length subunit 1 protein of the SDH of the invention from nucleotides 665-2,929 of SEQ ID NO:7 identified as SEQ ID NO:1; (15) the nucleotide sequence encoding the mature form of the subunit 1 protein of the SDH of the invention from nucleotides 767-2,929 of SEQ ID NO:7 identified as SEQ ID NO:22; (16) the nucleotide sequence encoding the full length subunit 2 protein of the SDH of the invention from nucleotides 2,964-4,400 of SEQ ID NO:7 identified as SEQ ID NO:2; (17) the nucleotide sequence encoding the mature form of the subunit 2 protein of the SDH of the invention from nucleotides3,072-4,400 of SEQ ID NO:7 identified as SEQ ID NO:23; (18) nucleotides 2,930-2,963 of SEQ ID NO:7 identified as SEQ ID NO:41; (19) nucleotides 4,401-4,451 of SEQ ID NO:7 identified as SEQ ID NO:42; (20) nucleotides 4,401-4,501 of SEQ ID NO:7 identified as SEQ ID NO:43; (21) nucleotides 4,401-4,551 of SEQ ID NO:7 identified as SEQ ID NO:44; (22) nucleotides 4,401-4,601 of SEQ ID NO:7 identified as SEQ ID NO:45; (23) nucleotides 4,401-4,651 of SEQ ID NO:7 identified as SEQ ID NO:46; (24) nucleotides 4,401-4,701 of SEQ ID NO:7 identified as SEQ ID NO:47; (25) nucleotides 4,401-4,751 of SEQ ID NO:7 identified as SEQ ID NO:48; (26) nucleotides 4,401-4,801 of SEQ ID NO:7 identified as SEQ ID NO:49; and (27) nucleotides 4,401-4,830 of SEQ ID NO:7 identified as SEQ ID NO:50.

The sequence obtained from the Cla I-#69 clone is presented in FIG.

10

and assigned SEQ ID NO:8. The complete coding sequence for the third subunit gene is located at position 1,384-2,304 (SEQ ID NO:3), with the signal sequence located at position 1,384-1,461, and the coding sequence for the mature protein of the SDH third subunit located at position 1,462-2,304 (SEQ ID NO:24).

Thus, another embodiment of the invention provides isolated nucleic acid molecules derived from sequence obtained from the Cla I-#69 clone that is presented in FIG.

10

and assigned SEQ ID NO:8. Such isolated nucleic acid molecules include the following: (1) nucleotides 1-1,383 of SEQ ID NO:8 identified as SEQ ID NO:51; (2)nucleotides 50-1,383 of SEQ ID NO:8 identified as SEQ ID NO:52; (3) nucleotides 100-1,383 of SEQ ID NO:8 identified as SEQ ID NO:53; (4) nucleotides 150-1,383 of SEQ ID NO:8 identified as SEQ ID NO:54; (5) nucleotides 200-1,383 of SEQ ID NO:8 identified as SEQ ID NO:55; (6) nucleotides 250-1,383 of SEQ ID NO:8 identified as SEQ ID NO:56; (7) nucleotides 300-1,383 of SEQ ID NO:8 identified as SEQ ID NO:57; (8) nucleotides 350-1,383 of SEQ ID NO:8 identified as SEQ ID NO:58; (9) nucleotides 400-1,383 of SEQ ID NO:8 identified as SEQ ID NO:59; (10) nucleotides 450-1,383 of SEQ ID NO:8 identified as SEQ ID NO:60; (11) nucleotides 500-1,383 of SEQ ID NO:8 identified as SEQ ID NO:61; (12) nucleotides 550-1,383 of SEQ ID NO:8 identified as SEQ ID NO:62; (13) nucleotides 600-1,383 of SEQ ID NO:8 identified as SEQ ID NO:63; (14) nucleotides 600-1,383 of SEQ ID NO:8 identified as SEQ ID NO:64; (15) nucleotides 650-1,383 of SEQ ID NO:8 identified as SEQ ID NO:65; (16) nucleotides 700-1,383 of SEQ ID NO:8 identified as SEQ ID NO:66; (17) nucleotides 750-1,383 of SEQ ID NO:8 identified as SEQ ID NO:67; (18) nucleotides 800-1,383 of SEQ ID NO:8 identified as SEQ ID NO:68; (19) nucleotides 850-1,383 of SEQ ID NO:8 identified as SEQ ID NO:69; (20) nucleotides 900-1,383 of SEQ ID NO:8 identified as SEQ ID NO:70; (21) nucleotides 950-1,383 of SEQ ID NO:8 identified as SEQ ID NO:71; (22) nucleotides 1,000-1,383 of SEQ ID NO:8 identified as SEQ ID NO:72; (23) nucleotides 1,050-1,383 of SEQ ID NO:8 identified as SEQ ID NO:73; (24) nucleotides 1,100-1,383 of SEQ ID NO:8 identified as SEQ ID NO:74; (25) nucleotides 1,150-1,383 of SEQ ID NO:8 identified as SEQ ID NO:75; (26) nucleotides 1,200-1,383 of SEQ ID NO:8 identified as SEQ ID NO:76; (27) nucleotides 1,250-1,383 of SEQ ID NO:8 identified as SEQ ID NO:77; (28) nucleotides 1,300-1,383 of SEQ ID NO:8 identified as SEQ ID NO:78; (29) nucleotides 1,350-1,383 of SEQ ID NO:8 identified as SEQ ID NO:79;(30) the nucleotide sequence encoding the full length SDH subunit 3 protein of the invention from nucleotides 1,384-1,461 of SEQ ID NO:8 identified as SEQ ID NO:3; (31) the nucleotide sequence encoding the mature form ofthe SDH subunit 3 protein of the invention from nucleotides 1,462-2,304 of SEQ ID NO:8 identified as SEQ ID NO:24; (32) nucleotides 2,305-2,355 of SEQ ID NO:8 identified as SEQ ID NO:80; (33) nucleotides 2,305-2,405 of SEQ ID NO:8 identified as SEQ ID NO:81; (34) nucleotides 2,305-2,455 of SEQ ID NO:8 identified as SEQ ID NO:82; (35) nucleotides 2,305-2,505 of SEQ ID NO:8 identified as SEQ ID NO:83; (32) nucleotides 2,305-2,555 of SEQ ID NO:8 identified as SEQ ID NO:84; (32) nucleotides 2,305-2,605 of SEQ ID NO:8 identified as SEQ ID NO:85; (32) nucleotides 2,305-2,655 of SEQ ID NO:8 identified as SEQ ID NO:86; and (33) nucleotides 2,305-2,700 of SEQ ID NO:8 identified as SEQ ID NO:87.

The invention also includes recombinant constructs comprising one or more of the sequences as broadly described above. The constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation. In a preferred aspect of this embodiment, the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art and are commercially available. The following vectors are provided by way of example: Bacterial-pET (Novagen), pQE70, pQE60, pQE-9 (Qiagen), pBs, phagescript, psiXl74, pBlueScript SK, pBsKS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK23 3-3, pDR540, pRIT5 (Pharmacia); and Eukaryotic-pWLneo, pSV2cat, pOG44, pXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia). Thus, these and any other plasmids or vectors may be used as long as they are replicable and viable in the host.

Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers. Two appropriate vectors are pKK232-8 and pCM7. Particular named bacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P

R

, P

L

and trp. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.

In another embodiment, the invention provides processes for producing the vectors described herein which comprises: (a) inserting the isolated nucleic acid molecule of the invention into a vector; and (b) selecting and propagating said vector in a host cell.

Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as Gluconobacter, Brevibacterium, Corynebacterim,

E. coli

, Streptomyces,

Salmonella typhimurium

, Acetobacter, Pseudomonas, Pseudogluconobacter, Bacillus and Agrobacterium cells; fungal and yeast organisms including Saccharomyces, Kluyveromyces, Aspergillus and Rhizopus; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS and Bowes melanoma cells; and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art.

4. Polypeptides of the Invention

The invention provides isolated polypeptide molecules for the SDH enzyme of the invention. Methods and techniques designed for the manipulation of isolated polypeptide molecules are well known in the art. For example, methods for the isolation and purification of polypeptide molecules are described Current Protocols in Protein Science, John E. Coligan et al. Eds., John Wiley & Sons, Inc., 1997, the disclosure of which is hereby incorporated by reference.

More particularly, the invention provides several isolated polypeptide molecules encoding the individual 75 kDa, 50 kDa, and 29 kDa subunit proteins of SDH enzyme of the invention. For the purposes of clarity, the particular isolated polypeptide molecules ofthe invention are described. Thereafter, specific properties and characteristics of these isolated polypeptide molecules are described in more detail.

In one embodiment, the invention provides an isolated polypeptide comprising a polypeptide sequence selected from the group consisting of: (a) the polypeptide sequence encoded in the polynucleotide sequence of SEQ ID NO:1; (b) the polypeptide sequence of SEQ ID NO:4; and (c) a polypeptide at least about 10 amino acids long from the polypeptide sequence of SEQ ID NO:4.

In another embodiment, the invention provides an isolated polypeptide comprising a polypeptide sequence selected from the group consisting of: (a) the polypeptide sequence encoded in the polynucleotide sequence of SEQ ID NO:2; (b) the polypeptide sequence of SEQ ID NO:5; and (c) a polypeptide at least about 10 amino acids long from the polypeptide sequence of SEQ ID NO:5.

In yet another embodiment, the invention provides an isolated polypeptide comprising a polypeptide sequence selected from the group consisting of: (a) the polypeptide sequence encoded in the polynucleotide sequence of SEQ ID NO:3; (b) the polypeptide sequence of SEQ ID NO:6; and (c) a polypeptide at least about 10 amino acids long from the polypeptide sequence of SEQ ID NO:6.

Other embodiments of the invention include an isolated polypeptide sequence comprising the polypeptide encoded by the isolated nucleic acid sequence SEQ ID NO:7; an isolated polypeptide sequence comprising the polypeptide encoded by the isolated nucleic acid sequence SEQ ID NO:8; an isolated polypeptide sequence comprising the polypeptide encoded by the DNA clone contained in KCTC Deposit No. 0593BP; and an isolated polypeptide sequence comprising the polypeptide encoded by the DNA clone contained in KCTC Deposit No. 0594BP.

The term “isolated polypeptide” is used herein to mean a polypeptide removed from its native environment. Thus, a polypeptide produced and/or contained within a recombinant host cell is considered isolated for purposes of the present invention. Also intended as an “isolated polypeptide” are polypeptides that have been purified, partially or substantially, from a recombinant host cell.

Polypeptides of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.

The isolated polypeptides of the invention also include variants of those polypeptides described above. The term “variants” is meant to include natural allelic variant polypeptide sequences possessing conservative or nonconservative amino acid substitutions, deletions or insertions. The term “variants” is also meant to include those isolated polypeptide sequences produced by the hand of man, through known mutagenesis techniques or through chemical synthesis methodology. Such man-made variants may include polypeptide sequences possessing conservative or non-conservative amino acid substitutions, deletions or insertions.

Whether a particular amino acid is conservative or non-conservative is well known to those skilled in the art. Conservative amino acid substitutions do not significantly affect the folding or activity of the protein. For exemplary purposes, Table 1 presents a list of conservative amino acid substitutions.

TABLE 1

Conservative Amino Acid Substitutions

Aromatic

Phenylalanine

Tryptophan

Tyrosine

Hydrophobic

Leucine

Isoleucine

Valine

Polar

Glutamine

Asparagine

Basic

Arginine

Lysine

Histidine

Acidic

Aspartic Acid

Glutamic Acid

Small

Alanine

Serine

Threonine

Methionine

Glycine

Amino acids in the protein of the present invention that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells,

Science

244:1081-1085 (1989)).

Isolated polypeptide molecules of the invention also include polypeptide sequences that are 95%, 96%, 97%, 98% and 99% identical to the isolated polypeptide molecules described herein. Computer programs such as the BestFit program (Wisconsin Sequence Analysis Package, Version 10 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711) may be used to determine whether any particular polypeptide molecule is at least 95%, 96%, 97%, 98% or 99% identical to the polypeptide sequences disclosed herein or the the polypeptide sequences encoded by the isolated DNA molecule of the deposited clones described herein. BestFit uses the local homology algorithm of Smith and Waterman,

Advances in Applied Mathematics

2: 482-489 (1981), to find the best segment of homology between two sequences.

By way of example, when a computer alignment program such as BestFit is utilized to determine 95% identity to a reference polypeptide sequence, the percentage of identity is calculated over the full length of the reference polypeptide sequence and gaps in homology of up to 5% of the total number of amino acids in the reference sequence are allowed. Thus, per 100 amino acids analyzed, 95% identity indicates that as many as 5 of 100 amino acids in the subject sequence may vary from the reference polypeptide sequence.

The invention also encompasses fragments of the polypeptide sequences and isolated polypeptide molecules described herein. In a preferred embodiment the invention provides for fragments that are at least 10 amino acids in length. The length of such fragments may be easily defined algebraically. For example, SEQ ID NO:4 provides for an isolated polypeptide molecule that is 754 amino acids in length. A fragment (F4) of SEQ ID NO:4 at least 10 amino acids in length may be defined as F4=10+X, wherein X is defined to be zero or any whole integer from 1 to 744. Similarly, fragments for other isolated polypeptide molecules described herein may be defined as follows: for SEQ ID NO:5 which is 478 amino acids in length, a fragment (F5) of SEQ ID NO:5 at least 10 amino acids in length may be defined as F5=10+X, wherein X is defined to be zero or any whole integer from 1 to 468; and for SEQ ID NO:6 which is 306 amino acids in length, a fragment (F6) of SEQ ID NO:6 at least 10 amino acids in length may be defined as F6=10+X, wherein X is defined to be zero or any whole integer from 1 to 296.

Particularly preferred embodiments of the invention provide isolated polypeptides such as the following: (1) the full length polypeptide the SDH subunit 1 of the invention, encoded by the isolated nucleic acid molecule of SEQ ID NO:1 and identified by SEQ ID NO:4; (2) the full length polypeptide the SDH subunit 2 of the invention, encoded by the isolated nucleic acid molecule of SEQ ID NO:2 and identified by SEQ ID NO:5; (3) the full length polypeptide the SDH subunit 3 of the invention, encoded by the isolated nucleic acid molecule of SEQ ID NO:3 and identified by SEQ ID NO:6; (4) the mature form of the SDH subunit 1 polypeptide of the invention, encoded by the isolated nucleic acid molecule of SEQ ID NO:22 and identified by SEQ ID NO:25; (5) the mature form of the SDH subunit 2 polypeptide of the invention, encoded by the isolated nucleic acid molecule of SEQ ID NO:24 and identified as SEQ ID NO:26; and the mature form of the SDH subunit 3 polypeptide of the invention, encoded by the isolated nucleic acid molecule of SEQ ID NO:23 and identified as SEQ ID NO:27.

The invention also provides a process for producing a polypeptide comprising: (a) growing the host cell containing the isolated nucleic acid molecule of SEQ ID NO:1 or variants thereof; (b) expressing the polypeptide encoded by said isolated nucleic acid molecule; and (c) isolating said polypeptide.

In another embodiment, the invention provides a process for producing a polypeptide comprising: (a) growing the host cell containing the isolated nucleic acid molecule of SEQ ID NO:2 or variants thereof; (b) expressing the polypeptide encoded by said isolated nucleic acid molecule; and (c) isolating said polypeptide.

Another process provided by the invention is for the production of a polypeptide which comprises: (a) growing the host cell containing the isolated nucleic acid molecule of SEQ ID NO:3 or variants thereof; (b) expressing the polypeptide encoded by said isolated nucleic acid molecule; and (c) isolating said polypeptide.

Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as Gluconobacter, Brevibacterium, Corynebacterim,

E. coli

, Streptomyces,

Salmonella typhimurium

, Acetobacter, Pseudomonas, Pseudogluconobacter, Bacillus and Agrobacterium cells; fungal and yeast organisms including Saccharomyces, Kluyveromyces, Aspergillus and Rhizopus; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS and Bowes melanoma cells; and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art.

The polypeptide may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. The addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others, are familiar and routine techniques in the art.

Polypeptides of the invention may be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography (“HPLC”) is employed for purification.

5. Production of L-Sorbose and 2-Keto-L-Gulonic Acid

The invention provides processes for the production of L-sorbose and 2-keto-L-gulonic acid, which are useful in the production of vitamin C.

In one embodiment, the invention provides a process for the production of L-sorbose from D-sorbitol comprising: (a) transforming a host cell with at least one isolated nucleotide sequence selected from the group consisting of: (i) a polynucleotide comprising the polynucleotide sequence of SEQ ID NO:1; (ii) a polynucleotide comprising the polynucleotide sequence of SEQ ID NO:2; and (iii) a polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 3; and (b) selecting and propagating said transformed host cell.

In another embodiment, the invention provides a process for the production of 2-keto-L-gulonic acid comprising: (a) transforming a host cell with at least one isolated nucleotide sequence selected from the group consisting of: (i) a polynucleotide comprising the polynucleotide sequence of SEQ ID NO:1; (ii) a polynucleotide comprising the polynucleotide sequence of SEQ ID NO:2; and (iii) a polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 3; and (b) selecting and propagating said transformed host cell.

Suitable bacteria for use as host cells in the processes provided herein for the production of L-sorbose and 2-keto-L-gulonic acid are known to those skilled in the art. Such bacteria include, but are not limited to,

Escherichia coli

, Brevibacterium, Corynebacterium, Gluconobacter, Acetobacter, Pseudomonas and Pseudogluconobacter.

Other host cells for expression of the SDH enzyme of the invention include: strains identified in U.S. Pat. No. 5,834,231

; Glucanobacter oxydans

DSM 4025 (U.S. Pat. No. 4,960,695);

Gluconobactor oxydans

TIOO (

Appl. Environ. Microbiol

. 63:454-460 (1997));

Pseudogluconobacter saccharoketogenes

IFO 14464 (European Patent No. 221 707);

Pseudomonas sorbosoxidans

(U.S. Pat. No. 4,933,289); and

Acetobacter liquefaciens

IFO 12258 (

Appl Environ. Microbiol

. 61:413-420 (1995)).

In other embodiments of the invention, a variety of fermentation techniques known in the art may be employed in processes of the invention drawn to the production of L-sorbose and 2-keto-L-gulonic acid. Generally, L-sorbose and 2-keto-L-gulonic acid may be produced by fermentation processes such as the batch type or of the fed-batch type. In batch type fermentations, all nutrients are added at the beginning of the fermentation. In fed-batch or extended fed-batch type fermentations one or a number of nutrients are continuously supplied to the culture, right from the beginning of the fermentation or after the culture has reached a certain age, or when the nutrient(s) which are fed were exhausted from the culture fluid. A variant of the extended batch of fed-batch type fermentation is the repeated fed-batch or fill-and-draw fermentation, where part of the contents of the fermenter is removed at some time, for instance when the fermenter is full, while feeding of a nutrient is continued. In this way a fermentation can be extended for a longer time.

Another type of fermentation, the continuous fermentation or chemostat culture, uses continuous feeding of a complete medium, while culture fluid is continuously or semi-continuously withdrawn in such a way that the volume of the broth in the fermenter remains approximately constant. A continuous fermentation can in principle be maintained for an infinite time.

In a batch fermentation an organism grows until one of the essential nutrients in the medium becomes exhausted, or until fermentation conditions become unfavorable (e.g. the pH decreases to a value inhibitory for microbial growth). In fed-batch fermentations measures are normally taken to maintain favorable growth conditions, e.g. by using pH control, and exhaustion of one or more essential nutrients is prevented by feeding these nutrient(s) to the culture. The microorganism will continue to grow, at a growth rate dictated by the rate of nutrient feed. Generally a single nutrient, very often the carbon source, will become limiting for growth. The same principle applies for a continuous fermentation, usually one nutrient in the medium feed is limiting, all other nutrients are in excess. The limiting nutrient will be present in the culture fluid at a very low concentration, often unmeasurably low. Different types of nutrient limitation can be employed. Carbon source limitation is most often used. Other examples are limitation by the nitrogen source, limitation by oxygen, limitation by a specific nutrient such as a vitamin or an amino acid (in case the microorganism is auxotrophic for such a compound), limitation by sulphur and limitation by phosphorous.

Illustrative examples of suitable supplemental carbon sources include, but are not limited to: other carbohydrates, such as glucose, fructose, mannitol, starch or starch hydrolysate, cellulose hydrolysate and molasses; organic acids, such as acetic acid, propionic acid, lactic acid, formic acid, malic acid, citric acid, and fumaric acid; and alcohols, such as glycerol.

Illustrative examples of suitable nitrogen sources include, but are not limited to: ammonia, including ammonia gas and aqueous ammonia; ammonium salts of inorganic or organic acids, such as ammonium chloride, ammonium nitrate, ammonium phosphate, ammonium sulfate and ammonium acetate; urea; nitrate or nitrite salts, and other nitrogen-containing materials, including amino acids as either pure or crude preparations, meat extract, peptone, fish meal, fish hydrolysate, corn steep liquor, casein hydrolysate, soybean cake hydrolysate, yeast extract, dried yeast, ethanol-yeast distillate, soybean flour, cottonseed meal, and the like.

