EP-4735617-A1 - CELL-FREE PRODUCTION OF CARMINIC ACID

Abstract

The invention is related to materials and methods for production of carminic acid from substrates. The invention provides methods and materials for cell-free bioproduction of carminic acid.

Inventors

• KIM, NEILL
• AGBAY, Anthony
• AMAYA, Jose
• LI, JINGYI
• BRIDEAU, Nicholas

Assignees

• Debut Biotechnology, Inc.

Dates

Publication Date

20260506

Application Date

20240627

Claims (20)

1. 1. A method for cell-free production of carminic acid, wherein the method comprises: providing one or more enzymes in a cell-free medium, wherein the one or more enzymes result in transformation of one or more substrates to carminic acid.
2. 2. The method of claim 1, wherein the substrate is kermesic acid.
3. 3. The method of claim 2, wherein the enzyme is C-glucosyltransferase (CGT).
4. 4. The method of claim 2, further comprising the cell-free medium comprises an activated sugar.
5. 5. The method of claim 4, wherein the activated sugar is UDP-glucose.
6. 6. The method of claim 5, wherein the UDP-glucose is added to the cell-free medium.
7. 7. The method of claim 5, wherein the UDP-glucose is synthesized in the cell-free medium by the one or more enzymes.
8. 8. The method of claim 7, wherein the UDP-glucose is synthesized from one or ingredients selected from the group consisting of: sucrose, glucose, UTP, UDP, ATP, glucose-6- phosophate, glucose- 1 -phosphate, and/or polyphosphate.
9. 9. The method of claim 8, wherein the one or more enzymes is selected from the group consisting of sucrose synthase (SuSy), glucokinase (GLK), hexokinase (HK), phosphoglucomutase (PGM), polyphosphate kinase (PPK), UTP — glucose- 1 -phosphate uridylyltransferase (UGP), and nucleoside diphosphate kinase (NDK).
10. 10. The method of claim 1, wherein the cell -free medium is a cell lysate.
11. 11 . The method of claim 10, wherein the cell lysate is a cell lysate from cells from a host organism expressing the one or more enzymes.
12. 12. The method of claim 11, wherein the host organism is selected from a group consisting of: bacteria, yeast, and/or mammalian cells.
13. 13. The method of claim 11, wherein the one or more enzymes are introduced in the host organism by integration into genome of the host organism or on a plasmid.
14. 14. The method of claim 13, wherein the host organisms expressing the one or more enzymes are cultured until a pre-determined biomass is achieved to produce the requisite quantity of the one or more enzymes.
15. 15. The method of claim 14, further comprising lysing of cells followed by removal of cell debris to generate a cell lysate for use in the cell-free medium for cell-free production of carminic acid.
16. 16. The method of any of claims 1-15, wherein the one or enzymes are selected from the group consisting of CGT, SuSy, GLK, HK, PGM, PPK, UGP, and NDK.
17. 17. The method of claim 16, wherein the one or more enzymes are present in the cell-free medium for cell-free production of carminic acid.
18. 18. The method of claim 16, wherein the method does not include separating and/or purifying one more enzymes for cell-free production of carminic acid.
19. 19. The method of any of claims 1-18, wherein the cell-free medium further comprises: buffer, kermesic acid, an activated sugar, magnesium chloride, cell lysate, sucrose, glucose, glucose- 1 -phosphate, glucose-6-phosphate, UDP, UTP, ATP, polyphosphate, and/or water.
20. 20. The method of claim 19, wherein the buffer is a phosphate buffer.

Description

Cell-free Production of Carminic Acid I. Field of Invention: The invention is related to materials and methods for production of carminic acid. The invention provides methods and materials for cell-free production of carminic acid. II. Reference to Sequence Listing The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file named DEBU-020-01WO.xml, created on June 26, 2024, which is 56 kilobytes in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety. III. Background Carminic acid is used as an additive in food. Carminic acid is one of the most frequently used dyes in food, medicine, cosmetics and textiles. Carminic acid is added to foods such as ketchup, strawberry milk, and candies. It is also added to cosmetics such as eye shadow, nail polish and lipstick. Carminic acid is a colorant, which can be extracted from the female insect bodies of Dactylopius coccus costa (alternative name Coccus cacti L.). The insects live on Nopalea coccinellifera, Opuntia fidus indica and other plants of the family Cactaceae cultivated for instance in the desert areas of Mexico, Central and South America and Canary Islands. Depending on the pH the colorant may be a color in a spectrum from orange over red to purple and is generally known as cochineal or cochineal color. Carmine colorant is widely used in foods and beverages. In relation to current industrial relevant production, carminic acid is harvested by extraction from the insect® dried bodies with water or alcohol. The insects (Dactylopius coccus) are cultured on cacti. IV. Summary of the Invention The conventional methods of industrial production of carminic acid involves extraction of carminic acid from insect’s bodies. In some other convention methods of production of carminic acid, the production of carminic acid may be conducted inside a host organisms and/or cells. As a result, the supply may therefore be relatively expensive and subject to undesirable variations and price fluctuations. The invention provides methods and compositions for cell-free production of carminic acid. The methods provided in the invention related to cell-free production of carminic acid are economic and reliable as compared to other conventional methods of production of carminic acid. In addition, the methods provided in the invention provide higher titer values of the carminic acid from these processes as compared to carminic acid produced from other conventional methods. In one aspect, the invention provides a cell-free production of carminic acid, wherein the method comprises: providing one or more enzymes in a cell-free medium, wherein the one or more enzymes result in transformation of one or more substrates to carminic acid. In certain embodiments, the substrate that is converted to carminic acid is kermesic acid. In certain embodiments, the enzyme that transforms kermesic acid to carminic acid is C-glucosyltransferase (CGT). In certain embodiments, the reaction medium for C-glucosyltransferase (CGT) mediated enzymatic transformation of kermesic acid to carminic acid further comprises an activated sugar. In certain embodiments, the activated sugar is uridine diphosphate glucose (UDP-glucose). In certain embodiments, UDP-glucose is an essential co-factor for the transformation of kermesic acid to carminic acid. An overview of the conversion of kermesic acid to carminic acid is provided in FIG. 1 In certain aspects, the activated sugar and/or UDP-glucose is added to the reaction medium. In certain aspects, the activated sugar in the reaction medium for cell-free production of carminic acid is UDP-glucose. In certain embodiments, the UDP-glucose for the reaction medium is generated from sucrose. In certain embodiments, the UDP-glucose is generated in the reaction medium by one or more enzymes. In certain embodiments, the generation of UDP-sugar is mediated by sucrose synthase (SuSy). In certain embodiments, sucrose synthase (SuSy) mediates the conversion of sucrose to UDP-glucose. Thus, in certain embodiments, the reaction medium comprises sucrose, which is subsequently converted to UDP-glucose in course of the reaction. An overview of the process where kermesic acid is converted to carminic acid and UDP-glucose is generated from sucrose is provided in FIG. 2. In certain beneficial aspects, the invention provides that the UDP -glucose used in the reaction is generated from other substrates in course of the reaction. In certain embodiments, the UDP moiety in UDP -glucose is recycled in course of the process. The recycling of UDP provides economic efficiency of the processes of the invention. Accordingly, in certain aspects, the production of carminic acid from kermesic acid catalyzed by CGT is conducted in conjunction with other methods for UDP-glucose production or recycling of UDP. In certain embodiments, the overview of the synthetic sche