Microbial polycultures and methods of use thereof

Patent metadata

Field	Value
Publication number	US-11965197-B2
Application number	US-202117197468-A
Country	US
Kind code	B2
Filing date	Mar 10, 2021
Priority date	Nov 2, 2015
Publication date	Apr 23, 2024
Grant date	Apr 23, 2024

Abstract

Disclosed herein are novel microbial polycultures of two or more cell strains, capable of producing flavanones, flavonoids, and anthocyanidin-3-O-glucosides, and methods of use thereof. Also disclosed is a microbial cell capable of producing phenylpropanoic acids, and methods of use thereof.

First claim

What is claimed is:

1. A microbial polyculture comprising a first module cell and at least a second module cell, the first and the at least a second module cell including:
a TAL module cell comprising an exogenous gene encoding for a tyrosine ammonia lyase (TAL);
a C5 module cell comprising an exogenous gene encoding for a 4-coumaroyl-CoA ligase (4CL), an exogenous gene encoding for a chalcone synthase (CHS), an exogenous gene encoding for a chalcone isomerase (CHI), or, the C5 module cell comprises an exogenous gene encoding for a 4-coumaroyl-CoA ligase (4CL), an exogenous gene encoding for a chalcone synthase (CHS), an exogenous gene encoding for a chalcone isomerase (CHI) and further comprises an exogenous gene encoding for malonyl-CoA synthetase (MatB) and an exogenous gene encoding for putative dicarboxylate carrier protein (MatC);
a p168 module cell comprising an exogenous gene encoding for a flavanone 3(3-hydroxylase (F3H), an exogenous gene encoding for a dihydroflavonol 4-reductase (DFR), and an exogenous gene encoding for a leucoanthocyanidin reductase (LAR); and
an Antho module cell comprising an exogenous gene encoding for an anthocyanidin synthase (ANS) and an exogenous gene encoding for a 3-glucosyl transferase (3GT);
wherein the exogenous gene encoding for TAL encodes a polypeptide having at least 85% amino acid identity with the amino acid sequence of Rhodotorula glutinis tyrosine ammonia lyase (RgTAL) of SEQ ID NO: 29, Rhodobacter capsulatus TAL of SEQ ID NO: 80, Rice TAL of SEQ ID NO: 81, Parsley TAL of SEQ ID NO: 82, Tomato TAL of SEQ ID NO: 83, Arabidopsis TAL of SEQ ID NO: 84, or a combination thereof, having TAL activity,
the exogenous gene encoding for 4CL encodes a polypeptide having at least 85% amino acid identity with the amino acid sequence of Arabidopsis thaliana 4-coumaroyl-CoA ligase (At4CL) of SEQ ID NO: 31 , Petroselinum crispum 4-coumaroyl-CoA ligase (Pc4CL) of SEQ ID NO: 33, Vitis vinifera 4-coumaroyl-CoA ligase (Vv4CL) of SEQ ID NO: 35, or a combination thereof, having 4CL activity,
the exogenous gene encoding for CHS encodes a polypeptide having at least 85% amino acid identity with the amino acid sequence of Petunia X hybrida chalcone synthase (PhCHS) of SEQ ID NO: 37 , Citrus maxima chalcone synthase (CmCHS) of SEQ ID NO: 39, or a combination thereof, having CHS activity,
the exogenous gene encoding for CHI encodes a polypeptide having at least 85% amino acid identity with the amino acid sequence of Medicago sativa chalcone isomerase (MsCHI) of SEQ ID NO: 43 , Citrus maxima chalcone isomerase (CmCRE) of SEQ ID NO: 41, or a combination thereof, having CHI activity,
the exogenous gene encoding for MatB encodes a polypeptide having at least 85% amino acid identity with the amino acid sequence of Rhizobium trifolii malonyl-CoA synthetase (RtMatB) of SEQ ID NO: 25 having MatB activity,
the exogenous gene encoding for MatC encodes a polypeptide having at least 85% amino acid identity with the amino acid sequence of Rhizobium trifolii putative dicarboxylate carrier protein (RtMatC) of SEQ ID NO: 26 having MatC activity,
