# US 9,040,259 B2 · Production of polyketides and other natural products

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- Number: US 9,040,259 B2
- Document ID: US9040259B2
- Status: Granted
- Kind: B2
- Application number: 13/046,304
- Filed: 2011-03-11
- Published: 2011-07-07
- Granted: 2015-05-26
- Earliest claimed: 2002-07-16
- Assignees: Buck Institute for Research on Aging
- Applicants: Matthew Alan Gregory; Sabine Gaisser; Hrvoje Petkovic; Steven Moss
- Inventors: Matthew Alan Gregory; Sabine Gaisser; Hrvoje Petkovic; Steven Moss
- Web page: https://patentgrep.com/patent/US9040259B2

## CPC

- C07D 498/18: Bridged systems
- A61P 29/00: Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
- A61P 31/00: Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P 31/10: Antimycotics
- A61P 35/00: Antineoplastic agents
- A61P 37/00: Drugs for immunological or allergic disorders
- A61P 37/04: Immunostimulants
- A61P 37/06: Immunosuppressants, e.g. drugs for graft rejection
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C07D 405/12: linked by a chain containing hetero atoms as chain links
- C07D 471/18: Bridged systems
- C07D 487/18: Bridged systems
- C07D 493/18: Bridged systems
- C07K 14/36: from Actinomyces; from Streptomyces (G)
- C12N 15/52: Genes encoding for enzymes or proenzymes
- C12N 15/70: Vectors or expression systems specially adapted for E. coli
- C12N 15/76: for Actinomyces; for Streptomyces
- C12P 17/188: {Heterocyclic compound containing in the condensed system at least one hetero ring having nitrogen atoms and oxygen atoms as the only ring heteroatoms (ergot-alcaloids C12P17/183)}
- C07D: HETEROCYCLIC COMPOUNDS (macromolecular compounds C08)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)}

## Abstract

The present invention relates to production of polyketides and other natural products and to libraries of compounds and individual novel compounds. One important area is the isolation and potential use of novel FKBP-ligand analogs and host cells that produce these compounds. The invention is particularly concerned with methods for the efficient transformation of strains that produce FKBP analogs and recombinant cells in which cloned genes or gene cassettes are expressed to generate novel compounds such as polyketide (especially rapamycin) FKBP-ligand analogs, and to processes for their preparation, and to means employed therein (e.g. nucleic acids, vectors, gene cassettes and genetically modified strains).

## Claims

### Claim 1

A method of generating analogues of FKBP-ligands which incorporate a non-natural starter unit, said method comprising:
(a) generating a recombinant strain in which at least the rapK homologue has been deleted or inactivated; and
(b) feeding a non-natural starter unit to said strain, wherein said starter unit is selected from the group consisting of 2-norbornane carboxylic acid, 2-(cis/trans)-hydroxycyclohexane carboxylic acid, 3-(cis/trans)-hydroxycyclohexane carboxylic acid, 4-(cis/trans)-hydroxycyclohexane carboxylic acid, 2-(cis/trans)-methylcyclohexane carboxylic acid, 4-(cis/trans)-methylcyclohexane carboxylic acid, 3-(cis/trans)methoxycyclohexane carboxylic acid, 4-(cis/trans)methoxycyclohexane carboxylic acid, 4-oxo cyclohexane carboxylic acid, 2-oxo cyclohexane carboxylic acid, 4-trans-n-pentylcyclohexane carboxylic acid, 2-trans-aminocyclohexane carboxylic acid, 4-cis-aminocyclohexane carboxylic acid, 4-(cis/trans)-aminomethylcyclohexane carboxylic acid, cyclopentane carboxylic acid, cyclobutane carboxylic acid, 1-methylcyclohexane carboxylic acid, 3-trans-hydroxy-4-cis-fluorocyclohexane carboxylic acid and 4-trans-hydroxy-3-cis-fluorocyclohexane carboxylic acid, 3-cis-hydroxy-4-trans-fluorocyclohexane carboxylic acid and 4-cis-hydroxy-3-trans-fluorocyclohexane carboxylic acid, 3-cis-hydroxy-4-trans-chlorocyclohexane carboxylic acid and 4-cis-hydroxy-3-trans-chlorocyclohexane carboxylic acid, 3-trans-hydroxy-4-cis-chlorocyclohexane carboxylic acid and 4-trans-hydroxy-3-cis-chlorocyclohexane carboxylic acid, 3-trans-cyclohexeneoxide carboxylic acid, 3-cis-cyclohexeneoxide carboxylic acid, 3,4-cis-dihydroxycyclohexane carboxylic acid and 3,4-trans-dihydroxycyclohexane carboxylic acid, cyclohexaneacetic acid, cyclohexanepropionic acid and 4-cis/trans-tert-butylcyclohexane carboxylic acid, and simple esters or salts thereof.