Illustrative examples of suitable inorganic salts include, but are not limited to: salts of potassium, calcium, sodium, magnesium, manganese, iron, cobalt, zinc, copper and other trace elements, and phosphoric acid.

Illustrative examples of appropriate trace nutrients, growth factors, and the like include, but are not limited to: coenzyme A, pantothenic acid, biotin, thiamine, riboflavin, flavine mononucleotide, flavine adenine dinucleotide, other vitamins, amino acids such as cysteine, sodium thiosulfate, p-aminobenzoic acid, niacinamide, and the like, either as pure or partially purified chemical compounds or as present in natural materials. Cultivation of the inventive microorganism strain may be accomplished using any of the submerged fermentation techniques known to those skilled in the art, such as airlift, traditional sparged-agitated designs, or in shaking culture.

The culture conditions employed, including temperature, pH, aeration rate, agitation rate, culture duration, and the like, may be determined empirically by one of skill in the art to maximize L-sorbose and 2-keto-L-gulonic acid production. The selection of specific culture conditions depends upon factors such as the particular inventive microorganism strain employed, medium composition and type, culture technique, and similar considerations.

Illustrative examples of suitable methods for recovering 2-KLG are described in U.S. Pat. Nos. 5,474,924; 5,312,741; 4,960,695; 4,935,359; 4,877,735; 4,933,289; 4,892,823; 3,043,749; 3,912,592; 3,907,639 and 3,234,105.

According to one such method, the microorganisms are first removed from the culture broth by known methods, such as centrifugation or filtration, and the resulting solution concentrated in vacuo. Crystalline 2-KGL is then recovered by filtration and, if desired, purified by recrystallization. Similarly, 2-KGL can be recovered using such known methods as the use of ion-exchange resins, solvent extraction, precipitation, salting out and the like.

When 2-KGL is recovered as a free acid, it can be converted to a salt, as desired, with sodium, potassium, calcium, ammonium or similar cations using conventional methods. Alternatively, when 2-KGL is recovered as a salt, it can be converted to its free form or to a different salt using conventional methods.

All patents and publications referred to herein are expressly incorporated by reference. Having now generally described the invention, the same will be more readily understood through reference to the following Examples which are provided by way of illustration, and are not intended to be limiting of the present invention, unless specified.

EXAMPLES

Example 1

Isolation and Characterization of SDHfrom

G. suboxydans

KCTC 2111

An improved method for the purification of the pyrroloquinoline quinone (PQQ)-dependent SDH of the invention from

G. suboxydans

KCTC 2111 is presented. Improvements over the original purification scheme (Choi, E. S. et al.,

FEMS Microbiol. Lett

., 125:45 (1995)) relate to greater subunit resolution and improved stability of enzyme activity.

Step 1: Cultivation of

G. suboxydans

KCTC 2111

G. suboxydans

KCTC 21 11 was inoculated into 5 ml of SYP medium (5% D-sorbitol, 1% BactoPeptone and 0.5% yeast extract) and incubated at 30° C. for 20 hours. One milliliter (ml) of this culture were transferred to 50 ml of the same medium in a 500 ml flask and cultivated at 30° C. for 20 hours on a rotary shaker (180 rpm). The culture thus prepared was used as an inoculum for a 5 L jar fermentor containing 3 L of the same medium, and the 3 L culture was grown to early stationary phase.

Step 2: Preparation of the Membrane Fraction

Cells were harvested by centrifugation at 12,000 g for 10 min, washed once with 10 mM sodium acetate buffer (pH 5.0) and disrupted with glass beads (0.1 mm in diameter) in a bead beater (Edmund Buhler, Vi 4) for 90 sec at 4° C. The homogenate thus prepared was centriftiged at 12,000 g for 5 min to remove cell debris and glass beads. The resulting supernatant was centrifuged at 100,000 g for 60 min, and a crude membrane fraction was obtained as a precipitate.

Step 3: Solubilization of SDH from the Membrane Fraction

The crude membrane fraction was resuspended at 40 mg of protein per ml and solubilized with 1.5% n-octyl glucoside by stirring for 2 hours at 4° C. The resultant suspension was centrifuged at 12,000 g for 10 min to give a supernatant, designated as the solubilized SDH fraction. All the solutions employed in the purification procedures contained 0.75% n-octyl glucoside.

Step 4: Enzyme Activity Assay

Enzyme activity was assayed spectrophotometrically using 2,6-dichlorophenol indophenol (DCIP) as an artificial electron acceptor and phenazine methosulphate (PMS) as an electron mediator. The reaction mixture contained 50 mM sodium acetate buffer (pH 5.0), 10 mM MgCl

2

, 5 mM CaCl

2

, 5 mM KCN, 0.1 mM PMS, 0.12 mM DCIP, 250 mM D-sorbitol, 0.75% n-octyl glucoside and enzyme solution in a total volume of 1.0 ml. The molar extinction coefficient of ε=5,600 cm

−1

M

−1

for DCIP at pH 5.0 was employed. One unit of enzyme activity was defined as the amount of enzyme catalyzing reduction of 1 μmol of DCIP per min.

Enzyme activity was determined also by the Ferric-Dupanol method (Wood, W.A. et al.,

Meth. Enzymol

. 5:287 (1962)) for the reconstituted subunits described in step 6 of Example 1. Subunit proteins were preincubated either singly or in different combinations for 5 min at 25° C. The reaction was started by the addition of 10 mM (final concentration) of potassium ferricyanide and 250 mM (final concentration) D-sorbitol. After an appropriate time, the reaction was stopped by adding the ferric sulfate-Dupanol solution (Fe

2

(SO

4

)

3

.nH

2

O 5 g/L, Dupanol (sodium lauryl sulfate) 3 g/L and 85% phosphoric acid 95 ml/L), and the absorbance of the Prussian color was determined at 660 nm in a spectrophotometer.

Step 5: Ion-exchange Chromatography

The solubilized fraction was loaded onto a CM-TSK 650 (S) (Merck) column (2.5×20 cm) equilibrated with 10 mM sodium acetate (pH 5.0). The CM-TSK column was eluted with a linear gradient (from 10 mM to 500 mM) of sodium acetate. Active fractions were pooled and concentrated by ultrafiltration using a membrane filter (Amicon, YM 10) and loaded onto a DEAE-TSK 650 (S) (Merck) column (2.5×20 cm). The DEAE-TSK column was eluted isocratically with a 10 mM sodium acetate buffer of either pH 5.0 or pH 6.0.

As shown in

FIG. 1

, when eluted at pH 6.0 (FIG.

1

-B), the resolution of the activity peaks was much higher than with an elution at pH 5.0 (FIG.

1

-A), and the enzyme activity peaks I and II were completely separated. When the enzyme activity of the fractions was measured immediately after the elution and one hour after the elution, it was found that the enzyme activity of the fractions in peaks I and II eluting together at pH 5.0 did not differ signficantly with time. In the case of the fractions in peaks I and II eluting separately at pH 6.0, however, the enzyme activity in peak I decreased to less than one tenth of the original value within one hour after the elution, whereas the enzyme activity in the peak II remained unchanged.

Fractions containing peaks I and II eluted at pH 6.0 on DEAE-TSK colunn were analyzed by SDS-PAGE (Laemmli, U. K.,

Nature

, 227, 680 (1970)) (FIG.

2

).

FIG. 2-A

shows fractions of peak I. Column M shows standard molecular weight markers. As shown in columns 1 through 4, the 75 kDa first subunit band and the 50 kDa second subunit band could be observed but the 29 kDa third subunit band was not observed. In this case, one hour after elution, the enzyme activity was reduced by more than ten fold.

FIG. 2-B

shows the fractions of peak II. Column M shows standard molecular weight markers. As shown in columns 5 through 8, it was observed that fractions of peak II contained the 29 kDa third subunit band in addition to the 75 kDa and 50 kDa subunits. This observation indicated that the 29 kDa third subunit might play an important role in the stability of SDH.

Step 6: SDH Reconstitution with Different Subunits

Since the increase of the pH of the elution buffer from 5.0 to 6.0 during DEAE column chromatography increased the resolution of the chromatographic peaks, the pH of the elution buffer was further increased to pH 6.5. Partially purified SDH provided by CM-TSK 650 (S) column chromatography as described in step 5 of Example 1 was loaded onto DEAE-TSK 650 (S) column (2.5×16.5 cm) pre-equilibrated with 20 mM sodium phosphate buffer (pH 6.5), and the column was isocratically eluted with 180 ml of the same buffer followed by a gradient elution with 160 ml each of 20 mM and 500 mM of sodium phosphate buffer (pH 6.5). Column fractions were analyzed by SDS-PAGE.

As shown

FIG. 3

, the first subunit of 75 kDa eluted first (peak I) followed by the third subunit of 29 kDa (peak II) during the isocratic elution, and the second subunit of 50 kDa eluted later during the gradient elution (peak III). As shown in

FIG. 4

, the SDS-PAGE analysis of the three peaks indicated that the three subunits were essentially pure and completely separated from each other.

Since the three subunits could be purified and separated into individual species under non-denaturing conditions, reconstitution experiments were conducted to determine the role of each subunit for catalytic activity of the SDH enzyme. Approximately 100 pmoles of each subunit obtained from the DEAE column by elution at pH 6.5 were preincubated either singly or in different combinations and assayed for the enzyme activity by Ferric-Dupanol method as described in step 4 of Example 1 (Table 2). Each subunit alone showed a very low level of activity. When the first and the second subunits were reconstituted, enzyme activity increased by two times compared with the first subunit alone. When the third subunit was added to the first subunit, and to the mixture of the first and the second subunits, the enzyme activity increased by about 30 and 20 times, respectively, These results indicated that the third subunit plays an important role for the catalytic activity of SDH as well as the stability of the enzyme.

TABLE 2

Subunit (kDa)

SDH enzyme activity (OD

660

)

75

0.029

50

0.002

29

0.004

75 + 50

0.051

75 + 29

0.944

75 + 50 + 29

0.692

Example 2

Determination of the N-terminal Amino Acid Sequences of the SHD Subunits

The purified SDH prepared in Example 1 was subjected to SDS-PAGE (12.5% gel), and the separated proteins were electroblotted onto apolyvinylidene difluoride (PVDF) membrane (Bollag, D. M. and Edelstein, S. J.,

Protein Methods

, Wiley-Liss, Inc.,8(1991)). After visualization with ponceau S stain, the section of membrane containing each SDH subunit was cut into pieces, and the membrane pieces for each subunit were applied directly to an amino acid sequence analyzer (Applied Biosystems, Model 477A) for N-terminal amino acid sequence analysis.

The resultant data for the first and the third subunits are shown in Table 3 (SEQ ID Nos:9 and 11). The N-terminal amino acid sequence of the second subunit could not be obtained, most likely because of a blocked N-terminus. A similar finding of ablocked N-terminus was reported for the cytochrome c subunit of the alcohol dehydrogenase complex of another acetic acid bacterium,

Acetobacter pasteurianus

(Takemura, H. et al.,

J Bacteriol

. 175, 21, 6857 (1993)), where the blockage of the N-terminus was ascribed to the modification of glutamate residue at the N-terminus to a pyroglutamate residue, which is recalcitrant to the Edman degradation during N-terminal amino acid sequencing.

Therefore, in order to obtain N-terminal sequence for the second subunit of purified SDH, the isolated SDH protein was first treated with pyroglutamate aminopeptidase to release the potentially blocked N-terminus. Briefly, about 50 pg of purified SDH from Example 1 was dissolved in a digestion buffer (100 mM sodium phosphate buffer, 10 mM EDTA, 5 mM dithiothreitol and 5% (v/v) glycerol, pH 8.0) and incubated with 3.75 μg of pyroglutarnate aminopeptidase (Boehringer Mannheim) at 4° C. for 18 hours, followed by an additional incubation for 4 hours at 25° C. After incubation, the reaction mixture was subjected to SDS-PAGE, electroblotted onto a PVDF membrane, and the second subunit band on the membrane was excised and analyzed for the N-terminal amino acid sequence as described above. Ten residues of N-terminal amino acid sequence is shown in Table 3 as Sequence ID. No:10.

TABLE 3

Sequence ID No.

N-terminal amino acid sequence

SEQ ID NO: 9

EDTGTAITNADQHPG

SEQ ID NO: 10

DADDALIQRG

SEQ ID NO: 11

AGTPLKIGVSFQEMNNPYFVTMKDA

Example 3

Determination of Internal Amino Acid Sequence for the Third Subunit

For the third subunit, internal amino acid sequences were also determined in addition to the N-terminal amino acid sequence for the facilitated cloning of the corresponding gene. About 7 μg of the third subunit protein isolated as described in step 6 of Example 1, was digested with trypsin (Boehringer Mannheim) as previously described (Matsudaira, P. T.,

A Practical Guide to Protein and Pept Purification for Mcrosequencing Academic Press

p. 37 (1989)). The tryptic digest was separated by HPLC using a Brownlee SPHERI-5 RP 18 column (0.2×22 cm), and the column was eluted with a linear gradient of 15-70% acetonitrile for 60 minutes at 210 microliter/minute. The elution was monitored at 214 nrn, and the well separated three peptide peaks were collected (designated 1, 5 and 7 in

FIG. 5

) and analyzed for their amino acid sequence as described in (Example 2). The internal amino acid sequences for the peaks, 1, 5 and 7 are shown below in Table 4 (SEQ ID NO: 12, 13, and 14), respectively.

TABLE 4

Sequence ID No.

Internal amino acid sequence

SEQ ID NO: 12

HSDIK

SEQ ID NO: 13

NYDAGFK

SEQ ID NO: 14

KWGAGVPK

Example 4

Primer Design and Isolation of a 1.53 kb DNA Fragment Containing a Portion of the SDH Gene

Based on the N-terminal amino acid sequence of the first subunit (SEQ ID NO:9), two degenerate primers, primer 1 (5′- CCGGAATTC GAA(G) GAT(C) ACI GGI ACI GC-3′) (SEQ ID NO:15) and primer 2 (5′-ATT(C,A) ACI AAT(C) GCI GAT(C) CAAG) CAT(C) CC-3′)(SEQ ID NO:16), were synthesized.

The genomic DNA isolated from

G. suboxydans

KCTC 2111 using the method of Takeda and Shimizu (Takeda and Shimizu,

J Ferm. Bioeng

., 72:1 (1991)) was partially digested with BamHI. The plasmid pBluescript SK (Stratagene) was restricted with Nael and BamHI and the BamHI partial digest of genomic DNA was ligated to the BamHI site of the plasmid.

Thirty cycles of polymerase chain reaction (PCR) was performed in accordance with the single specific primer PCR (SSP-PCR) method (White, B., SSP-PCR and genome walking, in

Method in Mol. Biol

., PCR protocols, Humana Press, 15:339 (1993)) to isolate a clone (#SDH2-1) 1.53 kb in size.

The ligation reaction mixture prepared above was amplified with a gene specific primer, the primer 1, and the T7 primer of the vector. Although the generic T7 primer anneals to the ends of all ligated fragments, the resulting products increase only linearly. However, simultaneous annealing of the gene specific primer 1 and T7 primer to the specific product results in exponential amplification of the specific primary product. Secondary PCR carried out with a nested primer, the gene specific primer 2, and the T7 primer generated a specific secondary product, which confirmed the specificity of the primary PCR.

The PCR product was ligated to plasmid pT7 Blue (Novagen) and transformed into

Escherichia coli

DH5a by SEM protocol (Inoue, H. et al.,

Gene

, 96:23 (1990)). Transformants were cultivated in an LB medium (1% Bacto-Tryptone, 0.5% yeast extract and 1% NaCl) supplemented with 100 μg/ml of ampicillin. Subsequently, the plasmid was extracted by alkaline lysis method (Sambrook, J. et al.,

Molecular Cloning

, CSH Press, p. 125 (1988)).

PCR with primer 1 or primer 2 together with the T7 primer using this plasmid as a template yielded a positive reaction. Partial nucleotide sequencing of #SDH2-1 fragment further confirmed that the derived amino acid sequence downstream of the primer 1 binding site matched with the experimentally determined N-terminal amino acid sequence. However, #SDH2-1 contained only a part of the first subunit gene.

Example 5

Isolation of Lambda GEM5-1 Clone Containing SDH Subunit Genes Using the 1.53 kb DNA Fragment as a Probe

The #SDH2-1 isolated in Example 4 was labeled with DIG Labeling and Detection Kit (The DIG System User's Guide for Filter Hybridization. p. 6-9, Boehringer Mannheim (1993)).

To construct a genomic DNA library of G. suboxydans KCTC 2111, the genomic DNA isolated from G. suboxydans KCTC 2111 using the method described in Example 4 was partially digested with Sau3AI. Partially digested DNA was electrophoresed in a 0.8% agarose gel and the DNA of 15 to 23 kb in size was eluted using QLAEX II Gel Extraction Kit (QIAGEN). The eluted DNA was then ligated into the BamHI site of Lambda GEM-11 vector (Promega). The ligation mixture was packaged into phage lambda particles using the Packagene In Vitro Packaging System (Promega) according to the instruction manual.

E. coli

LE392 cells were grown in TB medium (1% Bacto-Tryptone and 0.5% NaCI) supplemented with 0.2% maltose and 10 mM MgSO

4

, at 30° C. and stored at 4° C. when the OD

600

had reached 0.6. The packaging mixture was added to the cell suspension, and the mixture was incubated for 30 min at 37° C. to allow infection. To this mixture, 3 ml of molten (45° C.) TB top agar (0.8% Bacto-Agar in T13 medium containing 10 mM MgSO

4

) was added, vortexed gently and immediately poured onto LB plates. The plates were incubated inverted at 37° C. overnight.

The lambda phage plaques were immobilized on nylon membranes (Amersham) and the membranes were prehybridized in a hybridization oven (Hybaid) using a prehybridization solution (5×SSC, 1% (w/v) blocking reagent, 0.1% N-lauroylsarcosine, 0.02% SDS and 50% (v/v) formamide) at 42° C. for 3 hours. Then the membranes were hybridized using a hybridization solution (DIG-labeled #SDH2-1 probe diluted in the prehybridization solution) at 42° C. for 16 hours. Eleven plaques among about 20,000 plaques of the lambda phage gave positive signals by plaque hybridization (The DIG System User's Guide for Filter Hybridization. Boehringer Mannheim (1993)) with the DIG-labeled #SDH2-1 probe.

The lambda DNAs were isolated from positive lambda clones and purified with Lambda DNA Purification Kit (Stratagene). The isolated lambda DNAs were digested with BamHl and subjected to a 0.7% agarose gel electrophoresis. The DNA fragments separated on the gel were transferred onto a nylon membrane and analyzed again by, Southern hybridization with #SDH2-1 as probe under the same condition as described above. A clone which gave a positive signal was selected, and the insert DNA of 15 kb was excised with XhoI from this clone and subsequently cloned into the Xhol site of pbluescript SK give Lambda GEM 5-1. The Lambda GEM 5-1 clone was mapped by digestion with several different restriction enzymes.

FIG. 6

presents the restriction enzyme map of Lambda GEM 5-1.

Example 6

Determination of the Nucleotide Sequence of the 5.7 kb Pstl Fragment in the Lambda GEM 5-1 Clone

The positive clone, Lambda GEM 5-1, obtained in Example 5 was mapped with several different restriction enzymes and analyzed by Southern hybridization as described in Example 5. A 5.7 kb Pstl fragment hybridizing with #SDH2-1 was subcloned, and the nucleotide sequence was determined. To make the DNA sequencing simpler, three overlapping subclones, S1, S2 and S3, were constructed using restriction enzyme sets, KpnI-Pstl, Notl-Sacll and Pstl-SacI, respectively, as shown in

FIG. 7. A

set of deletion clones was prepared for each subclone using Exo III-Mungbean Deletion Kit (Stratagene). The nucleotide sequencing reaction was done by Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems), and the sequence was determined in an automatic DNA sequencer (Applied Biosystems, Model 373A).

FIG. 8

shows the nucleotide sequence of 4,830 bp in the 5.7 kb Pstl fragment (SEQ ID NO:7). The sequenced DNA contains two open reading frames (ORFs) of 2,265 and 1,437 nucleotides. The first ORF encodes the first subunit. The first subunit gene is preceded by a Shine-Dalgarno sequence, “AGGA” positioned at 651-654 bp. The 34 amino acid signal sequence of the first subunit is positioned at 665-766 bp of SEQ ID NO:7. The coding sequence of the mature part of the first subunit protein is positioned at 767-2,929 bp of SEQ ID NO:7, which encodes a 720 amino acid polypeptide whose derived N-terminal amino acid sequence is in perfect agreement with 15 amino acid residues obtained by N-terminal amino sequence analysis.

