Process for producing a glucuronide and genetically modified microorganisms useful in this process

Patent metadata

Field	Value
Publication number	US-12018307-B2
Application number	US-202217822733-A
Country	US
Kind code	B2
Filing date	Aug 26, 2022
Priority date	Oct 28, 2021
Publication date	Jun 25, 2024
Grant date	Jun 25, 2024

Abstract

The present invention relates to an in vitro or in vivo process for producing a glucuronide comprising a glucuronic acid moiety bound to a phenolic hydroxyl group or a phenolic carboxyl group. Also provided are expression vectors, nucleic acids, polypeptides, and recombinant microbial cells useful in carrying out the process and prodrugs produced by the process.

First claim

What is claimed is:

1. A process for producing a glucuronide from UDP-glucuronic acid and a phenolic compound that comprises a hydroxyl or a carboxyl group, wherein said glucuronide comprises a glucuronic acid moiety bound to the hydroxyl or the carboxyl group of said phenolic compound, wherein said process comprises the steps of:
(i) culturing a genetically modified microorganism in a medium that comprises the phenolic compound, wherein the genetically modified microorganism expresses a UDP-glucuronyltransferase (UGT) that comprises the amino acid sequence of SEQ ID NO:7 or an amino acid sequence at least 95% identical to SEQ ID NO:7, under conditions allowing the genetically modified microorganism to produce the glucuronide; and
(ii) optionally recovering the glucuronide from the medium.

2. The process of claim 1 , wherein the genetically modified microorganism expresses a UDP-glucose dehydrogenase that comprises SEQ ID NO: 6 and a UDP-glucose pyrophosphorylase that comprises SEQ ID NO:8.

3. The process of claim 1 , wherein the genetically modified microorganism has been transformed to express (i) a nucleic acid that encodes a UDP-glucose dehydrogenase having at least 95% sequence identity to the polypeptide of SEQ ID NO: 6, and/or (ii) a nucleic acid that encodes a UDP-glucose pyrophosphorylase having at least 95% sequence identity to the polypeptide of SEQ ID NO: 8.

4. The process of claim 1 , wherein the genetically modified microorganism is selected from the group consisting of bacteria, yeast, filamentous fungi, and microalgae.

5. The process of claim 4 , wherein the genetically modified microorganism is selected from the group consisting of Aspergillus, Trichoderma, Saccharomyces, Pichia, Phaffia, Candida, Hansenula, Salmonella, Bacillus, Acinectorhacter, Zymomonas, Agrobacterium, Erythrobacter, Chloroborium, Chlorella, Chromatium, Flavobacterium, Cytophaga, Rhodobacter, Rhodococcus, Streptomyces, Brevibacterium, Corynebacteria, Mycobacterium, Deinococcus, Escherichia, Erwinia, Pantoea, Pseudomonas, Sphingomonas, Methylomonas, Methylobacter, Methylococcus, Methylosinus, Methylomicrobium, Methylocystis, Alcaligenes, Synechocystis, Methanomonas, Synechococcus, Anabeana, Thiobacillus, Methanobacterium, Klebsiella , and Myxococcus.

6. The process of claim 1 , wherein the phenolic compound is selected from the group consisting of resveratrol, quercetin, ferulic acid, vanillic acid, curcumin, vanillin, chrysin, zearalenone, apigenin, doxorubicin, etoposide, morphine, ezetimibe, 2′-hydroxyflavone, and combinations thereof.

7. The process of claim 1 , wherein the phenolic compound is at a concentration of between 0.25 mM and 0.75 mM.

8. The process of claim 1 , wherein (a) the medium in maintained at a pH of between 5.5 and 7.5, (b) the medium is maintained at a temperature of between 35° C. and 45° C.; and/or (c) the genetically modified microorganism is cultured in the medium for a period of 2.5 hours and 3.5 hours prior to recovering the glucuronide from the medium.

9. The process of claim 1 , wherein the genetically modified microorganism has been transformed to express a UGT that comprises SEQ ID NO: 7.

10. The process of claim 9 , wherein the genetically modified microorganism has been transformed to express a UDP-glucose dehydrogenase that comprises SEQ ID NO: 6.

11. The process of claim 10 , wherein the genetically modified microorganism has been transformed to express a UDP-glucose pyrophosphorylase that comprises SEQ ID NO: 8.

Assignees

• Zhan Jixun
• Univ Utah State

Inventors

• Zhan Jixun

Classifications

• C12N9/1241 Primary

  Nucleotidyltransferases (2.7.7) · CPC title

• C12Y101/01022

  UDP-glucose 6-dehydrogenase (1.1.1.22) · CPC title

• C12Y204/01017

  Glucuronosyltransferase (2.4.1.17) · CPC title

• C12N15/63

  Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression · CPC title

• C12Y207/07009

  UTP-glucose-1-phosphate uridylyltransferase (2.7.7.9), i.e. UDP-glucose-pyrophosphorylase · CPC title