# US 2014/0154753 A1 · Testosteronan, a novel heparosan analog, testosteronan synthase, and methods of production and use thereof

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- Number: US 2014/0154753 A1
- Document ID: US20140154753A1
- Status: Published application
- Kind: A1
- Application number: 13/981,886
- Filed: 2012-01-31
- Published: 2014-06-05
- Earliest claimed: 2011-01-31
- Assignees: The Board of Regents of the University of Oklahoma
- Inventors: Paul L. DeAngelis; Nigel J. Otto
- Web page: https://patentgrep.com/patent/US20140154753A1

## CPC

- C12P 19/26: Preparation of nitrogen-containing carbohydrates
- C08B 37/0063: {Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan}
- C12N 9/0006: {acting on CH-OH groups as donors (1.1)}
- C12N 9/1051: {Hexosyltransferases (2.4.1)}
- C12N 9/1241: {Nucleotidyltransferases (2.7.7)}
- C12N 15/52: Genes encoding for enzymes or proenzymes
- C12P 19/04: Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
- C12Y 101/01022: UDP-glucose 6-dehydrogenase (1.1.1.22)
- C12Y 207/07009: UTP-glucose-1-phosphate uridylyltransferase (2.7.7.9), i.e. UDP-glucose-pyrophosphorylase
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C08B: POLYSACCHARIDES; DERIVATIVES THEREOF (polysaccharides containing less than six saccharide radicals attached to each other by glycosidic linkages C07H; fermentation or enzyme-using processes C12P19/00; sugar industry C13; production of cellulose D21)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES

## Abstract

Testosteronan, a heparosan analog having the structure [-4-D-GlcUA-α1,4-D-GlcNAc-α1-]n, is produced by testosteronan synthase, a single protein that is a dual-action catalyst that utilizes UDP-GlcUA and UDP-GlcNAc to synthesize a polysaccharide having the structure [-4-D-GlcUA-α1,4-D-GlcNAc-α1-]n.

## Claims

### Claim at position 1

1-4. (canceled)

### Claim 5

A recombinant vector comprising an isolated nucleotide sequence encoding an enzymatically active testosteronan synthase wherein the testosteronan synthase is a single protein that is a dual-action catalyst that utilizes UDP-GlcUA and UDP-GlcNAc to synthesize a polysaccharide having the repeat structure [-4-D-GlcUA-α1,4-D-GlcNAc-α1-]n, the isolated nucleotide sequence comprising at least one of:
(a) the nucleotide sequence of SEQ ID NO:2;
(b) a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:1;
(c) a nucleotide sequence that is at least 90% identical to SEQ ID NO:2;
(d) a nucleotide sequence encoding an amino acid sequence that is at least 90% identical to SEQ ID NO:1;
(e) a nucleotide sequence capable of hybridizing to a complement of SEQ ID NO:2 under hybridization conditions of at least one of:
(1) hybridization at a temperature of 68° C. in 5×SSC/5×Denhardt's solution/1.0% SDS, followed with washing in 3×SSC at 42° C.; and
(2) hybridization at a temperature of 30° C. in 5×SSC, 5×Denhardt's reagent, 30% formamide for about 20 hours followed by washing twice in 2×SSC, 0.1% SDS at about 30° C. for about 15 minutes followed by 0.5×SSC, 0.1% SDS at about 30° C. for about 30 minutes;
(f) a nucleotide sequence capable of hybridizing to a complement of a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:1 under hybridization conditions of at least one of (1) and (2) of (e) above; and
(g) a nucleotide sequence encoding an amino acid sequence having up to 50 amino acid insertions, deletions or substitutions when compared to SEQ ID NO:1; and
(h) a nucleotide sequence encoding an amino acid sequence having up to 25 amino acid insertions, deletions or substitutions when compared to SEQ ID NO:1.

### Claim 6 (depends on 5)

The recombinant vector of claim 5, wherein the recombinant vector is selected from the group consisting of a plasmid, cosmid, phage, integrated cassette and virus vector.

### Claim 7 (depends on 5)

The recombinant vector of claim 5, further comprising a promoter operatively linked to the coding region encoding enzymatically active testosteronan synthase.

### Claim 8 (depends on 5)

A recombinant host cell comprising the recombinant vector of claim 5.

### Claim 9 (depends on 8)

The recombinant host cell of claim 8, wherein the recombinant host cell is a prokaryotic cell.

### Claim 10 (depends on 8)

The recombinant host cell of claim 8, wherein the recombinant host cell is a eukaryotic cell.

### Claim 11 (depends on 8)

The recombinant host cell of claim 8, wherein the recombinant host cell produces a polymer having the repeat structure [-4-D-GlcUA-α1,4-D-GlcNAc-α1-]n.

### Claim 12 (depends on 8)

The recombinant host cell of claim 8, further comprising a purified nucleic acid segment having a coding region encoding an enzymatically active UDP-GlcUA biosynthetic pathway enzyme, wherein the enzymatically active UDP-GlcUA biosynthetic pathway enzyme is selected from the group consisting of UDP-glucose dehydrogenase, UDP-glucose pyrophosphorylase, and combinations thereof.