The first ORF was followed by the second ORF, the two ORF's being interrupted by a short intergenic region. The second ORF encodes the second subunit. The Shine-Dalgamo sequence (AGGA) of the second subunit gene is found at 2,950-2,953 bp SEQ ID NO:8, and the structural gene is positioned at 2,964-4,400 bp of SEQ ID NO:8. The second subunit gene encodes a 478 amino acid polypeptide, including a signal sequence of 36 amino acids. The derived amino acid sequence of the mature polypeptide showed a perfect match with the experimentally obtained sequence for the sample treated with pyroglutamate aminopeptidase. Downstream of the stop codon of the second subunit gene, inverted repeat sequences are found.

The calculated molecular weights of the mature proteins of the first and the second subunit are 79 kDa and 48 kDa, respectively, which are in good agreement with the experimental values of 75 kDa and 50 kDa, respectively, as determined by SDS-PAGE.

In addition it was also found that signature PQQ-binding sequences, consensus sequences appearing characteristically in the amino- and carboxy-terminals of PQQ-dependent dehydrogenases, are present in the first subunit gene. In Table 5, the signature PQQ-binding sequence in amino-terminal part is shown as Sequence ID NO:17, and the signature sequence existing in the carboxy-terminal part is shown as Sequence ID No:18 (Here, X represents an arbitrary amino acid.). The amino-terminal signature sequence occurs at position 812-898 bp, and the carboxy-terminal signature sequence occurs at position 1,490-1,555 bp. These data provide additional evidence that the first subunit contains pyrroloquinoline quinone (PQQ) as cofactor.

TABLE 5

[D/E/N]-W-X-X-X-G-[R/K]-X-X-X-X-X-X-[F/Y/W]-S-X-X-X-X-[L/I/V/M]-X-X-

[SEQ ID NO: 17]

X-N-X-X-X-L-[R/K]

W-X-X-X-X-Y-D-X-X-X-[D/N]-[L/I/V/M/F/Y]-[L/I/V/M/F/Y]-[L/I/N/M/F/Y]-

[SEQ ID NO: 18]

[L/I/V/M/F/Y]-X-X-G-X-X-[S/T/A]-P

In addition, it was also discovered that a single heme-binding sequence occured at position 2,612-2,626 bp in the first subunit gene (SEQ ID NO:19 in Table 6 below), and three heme-binding sequence occurred at the following positions: 3,129-3,143; 3,573-3,587 and 3,981-3,995 bp in the second subunit gene (Here, X

A

represents an arbitrary amino acid. X

B

, is an arbitrary amino acid different from X

A.

).

TABLE 6

C-X

A

-X

B

-C-H

[SEQ ID NO: 19]

A database search for homologous sequences determined that the DNA sequence of the first subunit gene showed a great degree of similarity to many dehydrogenases containing pyrroloquinoline quinone (PQQ) as cofactor: In particular, the alcohol dehydrogenases of

Acetobacter polyoxogenes

(Tamaki, T. et al.,

Biochim. Biophys. Acta

, 1088, 292 (1991)) and

Acetobacter aceti

(Inoue, T. et al.,

J Bacteriol

, 171, 3115 (1989)) are 77% and 70% identical, respectively, to the first subunit gene. A search of the database with the second subunit gene provided the greatest degree of similarity, matching the cytochrome c of

G. suboxydans

IFO 12528 (Takeda and Shimizu,

J Ferm. Bioeng

., 72:1 (1991)) with a nucleotide sequence identity of 83% and an amino acid sequence identity of 88%.

Example 7

Primer Design and PCR Cloning of 320 bp DNA Fragment Containing a Portion of the Third Subunit Gene

The nucleotide sequence analysis of the first and the second subunit genes described in Example 6 indicated that the isolated operon clone did not contain the third subunit gene. Further sequencing of the 5′ and 3′ flanking regions also failed to show the presence of the third subunit. Therefore, in order to isolate the gene encoding the third subunit, degenerate primers were synthesize based on the amino acid sequence information, in order to generate by PCR a short DNA fragment containing a portion of third subunit gene for use as probe.

Based on the N-terminal amino acid sequence (SEQ ID NO:1) of the mature third subunit protein obtained in Example 2 and one of the internal amino acid sequence (SEQ ID NO:13) of the tryptic peptides obtained in Example 3, two degenerate primers, primer 3 (5′-GGGAATTC TTT(C) CAA(G) GAA(G) ATG AAT(C) AA-3′) (SEQ ID NO:20) and primer 4(5′-GGGAATTC TT GAA A(G)CC NGC A(G)TC A(G)TA-3′)(SEQ ID NO:21), respectively, were synthesized.

A PCR reaction was done with primers 3 and 4 using genomic DNA of

G. suboxydans

KCTC 21 1 1 prepared by the method of Takeda and Shimizu (Takeda and Shimizu,

J. Ferm. Bioeng

., 72 1 (1991)) as template. The reaction generated a 320 bp DNA fragement. This 320 bp PCR product was ligated to pBluescript SK (Stratagene) and transformed into

E. coli

DH5α by the SEM protocol (Inoue, H. et al.,

Gene

. 96, 23 (1990)). Transformants were cultivated in an LB medium supplemented with 100 μg/ml of ampicillin. Subsequently, the plasmid was extracted by the alkaline lysis method (Sambrook, J. et al.,

Molecular Cloning

, CSH Press, (1988)). Subcloning was verified by performing a PCR reaction with primer 3 and primer 4 using this plasmid DNA as a template. Partial nucleotide sequencing of the 320 bp fragment further confirmed that the derived amino acid sequence downstream of the primer 3 binding site matched with the experimentally determined N-terminal amino acid sequence. However, the 320 bp fragment contained only the N-terminal part of the third subunit gene.

Example 8

Isolation of the third Subunit Gene Using 320 bp DNA Fragment as Probe

The 320 bp DNA fragment isolated in (Example 7) was labeled with DIG Labeling and Detection Kit (

The DIG System User's Guide for Filter Hybridzation

. p6-9, Boehringer Mannheim (1993)). The genomic DNA isolated from

G. suboxydans

KCTC 2111 using the method of Takeda and Shimizu (Takeda and Shimizu,

J. Ferm. Bioeng

., 72, ((1991)) was digested with BamHI, Clal, EcoRI, HindIII, PstI, or XhoI, electrophoresed in a 0.8% agarose gel and transferred to a nylon membrane (NYTRAN, Schleicher & Schuell) as described (Southern, E. M., J. Mol. Biol. 98,503 (1975)). The membrane was prehybridized in a hybridization oven (Hybaid) using a prehybridization solution (5×SSC, 1% (w/v) blocking reagent, 0.1% N-lauroylsarcosine, 0.2% SDS and 50% (v/v) formamide) at 42° C. for 2 hours. Then the membrane was hybridized using a hybridization solution (DIG-labeled probe diluted in the prehybridization solution) at 42° C. for 12 hours. Southern hybridization gave a strong discrete signal for each enzyme used. DNA corresponding to the positive signal at 4.5 kb ClaI was eluted and cloned into pBluescript SK to construct a mini-library. The mini-library was screened for the positive clone by repeating the Southern hybridization as described above. A clone which gave a positive signal was selected and designated ClaI-#69.

FIG. 9

presents the restriction enzyme map of ClaI-#69.

Example 9

Nucleotide Sequence Analysis of the 4.5 kb ClaI Fragment Containing the Third Subunit

The nucleotide sequence of the third subunit gene in the 4.5 kb ClaI-#69 clone was determined and analyzed. To facilitate the DNA sequencing, several overlapping restriction fragments were subcloned, and the nucleotide sequence of each clone determined. The nucleotide sequencing reaction was done by Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems), and the sequence was determined in an automatic DNA sequencer (Applied Biosystems, Model 373A).

FIG. 10

shows the nucleotide sequence of 2700 bp of the 4.5 kb ClaI fragment (SEQ ID NO:8). The sequenced DNA contains an open reading frame (ORF) of 921 nucleotides, which encodes the third subunit polypeptide. The third subunit gene is preceded by a potential Shine-Dalgarno sequence (AGG) positioned at 1,375-1,377 bp. The amino acid signal sequence of the third subunit polypeptide is positioned at 1,384-1,461 bp. The coding sequence of the mature part of the third subunit protein is positioned at 1,462-2,304 bp, encoding a 280 amino acid polypeptide whose derived N-terminal amino acid sequence was in perfect agreement with the 25 amino acid residues obtained by N-terminal amino sequence analysis. The calculated molecular weight of the mature protein of the third subunit is 29,552 Da, which was in good agreement with the experimental value of 29 kDa determined by SDS-PAGE.

87

1

2265

DNA

Gluconobacter suboxydans

1
atggtttctg gtctactgac gccgatcaac gttacgaaga agcgccttct gggttgcgct 60
gctgctctgg cattctgcgc cacctctcct gtcgccctgg ctgaggacac aggaacagcc 120
attacaaacg ccgaccagca tccgggtgac tggatgagct atggccggac ctattccgag 180
cagcgctaca gcccgctgga tcagatcacc aaggacaatg cgagcaatct gaagctggca 240
tggcactacg atctggatac caaccgtggt caggaaggta cgccgctgat cgttgatggc 300
gtcatgtacg ccaccacaaa ctggagcaag atgaaggctc tggatgcagc tacgggcaag 360
ctgctgtggt cttacgatcc aaaggttcca ggcaacatcg ccgaccgcgg ctgctgcgat 420
acggtcaacc gtggtgcagc ctactggaac ggcaaagtct atttcggcac cttcgacggt 480
cgcctgattg ccctggatgc caagaccggc aagctggtct ggagcgtcta tacggttccc 540
aaggaagcgc agctgggtca ccagcgctcc tacacggttg acggtgctcc ccgtatcgcc 600
aagggcaagg tcatcatcgg caacggcggt gcagagttcg gcgcccgtgg cttcgtgacg 660
gcgtatgacg ctgaaacggg aaagatggac tggcgcttct tcaccgttcc gaaccctgac 720
aacaagccgg acggcgcagc gtctgacgac gtgctgatgt ccaaggctta tccgacatgg 780
ggcaagggcg gcgcgtggaa gcagcagggc ggtggcggta ccgtctggga ttcgctgatc 840
tatgaccctg taacggatct cgtctatctg ggcgtcggta acggctcgcc atggaactac 900
aagttccgtt ctgaaggaaa aggcaacaac ctcttcctcg gcagcatcgt ggccatcaat 960
cctgacaccg gcaaatacgt ctggcatttc caggaaacgc caatggacca gtgggattat 1020
acctcggttc agcagatcat ggccctcgac atgccggtca atggcgaaat gcgccatgtg 1080
ctcgtgcatg cgccgaagaa cggcttcttc tatatcattg atgccaagac cggtaagttc 1140
atctccggca agccgtacac ctacgagaac tgggccaatg gcctcgatcc ggtaacgggt 1200
cgtccgaact acaatccaga tgctctctgg acgctgaacg gcaagccctg gtacggcatc 1260
cccggcgatc tgggtggtca taacttcgct gccatggctt acagcccaca gacgaagctg 1320
gtttacattc ccgcccagca ggttcccttc gtttacgatc cgcagaaggg tggcttcaag 1380
gctcaccacg acagctggaa ccttggcctc gacatgaaca agatcggcct gcttgatgac 1440
aacgatccac agcacaaggc tgacaaggcc cagttcctga aggatctgaa gggctggatc 1500
gttgcatggg atccgcagaa gcagcaggca gccttcacgg ttgaccacaa gggtccgtgg 1560
aatggcggtc ttctggcaac ggctggtggc gttctgttcc agggtctcgc caacggtgag 1620
ttccacgcct acgacgcgac gacgggtaag gatctcttca ccttcccagc acagagcgcc 1680
atcattgccc cgccagtcac ctacacagcc aacggcaagc agtatgttgc ggttgaagtg 1740
ggctggggcg gtatctatcc gttcttcctg ggcggcgtag cccgtacgtc cggctggacc 1800
gtcaaccact cccggatcat cgcgttcgct ctggacggca acgacaagct gccagccaag 1860
aacgagctcg gcttcgttcc agtgaagccg cctgagaaat gggatgaagc caagatcaag 1920
gacggctact tccagttcca gacctattgc gcagcctgcc atggtgacaa cggtatctcc 1980
ggcggtgttc tgccagacct gcgctggtcc ggtgcgatcc gtggagagga gaagttctac 2040
aagctcgtcg gcaagggtgc tctaacggcc tacggtatgg accgtttcga cacgtccatg 2100
tcgccagctg aaatcgaaga catccgcaac ttccttgtga agcgcgccaa cgagtcctac 2160
gcagacgaag tcaaggcccg aaagaatgag gcaggcgtcc ctaacggcga attcctcaac 2220
gtccctcagg gttcggttgc gcctgcaacg ccggaccatc cgtaa 2265

2

1437

DNA

Gluconobacter suboxydans

2
atgctcaagg cattaactcg ggacagactg gtatctgaga tgaaacaggg atggaaatac 60
gcggccgcag tcggcctcat ggcagtgtct ttcggtgctg cccaagccca ggacgctgat 120
gacgccctga ttcagcgcgg tgcctacgtg gcccgcctgt ctgactgcgt tgcctgccat 180
accgcactac acggccagcc ttttgctggt ggtctggaga tcaagagccc gatcggcacg 240
atctactcca ccaacatcac gcctgacccg aaatacggta tcggcaacta tacactcgaa 300
gatttcacga aggcgatccg taagggtatc cgcaaggacg gcgcgacggt ttatccggcc 360
atgccgtatc ctgagttcgc tcgcctgtct gatgacgaca tcaaggccat gtatgccttc 420
ttcatgcatg gcgtgaagcc ggtcgccctt cagaacaagc agccggacat ctcctggccg 480
atgaacatgc gctggccgtt ggccatctgg cgcgcgatgt ttgttccgac tgtcacacca 540
ggcctcgaca agagcatctc cgatccggaa gtggcgcgtg gcgaatacct cgtgaatggc 600
ccaggccatt gtggcgagtg tcatacgccc cgtggcatgg ccatgcaggt caagggctat 660
acggccaagg acggcaacgc ttacctctcc ggtggcgcac cgatcgacaa ctggattgct 720
cccagcctgc gtagcaatag cgacacgggt ctgggtcgct ggtctgaaga cgacattgcc 780
gagttcctga agagcggccg tatcgaccat tctgccgtct tcggtggcat ggctgacgtg 840
gtggcctaca gcacccagca ctggaccgac gacgatctgc acgcaacggc caagtacctg 900
aagagcatgc cggccgttcc ggaaggcaaa aacctgggtc aggatgacgg caaggccacg 960
gccctgctcg aagccggtgg caagggtgat gcaggcgcag aggtttacct ccacaactgt 1020
gccatctgcc atatgaacga tggcactggt gtcaaccgca tgttcccgcc gctggctggc 1080
aacccggtcg tcatcacgga caatgcaacc tcaatggcca acatcgtgac attcggcggt 1140
attctgcctc cgacgaatac ggcgccatct gctgttgcca tgccgggctt ccgcgatcat 1200
ctgtctgacc agcagatcgc cgatgttgtg aacttcatgc gcaagagctg gggcaaccag 1260
gctccgggaa ccctgtctgc ctcggatatc cgcaagctcc gcacatcggg tactgcggtt 1320
tccacggccg gctggaacgt ctcttccaag ggctggatgg cctacatgcc gcagccttat 1380
ggcgaaggct ggaccttctc cccgcagaca cacacgggcg tggatcaggc tcagtaa 1437

3

921

DNA

Gluconobacter suboxydans

3
atgaagacca agacgcttcc gctggccctt ctgtctctcg ctcttggcgg gacggccctg 60
tcagacccgg ctgccgcagc tggaaccccg ctgaaaattg gcgtttcctt tcaggaaatg 120
aacaatccgt acttcgtcac catgaaggac gcattgcagg aagccgctgg cacgatcggt 180
gcgaatgtca tcatcagtga tgcgcatcat gatgtttcca agcaggtgag tgacattgag 240
gacatgatcc agcagggcgc gcagatcatc atcatcaacc cgaccgatac agtaggcgtc 300
acgtccgtcg taaagagcgt tcatgacaag aacatcccga tcgtctcggt ggatgctcag 360
gctggcggtc cgctcgatgc gtttgtgggg tccaagaact atgatgccgg cttcaaggcc 420
tgcgagtatc tcgccacgac gatcaagagc ggcaatatcg gaatcatcga cggtatcccg 480
gtcgttccca ttcttgagcg tgtcaaaggc tgcaaaaacg ctatcgccaa gcattcagat 540
atcaagattg tcagcgttca gaacggcaag caggagcgcg atgaggctct gacggtggct 600
gaaaacatgc tccaggccaa cccggatctg aaaggtatct tcagcgtcaa tgacaacgga 660
tcgctcggtg tgctgtccgc tatcgaatcc agtggttcag acgtgaagct ggtcagcgtt 720
gatggcaacc cggaagccgt gaaggccatc tacaagccag gctctcattt catcgctacg 780
gctgcgcagt tcccccggca ggatatccgt ctggcactgg cgctcgccct tgccaggaaa 840
tggggcgcag gcgtgccgaa ggtcctgcct gttgatgtcg agctgatcga cgcgacgaaa 900
gccaagacgt tcagctggta a 921

4

754

PRT

Gluconobacter suboxydans

4
Met Val Ser Gly Leu Leu Thr Pro Ile Asn Val Thr Lys Lys Arg Leu
1 5 10 15
Leu Gly Cys Ala Ala Ala Leu Ala Phe Cys Ala Thr Ser Pro Val Ala
20 25 30
Leu Ala Glu Asp Thr Gly Thr Ala Ile Thr Asn Ala Asp Gln His Pro
35 40 45
Gly Asp Trp Met Ser Tyr Gly Arg Thr Tyr Ser Glu Gln Arg Tyr Ser
50 55 60
Pro Leu Asp Gln Ile Thr Lys Asp Asn Ala Ser Asn Leu Lys Leu Ala
65 70 75 80
Trp His Tyr Asp Leu Asp Thr Asn Arg Gly Gln Glu Gly Thr Pro Leu
85 90 95
Ile Val Asp Gly Val Met Tyr Ala Thr Thr Asn Trp Ser Lys Met Lys
100 105 110
Ala Leu Asp Ala Ala Thr Gly Lys Leu Leu Trp Ser Tyr Asp Pro Lys
115 120 125
Val Pro Gly Asn Ile Ala Asp Arg Gly Cys Cys Asp Thr Val Asn Arg
130 135 140
Gly Ala Ala Tyr Trp Asn Gly Lys Val Tyr Phe Gly Thr Phe Asp Gly
145 150 155 160
Arg Leu Ile Ala Leu Asp Ala Lys Thr Gly Lys Leu Val Trp Ser Val
165 170 175
Tyr Thr Val Pro Lys Glu Ala Gln Leu Gly His Gln Arg Ser Tyr Thr
180 185 190
Val Asp Gly Ala Pro Arg Ile Ala Lys Gly Lys Val Ile Ile Gly Asn
195 200 205
Gly Gly Ala Glu Phe Gly Ala Arg Gly Phe Val Thr Ala Tyr Asp Ala
210 215 220
Glu Thr Gly Lys Met Asp Trp Arg Phe Phe Thr Val Pro Asn Pro Asp
225 230 235 240
Asn Lys Pro Asp Gly Ala Ala Ser Asp Asp Val Leu Met Ser Lys Ala
245 250 255
Tyr Pro Thr Trp Gly Lys Gly Gly Ala Trp Lys Gln Gln Gly Gly Gly
260 265 270
Gly Thr Val Trp Asp Ser Leu Ile Tyr Asp Pro Val Thr Asp Leu Val
275 280 285
Tyr Leu Gly Val Gly Asn Gly Ser Pro Trp Asn Tyr Lys Phe Arg Ser
290 295 300
Glu Gly Lys Gly Asn Asn Leu Phe Leu Gly Ser Ile Val Ala Ile Asn
305 310 315 320
Pro Asp Thr Gly Lys Tyr Val Trp His Phe Gln Glu Thr Pro Met Asp
325 330 335
Gln Trp Asp Tyr Thr Ser Val Gln Gln Ile Met Ala Leu Asp Met Pro
340 345 350
Val Asn Gly Glu Met Arg His Val Leu Val His Ala Pro Lys Asn Gly
355 360 365
Phe Phe Tyr Ile Ile Asp Ala Lys Thr Gly Lys Phe Ile Ser Gly Lys
370 375 380
Pro Tyr Thr Tyr Glu Asn Trp Ala Asn Gly Leu Asp Pro Val Thr Gly
385 390 395 400
Arg Pro Asn Tyr Asn Pro Asp Ala Leu Trp Thr Leu Asn Gly Lys Pro
405 410 415
Trp Tyr Gly Ile Pro Gly Asp Leu Gly Gly His Asn Phe Ala Ala Met
420 425 430
Ala Tyr Ser Pro Gln Thr Lys Leu Val Tyr Ile Pro Ala Gln Gln Val
435 440 445
Pro Phe Val Tyr Asp Pro Gln Lys Gly Gly Phe Lys Ala His His Asp
450 455 460
Ser Trp Asn Leu Gly Leu Asp Met Asn Lys Ile Gly Leu Leu Asp Asp
465 470 475 480
Asn Asp Pro Gln His Lys Ala Asp Lys Ala Gln Phe Leu Lys Asp Leu
485 490 495
Lys Gly Trp Ile Val Ala Trp Asp Pro Gln Lys Gln Gln Ala Ala Phe
500 505 510
Thr Val Asp His Lys Gly Pro Trp Asn Gly Gly Leu Leu Ala Thr Ala
515 520 525
Gly Gly Val Leu Phe Gln Gly Leu Ala Asn Gly Glu Phe His Ala Tyr
530 535 540
Asp Ala Thr Thr Gly Lys Asp Leu Phe Thr Phe Pro Ala Gln Ser Ala
545 550 555 560
Ile Ile Ala Pro Pro Val Thr Tyr Thr Ala Asn Gly Lys Gln Tyr Val
565 570 575
Ala Val Glu Val Gly Trp Gly Gly Ile Tyr Pro Phe Phe Leu Gly Gly
580 585 590
Val Ala Arg Thr Ser Gly Trp Thr Val Asn His Ser Arg Ile Ile Ala
595 600 605
Phe Ala Leu Asp Gly Asn Asp Lys Leu Pro Ala Lys Asn Glu Leu Gly
610 615 620
Phe Val Pro Val Lys Pro Pro Glu Lys Trp Asp Glu Ala Lys Ile Lys
625 630 635 640
Asp Gly Tyr Phe Gln Phe Gln Thr Tyr Cys Ala Ala Cys His Gly Asp
645 650 655
Asn Gly Ile Ser Gly Gly Val Leu Pro Asp Leu Arg Trp Ser Gly Ala
660 665 670
Ile Arg Gly Glu Glu Lys Phe Tyr Lys Leu Val Gly Lys Gly Ala Leu
675 680 685
Thr Ala Tyr Gly Met Asp Arg Phe Asp Thr Ser Met Ser Pro Ala Glu
690 695 700
Ile Glu Asp Ile Arg Asn Phe Leu Val Lys Arg Ala Asn Glu Ser Tyr
705 710 715 720
Ala Asp Glu Val Lys Ala Arg Lys Asn Glu Ala Gly Val Pro Asn Gly
725 730 735
Glu Phe Leu Asn Val Pro Gln Gly Ser Val Ala Pro Ala Thr Pro Asp
740 745 750
His Pro

