# US 12,760,340 B2 · Methods and compositions for the production of rhamnolipid

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- Number: US 12,760,340 B2
- Document ID: US12760340B2
- Status: Granted
- Kind: B2
- Application number: 18/324,283
- Filed: 2023-05-26
- Published: 2023-12-14
- Granted: 2026-10-13
- Earliest claimed: 2022-05-27
- Assignees: The Regents of the University of California
- Applicants: The Regents of the University of California
- Inventors: Deepika Awasthi; Steven W. Singer
- Web page: https://patentgrep.com/patent/US12760340B2

## CPC

- C12P 7/6409: Fatty acids
- C12N 9/1029: {transferring groups other than amino-acyl groups (2.3.1)}
- C12N 9/1051: {Hexosyltransferases (2.4.1)}
- C12N 9/88: Lyases (4.)
- C12Y 203/01191: UDP-3-O-(3-hydroxymyristoyl)glucosamine N-acyltransferase (2.3.1.191)
- C12Y 204/01: Hexosyltransferases (2.4.1)
- C12Y 402/01055: 3-Hydroxybutyryl-CoA dehydratase (4.2.1.55)
- 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)}
- 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

The present invention provides for a method for producing a rhamnolipid, the method comprising: (a) providing a genetically modified host cell comprising one or more of RhlYZAB capable of expression in the host cell in a growth or culture medium; (b) growing or culturing the host cell such that the one or more of RhlYZAB are expressed and a rhamnolipid is produced; and (c) optionally recovering the rhamnolipid from the host cell or from the growth or culture medium. In some embodiments, the host cell is a methanotroph.

## Claims

### Claim 1

A genetically modified methanotroph comprising one or more of enoyl-CoA hydratase/isomerase (RhlY and RhlZ), 3-hydroxyacyl-ACP-O-3 hydroxyacyltransferase (RhlA), or rhamnosyl transferase (RhlB), and one or more of the following mutations:
#CHROM
POS
REF
ALT
NC 016112.1
257711
T
C
NC 016112.1
274084
A
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NC 016112.1
274102
A
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C
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NC 016112.1
274678
T
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NC 016112.1
274869
C
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NC 016112.1
275047
T
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NC 016112.1
275049
G
A
NC 016112.1
275058
C
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NC 016112.1
275059
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NC 016112.1
275081
C
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NC 016112.1
275093
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NC 016112.1
275110
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NC 016112.1
278205
C
G
NC 016112.1
278215
A
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NC 016112.1
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NC 016112.1
278240
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A
NC 016112.1
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A
NC 016112.1
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A
NC 016112.1
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NC 016112.1
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NC 016112.1
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NC 016112.1
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NC 016112.1
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A
NC 016112.1
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NC 016112.1
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A
NC 016112.1
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NC 016112.1
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NC 016112.1
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279721
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NC 016112.1
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NC 016112.1
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NC 016112.1
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A
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A
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NC 016112.1
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A
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NC 016112.1
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A
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C
T
NC 016112.1
4249287
A
T
NC 016112.1
4249322
C
T
NC 016112.1
4249335
A
G
NC 016112.1
4249392
G
A
NC 016112.1
4249608
T
C
NC 016112.1
4249620
G
T
NC 016112.1
4249623
T
C
NC 016112.1
4249634
A
G
NC 016112.1
4249635
A
G
NC 016112.1
4249670
A
G
NC 016112.1
4249692
C
T
NC 016112.1
4249695
C
T
NC 016112.1
4249717
C
T
NC 016112.1
4249809
T
C
NC 016112.1
4249824
G
A
NC 016112.1
4249842
T
C
NC 016112.1
4249845
C
G
NC 016112.1
4249860
T
C
NC 016112.1
4249874
T
C
NC 016112.1
4249883
A
G
NC 016112.1
4249893
C
T
NC 016112.1
4249902
T
C
NC 016112.1
4249908
A
G
NC 016112.1
4249911
A
G
NC 016112.1
4249912
A
C
NC 016112.1
4249914
C
T
NC 016112.1
4249917
A
G
NC 016112.1
4249926
T
C
NC 016112.1
4249986
A
G
NC 016112.1
4250010
T
C
NC 016112.1
4250103
G
A
NC 016112.1
4250110
A
C
NC 016112.1
4250113
C
T
NC 016112.1
4250115
T
C
NC 016112.1
4250119
T
A
NC 016112.1
4250142
A
G
NC 016112.1
4250148
T
C
NC 016112.1
4250154
G
A
NC 016112.1
4250157
G
A
NC 016112.1
4250166
C
T
NC 016112.1
4250169
C
T
NC 016112.1
4250172
G
A
NC 016112.1
4250199
T
C
NC 016112.1
4250200
T
G
NC 016112.1
4250205
T
C
NC 016112.1
4250235
G
A
NC 016112.1
4250253
T
G
NC 016112.1
4250319
G
A
NC 016112.1
4250546
G
A
NC 016112.1
4250584
T
C
NC 016112.1
4250678
C
T
NC 016112.1
4250712
A
T
NC 016112.1
4250808
A
G
NC 016112.1
4250821
C
T
NC 016112.1
4250837
G
C
NC 016112.1
4250863
T
A
NC 016112.1
4250879
A
G
NC 016112.1
4250924
A
G
NC 016112.1
4255137
C
T
NC 016112.1
4255200
C
T
NC 016112.1
4258918
T
A
NC 016112.1
4258932
A
G
NC 016112.1
4258938
G
A
NC 016112.1
4258963
A
T
NC 016112.1
4258972
C
T
NC 016112.1
4259013
A
G
NC 016112.1
4259401
C
T
NC 016112.1
4259421
T
C
NC 016112.1
4259727
T
C
NC 016112.1
4262921
A
G
NC 016112.1
4262948
A
G
NC 016112.1
4263098
C
T
NC 016112.1
4263149
G
T
NC 016112.1
4263405
T
G
NC 016112.1
4263776
T
A
NC 016112.1
4263947
C
G
NC 016112.1
4263958
T
C
NC 016112.1
4263968
C
T
NC 016112.1
4263979
A
G
NC 016112.1
4270962
C
T
NC 016112.1
4270965
A
G
NC 016112.1
4270968
T
C
NC 016112.1
4271667
C
A
NC 016112.1
4271679
C
G
NC 016112.1
4271701
T
A
NC 016112.1
4428308
G
C
NC 016112.1
4552963
T
C
NC 016112.1
4553104
G
A
NC 016112.1
4553506
C
T
NC 016112.1
4553743
A
G
NC 016112.1
4554354
T
C
NC 016108.1
128058
G
C;
wherein the genetically modified methanotroph is a Methylotuvimicrobium cell.

