ENZYMES, METHODS, AND HOST CELLS FOR PRODUCING CARMINIC ACID
The present invention is related to enzymatic pathways for production of carminic acid, host cells capable of production of carminic acid, and methods for the production of carminic acid and related compounds.
1 . A host cell for producing carminic acid, the host cell expressing an enzymatic pathway for biosynthesis of carminic acid from polyketide building blocks.
2 . The host cell of claim 1 , wherein the host cell is a yeast or bacteria.
3 . The host cell of claim 2 , wherein the host cell is a species of Saccharomyces, Pichia , or Yarrowia , which is optionally Saccharomyces cerevisiae, Pichia pastoris , and Yarrowia lipolytica.
4 . (canceled)
5 . The host cell of claim 2 , wherein the host cell is a bacteria selected from Escherichia spp., Bacillus spp Corynebacterium spp Rhodobacter spp Zymomonas spp Vibrio spp., and Pseudomonas spp., and which is optionally Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Rhodobacter capsulatus, Rhodobacter sphaeroides, Zymomonas mobilis, Vibrio natriegens , or Pseudomonas putida.
6 . (canceled)
7 . The host cell of claim 1 , wherein the host cell expresses:
a recombinant fatty acid synthase (FAS)/polyketide synthase (PKS) that converts Acetyl-CoA and/or Malonyl-CoA building blocks to flavokermesic anthrone (FKA);
a monooxygenase enzyme that converts FKA to flavokermesic acid (FK), and a monooxygenase enzyme that converts FK to kermesic acid (KA), where the monooxygenases can be the same or different; and
a C-UDP-glycosyltransferase (C-UGT) that glycosylates FK and/or KA substrate.
8 . The host cell of claim 1 , wherein the host cell expresses one or more enzymes of a bacteria, fungus, plant or insect species, or an engineered variant thereof.
9 . The host cell of claim 8 , wherein the host cell expresses one or more enzymes of Dactyopius coccus, Coccus hesperidum, Porphyrophora polonica, Porphyrophora hamelii, Palmicultor browni , or Pseudococcus longispinus.
10 . The host cell of claim 8 , wherein the host cell expresses one or more enzymes of Aloe arborescens, Hypericum perforatum, Streptomyces spp., Saccharomyces cerevisiae, Schizosaccharomyces pombe, Yarrowia lipolytica , and Escherichia coli.
11 . The host cell of claim 10 , wherein the host cell expresses one or more enzymes of Streptomyces coelicolor or Streptomyces sp. R1128.
12 . The host cell of claim 9 , wherein the FAS/PKS enzyme is an insect enzyme or engineered variant thereof, wherein the FAS/PKS enzyme is optionally an enzyme of Dactyopius coccus, Coccus hesperidum, Porphyrophora polonica, Porphyrophora hamelii, Palmicultor browni , or Pseudococcus longispinus , or an engineered variant thereof.
13 . The host cell of claim 8 , wherein the PKS enzyme is a plant, fungal, or bacterial enzyme that possesses the octaketide synthase and cyclase activities.
14 . The host cell of claim 13 , wherein the FAS/PKS enzyme comprises an enzyme of Aloe arborescens, Hypericum perforatum, Streptomyces spp., Saccharomyces cerevisiae, Schizosaccharomyces pombe, Yarrowia lipolytica , or Escherichia coli , or a catalytically active portion or derivative thereof.
15 . The host cell of claim 14 , wherein the FAS/PKS enzyme comprises an enzyme of Streptomyces coelicolor or Streptomyces sp. R1128, or a catalytically active portion or derivative thereof.
16 . The host cell of claim 7 , wherein modules of Type I and Type II polyketide synthases are assembled to create a polyketide synthase system capable of flavokermesic acid anthrone or flavokermesic acid biosynthesis.
17 . The host cell of claim 13 , wherein the PKS enzyme comprises an amino acid sequence selected from SEQ ID NO:10, SEQ ID NO:11, 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, SEQ ID NO: 12 and/or SEQ ID NO:13, or a catalytic portion and/or engineered variant thereof.
18 . (canceled)
19 . The host cell of claim 1 , wherein a single monooxygenase enzyme converts FKA to CA, through flavokermesic acid (FK).
20 . The host cell of claim 1 , wherein a first monooxygenase enzyme converts FKA to FK, and a second monooxygenase enzyme converts FK to CA.
21 - 23 . (canceled)
24 . The host cell of claim 19 , wherein one or more monooxygenase enzymes is an insect enzyme, optionally selected from Dactyopius coccus, Coccus hesperidum, Porphyrophora polonica, Porphyrophora hamelii, Palmicultor browni , or Pseudococcus longispinus ; or is an engineered variant thereof.
25 . The host cell of claim 1 , wherein the C-UGT comprises the amino acid sequence of SEQ ID NO:2, or an engineered variant thereof.
26 . A method for producing carminic acid, comprising, culturing the microbial cell of claim 1 under conditions suitable for producing carminic acid.
27 . (canceled)