5

478

PRT

Gluconobacter suboxydans

5
Met Leu Lys Ala Leu Thr Arg Asp Arg Leu Val Ser Glu Met Lys Gln
1 5 10 15
Gly Trp Lys Tyr Ala Ala Ala Val Gly Leu Met Ala Val Ser Phe Gly
20 25 30
Ala Ala Gln Ala Gln Asp Ala Asp Asp Ala Leu Ile Gln Arg Gly Ala
35 40 45
Tyr Val Ala Arg Leu Ser Asp Cys Val Ala Cys His Thr Ala Leu His
50 55 60
Gly Gln Pro Phe Ala Gly Gly Leu Glu Ile Lys Ser Pro Ile Gly Thr
65 70 75 80
Ile Tyr Ser Thr Asn Ile Thr Pro Asp Pro Lys Tyr Gly Ile Gly Asn
85 90 95
Tyr Thr Leu Glu Asp Phe Thr Lys Ala Ile Arg Lys Gly Ile Arg Lys
100 105 110
Asp Gly Ala Thr Val Tyr Pro Ala Met Pro Tyr Pro Glu Phe Ala Arg
115 120 125
Leu Ser Asp Asp Asp Ile Lys Ala Met Tyr Ala Phe Phe Met His Gly
130 135 140
Val Lys Pro Val Ala Leu Gln Asn Lys Gln Pro Asp Ile Ser Trp Pro
145 150 155 160
Met Asn Met Arg Trp Pro Leu Ala Ile Trp Arg Ala Met Phe Val Pro
165 170 175
Thr Val Thr Pro Gly Leu Asp Lys Ser Ile Ser Asp Pro Glu Val Ala
180 185 190
Arg Gly Glu Tyr Leu Val Asn Gly Pro Gly His Cys Gly Glu Cys His
195 200 205
Thr Pro Arg Gly Met Ala Met Gln Val Lys Gly Tyr Thr Ala Lys Asp
210 215 220
Gly Asn Ala Tyr Leu Ser Gly Gly Ala Pro Ile Asp Asn Trp Ile Ala
225 230 235 240
Pro Ser Leu Arg Ser Asn Ser Asp Thr Gly Leu Gly Arg Trp Ser Glu
245 250 255
Asp Asp Ile Ala Glu Phe Leu Lys Ser Gly Arg Ile Asp His Ser Ala
260 265 270
Val Phe Gly Gly Met Ala Asp Val Val Ala Tyr Ser Thr Gln His Trp
275 280 285
Thr Asp Asp Asp Leu His Ala Thr Ala Lys Tyr Leu Lys Ser Met Pro
290 295 300
Ala Val Pro Glu Gly Lys Asn Leu Gly Gln Asp Asp Gly Lys Ala Thr
305 310 315 320
Ala Leu Leu Glu Ala Gly Gly Lys Gly Asp Ala Gly Ala Glu Val Tyr
325 330 335
Leu His Asn Cys Ala Ile Cys His Met Asn Asp Gly Thr Gly Val Asn
340 345 350
Arg Met Phe Pro Pro Leu Ala Gly Asn Pro Val Val Ile Thr Asp Asn
355 360 365
Ala Thr Ser Met Ala Asn Ile Val Thr Phe Gly Gly Ile Leu Pro Pro
370 375 380
Thr Asn Thr Ala Pro Ser Ala Val Ala Met Pro Gly Phe Arg Asp His
385 390 395 400
Leu Ser Asp Gln Gln Ile Ala Asp Val Val Asn Phe Met Arg Lys Ser
405 410 415
Trp Gly Asn Gln Ala Pro Gly Thr Leu Ser Ala Ser Asp Ile Arg Lys
420 425 430
Leu Arg Thr Ser Gly Thr Ala Val Ser Thr Ala Gly Trp Asn Val Ser
435 440 445
Ser Lys Gly Trp Met Ala Tyr Met Pro Gln Pro Tyr Gly Glu Gly Trp
450 455 460
Thr Phe Ser Pro Gln Thr His Thr Gly Val Asp Gln Ala Gln
465 470 475

6

306

PRT

Gluconobacter suboxydans

6
Met Lys Thr Lys Thr Leu Pro Leu Ala Leu Leu Ser Leu Ala Leu Gly
1 5 10 15
Gly Thr Ala Leu Ser Asp Pro Ala Ala Ala Ala Gly Thr Pro Leu Lys
20 25 30
Ile Gly Val Ser Phe Gln Glu Met Asn Asn Pro Tyr Phe Val Thr Met
35 40 45
Lys Asp Ala Leu Gln Glu Ala Ala Gly Thr Ile Gly Ala Asn Val Ile
50 55 60
Ile Ser Asp Ala His His Asp Val Ser Lys Gln Val Ser Asp Ile Glu
65 70 75 80
Asp Met Ile Gln Gln Gly Ala Gln Ile Ile Ile Ile Asn Pro Thr Asp
85 90 95
Thr Val Gly Val Thr Ser Val Val Lys Ser Val His Asp Lys Asn Ile
100 105 110
Pro Ile Val Ser Val Asp Ala Gln Ala Gly Gly Pro Leu Asp Ala Phe
115 120 125
Val Gly Ser Lys Asn Tyr Asp Ala Gly Phe Lys Ala Cys Glu Tyr Leu
130 135 140
Ala Thr Thr Ile Lys Ser Gly Asn Ile Gly Ile Ile Asp Gly Ile Pro
145 150 155 160
Val Val Pro Ile Leu Glu Arg Val Lys Gly Cys Lys Asn Ala Ile Ala
165 170 175
Lys His Ser Asp Ile Lys Ile Val Ser Val Gln Asn Gly Lys Gln Glu
180 185 190
Arg Asp Glu Ala Leu Thr Val Ala Glu Asn Met Leu Gln Ala Asn Pro
195 200 205
Asp Leu Lys Gly Ile Phe Ser Val Asn Asp Asn Gly Ser Leu Gly Val
210 215 220
Leu Ser Ala Ile Glu Ser Ser Gly Ser Asp Val Lys Leu Val Ser Val
225 230 235 240
Asp Gly Asn Pro Glu Ala Val Lys Ala Ile Tyr Lys Pro Gly Ser His
245 250 255
Phe Ile Ala Thr Ala Ala Gln Phe Pro Arg Gln Asp Ile Arg Leu Ala
260 265 270
Leu Ala Leu Ala Leu Ala Arg Lys Trp Gly Ala Gly Val Pro Lys Val
275 280 285
Leu Pro Val Asp Val Glu Leu Ile Asp Ala Thr Lys Ala Lys Thr Phe
290 295 300
Ser Trp
305

7

4830

DNA

Gluconobacter suboxydans

7
cgagaacgga agcccgctga aatcgacccg ttccccatca aaatactttt cgagaagatc 60
acgaaccttc accaggagcg gcgtctcttc ctgatcgcgc ccccaccccc aatcgagagc 120
aacaatacgc ccgtcatctt cactgatggt cagggctccg agatgggaat ggcaggaaag 180
ctgtggcata cagatacgct gccccatccc ccggaaagcg tcaatcatgc ttccctaaaa 240
gagtccctga gaaaaaaata catgcgtgtc acgcatatgc agggaggccg gtattctcaa 300
ataacatatg ggatcatttt tgtatgattt catgaaatat tacgcacttt gttgagaaac 360
tgccattttt tgtgtcaaac ctgcgacaga cactaaagct gttttggttg tttggttatt 420
aagaataatt ctcatgtaat taagcgagcg attttacgcg gatagtgctc acggagacgt 480
cagaagccca cgtttccgac aaacaataaa ataagcgagt agtaagttca cgcgatgcta 540
cgttttccag acgacttgga gaaactgagg agcacctagg cacccacaga ggcgcctatc 600
aggacttgga ttacgtctga ataccattaa caggaacagt ctttgcaaaa aggacagtcg 660
gatcatggtt tctggtctac tgacgccgat caacgttacg aagaagcgcc ttctgggttg 720
cgctgctgct ctggcattct gcgccacctc tcctgtcgcc ctggctgagg acacaggaac 780
agccattaca aacgccgacc agcatccggg tgactggatg agctatggcc ggacctattc 840
cgagcagcgc tacagcccgc tggatcagat caccaaggac aatgcgagca atctgaagct 900
ggcatggcac tacgatctgg ataccaaccg tggtcaggaa ggtacgccgc tgatcgttga 960
tggcgtcatg tacgccacca caaactggag caagatgaag gctctggatg cagctacggg 1020
caagctgctg tggtcttacg atccaaaggt tccaggcaac atcgccgacc gcggctgctg 1080
cgatacggtc aaccgtggtg cagcctactg gaacggcaaa gtctatttcg gcaccttcga 1140
cggtcgcctg attgccctgg atgccaagac cggcaagctg gtctggagcg tctatacggt 1200
tcccaaggaa gcgcagctgg gtcaccagcg ctcctacacg gttgacggtg ctccccgtat 1260
cgccaagggc aaggtcatca tcggcaacgg cggtgcagag ttcggcgccc gtggcttcgt 1320
gacggcgtat gacgctgaaa cgggaaagat ggactggcgc ttcttcaccg ttccgaaccc 1380
tgacaacaag ccggacggcg cagcgtctga cgacgtgctg atgtccaagg cttatccgac 1440
atggggcaag ggcggcgcgt ggaagcagca gggcggtggc ggtaccgtct gggattcgct 1500
gatctatgac cctgtaacgg atctcgtcta tctgggcgtc ggtaacggct cgccatggaa 1560
ctacaagttc cgttctgaag gaaaaggcaa caacctcttc ctcggcagca tcgtggccat 1620
caatcctgac accggcaaat acgtctggca tttccaggaa acgccaatgg accagtggga 1680
ttatacctcg gttcagcaga tcatggccct cgacatgccg gtcaatggcg aaatgcgcca 1740
tgtgctcgtg catgcgccga agaacggctt cttctatatc attgatgcca agaccggtaa 1800
gttcatctcc ggcaagccgt acacctacga gaactgggcc aatggcctcg atccggtaac 1860
gggtcgtccg aactacaatc cagatgctct ctggacgctg aacggcaagc cctggtacgg 1920
catccccggc gatctgggtg gtcataactt cgctgccatg gcttacagcc cacagacgaa 1980
gctggtttac attcccgccc agcaggttcc cttcgtttac gatccgcaga agggtggctt 2040
caaggctcac cacgacagct ggaaccttgg cctcgacatg aacaagatcg gcctgcttga 2100
tgacaacgat ccacagcaca aggctgacaa ggcccagttc ctgaaggatc tgaagggctg 2160
gatcgttgca tgggatccgc agaagcagca ggcagccttc acggttgacc acaagggtcc 2220
gtggaatggc ggtcttctgg caacggctgg tggcgttctg ttccagggtc tcgccaacgg 2280
tgagttccac gcctacgacg cgacgacggg taaggatctc ttcaccttcc cagcacagag 2340
cgccatcatt gccccgccag tcacctacac agccaacggc aagcagtatg ttgcggttga 2400
agtgggctgg ggcggtatct atccgttctt cctgggcggc gtagcccgta cgtccggctg 2460
gaccgtcaac cactcccgga tcatcgcgtt cgctctggac ggcaacgaca agctgccagc 2520
caagaacgag ctcggcttcg ttccagtgaa gccgcctgag aaatgggatg aagccaagat 2580
caaggacggc tacttccagt tccagaccta ttgcgcagcc tgccatggtg acaacggtat 2640
ctccggcggt gttctgccag acctgcgctg gtccggtgcg atccgtggag aggagaagtt 2700
ctacaagctc gtcggcaagg gtgctctaac ggcctacggt atggaccgtt tcgacacgtc 2760
catgtcgcca gctgaaatcg aagacatccg caacttcctt gtgaagcgcg ccaacgagtc 2820
ctacgcagac gaagtcaagg cccgaaagaa tgaggcaggc gtccctaacg gcgaattcct 2880
caacgtccct cagggttcgg ttgcgcctgc aacgccggac catccgtaac gggaaaccgt 2940
cacgctgaaa ggaatgacgt gacatgctca aggcattaac tcgggacaga ctggtatctg 3000
agatgaaaca gggatggaaa tacgcggccg cagtcggcct catggcagtg tctttcggtg 3060
ctgcccaagc ccaggacgct gatgacgccc tgattcagcg cggtgcctac gtggcccgcc 3120
tgtctgactg cgttgcctgc cataccgcac tacacggcca gccttttgct ggtggtctgg 3180
agatcaagag cccgatcggc acgatctact ccaccaacat cacgcctgac ccgaaatacg 3240
gtatcggcaa ctatacactc gaagatttca cgaaggcgat ccgtaagggt atccgcaagg 3300
acggcgcgac ggtttatccg gccatgccgt atcctgagtt cgctcgcctg tctgatgacg 3360
acatcaaggc catgtatgcc ttcttcatgc atggcgtgaa gccggtcgcc cttcagaaca 3420
agcagccgga catctcctgg ccgatgaaca tgcgctggcc gttggccatc tggcgcgcga 3480
tgtttgttcc gactgtcaca ccaggcctcg acaagagcat ctccgatccg gaagtggcgc 3540
gtggcgaata cctcgtgaat ggcccaggcc attgtggcga gtgtcatacg ccccgtggca 3600
tggccatgca ggtcaagggc tatacggcca aggacggcaa cgcttacctc tccggtggcg 3660
caccgatcga caactggatt gctcccagcc tgcgtagcaa tagcgacacg ggtctgggtc 3720
gctggtctga agacgacatt gccgagttcc tgaagagcgg ccgtatcgac cattctgccg 3780
tcttcggtgg catggctgac gtggtggcct acagcaccca gcactggacc gacgacgatc 3840
tgcacgcaac ggccaagtac ctgaagagca tgccggccgt tccggaaggc aaaaacctgg 3900
gtcaggatga cggcaaggcc acggccctgc tcgaagccgg tggcaagggt gatgcaggcg 3960
cagaggttta cctccacaac tgtgccatct gccatatgaa cgatggcact ggtgtcaacc 4020
gcatgttccc gccgctggct ggcaacccgg tcgtcatcac ggacaatgca acctcaatgg 4080
ccaacatcgt gacattcggc ggtattctgc ctccgacgaa tacggcgcca tctgctgttg 4140
ccatgccggg cttccgcgat catctgtctg accagcagat cgccgatgtt gtgaacttca 4200
tgcgcaagag ctggggcaac caggctccgg gaaccctgtc tgcctcggat atccgcaagc 4260
tccgcacatc gggtactgcg gtttccacgg ccggctggaa cgtctcttcc aagggctgga 4320
tggcctacat gccgcagcct tatggcgaag gctggacctt ctccccgcag acacacacgg 4380
gcgtggatca ggctcagtaa gcctctccag accctgtcag tctgacagaa aagggcggtc 4440
cggatacggg ccgccttttt cttttgtatt caggccgttt tcacaggatg gcatcctgca 4500
ctacatatga aggcatgact cccttctcgt cttctgctcc gcagtcagtt tttcctttgg 4560
caacaggcgc aggccgcgca gcattgccat catgcgcgcc aggacccgga agcgccgagc 4620
ttctcaagag cctctgcggt tcccttcctg accctcgacg cgccacctgc gcagctgcgc 4680
catcaaggcg aagtgctgga tcatgccgtg gttctctggc ttcctggacc gaaatcctac 4740
accggcgaag acgggtgtcg aactccacct tcatgctgga cccgctgtta tcactcgcgt 4800
tgcggatgct ctgaccgatc tgggtgcacg 4830

8

2700

DNA

Gluconobacter suboxydans

8
cgaagcgatc gggacttttc agggagccgg gaagctggcc gcctgtctgg atcttctcgc 60
cacactgacg tctgctccgt tcaggacgct gtccctcaaa ggggagacca aggctctcaa 120
cagcccggaa atgcagcgga taagcggtgt catcacatcc ctgatggcca tggacccatc 180
cgaaatccgg catgaccatc tggcgcagga tctgggcctc tgcgcatcca ccttctctcg 240
ccagttccgt tctgcgacgg gcgacacatt catgtctttc ctgcatcggc tgcgggtgtg 300
tcacgcgtgc catctgctgg ccagttccac actctcaatc acggaaatcg gggctgcttc 360
cggcttcaac aacctgtcca attttaaccg catcttcctg cgcctgcgtg gctgcacgcc 420
acgggaatac cgccgtcatg cccgcgaaat gacagccctt tcgccgacag acgccgcaga 480
tttcctgaac tgaccgacaa gggaaaacag atcatgccaa cacttccaca acgtttttcc 540
ctcgatggtc gcaaagctct tgtcacgggt gcatcccgcg ggcttggtgt tacgatctgc 600
gacgttctga gtgctgcggg ggccgatatt gtcgccgttg cgcgttctga aaccgacatg 660
gccgccacat gccggatcgt ggaaggccat ggtcgtcaat gcctcacggt tgttgccgat 720
ctcagtgatc cgatggctcc ggacgctgtc gcgcagacag tgaacgcagc gtggggtggg 780
gtggatattg tcgtcaacaa tgctggcgtc agtttccctc gccctctggt ggaacagacc 840
gtcgaggagt gggacaccgt gcaggccatt aacctgcgtg cgccatggct tctcgcccgt 900
gtcttcgctc cgggcatgat tgaacgcaag cgtgggaaaa tcatcaacat cagttcccag 960
gccagctctg tcgcgctgat tgaccatggt gcttacgtcg catccaaggc cggtctgaac 1020
ggtctcacca aggtcatgac ggcggaatgg gcggctcata acatacaggc caatgccatc 1080
tgccccacag tcgtctggac gcccatgggt gaacgcgtct ggagcgttgg gaacaagctg 1140
gaaaagctac tggaaaagat ccccgctggc cgtgtcgcaa caccggaaga tgtcgcggat 1200
atagttctgt atctcgcctc cgacgcgtcg agcatggtca acgggcagga aatatttgtc 1260
gatggcggat acacagccct ttaggccgcc acatcttcaa ataaagacat gtgattttac 1320
ggttttaaca aggccatgtg cagggaatgg cctgcgcatt tcatgcagat caacaggtgt 1380
aacatgaaga ccaagacgct tccgctggcc cttctgtctc tcgctcttgg cgggacggcc 1440
ctgtcagacc cggctgccgc agctggaacc ccgctgaaaa ttggcgtttc ctttcaggaa 1500
atgaacaatc cgtacttcgt caccatgaag gacgcattgc aggaagccgc tggcacgatc 1560
ggtgcgaatg tcatcatcag tgatgcgcat catgatgttt ccaagcaggt gagtgacatt 1620
gaggacatga tccagcaggg cgcgcagatc atcatcatca acccgaccga tacagtaggc 1680
gtcacgtccg tcgtaaagag cgttcatgac aagaacatcc cgatcgtctc ggtggatgct 1740
caggctggcg gtccgctcga tgcgtttgtg gggtccaaga actatgatgc cggcttcaag 1800
gcctgcgagt atctcgccac gacgatcaag agcggcaata tcggaatcat cgacggtatc 1860
ccggtcgttc ccattcttga gcgtgtcaaa ggctgcaaaa acgctatcgc caagcattca 1920
gatatcaaga ttgtcagcgt tcagaacggc aagcaggagc gcgatgaggc tctgacggtg 1980
gctgaaaaca tgctccaggc caacccggat ctgaaaggta tcttcagcgt caatgacaac 2040
ggatcgctcg gtgtgctgtc cgctatcgaa tccagtggtt cagacgtgaa gctggtcagc 2100
gttgatggca acccggaagc cgtgaaggcc atctacaagc caggctctca tttcatcgct 2160
acggctgcgc agttcccccg gcaggatatc cgtctggcac tggcgctcgc ccttgccagg 2220
aaatggggcg caggcgtgcc gaaggtcctg cctgttgatg tcgagctgat cgacgcgacg 2280
aaagccaaga cgttcagctg gtaaattccg aaggcggccc cgaattccgg agggaacatt 2340
atgactgaat ccagtcagac atctccagaa cttcttctgg cgcttgaggg aatctccaag 2400
agttttccgg gagtccgggc gttgcggaat gtcagcctca gcctggagcg tggagaaatc 2460
catgctctgc tgggggaaaa cggcgctgga aaatccacga tcatcaagat catgggcggt 2520
atccagtctc aggatgaagg gcagatcttt ctcaacggaa aggagcgcca cttctccagc 2580
tacaaggatg ccatcagcgc aggtatcggg attgtttttc aggaattcag cctgattcct 2640
gaactcgatg ccgtggataa tattttcctc ggtcgtgaga tgcggaacgc tcttggcttt 2700