### Claim 2 (depends on 1)

The method of claim 1, which additionally comprises deleting one or more further auxiliary genes.

### Claim 3 (depends on 2)

The method of claim 2, which additionally comprises restoring by complementation one or more of the deleted auxiliary genes.

### Claim 4 (depends on 1)

The method of claim 1, which additionally comprises the step of isolating and purifying the FKBP-ligand analogues generated.

### Claim 5 (depends on 1)

The method of claim 1, wherein said non-natural starter unit is selected from the group consisting of 3-(cis/trans)-hydroxycyclohexane carboxylic acid, 4-(cis/trans)-hydroxycyclohexane carboxylic acid, 3-(cis/trans)-methoxycyclohexane carboxylic acid, 4-(cis/trans)-methoxycyclohexane carboxylic acid, 4-oxo cyclohexane carboxylic acid, cyclobutane carboxylic acid, 3-trans-hydroxy-4-cis-fluorocyclohexane carboxylic acid and 4-trans-hydroxy-3-cis-fluorocyclohexane carboxylic acid, 3-cis-hydroxy-4-trans-fluorocyclohexane carboxylic acid and 4-cis-hydroxy-3-trans-fluorocyclohexane carboxylic acid, 3-cis-hydroxy-4-trans-chlorocyclohexane carboxylic acid and 4-cis-hydroxy-3-trans-chlorocyclohexane carboxylic acid, 3-trans-hydroxy-4-cis-chlorocyclohexane carboxylic acid and 4-trans-hydroxy-3-cis-chlorocyclohexane carboxylic acid, 3-trans-cyclohexeneoxide carboxylic acid, 3-cis-cyclohexeneoxide carboxylic acid, 3,4-cis-dihydroxycyclohexane carboxylic acid and 3,4-trans-dihydroxycyclohexane carboxylic acid, cyclohexanepropionic acid, 4-cis/trans-tert-butylcyclohexane carboxylic acid, or simple esters or salts thereof.

### Claim 6 (depends on 1)

The method of claim 1 provided that the non-natural starter fed to said recombinant strain is not selected from the group consisting of: cyclohexane carboxylic acid, 3-cis,4-trans-dihydroxycyclohexane carboxylic acid, 1-cyclohexene carboxylic acid, 3-cyclohexene carboxylic acid, cycloheptane carboxylic acid, 3-(cis/trans)-methylcyclohexane carboxylic acid, 4-(cis/trans)-methylcyclohexane carboxylic acid, 1-cycloheptene carboxylic acid and 5-cis-hydroxyl-3-cyclohexene carboxylic acid.

### Claim 7 (depends on 1)

The method of claim 1, wherein the recombinant strain of part
(a) is made according to a method comprising:
(a) constructing a conjugative deletion plasmid in an E. coli strain that is dam−, dcm−, or dam− and dcm−;
(b) generating spores from said host strain suitable for conjugation wherein said strain is grown at a humidity of between 10% and 40% and the spores are harvested at between 5 and 30 days;
(c) conjugating the E. coli strain of step (a) with the spores from step (b) on a medium that comprises per liter:
i) 0.5 g to 5 g corn steep powder,
ii) 0.1 g to 5 g Yeast extract,
iii) 0.1 g to lOg calcium carbonate; and
iv) 0.01 g to 0.5 g iron sulphate;
said medium additionally containing BACTO-agar and starch and having been dried to result in 1-20% weight loss; and
(d) optionally culturing the recombinant strain under conditions suitable for polyketide production.