### Claim 13

(canceled)

### Claim 14 (depends on 12)

A method of producing a polymer, at least a portion of which has the structure [-4-D-GlcUA-α1,4-D-GlcNAc-α1-]n, the method comprising the steps of:
culturing the recombinant host cell of claim 12 under conditions that allow for expression of the enzymatically active testosteronan synthase;
isolating the recombinant, enzymatically active testosteronan synthase;
combining the recombinant, enzymatically active testosteronan synthase with at least one UDP-sugar and a functional acceptor, thereby elongating the functional acceptor to provide a polymer comprising a structure selected from the group consisting of [-4-D-GlcUA-α1,4-D-GlcNAc-α1-]n, GlcUA-α1,4-R and D-GlcNAc-α1-4-R—, wherein R comprises any chemical group, and wherein the functional acceptor comprises at least one sugar unit, and wherein the at least one UDP-sugar is at least one of UDP-GlcUA and UDP-GlcNAc.

### Claim 15 (depends on 14)

The method of claim 14, wherein the at least one UDP-sugar is provided in a stoichiometric ratio to the at least one functional acceptor such that the recombinant testosteronan synthase elongates the at least one functional acceptor to provide a polysaccharide having a desired size distribution such that the polysaccharide is substantially monodisperse in size and has a polydispersity value in a range of from 1.0 to 1.5, and wherein the desired size distribution is obtained by controlling the stoichiometric ratio of UDP-sugar to functional acceptor.

### Claim 16 (depends on 14)

The method of claim 14, wherein the functional acceptor comprises at least one sugar unit, and wherein the sugar unit comprises at least one of uronic acid and a uronic acid analog comprising a substitution at at least one of the C2 and C3 positions thereof.

### Claim 17 (depends on 14)

The method of claim 14, wherein the functional acceptor comprises at least two sugar units, and wherein at least one of the at least two sugar units is selected from the group consisting of uronic acid, a uronic acid analog comprising a substitution at at least one of the C2 and C3 positions thereof, a hexosamine and a hexosamine analog comprising a substitution at at least one of the C2 and C6 positions thereof.

### Claim 18 (depends on 17)

The method of claim 17, wherein uronic acid is further defined as a uronic acid selected from the group consisting of GlcUA, iduronic acid (IdoUA) and GalUA; hexosamine is further defined as a hexosamine selected from the group consisting of GlcNAc, GalNAc, GlcN, GalN; the uronic acid analog is further defined as a uronic acid analog selected from the group consisting of GlcNAcUA, GlcdiNAcUA, and 2-deoxy-2-fluoro-GlcUA; and the hexosamine analog is further defined as a hexosamine analog selected from the group consisting of GlcN, GlcNAcNAc, GlcN[TFA], GlcNBut, GlcNPro, and 6-F-6-deoxyGlcNAc.

### Claim 19 (depends on 14)

The method of claim 14, wherein the UDP-sugar is radioactive or nuclear magnetic resonance-active.

### Claim 20 (depends on 14)

The method of claim 14, further comprising the step of providing a divalent metal ion, wherein the divalent metal ion is selected from the group consisting of manganese, magnesium, cobalt, nickel and combinations thereof, and wherein the method is carried out in a buffer having a pH from about 4 to about 9.

### Claim 21 (depends on 14)

The method of claim 14, wherein the at least one functional acceptor further comprises a moiety selected from the group consisting of a fluorescent tag, a radioactive tag or therapeutic, an affinity tag, a detection probe, a medicant, a biologically active agent, a therapeutic agent, and combinations thereof.

### Claim 22 (depends on 14)

The method of claim 14, wherein the functional acceptor comprises at least one of:
a testosteronan oligosaccharide, polysaccharide and/or polymer;
a heparosan oligosaccharide, polysaccharide and/or polymer; and
a GlcUA-based or GlcUA-analog glycoside.

### Claim 23 (depends on 8)

A method of producing a polymer, at least a portion of which has the repeat structure [-4-D-GlcUA-α1,4-D-GlcNAc-α1-]n the method comprising the steps of:
culturing the recombinant host cell of claim 8 under conditions that allow for the production of a polymer having the repeat structure [-4-D-GlcUA-α1,4-D-GlcNAc-α1-]n.

### Claim 24 (depends on 23)

The method of claim 23, further comprising the step of isolating the polymer having the repeat structure [-4-D-GlcUA-α1,4-D-GlcNAc-α1-]n.

### Claim 25 (depends on 24)

The method of claim 24, wherein the polymer is isolated from a culture medium.

### Claim 26 (depends on 23)

The method of claim 23, wherein the recombinant host cell comprises nucleic acid segments encoding enzymes which produce UDP-GlcUA and UDP-GlcNAc.

### Claim 27 (depends on 23)

The method of claim 23, wherein UDP-GlcUA and UDP-GlcNAc are supplied to the recombinant host cell.

### Claim at position 25

28-30. (canceled)