9

15

PRT

Gluconobacter suboxydans

9
Glu Asp Thr Gly Thr Ala Ile Thr Asn Ala Asp Gln His Pro Gly
1 5 10 15

10

10

PRT

Gluconobacter suboxydans

10
Asp Ala Asp Asp Ala Leu Ile Gln Arg Gly
1 5 10

11

25

PRT

Gluconobacter suboxydans

11
Ala Gly Thr Pro Leu Lys Ile Gly Val Ser Phe Gln Glu Met Asn Asn
1 5 10 15
Pro Tyr Phe Val Thr Met Lys Asp Ala
20 25

12

5

PRT

Gluconobacter suboxydans

12
His Ser Asp Ile Lys
1 5

13

7

PRT

Gluconobacter suboxydans

13
Asn Tyr Asp Ala Gly Phe Lys
1 5

14

8

PRT

Gluconobacter suboxydans

14
Lys Trp Gly Ala Gly Val Pro Lys
1 5

15

26

DNA

Gluconobacter suboxydans

modified_base

(18)

i

15
ccggaattcg argayacngg nacngc 26

16

23

DNA

Gluconobacter suboxydans

modified_base

(6)

i

16
athacnaayg cngaycarca rcc 23

17

29

PRT

Gluconobacter suboxydans

UNSURE

(1)

may be D, E, or N

17
Xaa Trp Xaa Xaa Xaa Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Xaa
1 5 10 15
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn Xaa Xaa Xaa Leu Xaa
20 25

18

22

PRT

Gluconobacter suboxydans

UNSURE

(2)..(5)

may be any amino acid

18
Trp Xaa Xaa Xaa Xaa Tyr Asp Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
1 5 10 15
Xaa Gly Xaa Xaa Xaa Pro
20

19

5

PRT

Gluconobacter suboxydans

UNSURE

(2)

may be any amino acid

19
Cys Xaa Xaa Cys His
1 5

20

25

DNA

Gluconobacter suboxydans

20
gggaattctt ycargaratg aayaa 25

21

25

DNA

Gluconobacter suboxydans

21
gggaattctt gaarccngcr tcrta 25

22

2163

DNA

Gluconobacter suboxydans

22
gaggacacag gaacagccat tacaaacgcc gaccagcatc cgggtgactg gatgagctat 60
ggccggacct attccgagca gcgctacagc ccgctggatc agatcaccaa ggacaatgcg 120
agcaatctga agctggcatg gcactacgat ctggatacca accgtggtca ggaaggtacg 180
ccgctgatcg ttgatggcgt catgtacgcc accacaaact ggagcaagat gaaggctctg 240
gatgcagcta cgggcaagct gctgtggtct tacgatccaa aggttccagg caacatcgcc 300
gaccgcggct gctgcgatac ggtcaaccgt ggtgcagcct actggaacgg caaagtctat 360
ttcggcacct tcgacggtcg cctgattgcc ctggatgcca agaccggcaa gctggtctgg 420
agcgtctata cggttcccaa ggaagcgcag ctgggtcacc agcgctccta cacggttgac 480
ggtgctcccc gtatcgccaa gggcaaggtc atcatcggca acggcggtgc agagttcggc 540
gcccgtggct tcgtgacggc gtatgacgct gaaacgggaa agatggactg gcgcttcttc 600
accgttccga accctgacaa caagccggac ggcgcagcgt ctgacgacgt gctgatgtcc 660
aaggcttatc cgacatgggg caagggcggc gcgtggaagc agcagggcgg tggcggtacc 720
gtctgggatt cgctgatcta tgaccctgta acggatctcg tctatctggg cgtcggtaac 780
ggctcgccat ggaactacaa gttccgttct gaaggaaaag gcaacaacct cttcctcggc 840
agcatcgtgg ccatcaatcc tgacaccggc aaatacgtct ggcatttcca ggaaacgcca 900
atggaccagt gggattatac ctcggttcag cagatcatgg ccctcgacat gccggtcaat 960
ggcgaaatgc gccatgtgct cgtgcatgcg ccgaagaacg gcttcttcta tatcattgat 1020
gccaagaccg gtaagttcat ctccggcaag ccgtacacct acgagaactg ggccaatggc 1080
ctcgatccgg taacgggtcg tccgaactac aatccagatg ctctctggac gctgaacggc 1140
aagccctggt acggcatccc cggcgatctg ggtggtcata acttcgctgc catggcttac 1200
agcccacaga cgaagctggt ttacattccc gcccagcagg ttcccttcgt ttacgatccg 1260
cagaagggtg gcttcaaggc tcaccacgac agctggaacc ttggcctcga catgaacaag 1320
atcggcctgc ttgatgacaa cgatccacag cacaaggctg acaaggccca gttcctgaag 1380
gatctgaagg gctggatcgt tgcatgggat ccgcagaagc agcaggcagc cttcacggtt 1440
gaccacaagg gtccgtggaa tggcggtctt ctggcaacgg ctggtggcgt tctgttccag 1500
ggtctcgcca acggtgagtt ccacgcctac gacgcgacga cgggtaagga tctcttcacc 1560
ttcccagcac agagcgccat cattgccccg ccagtcacct acacagccaa cggcaagcag 1620
tatgttgcgg ttgaagtggg ctggggcggt atctatccgt tcttcctggg cggcgtagcc 1680
cgtacgtccg gctggaccgt caaccactcc cggatcatcg cgttcgctct ggacggcaac 1740
gacaagctgc cagccaagaa cgagctcggc ttcgttccag tgaagccgcc tgagaaatgg 1800
gatgaagcca agatcaagga cggctacttc cagttccaga cctattgcgc agcctgccat 1860
ggtgacaacg gtatctccgg cggtgttctg ccagacctgc gctggtccgg tgcgatccgt 1920
ggagaggaga agttctacaa gctcgtcggc aagggtgctc taacggccta cggtatggac 1980
cgtttcgaca cgtccatgtc gccagctgaa atcgaagaca tccgcaactt ccttgtgaag 2040
cgcgccaacg agtcctacgc agacgaagtc aaggcccgaa agaatgaggc aggcgtccct 2100
aacggcgaat tcctcaacgt ccctcagggt tcggttgcgc ctgcaacgcc ggaccatccg 2160
taa 2163

23

1329

DNA

Gluconobacter suboxydans

23
caggacgctg atgacgccct gattcagcgc ggtgcctacg tggcccgcct gtctgactgc 60
gttgcctgcc ataccgcact acacggccag ccttttgctg gtggtctgga gatcaagagc 120
ccgatcggca cgatctactc caccaacatc acgcctgacc cgaaatacgg tatcggcaac 180
tatacactcg aagatttcac gaaggcgatc cgtaagggta tccgcaagga cggcgcgacg 240
gtttatccgg ccatgccgta tcctgagttc gctcgcctgt ctgatgacga catcaaggcc 300
atgtatgcct tcttcatgca tggcgtgaag ccggtcgccc ttcagaacaa gcagccggac 360
atctcctggc cgatgaacat gcgctggccg ttggccatct ggcgcgcgat gtttgttccg 420
actgtcacac caggcctcga caagagcatc tccgatccgg aagtggcgcg tggcgaatac 480
ctcgtgaatg gcccaggcca ttgtggcgag tgtcatacgc cccgtggcat ggccatgcag 540
gtcaagggct atacggccaa ggacggcaac gcttacctct ccggtggcgc accgatcgac 600
aactggattg ctcccagcct gcgtagcaat agcgacacgg gtctgggtcg ctggtctgaa 660
gacgacattg ccgagttcct gaagagcggc cgtatcgacc attctgccgt cttcggtggc 720
atggctgacg tggtggccta cagcacccag cactggaccg acgacgatct gcacgcaacg 780
gccaagtacc tgaagagcat gccggccgtt ccggaaggca aaaacctggg tcaggatgac 840
ggcaaggcca cggccctgct cgaagccggt ggcaagggtg atgcaggcgc agaggtttac 900
ctccacaact gtgccatctg ccatatgaac gatggcactg gtgtcaaccg catgttcccg 960
ccgctggctg gcaacccggt cgtcatcacg gacaatgcaa cctcaatggc caacatcgtg 1020
acattcggcg gtattctgcc tccgacgaat acggcgccat ctgctgttgc catgccgggc 1080
ttccgcgatc atctgtctga ccagcagatc gccgatgttg tgaacttcat gcgcaagagc 1140
tggggcaacc aggctccggg aaccctgtct gcctcggata tccgcaagct ccgcacatcg 1200
ggtactgcgg tttccacggc cggctggaac gtctcttcca agggctggat ggcctacatg 1260
ccgcagcctt atggcgaagg ctggaccttc tccccgcaga cacacacggg cgtggatcag 1320
gctcagtaa 1329

24

843

DNA

Gluconobacter suboxydans

24
gctggaaccc cgctgaaaat tggcgtttcc tttcaggaaa tgaacaatcc gtacttcgtc 60
accatgaagg acgcattgca ggaagccgct ggcacgatcg gtgcgaatgt catcatcagt 120
gatgcgcatc atgatgtttc caagcaggtg agtgacattg aggacatgat ccagcagggc 180
gcgcagatca tcatcatcaa cccgaccgat acagtaggcg tcacgtccgt cgtaaagagc 240
gttcatgaca agaacatccc gatcgtctcg gtggatgctc aggctggcgg tccgctcgat 300
gcgtttgtgg ggtccaagaa ctatgatgcc ggcttcaagg cctgcgagta tctcgccacg 360
acgatcaaga gcggcaatat cggaatcatc gacggtatcc cggtcgttcc cattcttgag 420
cgtgtcaaag gctgcaaaaa cgctatcgcc aagcattcag atatcaagat tgtcagcgtt 480
cagaacggca agcaggagcg cgatgaggct ctgacggtgg ctgaaaacat gctccaggcc 540
aacccggatc tgaaaggtat cttcagcgtc aatgacaacg gatcgctcgg tgtgctgtcc 600
gctatcgaat ccagtggttc agacgtgaag ctggtcagcg ttgatggcaa cccggaagcc 660
gtgaaggcca tctacaagcc aggctctcat ttcatcgcta cggctgcgca gttcccccgg 720
caggatatcc gtctggcact ggcgctcgcc cttgccagga aatggggcgc aggcgtgccg 780
aaggtcctgc ctgttgatgt cgagctgatc gacgcgacga aagccaagac gttcagctgg 840
taa 843

25

720

PRT

Gluconobacter suboxydans

25
Glu Asp Thr Gly Thr Ala Ile Thr Asn Ala Asp Gln His Pro Gly Asp
1 5 10 15
Trp Met Ser Tyr Gly Arg Thr Tyr Ser Glu Gln Arg Tyr Ser Pro Leu
20 25 30
Asp Gln Ile Thr Lys Asp Asn Ala Ser Asn Leu Lys Leu Ala Trp His
35 40 45
Tyr Asp Leu Asp Thr Asn Arg Gly Gln Glu Gly Thr Pro Leu Ile Val
50 55 60
Asp Gly Val Met Tyr Ala Thr Thr Asn Trp Ser Lys Met Lys Ala Leu
65 70 75 80
Asp Ala Ala Thr Gly Lys Leu Leu Trp Ser Tyr Asp Pro Lys Val Pro
85 90 95
Gly Asn Ile Ala Asp Arg Gly Cys Cys Asp Thr Val Asn Arg Gly Ala
100 105 110
Ala Tyr Trp Asn Gly Lys Val Tyr Phe Gly Thr Phe Asp Gly Arg Leu
115 120 125
Ile Ala Leu Asp Ala Lys Thr Gly Lys Leu Val Trp Ser Val Tyr Thr
130 135 140
Val Pro Lys Glu Ala Gln Leu Gly His Gln Arg Ser Tyr Thr Val Asp
145 150 155 160
Gly Ala Pro Arg Ile Ala Lys Gly Lys Val Ile Ile Gly Asn Gly Gly
165 170 175
Ala Glu Phe Gly Ala Arg Gly Phe Val Thr Ala Tyr Asp Ala Glu Thr
180 185 190
Gly Lys Met Asp Trp Arg Phe Phe Thr Val Pro Asn Pro Asp Asn Lys
195 200 205
Pro Asp Gly Ala Ala Ser Asp Asp Val Leu Met Ser Lys Ala Tyr Pro
210 215 220
Thr Trp Gly Lys Gly Gly Ala Trp Lys Gln Gln Gly Gly Gly Gly Thr
225 230 235 240
Val Trp Asp Ser Leu Ile Tyr Asp Pro Val Thr Asp Leu Val Tyr Leu
245 250 255
Gly Val Gly Asn Gly Ser Pro Trp Asn Tyr Lys Phe Arg Ser Glu Gly
260 265 270
Lys Gly Asn Asn Leu Phe Leu Gly Ser Ile Val Ala Ile Asn Pro Asp
275 280 285
Thr Gly Lys Tyr Val Trp His Phe Gln Glu Thr Pro Met Asp Gln Trp
290 295 300
Asp Tyr Thr Ser Val Gln Gln Ile Met Ala Leu Asp Met Pro Val Asn
305 310 315 320
Gly Glu Met Arg His Val Leu Val His Ala Pro Lys Asn Gly Phe Phe
325 330 335
Tyr Ile Ile Asp Ala Lys Thr Gly Lys Phe Ile Ser Gly Lys Pro Tyr
340 345 350
Thr Tyr Glu Asn Trp Ala Asn Gly Leu Asp Pro Val Thr Gly Arg Pro
355 360 365
Asn Tyr Asn Pro Asp Ala Leu Trp Thr Leu Asn Gly Lys Pro Trp Tyr
370 375 380
Gly Ile Pro Gly Asp Leu Gly Gly His Asn Phe Ala Ala Met Ala Tyr
385 390 395 400
Ser Pro Gln Thr Lys Leu Val Tyr Ile Pro Ala Gln Gln Val Pro Phe
405 410 415
Val Tyr Asp Pro Gln Lys Gly Gly Phe Lys Ala His His Asp Ser Trp
420 425 430
Asn Leu Gly Leu Asp Met Asn Lys Ile Gly Leu Leu Asp Asp Asn Asp
435 440 445
Pro Gln His Lys Ala Asp Lys Ala Gln Phe Leu Lys Asp Leu Lys Gly
450 455 460
Trp Ile Val Ala Trp Asp Pro Gln Lys Gln Gln Ala Ala Phe Thr Val
465 470 475 480
Asp His Lys Gly Pro Trp Asn Gly Gly Leu Leu Ala Thr Ala Gly Gly
485 490 495
Val Leu Phe Gln Gly Leu Ala Asn Gly Glu Phe His Ala Tyr Asp Ala
500 505 510
Thr Thr Gly Lys Asp Leu Phe Thr Phe Pro Ala Gln Ser Ala Ile Ile
515 520 525
Ala Pro Pro Val Thr Tyr Thr Ala Asn Gly Lys Gln Tyr Val Ala Val
530 535 540
Glu Val Gly Trp Gly Gly Ile Tyr Pro Phe Phe Leu Gly Gly Val Ala
545 550 555 560
Arg Thr Ser Gly Trp Thr Val Asn His Ser Arg Ile Ile Ala Phe Ala
565 570 575
Leu Asp Gly Asn Asp Lys Leu Pro Ala Lys Asn Glu Leu Gly Phe Val
580 585 590
Pro Val Lys Pro Pro Glu Lys Trp Asp Glu Ala Lys Ile Lys Asp Gly
595 600 605
Tyr Phe Gln Phe Gln Thr Tyr Cys Ala Ala Cys His Gly Asp Asn Gly
610 615 620
Ile Ser Gly Gly Val Leu Pro Asp Leu Arg Trp Ser Gly Ala Ile Arg
625 630 635 640
Gly Glu Glu Lys Phe Tyr Lys Leu Val Gly Lys Gly Ala Leu Thr Ala
645 650 655
Tyr Gly Met Asp Arg Phe Asp Thr Ser Met Ser Pro Ala Glu Ile Glu
660 665 670
Asp Ile Arg Asn Phe Leu Val Lys Arg Ala Asn Glu Ser Tyr Ala Asp
675 680 685
Glu Val Lys Ala Arg Lys Asn Glu Ala Gly Val Pro Asn Gly Glu Phe
690 695 700
Leu Asn Val Pro Gln Gly Ser Val Ala Pro Ala Thr Pro Asp His Pro
705 710 715 720

26

442

PRT

Gluconobacter suboxydans

26
Gln Asp Ala Asp Asp Ala Leu Ile Gln Arg Gly Ala Tyr Val Ala Arg
1 5 10 15
Leu Ser Asp Cys Val Ala Cys His Thr Ala Leu His Gly Gln Pro Phe
20 25 30
Ala Gly Gly Leu Glu Ile Lys Ser Pro Ile Gly Thr Ile Tyr Ser Thr
35 40 45
Asn Ile Thr Pro Asp Pro Lys Tyr Gly Ile Gly Asn Tyr Thr Leu Glu
50 55 60
Asp Phe Thr Lys Ala Ile Arg Lys Gly Ile Arg Lys Asp Gly Ala Thr
65 70 75 80
Val Tyr Pro Ala Met Pro Tyr Pro Glu Phe Ala Arg Leu Ser Asp Asp
85 90 95
Asp Ile Lys Ala Met Tyr Ala Phe Phe Met His Gly Val Lys Pro Val
100 105 110
Ala Leu Gln Asn Lys Gln Pro Asp Ile Ser Trp Pro Met Asn Met Arg
115 120 125
Trp Pro Leu Ala Ile Trp Arg Ala Met Phe Val Pro Thr Val Thr Pro
130 135 140
Gly Leu Asp Lys Ser Ile Ser Asp Pro Glu Val Ala Arg Gly Glu Tyr
145 150 155 160
Leu Val Asn Gly Pro Gly His Cys Gly Glu Cys His Thr Pro Arg Gly
165 170 175
Met Ala Met Gln Val Lys Gly Tyr Thr Ala Lys Asp Gly Asn Ala Tyr
180 185 190
Leu Ser Gly Gly Ala Pro Ile Asp Asn Trp Ile Ala Pro Ser Leu Arg
195 200 205
Ser Asn Ser Asp Thr Gly Leu Gly Arg Trp Ser Glu Asp Asp Ile Ala
210 215 220
Glu Phe Leu Lys Ser Gly Arg Ile Asp His Ser Ala Val Phe Gly Gly
225 230 235 240
Met Ala Asp Val Val Ala Tyr Ser Thr Gln His Trp Thr Asp Asp Asp
245 250 255
Leu His Ala Thr Ala Lys Tyr Leu Lys Ser Met Pro Ala Val Pro Glu
260 265 270
Gly Lys Asn Leu Gly Gln Asp Asp Gly Lys Ala Thr Ala Leu Leu Glu
275 280 285
Ala Gly Gly Lys Gly Asp Ala Gly Ala Glu Val Tyr Leu His Asn Cys
290 295 300
Ala Ile Cys His Met Asn Asp Gly Thr Gly Val Asn Arg Met Phe Pro
305 310 315 320
Pro Leu Ala Gly Asn Pro Val Val Ile Thr Asp Asn Ala Thr Ser Met
325 330 335
Ala Asn Ile Val Thr Phe Gly Gly Ile Leu Pro Pro Thr Asn Thr Ala
340 345 350
Pro Ser Ala Val Ala Met Pro Gly Phe Arg Asp His Leu Ser Asp Gln
355 360 365
Gln Ile Ala Asp Val Val Asn Phe Met Arg Lys Ser Trp Gly Asn Gln
370 375 380
Ala Pro Gly Thr Leu Ser Ala Ser Asp Ile Arg Lys Leu Arg Thr Ser
385 390 395 400
Gly Thr Ala Val Ser Thr Ala Gly Trp Asn Val Ser Ser Lys Gly Trp
405 410 415
Met Ala Tyr Met Pro Gln Pro Tyr Gly Glu Gly Trp Thr Phe Ser Pro
420 425 430
Gln Thr His Thr Gly Val Asp Gln Ala Gln
435 440