### Claim 8 (depends on 7)

The method of claim 7, wherein the strain is selected from the group consisting of Streptomyces hygroscopicus subsp. hygroscopicus NRRL 5491, Actinoplanes sp. N902-109 FERM BP-3832, Streptomyces sp. AA6554, Streptomyces hygroscopicus var. ascomyceticus MA 6475 ATCC 14891, Streptomyces hygroscopicus var. ascomyceticus MA 6678 ATCC 55087, Streptomyces hygroscopicus var. ascomyceticus MA 6674, Streptomyces hygroscopicus var. ascomyceticus ATCC 55276, Streptomyces hygroscopicus subsp. ascomyceticus ATCC 14891, Streptomyces tsukubaensis No. 9993 FERM BP-927, Streptomyces hygroscopicus subsp. yakushimaensis, Streptomyces sp. DSM 4137, Streptomyces sp. DSM 7348, Micromonospora n.sp. A92-306401 DSM 8429 and Streptomyces sp. MA 6858 ATCC 55098.

### Claim 9 (depends on 8)

The method of claim 8, wherein the strains are selected from the group consisting of: S. hygroscopicus subsp. hygroscopicus NRRL 5491 and S. hygroscopicus var. ascomyceticus ATCC 14891.

### Claim 10 (depends on 9)

The method of claim 9, wherein the strain is the rapamycin producer S. hygroscopicus subsp. hygroscopicus NRRL 5491.

### Claim 11 (depends on 3)

The method of claim 3, wherein the process of restoring the deleted genes comprises:
(a) constructing a gene cassette which contains one or more of the deleted genes; and
(b) transforming said recombinant strain that contains biosynthetic clusters that encode FKBP-ligands with said gene cassette.

### Claim 12 (depends on 11)

The method of claim 11, wherein said gene cassette is assembled directly in an expression vector.

### Claim 13 (depends on 4)

The method of claim 4, wherein the complementation is homologous.

### Claim 14 (depends on 5)

The method of claim 5, wherein the complementation is heterologous.

### Claim 15 (depends on 2)

The method of claim 2, wherein said one or more deleted or inactivated auxiliary genes are selected from the group consisting of starter unit supply genes, amino acid precursor supply genes, cytochrome P-450 monooxygenases, ferredoxins, and SAM-dependent O-methyltransferases.

### Claim 16 (depends on 15)

The method of claim 15, wherein the deleted or inactivated genes are selected from the group consisting of rapL, rapN, rapO, rapM, rapQ, rapI, and rapt.

### Claim 17 (depends on 1)

The method of claim 1, wherein said rapK homologue is the fkbO gene or rapK gene.

### Claim 18 (depends on 1)

The method of claim 1, wherein said FKBP-ligand is selected from the group consisting of rapamycin, FK506, FK520, FK525, antascomicin, FK523, tsukubamycin, and hygromicin.

### Claim 19 (depends on 18)

The method of claim 18, wherein said FKBP-ligand is selected from the group consisting of FK520, FK506, and rapamycin.

### Claim 20 (depends on 1)

The method of claim 1, wherein said non-natural starter unit comprises 2-norbornane carboxylic acid.

### Claim 21 (depends on 1)

The method of claim 1, wherein said non-natural starter unit comprises 2-(cis/trans)-hydroxycyclohexane carboxylic acid.

### Claim 22 (depends on 1)

The method of claim 1, wherein said non-natural starter unit comprises 3-(cis/trans)-hydroxycyclohexane carboxylic acid.

### Claim 23 (depends on 1)

The method of claim 1, wherein said non-natural starter unit comprises 4-(cis/trans)-hydroxycyclohexane carboxylic acid.

