# US 2019/0169664 A1 · Microbial platform for production of glycosylated compounds

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 2019/0169664 A1
- Document ID: US20190169664A1
- Status: Published application
- Kind: A1
- Application number: 16/099,032
- Filed: 2017-05-05
- Published: 2019-06-06
- Earliest claimed: 2016-05-06
- Assignees: University of Georgia Research Foundation, Inc.
- Applicants: University of Georgia Research Foundation, Inc.
- Inventors: Yajun YAN; Yifei WU; Xinxiao SUN; Yuheng LIN
- Web page: https://patentgrep.com/patent/US20190169664A1

## CPC

- C12P 19/04: Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
- C12N 1/20: Bacteria; Culture media therefor
- C12N 1/32: Processes using, or culture media containing, lower alkanols, i.e. C1 to C6
- C12N 15/52: Genes encoding for enzymes or proenzymes
- C12P 19/12: Disaccharides
- C12P 19/34: Polynucleotides, e.g. nucleic acids, oligoribonucleotides
- C12P 21/005: {Glycopeptides, glycoproteins}
- 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)

## Abstract

Host cells are metabolically engineered to consume glucose and glycerol simultaneously, and to divert glucose from catabolic to anabolic pathways without adversely affecting glucose uptake.

## Claims

### Claim 1

A genetically engineered microbe comprising:
at least one metabolic pathway modification that disrupts glucose catabolism; and
at least one metabolic pathway modification that metabolically redirects phosphoenolpyruvate (PEP) for enhanced uptake of glucose.

### Claim 2 (depends on 1)

The genetically engineered microbe of claim 1, wherein the metabolic pathway modification that disrupts glucose catabolism comprises a modification of the glycolysis pathway, or a modification in the pentose phosphate pathway, or both.

### Claim 3 (depends on 1)

The genetically engineered microbe of claim 1, wherein the metabolic pathway modification that metabolically redirects PEP comprises a modification that disrupts a PEP-dependent glycerol assimilation pathway.

### Claim 4 (depends on 1)

The genetically engineered microbe of claim 1, further comprising at least one metabolic pathway modification selected from the group consisting of:
(a) a metabolic pathway modification that disrupts the conversion of UDP-glucose to UDP glucuronic acid;
(b) a metabolic pathway modification that eliminates the conversion of glucose-1-phosphate to glucolactone;
(c) a metabolic pathway modification that enhances the UDP-glucose biosynthetic pathway so as to direct more glucose into UDP-glucose;
(d) a metabolic pathway modification that enhances the consumption or conversion of glucose-6-phosphate;
(e) a metabolic pathway modification that disrupts a metabolic pathway associated with degradation of a glycosylated compound or a metabolic pathway that diverts a precursor away from the glycosylated compound; and
(f) a metabolic pathway modification that disrupts a metabolic pathway associated with degradation of a glycosylated compound or a metabolic pathway that diverts a precursor away from the glycosylated compound.

### Claim at position 5

-9. (canceled)

### Claim 10 (depends on 1)

The genetically engineered microbe of claim 1, which simultaneously utilizes glucose and a secondary sugar as carbon sources.

### Claim 11 (depends on 10)

The genetically engineered microbe of claim 10, wherein the secondary sugar comprises at least one of glycerol or xylose.

### Claim 12

(canceled)

### Claim 13 (depends on 10)

The genetically engineered microbe of claim 10, wherein phosphoenolpyruvate (PEP) generated from consumption of the secondary sugar is utilized by the phosphotransferase system (PTS) so as to drive glucose uptake for production of a glycosylated compound.

### Claim 14 (depends on 1)

The genetically engineered microbe of claim 1 comprising a synergetic carbon utilization mechanism that
(a) decouples glucose uptake from glucose catabolism by using glycerol as a carbon source to generate phosphoenolpyruvate (PEP) for operating the phosphotransferase system; or
(b) couples glucose uptake with glycerol catabolism via the phosphoenolpyruvate (PEP) as a driving force for glucose transport;
or both (a) and (b).

### Claim at position 11

-17. (canceled)

### Claim 18 (depends on 1)

The genetically engineered microbe of claim 1, comprising at least one mutation selected from the group consisting of Δpgi, Δzwf, ΔpykA, ΔpykF, ΔgldA, Δugd, and Δgcd (E. coli) or their counterparts in other microbes.

### Claim 19 (depends on 1)

The genetically engineered microbe of claim 1, wherein the microbe expresses or overexpresses at least one enzyme encoded by galU or pgm (E. coli) or counterparts in other microbes.

### Claim 20 (depends on 1)

The genetically engineered microbe of claim 1, wherein the microbe produces trehalose, and wherein the microbe further comprises at least one mutation selected from the consisting of ΔtreA, ΔtreC, and ΔtreF (E. coli) or counterparts in other microbes.

### Claim 21 (depends on 1)

The genetically engineered microbe of claim 1, wherein the microbe expresses or overexpresses at least one enzyme encoded by otsA or otsB (E. coli) or counterparts in other microbes.

### Claim 22 (depends on 1)

The genetically engineered microbe of claim 1, comprising an E. coli cell comprising at least one deletion mutation selected from the group consisting of
(a) ΔpgiΔzwf,
(b) ΔpykAFΔgldA;
(c) ΔtreACF;
(d) Δglk;
(e) ΔugdΔgcd;
(f) Δppc; and
(g) any combination thereof.

### Claim 23 (depends on 22)

The genetically engineered microbe of claim 22, comprising an E. coli cell comprising at least one plasmid expressing at least one enzyme operably encoded by at least one member of the group consisting of otsA, otsB, pgm, and galU.

### Claim at position 18

-26. (canceled)

### Claim 27 (depends on 22)

The genetically engineered E. coli cell of claim 22, which is further metabolically engineered to enhance expression of phosphoglucomutase (pgm) or UTP-glucose-1-phosphate uridylyltransferase) (galU) or both.

### Claim 28

(canceled)

### Claim 29 (depends on 1)

A method for producing a glycosylated compound comprising culturing the microbe of claim 1 under conditions to produce the glycosylated compound.

### Claim 30 (depends on 29)

The method of claim 29, wherein the glycosylated compound is selected from the group consisting of a glycoprotein, glycopeptide, glycolipid, proteoglycan, antibody, glycan, glycoside, polysaccharide, nucleotide and nucleic acid.

### Claim 31 (depends on 30)

The method of claim 30, wherein the polysaccharide comprises trehalose, chondroitin or heparin.

### Claim at position 24

-33. (canceled)

### Claim 34 (depends on 30)

The method of claim 30, wherein glucose and at least one of glycerol or xylose are supplied as carbon sources.

### Claim at position 26

-37. (canceled)

### Claim 38 (depends on 1)

The genetically engineered microbe of claim 1, wherein the microbe is a bacterial cell or a yeast cell.

### Claim at position 28

-40. (canceled)