27

280

PRT

Gluconobacter suboxydans

27
Ala Gly Thr Pro Leu Lys Ile Gly Val Ser Phe Gln Glu Met Asn Asn
1 5 10 15
Pro Tyr Phe Val Thr Met Lys Asp Ala Leu Gln Glu Ala Ala Gly Thr
20 25 30
Ile Gly Ala Asn Val Ile Ile Ser Asp Ala His His Asp Val Ser Lys
35 40 45
Gln Val Ser Asp Ile Glu Asp Met Ile Gln Gln Gly Ala Gln Ile Ile
50 55 60
Ile Ile Asn Pro Thr Asp Thr Val Gly Val Thr Ser Val Val Lys Ser
65 70 75 80
Val His Asp Lys Asn Ile Pro Ile Val Ser Val Asp Ala Gln Ala Gly
85 90 95
Gly Pro Leu Asp Ala Phe Val Gly Ser Lys Asn Tyr Asp Ala Gly Phe
100 105 110
Lys Ala Cys Glu Tyr Leu Ala Thr Thr Ile Lys Ser Gly Asn Ile Gly
115 120 125
Ile Ile Asp Gly Ile Pro Val Val Pro Ile Leu Glu Arg Val Lys Gly
130 135 140
Cys Lys Asn Ala Ile Ala Lys His Ser Asp Ile Lys Ile Val Ser Val
145 150 155 160
Gln Asn Gly Lys Gln Glu Arg Asp Glu Ala Leu Thr Val Ala Glu Asn
165 170 175
Met Leu Gln Ala Asn Pro Asp Leu Lys Gly Ile Phe Ser Val Asn Asp
180 185 190
Asn Gly Ser Leu Gly Val Leu Ser Ala Ile Glu Ser Ser Gly Ser Asp
195 200 205
Val Lys Leu Val Ser Val Asp Gly Asn Pro Glu Ala Val Lys Ala Ile
210 215 220
Tyr Lys Pro Gly Ser His Phe Ile Ala Thr Ala Ala Gln Phe Pro Arg
225 230 235 240
Gln Asp Ile Arg Leu Ala Leu Ala Leu Ala Leu Ala Arg Lys Trp Gly
245 250 255
Ala Gly Val Pro Lys Val Leu Pro Val Asp Val Glu Leu Ile Asp Ala
260 265 270
Thr Lys Ala Lys Thr Phe Ser Trp
275 280

28

664

DNA

Gluconobacter suboxydans

28
cgagaacgga agcccgctga aatcgacccg ttccccatca aaatactttt cgagaagatc 60
acgaaccttc accaggagcg gcgtctcttc ctgatcgcgc ccccaccccc aatcgagagc 120
aacaatacgc ccgtcatctt cactgatggt cagggctccg agatgggaat ggcaggaaag 180
ctgtggcata cagatacgct gccccatccc ccggaaagcg tcaatcatgc ttccctaaaa 240
gagtccctga gaaaaaaata catgcgtgtc acgcatatgc agggaggccg gtattctcaa 300
ataacatatg ggatcatttt tgtatgattt catgaaatat tacgcacttt gttgagaaac 360
tgccattttt tgtgtcaaac ctgcgacaga cactaaagct gttttggttg tttggttatt 420
aagaataatt ctcatgtaat taagcgagcg attttacgcg gatagtgctc acggagacgt 480
cagaagccca cgtttccgac aaacaataaa ataagcgagt agtaagttca cgcgatgcta 540
cgttttccag acgacttgga gaaactgagg agcacctagg cacccacaga ggcgcctatc 600
aggacttgga ttacgtctga ataccattaa caggaacagt ctttgcaaaa aggacagtcg 660
gatc 664

29

615

DNA

Gluconobacter suboxydans

29
tcgagaagat cacgaacctt caccaggagc ggcgtctctt cctgatcgcg cccccacccc 60
caatcgagag caacaatacg cccgtcatct tcactgatgg tcagggctcc gagatgggaa 120
tggcaggaaa gctgtggcat acagatacgc tgccccatcc cccggaaagc gtcaatcatg 180
cttccctaaa agagtccctg agaaaaaaat acatgcgtgt cacgcatatg cagggaggcc 240
ggtattctca aataacatat gggatcattt ttgtatgatt tcatgaaata ttacgcactt 300
tgttgagaaa ctgccatttt ttgtgtcaaa cctgcgacag acactaaagc tgttttggtt 360
gtttggttat taagaataat tctcatgtaa ttaagcgagc gattttacgc ggatagtgct 420
cacggagacg tcagaagccc acgtttccga caaacaataa aataagcgag tagtaagttc 480
acgcgatgct acgttttcca gacgacttgg agaaactgag gagcacctag gcacccacag 540
aggcgcctat caggacttgg attacgtctg aataccatta acaggaacag tctttgcaaa 600
aaggacagtc ggatc 615

30

565

DNA

Gluconobacter suboxydans

30
cccccacccc caatcgagag caacaatacg cccgtcatct tcactgatgg tcagggctcc 60
gagatgggaa tggcaggaaa gctgtggcat acagatacgc tgccccatcc cccggaaagc 120
gtcaatcatg cttccctaaa agagtccctg agaaaaaaat acatgcgtgt cacgcatatg 180
cagggaggcc ggtattctca aataacatat gggatcattt ttgtatgatt tcatgaaata 240
ttacgcactt tgttgagaaa ctgccatttt ttgtgtcaaa cctgcgacag acactaaagc 300
tgttttggtt gtttggttat taagaataat tctcatgtaa ttaagcgagc gattttacgc 360
ggatagtgct cacggagacg tcagaagccc acgtttccga caaacaataa aataagcgag 420
tagtaagttc acgcgatgct acgttttcca gacgacttgg agaaactgag gagcacctag 480
gcacccacag aggcgcctat caggacttgg attacgtctg aataccatta acaggaacag 540
tctttgcaaa aaggacagtc ggatc 565

31

515

DNA

Gluconobacter suboxydans

31
tcagggctcc gagatgggaa tggcaggaaa gctgtggcat acagatacgc tgccccatcc 60
cccggaaagc gtcaatcatg cttccctaaa agagtccctg agaaaaaaat acatgcgtgt 120
cacgcatatg cagggaggcc ggtattctca aataacatat gggatcattt ttgtatgatt 180
tcatgaaata ttacgcactt tgttgagaaa ctgccatttt ttgtgtcaaa cctgcgacag 240
acactaaagc tgttttggtt gtttggttat taagaataat tctcatgtaa ttaagcgagc 300
gattttacgc ggatagtgct cacggagacg tcagaagccc acgtttccga caaacaataa 360
aataagcgag tagtaagttc acgcgatgct acgttttcca gacgacttgg agaaactgag 420
gagcacctag gcacccacag aggcgcctat caggacttgg attacgtctg aataccatta 480
acaggaacag tctttgcaaa aaggacagtc ggatc 515

32

465

DNA

Gluconobacter suboxydans

32
tgccccatcc cccggaaagc gtcaatcatg cttccctaaa agagtccctg agaaaaaaat 60
acatgcgtgt cacgcatatg cagggaggcc ggtattctca aataacatat gggatcattt 120
ttgtatgatt tcatgaaata ttacgcactt tgttgagaaa ctgccatttt ttgtgtcaaa 180
cctgcgacag acactaaagc tgttttggtt gtttggttat taagaataat tctcatgtaa 240
ttaagcgagc gattttacgc ggatagtgct cacggagacg tcagaagccc acgtttccga 300
caaacaataa aataagcgag tagtaagttc acgcgatgct acgttttcca gacgacttgg 360
agaaactgag gagcacctag gcacccacag aggcgcctat caggacttgg attacgtctg 420
aataccatta acaggaacag tctttgcaaa aaggacagtc ggatc 465

33

415

DNA

Gluconobacter suboxydans

33
agaaaaaaat acatgcgtgt cacgcatatg cagggaggcc ggtattctca aataacatat 60
gggatcattt ttgtatgatt tcatgaaata ttacgcactt tgttgagaaa ctgccatttt 120
ttgtgtcaaa cctgcgacag acactaaagc tgttttggtt gtttggttat taagaataat 180
tctcatgtaa ttaagcgagc gattttacgc ggatagtgct cacggagacg tcagaagccc 240
acgtttccga caaacaataa aataagcgag tagtaagttc acgcgatgct acgttttcca 300
gacgacttgg agaaactgag gagcacctag gcacccacag aggcgcctat caggacttgg 360
attacgtctg aataccatta acaggaacag tctttgcaaa aaggacagtc ggatc 415

34

365

DNA

Gluconobacter suboxydans

34
aataacatat gggatcattt ttgtatgatt tcatgaaata ttacgcactt tgttgagaaa 60
ctgccatttt ttgtgtcaaa cctgcgacag acactaaagc tgttttggtt gtttggttat 120
taagaataat tctcatgtaa ttaagcgagc gattttacgc ggatagtgct cacggagacg 180
tcagaagccc acgtttccga caaacaataa aataagcgag tagtaagttc acgcgatgct 240
acgttttcca gacgacttgg agaaactgag gagcacctag gcacccacag aggcgcctat 300
caggacttgg attacgtctg aataccatta acaggaacag tctttgcaaa aaggacagtc 360
ggatc 365

35

315

DNA

Gluconobacter suboxydans

35
tgttgagaaa ctgccatttt ttgtgtcaaa cctgcgacag acactaaagc tgttttggtt 60
gtttggttat taagaataat tctcatgtaa ttaagcgagc gattttacgc ggatagtgct 120
cacggagacg tcagaagccc acgtttccga caaacaataa aataagcgag tagtaagttc 180
acgcgatgct acgttttcca gacgacttgg agaaactgag gagcacctag gcacccacag 240
aggcgcctat caggacttgg attacgtctg aataccatta acaggaacag tctttgcaaa 300
aaggacagtc ggatc 315

36

265

DNA

Gluconobacter suboxydans

36
tgttttggtt gtttggttat taagaataat tctcatgtaa ttaagcgagc gattttacgc 60
ggatagtgct cacggagacg tcagaagccc acgtttccga caaacaataa aataagcgag 120
tagtaagttc acgcgatgct acgttttcca gacgacttgg agaaactgag gagcacctag 180
gcacccacag aggcgcctat caggacttgg attacgtctg aataccatta acaggaacag 240
tctttgcaaa aaggacagtc ggatc 265

37

215

DNA

Gluconobacter suboxydans

37
gattttacgc ggatagtgct cacggagacg tcagaagccc acgtttccga caaacaataa 60
aataagcgag tagtaagttc acgcgatgct acgttttcca gacgacttgg agaaactgag 120
gagcacctag gcacccacag aggcgcctat caggacttgg attacgtctg aataccatta 180
acaggaacag tctttgcaaa aaggacagtc ggatc 215

38

165

DNA

Gluconobacter suboxydans

38
caaacaataa aataagcgag tagtaagttc acgcgatgct acgttttcca gacgacttgg 60
agaaactgag gagcacctag gcacccacag aggcgcctat caggacttgg attacgtctg 120
aataccatta acaggaacag tctttgcaaa aaggacagtc ggatc 165

39

115

DNA

Gluconobacter suboxydans

39
gacgacttgg agaaactgag gagcacctag gcacccacag aggcgcctat caggacttgg 60
attacgtctg aataccatta acaggaacag tctttgcaaa aaggacagtc ggatc 115

40

65

DNA

Gluconobacter suboxydans

40
caggacttgg attacgtctg aataccatta acaggaacag tctttgcaaa aaggacagtc 60
ggatc 65

41

34

DNA

Gluconobacter suboxydans

41
cgggaaaccg tcacgctgaa aggaatgacg tgac 34

42

51

DNA

Gluconobacter suboxydans

42
gcctctccag accctgtcag tctgacagaa aagggcggtc cggatacggg c 51

43

101

DNA

Gluconobacter suboxydans

43
gcctctccag accctgtcag tctgacagaa aagggcggtc cggatacggg ccgccttttt 60
cttttgtatt caggccgttt tcacaggatg gcatcctgca c 101

44

151

DNA

Gluconobacter suboxydans

44
gcctctccag accctgtcag tctgacagaa aagggcggtc cggatacggg ccgccttttt 60
cttttgtatt caggccgttt tcacaggatg gcatcctgca ctacatatga aggcatgact 120
cccttctcgt cttctgctcc gcagtcagtt t 151

45

201

DNA

Gluconobacter suboxydans

45
gcctctccag accctgtcag tctgacagaa aagggcggtc cggatacggg ccgccttttt 60
cttttgtatt caggccgttt tcacaggatg gcatcctgca ctacatatga aggcatgact 120
cccttctcgt cttctgctcc gcagtcagtt tttcctttgg caacaggcgc aggccgcgca 180
gcattgccat catgcgcgcc a 201

46

251

DNA

Gluconobacter suboxydans

46
gcctctccag accctgtcag tctgacagaa aagggcggtc cggatacggg ccgccttttt 60
cttttgtatt caggccgttt tcacaggatg gcatcctgca ctacatatga aggcatgact 120
cccttctcgt cttctgctcc gcagtcagtt tttcctttgg caacaggcgc aggccgcgca 180
gcattgccat catgcgcgcc aggacccgga agcgccgagc ttctcaagag cctctgcggt 240
tcccttcctg a 251

47

301

DNA

Gluconobacter suboxydans

47
gcctctccag accctgtcag tctgacagaa aagggcggtc cggatacggg ccgccttttt 60
cttttgtatt caggccgttt tcacaggatg gcatcctgca ctacatatga aggcatgact 120
cccttctcgt cttctgctcc gcagtcagtt tttcctttgg caacaggcgc aggccgcgca 180
gcattgccat catgcgcgcc aggacccgga agcgccgagc ttctcaagag cctctgcggt 240
tcccttcctg accctcgacg cgccacctgc gcagctgcgc catcaaggcg aagtgctgga 300
t 301

48

351

DNA

Gluconobacter suboxydans

48
gcctctccag accctgtcag tctgacagaa aagggcggtc cggatacggg ccgccttttt 60
cttttgtatt caggccgttt tcacaggatg gcatcctgca ctacatatga aggcatgact 120
cccttctcgt cttctgctcc gcagtcagtt tttcctttgg caacaggcgc aggccgcgca 180
gcattgccat catgcgcgcc aggacccgga agcgccgagc ttctcaagag cctctgcggt 240
tcccttcctg accctcgacg cgccacctgc gcagctgcgc catcaaggcg aagtgctgga 300
tcatgccgtg gttctctggc ttcctggacc gaaatcctac accggcgaag a 351

49

401

DNA

Gluconobacter suboxydans

49
gcctctccag accctgtcag tctgacagaa aagggcggtc cggatacggg ccgccttttt 60
cttttgtatt caggccgttt tcacaggatg gcatcctgca ctacatatga aggcatgact 120
cccttctcgt cttctgctcc gcagtcagtt tttcctttgg caacaggcgc aggccgcgca 180
gcattgccat catgcgcgcc aggacccgga agcgccgagc ttctcaagag cctctgcggt 240
tcccttcctg accctcgacg cgccacctgc gcagctgcgc catcaaggcg aagtgctgga 300
tcatgccgtg gttctctggc ttcctggacc gaaatcctac accggcgaag acgggtgtcg 360
aactccacct tcatgctgga cccgctgtta tcactcgcgt t 401

50

430

DNA

Gluconobacter suboxydans

50
gcctctccag accctgtcag tctgacagaa aagggcggtc cggatacggg ccgccttttt 60
cttttgtatt caggccgttt tcacaggatg gcatcctgca ctacatatga aggcatgact 120
cccttctcgt cttctgctcc gcagtcagtt tttcctttgg caacaggcgc aggccgcgca 180
gcattgccat catgcgcgcc aggacccgga agcgccgagc ttctcaagag cctctgcggt 240
tcccttcctg accctcgacg cgccacctgc gcagctgcgc catcaaggcg aagtgctgga 300
tcatgccgtg gttctctggc ttcctggacc gaaatcctac accggcgaag acgggtgtcg 360
aactccacct tcatgctgga cccgctgtta tcactcgcgt tgcggatgct ctgaccgatc 420
tgggtgcacg 430

51

1383

DNA

Gluconobacter suboxydans

51
cgaagcgatc gggacttttc agggagccgg gaagctggcc gcctgtctgg atcttctcgc 60
cacactgacg tctgctccgt tcaggacgct gtccctcaaa ggggagacca aggctctcaa 120
cagcccggaa atgcagcgga taagcggtgt catcacatcc ctgatggcca tggacccatc 180
cgaaatccgg catgaccatc tggcgcagga tctgggcctc tgcgcatcca ccttctctcg 240
ccagttccgt tctgcgacgg gcgacacatt catgtctttc ctgcatcggc tgcgggtgtg 300
tcacgcgtgc catctgctgg ccagttccac actctcaatc acggaaatcg gggctgcttc 360
cggcttcaac aacctgtcca attttaaccg catcttcctg cgcctgcgtg gctgcacgcc 420
acgggaatac cgccgtcatg cccgcgaaat gacagccctt tcgccgacag acgccgcaga 480
tttcctgaac tgaccgacaa gggaaaacag atcatgccaa cacttccaca acgtttttcc 540
ctcgatggtc gcaaagctct tgtcacgggt gcatcccgcg ggcttggtgt tacgatctgc 600
gacgttctga gtgctgcggg ggccgatatt gtcgccgttg cgcgttctga aaccgacatg 660
gccgccacat gccggatcgt ggaaggccat ggtcgtcaat gcctcacggt tgttgccgat 720
ctcagtgatc cgatggctcc ggacgctgtc gcgcagacag tgaacgcagc gtggggtggg 780
gtggatattg tcgtcaacaa tgctggcgtc agtttccctc gccctctggt ggaacagacc 840
gtcgaggagt gggacaccgt gcaggccatt aacctgcgtg cgccatggct tctcgcccgt 900
gtcttcgctc cgggcatgat tgaacgcaag cgtgggaaaa tcatcaacat cagttcccag 960
gccagctctg tcgcgctgat tgaccatggt gcttacgtcg catccaaggc cggtctgaac 1020
ggtctcacca aggtcatgac ggcggaatgg gcggctcata acatacaggc caatgccatc 1080
tgccccacag tcgtctggac gcccatgggt gaacgcgtct ggagcgttgg gaacaagctg 1140
gaaaagctac tggaaaagat ccccgctggc cgtgtcgcaa caccggaaga tgtcgcggat 1200
atagttctgt atctcgcctc cgacgcgtcg agcatggtca acgggcagga aatatttgtc 1260
gatggcggat acacagccct ttaggccgcc acatcttcaa ataaagacat gtgattttac 1320
ggttttaaca aggccatgtg cagggaatgg cctgcgcatt tcatgcagat caacaggtgt 1380
aac 1383

52

1334

DNA

Gluconobacter suboxydans

52
gatcttctcg ccacactgac gtctgctccg ttcaggacgc tgtccctcaa aggggagacc 60
aaggctctca acagcccgga aatgcagcgg ataagcggtg tcatcacatc cctgatggcc 120
atggacccat ccgaaatccg gcatgaccat ctggcgcagg atctgggcct ctgcgcatcc 180
accttctctc gccagttccg ttctgcgacg ggcgacacat tcatgtcttt cctgcatcgg 240
ctgcgggtgt gtcacgcgtg ccatctgctg gccagttcca cactctcaat cacggaaatc 300
ggggctgctt ccggcttcaa caacctgtcc aattttaacc gcatcttcct gcgcctgcgt 360
ggctgcacgc cacgggaata ccgccgtcat gcccgcgaaa tgacagccct ttcgccgaca 420
gacgccgcag atttcctgaa ctgaccgaca agggaaaaca gatcatgcca acacttccac 480
aacgtttttc cctcgatggt cgcaaagctc ttgtcacggg tgcatcccgc gggcttggtg 540
ttacgatctg cgacgttctg agtgctgcgg gggccgatat tgtcgccgtt gcgcgttctg 600
aaaccgacat ggccgccaca tgccggatcg tggaaggcca tggtcgtcaa tgcctcacgg 660
ttgttgccga tctcagtgat ccgatggctc cggacgctgt cgcgcagaca gtgaacgcag 720
cgtggggtgg ggtggatatt gtcgtcaaca atgctggcgt cagtttccct cgccctctgg 780
tggaacagac cgtcgaggag tgggacaccg tgcaggccat taacctgcgt gcgccatggc 840
ttctcgcccg tgtcttcgct ccgggcatga ttgaacgcaa gcgtgggaaa atcatcaaca 900
tcagttccca ggccagctct gtcgcgctga ttgaccatgg tgcttacgtc gcatccaagg 960
ccggtctgaa cggtctcacc aaggtcatga cggcggaatg ggcggctcat aacatacagg 1020
ccaatgccat ctgccccaca gtcgtctgga cgcccatggg tgaacgcgtc tggagcgttg 1080
ggaacaagct ggaaaagcta ctggaaaaga tccccgctgg ccgtgtcgca acaccggaag 1140
atgtcgcgga tatagttctg tatctcgcct ccgacgcgtc gagcatggtc aacgggcagg 1200
aaatatttgt cgatggcgga tacacagccc tttaggccgc cacatcttca aataaagaca 1260
tgtgatttta cggttttaac aaggccatgt gcagggaatg gcctgcgcat ttcatgcaga 1320
tcaacaggtg taac 1334