### Claim 24 (depends on 1)

The method of claim 1, wherein said non-natural starter unit comprises 2-(cis/trans)-methylcyclohexane carboxylic acid.

### Claim 25 (depends on 1)

The method of claim 1, wherein said non-natural starter unit comprises 4-(cis/trans)-methylcyclohexane carboxylic acid.

### Claim 26 (depends on 1)

The method of claim 1, wherein said non-natural starter unit comprises 3-(cis/trans)methoxycyclohexane carboxylic acid.

### Claim 27 (depends on 1)

The method of claim 1, wherein said non-natural starter unit comprises 4-(cis/trans)methoxycyclohexane carboxylic acid.

### Claim 28 (depends on 1)

The method of claim 1, wherein said non-natural starter unit comprises 4-oxo cyclohexane carboxylic acid.

### Claim 29 (depends on 1)

The method of claim 1, wherein said non-natural starter unit comprises 2-oxo cyclohexane carboxylic acid.

### Claim 30 (depends on 1)

The method of claim 1, wherein said non-natural starter unit comprises 4-trans-n-pentylcyclohexane carboxylic acid.

### Claim 31 (depends on 1)

The method of claim 1, wherein said non-natural starter unit comprises 2-trans-aminocyclohexane carboxylic acid.

### Claim 32 (depends on 1)

The method of claim 1, wherein said non-natural starter unit comprises 4-cis-aminocyclohexane carboxylic acid.

### Claim 33 (depends on 1)

The method of claim 1, wherein said non-natural starter unit comprises 4-(cis/trans)-aminomethylcyclohexane carboxylic acid.

### Claim 34 (depends on 1)

The method of claim 1, wherein said non-natural starter unit comprises cyclopentane carboxylic acid.

### Claim 35 (depends on 1)

The method of claim 1, wherein said non-natural starter unit comprises cyclobutane carboxylic acid.

### Claim 36 (depends on 1)

The method of claim 1, wherein said non-natural starter unit comprises 1-methylcyclohexane carboxylic acid.

### Claim 37 (depends on 1)

The method of claim 1, wherein said non-natural starter unit comprises 3-trans-hydroxy-4-cis-fluorocyclohexane carboxylic acid and 4-trans-hydroxy-3-cis-fluorocyclohexane carboxylic acid.

### Claim 38 (depends on 1)

The method of claim 1, wherein said non-natural starter unit comprises 3-cis-hydroxy-4-trans-fluorocyclohexane carboxylic acid and 4-cis-hydroxy-3-trans-fluorocyclohexane carboxylic acid.

### Claim 39 (depends on 1)

The method of claim 1, wherein said non-natural starter unit comprises 3-cis-hydroxy-4-trans-chlorocyclohexane carboxylic acid and 4-cis-hydroxy-3-trans-chlorocyclohexane carboxylic acid.

### Claim 40 (depends on 1)

The method of claim 1, wherein said non-natural starter unit comprises 3-trans-hydroxy-4-cis-chlorocyclohexane carboxylic acid and 4-trans-hydroxy-3-cis-chlorocyclohexane carboxylic acid.

### Claim 41 (depends on 1)

The method of claim 1, wherein said non-natural starter unit comprises 3-trans-cyclohexeneoxide carboxylic acid.

### Claim 42 (depends on 1)

The method of claim 1, wherein said non-natural starter unit comprises 3-cis-cyclohexeneoxide carboxylic acid.

### Claim 43 (depends on 1)

The method of claim 1, wherein said non-natural starter unit comprises 3,4-cis-dihydroxycyclohexane carboxylic acid and 3,4-trans-dihydroxycyclohexane carboxylic acid.

### Claim 44 (depends on 1)

The method of claim 1, wherein said non-natural starter unit comprises cyclohexaneacetic acid.

### Claim 45 (depends on 1)

The method of claim 1, wherein said non-natural starter unit comprises cyclohexanepropionic acid and 4-cis/trans-tert-butylcyclohexane carboxylic acid.