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EP-4735589-A2 - ENZYMES AND METHODS FOR THE SYNTHESIS OF BAKUCHIOL AND DERIVATIVES

EP4735589A2EP 4735589 A2EP4735589 A2EP 4735589A2EP-4735589-A2

Abstract

Disclosed herein are prenyltransferases capable of producing Bakuchiol or analogs thereof from geranyl pyrophosphate (GPP) and cinnamic acid, coumaric acid, caffeic acid, and/or ferulic acid. Also disclosed herein are engineered cells which express a prenyltransferase capable of producing bakuchiol or analogs thereof and method of using the prenyltransferase and cells for producing bakuchiol or analogs thereof.

Inventors

  • KAMBOURAKIS, SPIROS
  • CAIAZZA, Nicky, Christopher
  • URANO, JUN
  • KOMOR, Russell, Scott

Assignees

  • Cellibre, Inc.

Dates

Publication Date
20260506
Application Date
20240701

Claims (20)

  1. 1. A mutant membrane-bound prenyltransferase (MPT) comprising an amino acid sequence with at least 85% identity to the amino acid sequence of SEQ ID NO. 35, 2 or 1, having at least one amino acid modification at a position selected from the group consisting of 78, 99, 123, 282, and 328 of SEQ ID NO: 35, 2 or 1, wherein the mutant MPT is capable of producing bakuchiol or analogs thereof from geranyl pyrophosphate (GPP) and at least one of cinnamic acid, coumaric acid, caffeic acid, and ferulic acid.
  2. 2. The mutant membrane-bound prenyltransferase of claim 1, further comprising at least one additional amino acid modification at a position selected from the group consisting of 106, 111, 209, 323, 344, 385, and 400 of SEQ ID NO: 35, 2 or 1.
  3. 3. The mutant membrane-bound prenyltransferase of claim 1, wherein the bakuchiol analog is selected from the group consisting of dehydrobakuchiol, 3-hydroxy-Bakuchiol, and 3-methoxy-bakuchiol.
  4. 4. The mutant membrane-bound prenyltransferase of any one of claims 1-3, wherein the mutant MPT comprises an amino acid sequence with at least 90% identity to the amino acid sequence of SEQ ID NO: 35, 2, or 1.
  5. 5. The mutant membrane-bound prenyltransferase of any one of claims 1-3, wherein the mutant MPT comprises an amino acid sequence with at least 97% identity to the amino acid sequence of SEQ ID NO: 35, 2, or 1.
  6. 6. The mutant membrane-bound prenyltransferase of any one of claims 1-3, wherein the mutant MPT comprises an amino acid sequence with at least 90% identity to the amino acid sequence of SEQ ID NO: 6, 7, 8, 9, 10, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34.
  7. 7. The mutant membrane-bound prenyltransferase of any one of claims 1-3, wherein the MPT has an amino acid sequence which comprises an amino acid sequence with between 98.5% and 99.8% identity to the amino acid sequence of SEQ ID NO: 35, 2, or 1. 4891 -1065-4412, v. 1
  8. 8. The mutant membrane-bound prenyltransferase (MPT) of claim 1, wherein the mutant MPT transfers geranyl pyrophosphate (GPP) to cinnamic acid, coumaric acid, caffeic acid, and/or ferulic acid to produce dehydrobakuchiol, bakuchiol, 3-hydroxy-bakuchiol, and/or 3-methoxy- bakuchiol, respectively, with a higher efficiency compared to either of enzymes MPT85 (SEQ ID NO: 1) or MPT94 (SEQ ID NO: 2).
  9. 9. The mutant membrane-bound prenyltransferase of claim 7, wherein the higher efficiency of transferring GPP to cinnamic acid, coumaric acid, caffeic acid, and/or ferulic acid comprises at least 1.5 times higher efficiency compared to either of enzymes MPT85 (SEQ ID NO: 1) or MPT94 (SEQ ID NO: 2).
  10. 10. The mutant membrane-bound prenyltransferase of any one of claims 1-3, wherein the at least one amino acid modification and at least one additional amino acid modification are individually selected from substitutions, deletions or insertions.
  11. 11. The mutant membrane -bound prenyltransferase of any one of claims 1-3, further comprising one or more substitutions, deletions, and/or insertions at a position selected from R 121, V124, N165-N172, D176-K184, Y229- Y236, K246-F251, C255-R260, Y298-D306, 1310-G317, and Y391-W395 of SEQ ID NO: 35, 2, or 1.
  12. 12. The mutant membrane-bound prenyltransferase of any one of claims 1-3, wherein the mutant MPT comprises an amino terminal truncation comprising deletion of between 1 and 73 amino acids corresponding to amino acids in positions 2-74 of the amino acid sequence of SEQ ID NO: 35, 2 or 1.
  13. 13. The mutant membrane-bound prenyltransferase of claim 12, wherein the mutant MPT comprises an amino acid sequence with at least 90% identity to an amino acid sequence selected from SEQ ID NO: 6-10, 13, and 23-33. 33 and 44-46.
  14. 14. An engineered cell that expresses the mutant membrane-bound prenyltransferase of any one of claims 1-13 or an MPT comprising an amino acid sequence of MPT85 (SEQ ID NO: 1) or MPT94 (SEQ ID NO: 4891 -1065-4412, v. 1 2), wherein the cell is capable of producing bakuchiol and/or analogs thereof in the presence of geranyl pyrophosphate (GPP) and at least one of cinnamic acid, coumaric acid, caffeic acid, and ferulic acid.
  15. 15. The cell of claim 14, wherein the cell has been engineered to provide greater flux of GPP through the GPP pathway.
  16. 16. The cell of claim 14 or 15, wherein the cell has been engineered to provide greater flux of tyrosine or phenylalanine through the respective aromatic amino acid synthesis pathways.
  17. 17. The cell of claim 16, wherein the greater flux through an aromatic amino acid synthesis pathway is achieved by overexpressing an Aro4 enzyme (EC 2.5.1.54) comprising an amino acid sequence having at least 95% identity to SEQ ID NO:4.
  18. 18. The cell of claim 16, wherein the greater flux through an aromatic amino acid synthesis pathway is achieved by expressing a feedback insensitive Aro4 enzyme having a sequence with at least 95% identity to SEQ ID NO: 5.
  19. 19. The cell of claim 14 or 15, wherein the cell is a yeast cell, an algal cell, or a bacterial cell.
  20. 20. The cell of claim 19, wherein the yeast cell is Saccharomyces, Pichia, or Yarrowia.