### Claim 2 (depends on 1)

The genetically modified methanotroph of claim 1, wherein the Methylotuvimicrobium cell is Methylotuvimicrobium buryatense or Methylotuvimicrobium alcaliphilum (syn. Methylomicrobium alcaliphilum).

### Claim 3 (depends on 2)

The genetically modified methanotroph of claim 2, wherein the methanotroph is Methylotuvimicrobium alcaliphilum (syn. Methylomicrobium alcaliphilum).

### Claim 4 (depends on 1)

The genetically modified methanotroph of claim 1, wherein methanotroph comprises mutations in one or more of the following genes: fabG, murA, dxs, tssJ, and tkt.

### Claim 5 (depends on 1)

The genetically modified methanotroph of claim 1, wherein methanotroph comprises mutations in the genes of one or more, or all, of the following: IS3 family transposase (gene-MEALZ_RS03460, gene-MEALZ_RS08615, gene-MEALZ_RS11580); autotransporter outer membrane beta-barrel domain-containing protein (gene-MEALZ_RS22650); Crp/Fnr family transcriptional regulator (gene-MEALZ_RS12360); multicopper oxidase domain-containing protein (gene-MEALZ_RS14455); type IV pilus secretin PilQ (pilQ; gene-MEALZ_RS15795); and, FAD-dependent oxidoreductase/hypothetical protein/NAD(P)/FAD-dependent oxidoreductase (gene-MEALZ_RS18045).

### Claim 6 (depends on 1)

The genetically modified methanotroph of claim 1, wherein the host cell or methanotroph in its unmodified form is sensitive, or unable to grow, in a medium having rhamnolipid at a concentration equal to or more than about 0.05 g/L.

### Claim 7 (depends on 1)

The genetically modified methanotroph of claim 1, wherein the genetically modified host cell or methanotroph is resistant, or able to grow, in a medium having rhamnolipid at a concentration equal to or less than about 5.0 g/L.

### Claim 8 (depends on 1)

The genetically modified methanotroph of claim 1, wherein the genetically modified host cell or methanotroph is capable of producing more rhamnolipid or fatty acids than the host cell or methanotroph in its unmodified form.

### Claim 9 (depends on 1)

The genetically modified methanotroph of claim 1, wherein the RhlY, RhlZ, RhlA, and RhlB are Pseudomonas aeruginosa RhlY, RhlZ, RhlA, and RhlB.