BIDIRECTIONAL MULTI-ENZYMATIC SCAFFOLDS FOR BIOSYNTHESIZING CANNABINOIDS
This document relates to using bidirectional, multi-enzymatic scaffolds to biosynthesize cannabinoids in recombinant hosts.
1 . A host cell comprising:
(a) a first exogenous nucleic acid encoding a first polypeptide having ATP citrate lyase activity and comprising a first heterologous interaction domain,
(b) a second exogenous nucleic acid encoding a second polypeptide having acetyl-CoA acetyltransferase activity and comprising a second heterologous interaction domain, and
(c) a third exogenous nucleic acid encoding a polypeptide scaffold comprising a peptide ligand for each of said first and second heterologous interaction domains,
wherein each of said first and second heterologous interaction domains is different,
wherein each peptide ligand for each of said first and second heterologous interaction domains is different,
wherein said polypeptide scaffold comprises, in an order extending from amino terminus to carboxy terminus, a first copy of said peptide ligand for said first heterologous interaction domain, a first copy of said peptide ligand for said second heterologous interaction domain, a second copy of said peptide ligand for said second heterologous interaction domain, and a second copy of said peptide ligand for said first heterologous interaction domain.
2 . The host cell of claim 1 , wherein said host cell is a bacterial or a yeast host cell.
3 . The host cell of claim 2 , wherein said bacterial cell is selected from the group consisting of Escherichia coli, Bacillus, Brevibacterium, Streptomyces , and Pseudomonas cells.
4 . The host cell of claim 2 , wherein said yeast cell is selected from the group consisting of Pichia pastoris, Saccharomyces cerevisiae, Yarrowia lipolytica, Kluyveromyces marxianus , and Komagataella phaffii cells.
5 . The host cell of claim 1 , wherein said host cell is an algae or a plant cell.
6 . The host cell of claim 5 , wherein said algae is Dunaliella sp., Chlorella variabilis, Euglena mutabilis , or Chlamydomonas reinhardtii cells.
7 . The host cell of claim 5 , wherein said plant cell is a Cannabis or tobacco cell.
8 . The host cell of claim 1 , wherein each of said first and second polypeptides is of the formula: enzyme—linker 1 —spacer—linker 2 —motif 1 —linker 3 —motif 2 , wherein linker 1 , linker 2 , and linker 3 are the same or different, wherein motif 1 and motif 2 are the same or different, and wherein motif 1 and motif 2 form said heterologous interaction domain.
9 . The host cell of claim 8 , wherein said scaffold polypeptide comprises a linker between each adjacent peptide ligand.
10 . The host cell of claim 9 , wherein said scaffold polypeptide is tagged with a MYC tag, FLAG tag, or HA tag.
11 . The host cell of claim 9 , wherein said linker is a flexible GS-rich sequence flanking a rigid α-helical moiety.
12 . The host cell of claim 9 , wherein said spacer is the cTPR6 spacer.
13 . The host cell of claim 1 , wherein a constitutive promoter is operably linked to one or both of said exogenous nucleic acids encoding said polypeptides or to said third exogenous nucleic acid encoding said polypeptide scaffold.
14 . The host cell of claim 1 , wherein a first constitutive promoter is operably linked to one or both of said exogenous nucleic acids encoding said polypeptides and a second constitutive promoter is operably linked to said third exogenous nucleic acid encoding said polypeptide scaffold.
15 . The host cell of claim 14 , wherein said constitutive promoter used to express said polypeptide scaffold has weaker constitutive activity level than said constitutive promoter used to express said polypeptides.
16 . The host cell of claim 1 , wherein each said exogenous nucleic acid comprises an inducible promoter operably linked to the sequence encoding said polypeptide or said polypeptide scaffold.
17 . The host cell of claim 16 , wherein said promoter is the GAL1-10 promoter.