53

1284

DNA

Gluconobacter suboxydans

53
aggggagacc aaggctctca acagcccgga aatgcagcgg ataagcggtg tcatcacatc 60
cctgatggcc atggacccat ccgaaatccg gcatgaccat ctggcgcagg atctgggcct 120
ctgcgcatcc accttctctc gccagttccg ttctgcgacg ggcgacacat tcatgtcttt 180
cctgcatcgg ctgcgggtgt gtcacgcgtg ccatctgctg gccagttcca cactctcaat 240
cacggaaatc ggggctgctt ccggcttcaa caacctgtcc aattttaacc gcatcttcct 300
gcgcctgcgt ggctgcacgc cacgggaata ccgccgtcat gcccgcgaaa tgacagccct 360
ttcgccgaca gacgccgcag atttcctgaa ctgaccgaca agggaaaaca gatcatgcca 420
acacttccac aacgtttttc cctcgatggt cgcaaagctc ttgtcacggg tgcatcccgc 480
gggcttggtg ttacgatctg cgacgttctg agtgctgcgg gggccgatat tgtcgccgtt 540
gcgcgttctg aaaccgacat ggccgccaca tgccggatcg tggaaggcca tggtcgtcaa 600
tgcctcacgg ttgttgccga tctcagtgat ccgatggctc cggacgctgt cgcgcagaca 660
gtgaacgcag cgtggggtgg ggtggatatt gtcgtcaaca atgctggcgt cagtttccct 720
cgccctctgg tggaacagac cgtcgaggag tgggacaccg tgcaggccat taacctgcgt 780
gcgccatggc ttctcgcccg tgtcttcgct ccgggcatga ttgaacgcaa gcgtgggaaa 840
atcatcaaca tcagttccca ggccagctct gtcgcgctga ttgaccatgg tgcttacgtc 900
gcatccaagg ccggtctgaa cggtctcacc aaggtcatga cggcggaatg ggcggctcat 960
aacatacagg ccaatgccat ctgccccaca gtcgtctgga cgcccatggg tgaacgcgtc 1020
tggagcgttg ggaacaagct ggaaaagcta ctggaaaaga tccccgctgg ccgtgtcgca 1080
acaccggaag atgtcgcgga tatagttctg tatctcgcct ccgacgcgtc gagcatggtc 1140
aacgggcagg aaatatttgt cgatggcgga tacacagccc tttaggccgc cacatcttca 1200
aataaagaca tgtgatttta cggttttaac aaggccatgt gcagggaatg gcctgcgcat 1260
ttcatgcaga tcaacaggtg taac 1284

54

1234

DNA

Gluconobacter suboxydans

54
tcatcacatc cctgatggcc atggacccat ccgaaatccg gcatgaccat ctggcgcagg 60
atctgggcct ctgcgcatcc accttctctc gccagttccg ttctgcgacg ggcgacacat 120
tcatgtcttt cctgcatcgg ctgcgggtgt gtcacgcgtg ccatctgctg gccagttcca 180
cactctcaat cacggaaatc ggggctgctt ccggcttcaa caacctgtcc aattttaacc 240
gcatcttcct gcgcctgcgt ggctgcacgc cacgggaata ccgccgtcat gcccgcgaaa 300
tgacagccct ttcgccgaca gacgccgcag atttcctgaa ctgaccgaca agggaaaaca 360
gatcatgcca acacttccac aacgtttttc cctcgatggt cgcaaagctc ttgtcacggg 420
tgcatcccgc gggcttggtg ttacgatctg cgacgttctg agtgctgcgg gggccgatat 480
tgtcgccgtt gcgcgttctg aaaccgacat ggccgccaca tgccggatcg tggaaggcca 540
tggtcgtcaa tgcctcacgg ttgttgccga tctcagtgat ccgatggctc cggacgctgt 600
cgcgcagaca gtgaacgcag cgtggggtgg ggtggatatt gtcgtcaaca atgctggcgt 660
cagtttccct cgccctctgg tggaacagac cgtcgaggag tgggacaccg tgcaggccat 720
taacctgcgt gcgccatggc ttctcgcccg tgtcttcgct ccgggcatga ttgaacgcaa 780
gcgtgggaaa atcatcaaca tcagttccca ggccagctct gtcgcgctga ttgaccatgg 840
tgcttacgtc gcatccaagg ccggtctgaa cggtctcacc aaggtcatga cggcggaatg 900
ggcggctcat aacatacagg ccaatgccat ctgccccaca gtcgtctgga cgcccatggg 960
tgaacgcgtc tggagcgttg ggaacaagct ggaaaagcta ctggaaaaga tccccgctgg 1020
ccgtgtcgca acaccggaag atgtcgcgga tatagttctg tatctcgcct ccgacgcgtc 1080
gagcatggtc aacgggcagg aaatatttgt cgatggcgga tacacagccc tttaggccgc 1140
cacatcttca aataaagaca tgtgatttta cggttttaac aaggccatgt gcagggaatg 1200
gcctgcgcat ttcatgcaga tcaacaggtg taac 1234

55

1184

DNA

Gluconobacter suboxydans

55
ctggcgcagg atctgggcct ctgcgcatcc accttctctc gccagttccg ttctgcgacg 60
ggcgacacat tcatgtcttt cctgcatcgg ctgcgggtgt gtcacgcgtg ccatctgctg 120
gccagttcca cactctcaat cacggaaatc ggggctgctt ccggcttcaa caacctgtcc 180
aattttaacc gcatcttcct gcgcctgcgt ggctgcacgc cacgggaata ccgccgtcat 240
gcccgcgaaa tgacagccct ttcgccgaca gacgccgcag atttcctgaa ctgaccgaca 300
agggaaaaca gatcatgcca acacttccac aacgtttttc cctcgatggt cgcaaagctc 360
ttgtcacggg tgcatcccgc gggcttggtg ttacgatctg cgacgttctg agtgctgcgg 420
gggccgatat tgtcgccgtt gcgcgttctg aaaccgacat ggccgccaca tgccggatcg 480
tggaaggcca tggtcgtcaa tgcctcacgg ttgttgccga tctcagtgat ccgatggctc 540
cggacgctgt cgcgcagaca gtgaacgcag cgtggggtgg ggtggatatt gtcgtcaaca 600
atgctggcgt cagtttccct cgccctctgg tggaacagac cgtcgaggag tgggacaccg 660
tgcaggccat taacctgcgt gcgccatggc ttctcgcccg tgtcttcgct ccgggcatga 720
ttgaacgcaa gcgtgggaaa atcatcaaca tcagttccca ggccagctct gtcgcgctga 780
ttgaccatgg tgcttacgtc gcatccaagg ccggtctgaa cggtctcacc aaggtcatga 840
cggcggaatg ggcggctcat aacatacagg ccaatgccat ctgccccaca gtcgtctgga 900
cgcccatggg tgaacgcgtc tggagcgttg ggaacaagct ggaaaagcta ctggaaaaga 960
tccccgctgg ccgtgtcgca acaccggaag atgtcgcgga tatagttctg tatctcgcct 1020
ccgacgcgtc gagcatggtc aacgggcagg aaatatttgt cgatggcgga tacacagccc 1080
tttaggccgc cacatcttca aataaagaca tgtgatttta cggttttaac aaggccatgt 1140
gcagggaatg gcctgcgcat ttcatgcaga tcaacaggtg taac 1184

56

1134

DNA

Gluconobacter suboxydans

56
ttctgcgacg ggcgacacat tcatgtcttt cctgcatcgg ctgcgggtgt gtcacgcgtg 60
ccatctgctg gccagttcca cactctcaat cacggaaatc ggggctgctt ccggcttcaa 120
caacctgtcc aattttaacc gcatcttcct gcgcctgcgt ggctgcacgc cacgggaata 180
ccgccgtcat gcccgcgaaa tgacagccct ttcgccgaca gacgccgcag atttcctgaa 240
ctgaccgaca agggaaaaca gatcatgcca acacttccac aacgtttttc cctcgatggt 300
cgcaaagctc ttgtcacggg tgcatcccgc gggcttggtg ttacgatctg cgacgttctg 360
agtgctgcgg gggccgatat tgtcgccgtt gcgcgttctg aaaccgacat ggccgccaca 420
tgccggatcg tggaaggcca tggtcgtcaa tgcctcacgg ttgttgccga tctcagtgat 480
ccgatggctc cggacgctgt cgcgcagaca gtgaacgcag cgtggggtgg ggtggatatt 540
gtcgtcaaca atgctggcgt cagtttccct cgccctctgg tggaacagac cgtcgaggag 600
tgggacaccg tgcaggccat taacctgcgt gcgccatggc ttctcgcccg tgtcttcgct 660
ccgggcatga ttgaacgcaa gcgtgggaaa atcatcaaca tcagttccca ggccagctct 720
gtcgcgctga ttgaccatgg tgcttacgtc gcatccaagg ccggtctgaa cggtctcacc 780
aaggtcatga cggcggaatg ggcggctcat aacatacagg ccaatgccat ctgccccaca 840
gtcgtctgga cgcccatggg tgaacgcgtc tggagcgttg ggaacaagct ggaaaagcta 900
ctggaaaaga tccccgctgg ccgtgtcgca acaccggaag atgtcgcgga tatagttctg 960
tatctcgcct ccgacgcgtc gagcatggtc aacgggcagg aaatatttgt cgatggcgga 1020
tacacagccc tttaggccgc cacatcttca aataaagaca tgtgatttta cggttttaac 1080
aaggccatgt gcagggaatg gcctgcgcat ttcatgcaga tcaacaggtg taac 1134

57

1084

DNA

Gluconobacter suboxydans

57
gtcacgcgtg ccatctgctg gccagttcca cactctcaat cacggaaatc ggggctgctt 60
ccggcttcaa caacctgtcc aattttaacc gcatcttcct gcgcctgcgt ggctgcacgc 120
cacgggaata ccgccgtcat gcccgcgaaa tgacagccct ttcgccgaca gacgccgcag 180
atttcctgaa ctgaccgaca agggaaaaca gatcatgcca acacttccac aacgtttttc 240
cctcgatggt cgcaaagctc ttgtcacggg tgcatcccgc gggcttggtg ttacgatctg 300
cgacgttctg agtgctgcgg gggccgatat tgtcgccgtt gcgcgttctg aaaccgacat 360
ggccgccaca tgccggatcg tggaaggcca tggtcgtcaa tgcctcacgg ttgttgccga 420
tctcagtgat ccgatggctc cggacgctgt cgcgcagaca gtgaacgcag cgtggggtgg 480
ggtggatatt gtcgtcaaca atgctggcgt cagtttccct cgccctctgg tggaacagac 540
cgtcgaggag tgggacaccg tgcaggccat taacctgcgt gcgccatggc ttctcgcccg 600
tgtcttcgct ccgggcatga ttgaacgcaa gcgtgggaaa atcatcaaca tcagttccca 660
ggccagctct gtcgcgctga ttgaccatgg tgcttacgtc gcatccaagg ccggtctgaa 720
cggtctcacc aaggtcatga cggcggaatg ggcggctcat aacatacagg ccaatgccat 780
ctgccccaca gtcgtctgga cgcccatggg tgaacgcgtc tggagcgttg ggaacaagct 840
ggaaaagcta ctggaaaaga tccccgctgg ccgtgtcgca acaccggaag atgtcgcgga 900
tatagttctg tatctcgcct ccgacgcgtc gagcatggtc aacgggcagg aaatatttgt 960
cgatggcgga tacacagccc tttaggccgc cacatcttca aataaagaca tgtgatttta 1020
cggttttaac aaggccatgt gcagggaatg gcctgcgcat ttcatgcaga tcaacaggtg 1080
taac 1084

58

1034

DNA

Gluconobacter suboxydans

58
ggggctgctt ccggcttcaa caacctgtcc aattttaacc gcatcttcct gcgcctgcgt 60
ggctgcacgc cacgggaata ccgccgtcat gcccgcgaaa tgacagccct ttcgccgaca 120
gacgccgcag atttcctgaa ctgaccgaca agggaaaaca gatcatgcca acacttccac 180
aacgtttttc cctcgatggt cgcaaagctc ttgtcacggg tgcatcccgc gggcttggtg 240
ttacgatctg cgacgttctg agtgctgcgg gggccgatat tgtcgccgtt gcgcgttctg 300
aaaccgacat ggccgccaca tgccggatcg tggaaggcca tggtcgtcaa tgcctcacgg 360
ttgttgccga tctcagtgat ccgatggctc cggacgctgt cgcgcagaca gtgaacgcag 420
cgtggggtgg ggtggatatt gtcgtcaaca atgctggcgt cagtttccct cgccctctgg 480
tggaacagac cgtcgaggag tgggacaccg tgcaggccat taacctgcgt gcgccatggc 540
ttctcgcccg tgtcttcgct ccgggcatga ttgaacgcaa gcgtgggaaa atcatcaaca 600
tcagttccca ggccagctct gtcgcgctga ttgaccatgg tgcttacgtc gcatccaagg 660
ccggtctgaa cggtctcacc aaggtcatga cggcggaatg ggcggctcat aacatacagg 720
ccaatgccat ctgccccaca gtcgtctgga cgcccatggg tgaacgcgtc tggagcgttg 780
ggaacaagct ggaaaagcta ctggaaaaga tccccgctgg ccgtgtcgca acaccggaag 840
atgtcgcgga tatagttctg tatctcgcct ccgacgcgtc gagcatggtc aacgggcagg 900
aaatatttgt cgatggcgga tacacagccc tttaggccgc cacatcttca aataaagaca 960
tgtgatttta cggttttaac aaggccatgt gcagggaatg gcctgcgcat ttcatgcaga 1020
tcaacaggtg taac 1034

59

984

DNA

Gluconobacter suboxydans

59
gcgcctgcgt ggctgcacgc cacgggaata ccgccgtcat gcccgcgaaa tgacagccct 60
ttcgccgaca gacgccgcag atttcctgaa ctgaccgaca agggaaaaca gatcatgcca 120
acacttccac aacgtttttc cctcgatggt cgcaaagctc ttgtcacggg tgcatcccgc 180
gggcttggtg ttacgatctg cgacgttctg agtgctgcgg gggccgatat tgtcgccgtt 240
gcgcgttctg aaaccgacat ggccgccaca tgccggatcg tggaaggcca tggtcgtcaa 300
tgcctcacgg ttgttgccga tctcagtgat ccgatggctc cggacgctgt cgcgcagaca 360
gtgaacgcag cgtggggtgg ggtggatatt gtcgtcaaca atgctggcgt cagtttccct 420
cgccctctgg tggaacagac cgtcgaggag tgggacaccg tgcaggccat taacctgcgt 480
gcgccatggc ttctcgcccg tgtcttcgct ccgggcatga ttgaacgcaa gcgtgggaaa 540
atcatcaaca tcagttccca ggccagctct gtcgcgctga ttgaccatgg tgcttacgtc 600
gcatccaagg ccggtctgaa cggtctcacc aaggtcatga cggcggaatg ggcggctcat 660
aacatacagg ccaatgccat ctgccccaca gtcgtctgga cgcccatggg tgaacgcgtc 720
tggagcgttg ggaacaagct ggaaaagcta ctggaaaaga tccccgctgg ccgtgtcgca 780
acaccggaag atgtcgcgga tatagttctg tatctcgcct ccgacgcgtc gagcatggtc 840
aacgggcagg aaatatttgt cgatggcgga tacacagccc tttaggccgc cacatcttca 900
aataaagaca tgtgatttta cggttttaac aaggccatgt gcagggaatg gcctgcgcat 960
ttcatgcaga tcaacaggtg taac 984

60

934

DNA

Gluconobacter suboxydans

60
tgacagccct ttcgccgaca gacgccgcag atttcctgaa ctgaccgaca agggaaaaca 60
gatcatgcca acacttccac aacgtttttc cctcgatggt cgcaaagctc ttgtcacggg 120
tgcatcccgc gggcttggtg ttacgatctg cgacgttctg agtgctgcgg gggccgatat 180
tgtcgccgtt gcgcgttctg aaaccgacat ggccgccaca tgccggatcg tggaaggcca 240
tggtcgtcaa tgcctcacgg ttgttgccga tctcagtgat ccgatggctc cggacgctgt 300
cgcgcagaca gtgaacgcag cgtggggtgg ggtggatatt gtcgtcaaca atgctggcgt 360
cagtttccct cgccctctgg tggaacagac cgtcgaggag tgggacaccg tgcaggccat 420
taacctgcgt gcgccatggc ttctcgcccg tgtcttcgct ccgggcatga ttgaacgcaa 480
gcgtgggaaa atcatcaaca tcagttccca ggccagctct gtcgcgctga ttgaccatgg 540
tgcttacgtc gcatccaagg ccggtctgaa cggtctcacc aaggtcatga cggcggaatg 600
ggcggctcat aacatacagg ccaatgccat ctgccccaca gtcgtctgga cgcccatggg 660
tgaacgcgtc tggagcgttg ggaacaagct ggaaaagcta ctggaaaaga tccccgctgg 720
ccgtgtcgca acaccggaag atgtcgcgga tatagttctg tatctcgcct ccgacgcgtc 780
gagcatggtc aacgggcagg aaatatttgt cgatggcgga tacacagccc tttaggccgc 840
cacatcttca aataaagaca tgtgatttta cggttttaac aaggccatgt gcagggaatg 900
gcctgcgcat ttcatgcaga tcaacaggtg taac 934

61

884

DNA

Gluconobacter suboxydans

61
agggaaaaca gatcatgcca acacttccac aacgtttttc cctcgatggt cgcaaagctc 60
ttgtcacggg tgcatcccgc gggcttggtg ttacgatctg cgacgttctg agtgctgcgg 120
gggccgatat tgtcgccgtt gcgcgttctg aaaccgacat ggccgccaca tgccggatcg 180
tggaaggcca tggtcgtcaa tgcctcacgg ttgttgccga tctcagtgat ccgatggctc 240
cggacgctgt cgcgcagaca gtgaacgcag cgtggggtgg ggtggatatt gtcgtcaaca 300
atgctggcgt cagtttccct cgccctctgg tggaacagac cgtcgaggag tgggacaccg 360
tgcaggccat taacctgcgt gcgccatggc ttctcgcccg tgtcttcgct ccgggcatga 420
ttgaacgcaa gcgtgggaaa atcatcaaca tcagttccca ggccagctct gtcgcgctga 480
ttgaccatgg tgcttacgtc gcatccaagg ccggtctgaa cggtctcacc aaggtcatga 540
cggcggaatg ggcggctcat aacatacagg ccaatgccat ctgccccaca gtcgtctgga 600
cgcccatggg tgaacgcgtc tggagcgttg ggaacaagct ggaaaagcta ctggaaaaga 660
tccccgctgg ccgtgtcgca acaccggaag atgtcgcgga tatagttctg tatctcgcct 720
ccgacgcgtc gagcatggtc aacgggcagg aaatatttgt cgatggcgga tacacagccc 780
tttaggccgc cacatcttca aataaagaca tgtgatttta cggttttaac aaggccatgt 840
gcagggaatg gcctgcgcat ttcatgcaga tcaacaggtg taac 884

