Search

WO-2026096786-A1 - METHODS AND SYSTEMS FOR PREPARING PANTHENOL

WO2026096786A1WO 2026096786 A1WO2026096786 A1WO 2026096786A1WO-2026096786-A1

Abstract

Methods and systems for bioproduction of panthenol in microbial hosts and cell-free are provided, which include a pantothenate synthetase (PanC) enzyme that is engineered for improved activity and increased panthenol production.

Inventors

  • MOXLEY, William, Chris
  • BAUTISTA, Angelica, Zabala
  • CONNOLLY, Morgan, P.

Assignees

  • Debut Biotechnology, Inc.

Dates

Publication Date
20260507
Application Date
20251030
Priority Date
20241031

Claims (20)

  1. 1. A composition for preparing panthenol, comprising: a variant of pantothenate synthetase (PanC), wherein the variant of PanC is engineered for increased activity for panthenol production as compared to wild-type PanC; (7?)-pantoate; and 3 -amino- 1 -propanol, wherein the engineered variant of PanC contains one or more mutations, each mutation at a position corresponding to E66, V109, R121, H124, T175, K184, R187, R196, 1255, K269, or R271 in SEQ ID NO:9.
  2. 2. The composition of claim 1, wherein the engineered variant of PanC comprises an amino acid sequence having at least 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO: 1, SEQ ID NOT, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID N0:9, or SEQ ID NO: 10.
  3. 3. The composition of claim 1, wherein the engineered variant of PanC comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NOT, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NOTO.
  4. 4. The composition of claim 1, wherein the engineered variant of PanC comprises an amino acid sequence having at least 99% identity to SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NOT, SEQ ID NOT, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO: 10.
  5. 5. The composition of claim 1, wherein the engineered variant of PanC comprises an amino acid sequencing having at least 99% identity to SEQ ID NOT, SEQ ID NOT, SEQ ID NO:9, or SEQ ID NO: 10.
  6. 6. The composition of claim 1, wherein the one or more mutations are selected from the group consisting of E66D, V109T, V109C, V109F, V109L, R121W, R121D, H124V, H124I, H124L, Attorney Docket No. DEBU-036/01WO 37396/184 H124M, T175I, T175V, K184F, K184S, R187A, R187C, R187D, R187E, R187F, R187G, R187H, R187L, R187M, R187P, R187S, R187T, R187V, R187W, R187Y, R196Y, K269Y, I255T, I255D, I255R, I255K, K269Y, R271A, R271F, R271G, R271P, and R271T.
  7. 7. The composition of claim 1, wherein the engineered variant of PanC contains a combination of two or more mutations, wherein the combination is one of the combinations listed in Table 3. Table 3
  8. 8. The composition of any one of claims 1-7, wherein the composition further comprises a microbial host strain overexpressing the engineered variant of PanC.
  9. 9. The composition of claim 8, wherein the microbial host strain comprises bacteria, yeast, and/or fungal cells.
  10. 10. The composition of claim 9, wherein the microbial host strain is modified to overexpress (i) one or more variants of ketopantoate hydroxymethyltransferase; (ii) an acetolactate synthase; (iii) 2-dehydropantoate 2-reductase; or (v) any combination thereof.
  11. 11. The composition of claim 8, wherein the host strain is in a defined M9 minimal media. Attorney Docket No. DEBU-036/01WO 37396/184
  12. 12. The composition of claim 8, wherein the (7?)-pantoate concentration is about 0.1 to about 1 mM.
  13. 13. The composition of claim 8, wherein the 3 -amino- 1 -propanol concentration is about 1 to about 200 mM.
  14. 14. The composition of claim 1, wherein the composition is cell-free.
  15. 15. The composition of claim 14, wherein the engineered variant of PanC is in lysate or purified form.
  16. 16. The composition of claim 15, wherein the engineered variant of PanC is a lysate and has a concentration of about 0.1 to about 20 vol%.
  17. 17. The composition of claim 14, wherein the (A)-pantoate concentration is about 1 to about 5 mM.
  18. 18. The composition of claim 14, wherein the 3 -amino- 1 -propanol concentration is about 1 to about 200 mM.
  19. 19. The composition of claim 14, further comprising a buffer at a concentration of about 20 to about 250 mM.
  20. 20. The composition of claim 10, wherein the buffer is Tris pH 7.5.