Description

ENZYMES AND METHODS FOR THE SYNTHESIS OF BAKUCHIOL AND DERIVATIVES RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63/524,506, filed on June 30, 2023, and U.S. Provisional Application No. 63/525,294, filed on July 6, 2023, the entire teachings of which are incorporated herein by reference. BACKGROUND OF THE INVENTION The Psoralea corylifolia plant has been used in Asia, China and India, as a medicinal herb for the treatment of a variety of conditions, including cardiotonic, vasodilator, antitumor, antibacterial and cytotoxic conditions and, more specifically, for the treatment of skin conditions such as alopecia, leukoderma, leprosy, psoriasis and eczema.(l) The plant produces a multitude of chemicals primarily concentrated in the seed and fruit, with about 90 compounds isolated and characterized thus far. (2) One of the most abundant chemicals is Bakuchiol, which accumulates to ~6 % w/w in dried seeds of the plant. Other compounds found in lower concentrations in the seed include 3-hydroxy-bakuchiol and certain coumarins such as psoralidin, psoralen, isopsoralen and angelicin.(3) Being the most abundant compound in the plant’s seeds, bakuchiol constitutes both the most studied and the only commercially available product isolated from the seeds of Psoralea corylifolia. Bakuchiol has been shown to have many of the clinical effects that have been associated with the plant, specifically its activity in skin conditions (anti- aging, anti-pigmentation, acne, etc.). As a result, it is used as a retinol alternative in various creams and other skin care products. (4, 5, 6) Other bakuchiol analogs described herein are poorly available due to the lack of a ready source for their isolation but may also have beneficial properties. Currently, Bakuchiol is commercially available only from plant extracts, resulting in low availability and a correspondingly high price tag (>$2000 / Kg). In addition, Bakuchiol is co-extracted with other plant compounds, such as phototoxic psoralen and isopsoralen that must be removed because they can cause skin irritation and dermatitis if present in cosmetic skin formulations. (7) Thus, an alternative low- cost method for the production of a less toxic and high purity bakuchiol-containing composition is required. SUMMARY OF THE INVENTION Described herein are enzymes capable of prenylating hydroxycinnamic acids to efficiently obtain bakuchiol and one or more analogs thereof. These enzymes can be expressed in engineered cells to efficiently produce bakuchiol and analogs thereof when fermented under the correct conditions. Also disclosed are methods for providing an alternative source of bakuchiol and its derivatives. Specifically, fermentation methods for cost-effectively producing a high yield of bakuchiol which is substantially free from the toxic compounds found in extracts from Psoralea corylifolia are disclosed. These and other aspects of the invention will be disclosed in more detail herein. Some aspects of the present disclosure are directed to a mutant membranebound prenyltransferase comprising an amino acid sequence with at least 85% identity to the amino acid sequence of SEQ ID NOS. 35, 2 or 1, having at least one amino acid modification at a position selected from the group consisting of 78, 99, 123, 282, and 328 of SEQ ID NO: 35, 2 or 1, wherein the mutant membrane - prenyltransferase is capable of producing bakuchiol or analogs thereof from geranyl pyrophosphate (GPP) and at least one of cinnamic acid, coumaric acid, caffeic acid, and ferulic acid. In some embodiments, the mutant membrane-bound prenyltransferase further comprises at least one additional amino acid modification at a position selected from the group consisting of 106, 111, 209, 323, 344, 385, and 400 of SEQ ID NO: 35, 2 or 1. In some embodiments, the bakuchiol analog is selected from the group consisting of dehydrobakuchiol, 3-hydroxy-Bakuchiol, and 3- methoxy -bakuchiol. In some embodiments, the membrane -bound prenyltransferase comprises an amino acid sequence with at least 90% identity to the amino acid sequence of SEQ ID NO: 35, 2, or 1. In some embodiments, the membrane-bound prenyltransferase comprises an amino acid sequence with at least 97% identify to the amino acid sequence of SEQ ID NO: 35, 2, or 1. 6. In some embodiments, the mutant membrane-bound prenyltransferase comprises an amino acid sequence with at least 4891 -1065-4412, v. 1 90% identity to the amino acid sequence of SEQ ID NO: 6, 7, 8, 9, 10, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34. In some embodiments, the mutant membrane-bound prenyltransferase comprises an amino acid sequence with between 98.5% and 99.8% identity to the amino acid sequence of SEQ ID NO: 35, 2, or 1. In some embodiments, the mutant membrane-bound prenyltransferase (MPT) transfers geranyl pyrophosphate (GPP) to cinnamic acid, coumaric acid, caffeic acid, and/or ferulic acid to p