62

834

DNA

Gluconobacter suboxydans

62
cgcaaagctc ttgtcacggg tgcatcccgc gggcttggtg ttacgatctg cgacgttctg 60
agtgctgcgg gggccgatat tgtcgccgtt gcgcgttctg aaaccgacat ggccgccaca 120
tgccggatcg tggaaggcca tggtcgtcaa tgcctcacgg ttgttgccga tctcagtgat 180
ccgatggctc cggacgctgt cgcgcagaca gtgaacgcag cgtggggtgg ggtggatatt 240
gtcgtcaaca atgctggcgt cagtttccct cgccctctgg tggaacagac cgtcgaggag 300
tgggacaccg tgcaggccat taacctgcgt gcgccatggc ttctcgcccg tgtcttcgct 360
ccgggcatga ttgaacgcaa gcgtgggaaa atcatcaaca tcagttccca ggccagctct 420
gtcgcgctga ttgaccatgg tgcttacgtc gcatccaagg ccggtctgaa cggtctcacc 480
aaggtcatga cggcggaatg ggcggctcat aacatacagg ccaatgccat ctgccccaca 540
gtcgtctgga cgcccatggg tgaacgcgtc tggagcgttg ggaacaagct ggaaaagcta 600
ctggaaaaga tccccgctgg ccgtgtcgca acaccggaag atgtcgcgga tatagttctg 660
tatctcgcct ccgacgcgtc gagcatggtc aacgggcagg aaatatttgt cgatggcgga 720
tacacagccc tttaggccgc cacatcttca aataaagaca tgtgatttta cggttttaac 780
aaggccatgt gcagggaatg gcctgcgcat ttcatgcaga tcaacaggtg taac 834

63

784

DNA

Gluconobacter suboxydans

63
cgacgttctg agtgctgcgg gggccgatat tgtcgccgtt gcgcgttctg aaaccgacat 60
ggccgccaca tgccggatcg tggaaggcca tggtcgtcaa tgcctcacgg ttgttgccga 120
tctcagtgat ccgatggctc cggacgctgt cgcgcagaca gtgaacgcag cgtggggtgg 180
ggtggatatt gtcgtcaaca atgctggcgt cagtttccct cgccctctgg tggaacagac 240
cgtcgaggag tgggacaccg tgcaggccat taacctgcgt gcgccatggc ttctcgcccg 300
tgtcttcgct ccgggcatga ttgaacgcaa gcgtgggaaa atcatcaaca tcagttccca 360
ggccagctct gtcgcgctga ttgaccatgg tgcttacgtc gcatccaagg ccggtctgaa 420
cggtctcacc aaggtcatga cggcggaatg ggcggctcat aacatacagg ccaatgccat 480
ctgccccaca gtcgtctgga cgcccatggg tgaacgcgtc tggagcgttg ggaacaagct 540
ggaaaagcta ctggaaaaga tccccgctgg ccgtgtcgca acaccggaag atgtcgcgga 600
tatagttctg tatctcgcct ccgacgcgtc gagcatggtc aacgggcagg aaatatttgt 660
cgatggcgga tacacagccc tttaggccgc cacatcttca aataaagaca tgtgatttta 720
cggttttaac aaggccatgt gcagggaatg gcctgcgcat ttcatgcaga tcaacaggtg 780
taac 784

64

784

DNA

Gluconobacter suboxydans

64
cgacgttctg agtgctgcgg gggccgatat tgtcgccgtt gcgcgttctg aaaccgacat 60
ggccgccaca tgccggatcg tggaaggcca tggtcgtcaa tgcctcacgg ttgttgccga 120
tctcagtgat ccgatggctc cggacgctgt cgcgcagaca gtgaacgcag cgtggggtgg 180
ggtggatatt gtcgtcaaca atgctggcgt cagtttccct cgccctctgg tggaacagac 240
cgtcgaggag tgggacaccg tgcaggccat taacctgcgt gcgccatggc ttctcgcccg 300
tgtcttcgct ccgggcatga ttgaacgcaa gcgtgggaaa atcatcaaca tcagttccca 360
ggccagctct gtcgcgctga ttgaccatgg tgcttacgtc gcatccaagg ccggtctgaa 420
cggtctcacc aaggtcatga cggcggaatg ggcggctcat aacatacagg ccaatgccat 480
ctgccccaca gtcgtctgga cgcccatggg tgaacgcgtc tggagcgttg ggaacaagct 540
ggaaaagcta ctggaaaaga tccccgctgg ccgtgtcgca acaccggaag atgtcgcgga 600
tatagttctg tatctcgcct ccgacgcgtc gagcatggtc aacgggcagg aaatatttgt 660
cgatggcgga tacacagccc tttaggccgc cacatcttca aataaagaca tgtgatttta 720
cggttttaac aaggccatgt gcagggaatg gcctgcgcat ttcatgcaga tcaacaggtg 780
taac 784

65

734

DNA

Gluconobacter suboxydans

65
aaaccgacat ggccgccaca tgccggatcg tggaaggcca tggtcgtcaa tgcctcacgg 60
ttgttgccga tctcagtgat ccgatggctc cggacgctgt cgcgcagaca gtgaacgcag 120
cgtggggtgg ggtggatatt gtcgtcaaca atgctggcgt cagtttccct cgccctctgg 180
tggaacagac cgtcgaggag tgggacaccg tgcaggccat taacctgcgt gcgccatggc 240
ttctcgcccg tgtcttcgct ccgggcatga ttgaacgcaa gcgtgggaaa atcatcaaca 300
tcagttccca ggccagctct gtcgcgctga ttgaccatgg tgcttacgtc gcatccaagg 360
ccggtctgaa cggtctcacc aaggtcatga cggcggaatg ggcggctcat aacatacagg 420
ccaatgccat ctgccccaca gtcgtctgga cgcccatggg tgaacgcgtc tggagcgttg 480
ggaacaagct ggaaaagcta ctggaaaaga tccccgctgg ccgtgtcgca acaccggaag 540
atgtcgcgga tatagttctg tatctcgcct ccgacgcgtc gagcatggtc aacgggcagg 600
aaatatttgt cgatggcgga tacacagccc tttaggccgc cacatcttca aataaagaca 660
tgtgatttta cggttttaac aaggccatgt gcagggaatg gcctgcgcat ttcatgcaga 720
tcaacaggtg taac 734

66

684

DNA

Gluconobacter suboxydans

66
tgcctcacgg ttgttgccga tctcagtgat ccgatggctc cggacgctgt cgcgcagaca 60
gtgaacgcag cgtggggtgg ggtggatatt gtcgtcaaca atgctggcgt cagtttccct 120
cgccctctgg tggaacagac cgtcgaggag tgggacaccg tgcaggccat taacctgcgt 180
gcgccatggc ttctcgcccg tgtcttcgct ccgggcatga ttgaacgcaa gcgtgggaaa 240
atcatcaaca tcagttccca ggccagctct gtcgcgctga ttgaccatgg tgcttacgtc 300
gcatccaagg ccggtctgaa cggtctcacc aaggtcatga cggcggaatg ggcggctcat 360
aacatacagg ccaatgccat ctgccccaca gtcgtctgga cgcccatggg tgaacgcgtc 420
tggagcgttg ggaacaagct ggaaaagcta ctggaaaaga tccccgctgg ccgtgtcgca 480
acaccggaag atgtcgcgga tatagttctg tatctcgcct ccgacgcgtc gagcatggtc 540
aacgggcagg aaatatttgt cgatggcgga tacacagccc tttaggccgc cacatcttca 600
aataaagaca tgtgatttta cggttttaac aaggccatgt gcagggaatg gcctgcgcat 660
ttcatgcaga tcaacaggtg taac 684

67

634

DNA

Gluconobacter suboxydans

67
cgcgcagaca gtgaacgcag cgtggggtgg ggtggatatt gtcgtcaaca atgctggcgt 60
cagtttccct cgccctctgg tggaacagac cgtcgaggag tgggacaccg tgcaggccat 120
taacctgcgt gcgccatggc ttctcgcccg tgtcttcgct ccgggcatga ttgaacgcaa 180
gcgtgggaaa atcatcaaca tcagttccca ggccagctct gtcgcgctga ttgaccatgg 240
tgcttacgtc gcatccaagg ccggtctgaa cggtctcacc aaggtcatga cggcggaatg 300
ggcggctcat aacatacagg ccaatgccat ctgccccaca gtcgtctgga cgcccatggg 360
tgaacgcgtc tggagcgttg ggaacaagct ggaaaagcta ctggaaaaga tccccgctgg 420
ccgtgtcgca acaccggaag atgtcgcgga tatagttctg tatctcgcct ccgacgcgtc 480
gagcatggtc aacgggcagg aaatatttgt cgatggcgga tacacagccc tttaggccgc 540
cacatcttca aataaagaca tgtgatttta cggttttaac aaggccatgt gcagggaatg 600
gcctgcgcat ttcatgcaga tcaacaggtg taac 634

68

584

DNA

Gluconobacter suboxydans

68
atgctggcgt cagtttccct cgccctctgg tggaacagac cgtcgaggag tgggacaccg 60
tgcaggccat taacctgcgt gcgccatggc ttctcgcccg tgtcttcgct ccgggcatga 120
ttgaacgcaa gcgtgggaaa atcatcaaca tcagttccca ggccagctct gtcgcgctga 180
ttgaccatgg tgcttacgtc gcatccaagg ccggtctgaa cggtctcacc aaggtcatga 240
cggcggaatg ggcggctcat aacatacagg ccaatgccat ctgccccaca gtcgtctgga 300
cgcccatggg tgaacgcgtc tggagcgttg ggaacaagct ggaaaagcta ctggaaaaga 360
tccccgctgg ccgtgtcgca acaccggaag atgtcgcgga tatagttctg tatctcgcct 420
ccgacgcgtc gagcatggtc aacgggcagg aaatatttgt cgatggcgga tacacagccc 480
tttaggccgc cacatcttca aataaagaca tgtgatttta cggttttaac aaggccatgt 540
gcagggaatg gcctgcgcat ttcatgcaga tcaacaggtg taac 584

69

534

DNA

Gluconobacter suboxydans

69
tgggacaccg tgcaggccat taacctgcgt gcgccatggc ttctcgcccg tgtcttcgct 60
ccgggcatga ttgaacgcaa gcgtgggaaa atcatcaaca tcagttccca ggccagctct 120
gtcgcgctga ttgaccatgg tgcttacgtc gcatccaagg ccggtctgaa cggtctcacc 180
aaggtcatga cggcggaatg ggcggctcat aacatacagg ccaatgccat ctgccccaca 240
gtcgtctgga cgcccatggg tgaacgcgtc tggagcgttg ggaacaagct ggaaaagcta 300
ctggaaaaga tccccgctgg ccgtgtcgca acaccggaag atgtcgcgga tatagttctg 360
tatctcgcct ccgacgcgtc gagcatggtc aacgggcagg aaatatttgt cgatggcgga 420
tacacagccc tttaggccgc cacatcttca aataaagaca tgtgatttta cggttttaac 480
aaggccatgt gcagggaatg gcctgcgcat ttcatgcaga tcaacaggtg taac 534

70

484

DNA

Gluconobacter suboxydans

70
tgtcttcgct ccgggcatga ttgaacgcaa gcgtgggaaa atcatcaaca tcagttccca 60
ggccagctct gtcgcgctga ttgaccatgg tgcttacgtc gcatccaagg ccggtctgaa 120
cggtctcacc aaggtcatga cggcggaatg ggcggctcat aacatacagg ccaatgccat 180
ctgccccaca gtcgtctgga cgcccatggg tgaacgcgtc tggagcgttg ggaacaagct 240
ggaaaagcta ctggaaaaga tccccgctgg ccgtgtcgca acaccggaag atgtcgcgga 300
tatagttctg tatctcgcct ccgacgcgtc gagcatggtc aacgggcagg aaatatttgt 360
cgatggcgga tacacagccc tttaggccgc cacatcttca aataaagaca tgtgatttta 420
cggttttaac aaggccatgt gcagggaatg gcctgcgcat ttcatgcaga tcaacaggtg 480
taac 484

71

434

DNA

Gluconobacter suboxydans

71
tcagttccca ggccagctct gtcgcgctga ttgaccatgg tgcttacgtc gcatccaagg 60
ccggtctgaa cggtctcacc aaggtcatga cggcggaatg ggcggctcat aacatacagg 120
ccaatgccat ctgccccaca gtcgtctgga cgcccatggg tgaacgcgtc tggagcgttg 180
ggaacaagct ggaaaagcta ctggaaaaga tccccgctgg ccgtgtcgca acaccggaag 240
atgtcgcgga tatagttctg tatctcgcct ccgacgcgtc gagcatggtc aacgggcagg 300
aaatatttgt cgatggcgga tacacagccc tttaggccgc cacatcttca aataaagaca 360
tgtgatttta cggttttaac aaggccatgt gcagggaatg gcctgcgcat ttcatgcaga 420
tcaacaggtg taac 434

72

384

DNA

Gluconobacter suboxydans

72
gcatccaagg ccggtctgaa cggtctcacc aaggtcatga cggcggaatg ggcggctcat 60
aacatacagg ccaatgccat ctgccccaca gtcgtctgga cgcccatggg tgaacgcgtc 120
tggagcgttg ggaacaagct ggaaaagcta ctggaaaaga tccccgctgg ccgtgtcgca 180
acaccggaag atgtcgcgga tatagttctg tatctcgcct ccgacgcgtc gagcatggtc 240
aacgggcagg aaatatttgt cgatggcgga tacacagccc tttaggccgc cacatcttca 300
aataaagaca tgtgatttta cggttttaac aaggccatgt gcagggaatg gcctgcgcat 360
ttcatgcaga tcaacaggtg taac 384

73

334

DNA

Gluconobacter suboxydans

73
ggcggctcat aacatacagg ccaatgccat ctgccccaca gtcgtctgga cgcccatggg 60
tgaacgcgtc tggagcgttg ggaacaagct ggaaaagcta ctggaaaaga tccccgctgg 120
ccgtgtcgca acaccggaag atgtcgcgga tatagttctg tatctcgcct ccgacgcgtc 180
gagcatggtc aacgggcagg aaatatttgt cgatggcgga tacacagccc tttaggccgc 240
cacatcttca aataaagaca tgtgatttta cggttttaac aaggccatgt gcagggaatg 300
gcctgcgcat ttcatgcaga tcaacaggtg taac 334

74

284

DNA

Gluconobacter suboxydans

74
cgcccatggg tgaacgcgtc tggagcgttg ggaacaagct ggaaaagcta ctggaaaaga 60
tccccgctgg ccgtgtcgca acaccggaag atgtcgcgga tatagttctg tatctcgcct 120
ccgacgcgtc gagcatggtc aacgggcagg aaatatttgt cgatggcgga tacacagccc 180
tttaggccgc cacatcttca aataaagaca tgtgatttta cggttttaac aaggccatgt 240
gcagggaatg gcctgcgcat ttcatgcaga tcaacaggtg taac 284

75

234

DNA

Gluconobacter suboxydans

75
ctggaaaaga tccccgctgg ccgtgtcgca acaccggaag atgtcgcgga tatagttctg 60
tatctcgcct ccgacgcgtc gagcatggtc aacgggcagg aaatatttgt cgatggcgga 120
tacacagccc tttaggccgc cacatcttca aataaagaca tgtgatttta cggttttaac 180
aaggccatgt gcagggaatg gcctgcgcat ttcatgcaga tcaacaggtg taac 234

76

184

DNA

Gluconobacter suboxydans

76
tatagttctg tatctcgcct ccgacgcgtc gagcatggtc aacgggcagg aaatatttgt 60
cgatggcgga tacacagccc tttaggccgc cacatcttca aataaagaca tgtgatttta 120
cggttttaac aaggccatgt gcagggaatg gcctgcgcat ttcatgcaga tcaacaggtg 180
taac 184

77

134

DNA

Gluconobacter suboxydans

77
aaatatttgt cgatggcgga tacacagccc tttaggccgc cacatcttca aataaagaca 60
tgtgatttta cggttttaac aaggccatgt gcagggaatg gcctgcgcat ttcatgcaga 120
tcaacaggtg taac 134

78

84

DNA

Gluconobacter suboxydans

78
aataaagaca tgtgatttta cggttttaac aaggccatgt gcagggaatg gcctgcgcat 60
ttcatgcaga tcaacaggtg taac 84

79

34

DNA

Gluconobacter suboxydans

79
gcctgcgcat ttcatgcaga tcaacaggtg taac 34

80

51

DNA

Gluconobacter suboxydans

80
attccgaagg cggccccgaa ttccggaggg aacattatga ctgaatccag t 51

81

101

DNA

Gluconobacter suboxydans

81
attccgaagg cggccccgaa ttccggaggg aacattatga ctgaatccag tcagacatct 60
ccagaacttc ttctggcgct tgagggaatc tccaagagtt t 101

82

151

DNA

Gluconobacter suboxydans

82
attccgaagg cggccccgaa ttccggaggg aacattatga ctgaatccag tcagacatct 60
ccagaacttc ttctggcgct tgagggaatc tccaagagtt ttccgggagt ccgggcgttg 120
cggaatgtca gcctcagcct ggagcgtgga g 151

83

201

DNA

Gluconobacter suboxydans

83
attccgaagg cggccccgaa ttccggaggg aacattatga ctgaatccag tcagacatct 60
ccagaacttc ttctggcgct tgagggaatc tccaagagtt ttccgggagt ccgggcgttg 120
cggaatgtca gcctcagcct ggagcgtgga gaaatccatg ctctgctggg ggaaaacggc 180
gctggaaaat ccacgatcat c 201

84

251

DNA

Gluconobacter suboxydans

84
attccgaagg cggccccgaa ttccggaggg aacattatga ctgaatccag tcagacatct 60
ccagaacttc ttctggcgct tgagggaatc tccaagagtt ttccgggagt ccgggcgttg 120
cggaatgtca gcctcagcct ggagcgtgga gaaatccatg ctctgctggg ggaaaacggc 180
gctggaaaat ccacgatcat caagatcatg ggcggtatcc agtctcagga tgaagggcag 240
atctttctca a 251

85

301

DNA

Gluconobacter suboxydans

85
attccgaagg cggccccgaa ttccggaggg aacattatga ctgaatccag tcagacatct 60
ccagaacttc ttctggcgct tgagggaatc tccaagagtt ttccgggagt ccgggcgttg 120
cggaatgtca gcctcagcct ggagcgtgga gaaatccatg ctctgctggg ggaaaacggc 180
gctggaaaat ccacgatcat caagatcatg ggcggtatcc agtctcagga tgaagggcag 240
atctttctca acggaaagga gcgccacttc tccagctaca aggatgccat cagcgcaggt 300
a 301

86

351

DNA

Gluconobacter suboxydans

86
attccgaagg cggccccgaa ttccggaggg aacattatga ctgaatccag tcagacatct 60
ccagaacttc ttctggcgct tgagggaatc tccaagagtt ttccgggagt ccgggcgttg 120
cggaatgtca gcctcagcct ggagcgtgga gaaatccatg ctctgctggg ggaaaacggc 180
gctggaaaat ccacgatcat caagatcatg ggcggtatcc agtctcagga tgaagggcag 240
atctttctca acggaaagga gcgccacttc tccagctaca aggatgccat cagcgcaggt 300
atcgggattg tttttcagga attcagcctg attcctgaac tcgatgccgt g 351

87

396

DNA

Gluconobacter suboxydans

87
attccgaagg cggccccgaa ttccggaggg aacattatga ctgaatccag tcagacatct 60
ccagaacttc ttctggcgct tgagggaatc tccaagagtt ttccgggagt ccgggcgttg 120
cggaatgtca gcctcagcct ggagcgtgga gaaatccatg ctctgctggg ggaaaacggc 180
gctggaaaat ccacgatcat caagatcatg ggcggtatcc agtctcagga tgaagggcag 240
atctttctca acggaaagga gcgccacttc tccagctaca aggatgccat cagcgcaggt 300
atcgggattg tttttcagga attcagcctg attcctgaac tcgatgccgt ggataatatt 360
ttcctcggtc gtgagatgcg gaacgctctt ggcttt 396

Number	Name	Date
4935359	Yin et al.	Jun 1990
4960695	Hoshino et al.	Oct 1990
5312741	Hoshino et al.	May 1994
5541108	Fujiwara et al.	Jul 1996
5747301	Hoshino et al.	May 1998
5753481	Niwa et al.	May 1998
5834263	Niwa et al.	Nov 1998
5861292	Niwa et al.	Jan 1999
5888786	Niwa et al.	Mar 1999

Number	Date	Country
0 897 984 A2	Feb 1999	EP
98-069057	Oct 1998	KR

Gluconobacter suboxydans sorbitol dehydrogenase, genes and methods of use thereof

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Entry
GenBank Accession No. E03223 (Jun. 1997).*
GenBank Accession No. E04355 (Jun. 1997).*
GenBank Accession No. AC005387 (Aug. 1998).*
GenBank Accession No. D86375 (Apr. 1997).*
Choi, E.-S. et al. “Purification of a membrane-bound sorbitol dehydrogenase from Gluoconobacter suboxydans,”FEMS Microbiol. Letts. 125:45-50 (1995).
Shinagawa, E. et al. “Purification and Characterization of D-Sorbitol Dehydrogenase from Membrane of Gluconobacter suboxydans ver α,” Agric. Biol. Chem. 46:135-141 (1982).
Kondo, K. and S. Horinouchi, “Characterization of the Genes Encoding the Three-Component Membrane-Bound Alcohol Dehydrogenase from Gluconobacter suboxydans and Their Expression in Acetobacter pasteurianus,” Appl. Environ. Microbiol. 63:1131-1138 (Mar. 1997).
International Search Report for International Application No. PCT/IB99/00736, mailed Feb. 18, 2000.