Description

Attorney Docket No. DEBU-036/01WO 37396/184 METHODS AND SYSTEMS FOR PREPARING PANTHENOL SEQUENCE LISTING This application incorporates by reference a sequence listing submitted as a text file entitled “DEBU-036-01WO-Seq-Listing.xml” created October 29, 2025 and having a size of 23 kilobytes. BACKGROUND Panthenol is the alcohol analog of pantothenic acid, and exists in two optically active forms that can be present individually or as a racemic mixture (DL-panthenol, CAS Registry Number® 16485-10-2). The dextrorotatory form of panthenol (D-panthenol, CAS Registry Number® 81-13-0; synonyms: (A)-(+)-2,4-dihydroxy-N-(3-hydroxypropyl)-3,3- dimethylbutyramide, ( )-2,4-dihydroxy -3, 3 -dimethylbutyric 3-hydroxypropylamide, D- pantothenyl alcohol, dexpanthenol, provitamin B5) is oxidized to produce D-pantothenic acid/pantothentate (Vitamin B5), which is one of the water-soluble B-complex vitamins important for protein metabolism and synthesis of red blood cells. Panthenol (D-panthenol or DL-panthenol) is useful in a variety of pharmaceutical and cosmetic products, including but not limited to topical preparations (ointments, creams, lotions, etc.) with moisturizing, wound healing, anti-aging, anti-inflammatory, and/or anti-oxidant effects. Panthenol is typically manufactured via chemical synthesis. However, improved methods of panthenol production are needed in the art. SUMMARY The present invention provides, in various embodiments, methods and systems for bioproduction of panthenol in microbial hosts or cell-free. In some embodiments, the invention provides a composition for preparing panthenol, comprising: a variant of pantothenate synthetase (PanC), wherein the variant of PanC is engineered for increased activity for panthenol production as compared to wild-type PanC; ( )- pantoate; and 3-amino-l -propanol, wherein the engineered variant of PanC contains one or more mutations, each mutation at a position corresponding to E66, V109, R121, H124, T175, K184, R187, R196, 1255, K269, or R271 in SEQ ID NOV. Attorney Docket No. DEBU-036/01WO 37396/184 In some embodiments, the engineered variant of PanC comprises an amino acid sequence having at least 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO: 10. In some embodiments, the engineered variant of PanC comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO: 10. In some embodiments, the engineered variant of PanC comprises an amino acid sequence having at least 99% identity to SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO: 10. In some embodiments, the engineered variant of PanC comprises an amino acid sequencing having at least 99% identity to SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO: 10. In some embodiments, the one or more mutations are selected from the group consisting of E66D, V109T, V109C, V109F, V109L, R121W, R121D, H124V, H124I, H124L, H124M, T175I, T175V, K184F, K184S, R187A, R187C, R187D, R187E, R187F, R187G, R187H, R187L, R187M, R187P, R187S, R187T, R187V, R187W, R187Y, R196Y, K269Y, I255T, I255D, I255R, I255K, K269Y, R271A, R271F, R271G, R271P, and R271T. In some embodiments, the engineered variant of PanC contains a combination of two or more mutations, wherein the combination is one of the combinations listed in Table 3. In some embodiments, the composition further comprises a microbial host strain overexpressing the engineered variant of PanC. In some embodiments, the microbial host strain comprises bacteria, yeast, and/or fungal cells. In some embodiments, the microbial host strain is engineered to include modifications that enhance production of (7? -pantoate, including but not limited to overexpression of panB, panE, and ALS. In some embodiments, the microbial host strain is modified to overexpress (i) one or more variants of ketopantoate hydroxymethyltransferase; (ii) an acetolactate synthase; (iii) 2- dehydropantoate 2-reductase; or (v) any combination thereof. In some embodiments, the host strain is in a defined M9 minimal media. Attorney Docket No. DEBU-036/01WO 37396/184 In some embodiments, the (7?)-pantoate concentration is about 0.1 to about 1 mM. In some embodiments, the 3 -amino- 1 -propanol concentration is about 1 to about 200 mM. In some embodiments, the composition is cell-free. In some embodiments, the engineered variant of PanC is in lysate or purified form. In some embodiments, the engineered variant of PanC is a lysate and has a concentration of about 0.1 to about 20 vol%. In some embodiments, the (A)-pantoate concentration is about 1 to about 5 mM. In some embodiments, the 3 -amino- 1 -propanol concentration is about 1 to about 200 mM. In some embodiments, the composition further comprises a buffer at