the exogenous gene encoding for F3H encodes a polypeptide having at least 85% amino acid identity with the amino acid sequence of Camellia sinensis flavanone 3(3-hydroxylase (CsF3H) of SEQ ID NO: 45, Malus domestica flavanone 33-hydroxylase (MdF3H) of SEQ ID NO: 47 , Petroselinum crispum flavanone 33-hydroxylase (PcF3H) of SEQ ID NO: 49, or a combination thereof, having F3H activity,
the exogenous gene encoding for DFR encodes a polypeptide having at least 85% amino acid identity with the amino acid sequence of Anthrium andraeanum dihydroflavonol 4-reductase (AaDFR) of SEQ ID NO: 51, Camellia sinensis dihydroflavonol 4-reductase (CsDFR) of SEQ ID NO: 53 , Fragaria x ananassa dihydroflavonol 4-reductase (FaDFR) of SEQ ID NO: 55, or a combination thereof, having DFR activity,
the exogenous gene encoding for LAR encodes a polypeptide having at least 85% amino acid identity with the amino acid sequence of Camellia sinensis leucoanthocyanidin reductase (CsLAR) of SEQ ID NO: 57, Desmodium uncinatum leucoanthocyanidin reductase (DuLAR) of SEQ ID NO: 59, or a combination thereof, having LAR activity,
the exogenous gene encoding for ANS encodes a polypeptide having at least 85% amino acid identity with the amino acid sequence of Petunia X hybrida anthocyanidin synthase (PhANS) of SEQ ID NO: 61, Malus domestica ANS of SEQ ID NO: 70, Pyrus communis ANS of SEQ ID NO: 71, Prunus avium ANS of SEQ ID NO: 72 , Fragaria x ananassa ANS of SEQ ID NO: 73, Vitis vinifera ANS of SEQ ID NO: 74, Ipomoea purpurea anthocyanidin synthase of SEQ ID NO: 75, Camellia sinensis ANS of SEQ ID NO: 76, Citrus sinensis anthocyanidin synthase (ANS) of SEQ ID NO: 77, Vaccinium ashei ANS of SEQ ID NO: 78, Populus trichocarpa ANS of SEQ ID NO: 79, or combinations thereof, having ANS activity, and
the exogenous gene encoding for 3GT encodes a polypeptide having at least 85% amino acid identity with the amino acid sequence of Arabidopsis thaliana 3-glucosyl transferase (At3GT) of SEQ ID NO: 63 , Fragaria x ananassa 3GT of SEQ ID NO: 64, Vitis vinifera 3GT of SEQ ID NO: 65, Forsynthia 3GT of SEQ ID NO: 66, Eggplant 3GT of SEQ ID NO: 67, Gentian 3GT of SEQ ID NO: 68, Petunia x hybrida 3GT of SEQ ID NO: 69, or a combination thereof, having 3GT activity;
with a proviso that:
the first module cell in the microbial polyculture comprises the TAL module cell and the second module cell is the C5 module cell; or
the microbial polyculture comprises the C5 module cell and the p168 module cell; or
the microbial polyculture comprises the p168 module cell and the Antho module cell; or
the microbial polyculture comprises the TAL module cell, the C5 module cell, and the p168 module cell; or
the microbial polyculture comprises the C5 module cell, the p168 module cell, and the Antho module cell; or
the microbial polyculture comprises the TAL module cell, the C5 module cell, the p168 module cell, and the Antho module cell.

2. The microbial polyculture of claim 1 , wherein the exogenous gene encoding for the tyrosine ammonia lyase (TAL) is a gene encoding for Rhodotorula glutinis tyrosine ammonia lyase (RgTAL).

3. The microbial polyculture of claim 1 , wherein:
the exogenous gene encoding for the 4-coumaroyl-CoA ligase (4CL) is a gene encoding for Arabidopsis thaliana 4-coumaroyl-CoA ligase (At4CL);
the exogenous gene encoding for the chalcone synthase (CHS) is a gene encoding for Petunia X hybrida chalcone synthase (PhCHS);
the exogenous gene encoding for the chalcone isomerase (CHI) is a gene encoding for Citrus maxima chalcone isomerase (CmCHI);
the exogenous gene encoding for the malonyl-CoA synthetase (MatB) is a gene encoding for Rhizobium trifolii malonyl-CoA synthetase (RtMatB); and
the exogenous gene encoding for the dicarboxylate carrier protein (MatC) is a gene encoding for Rhizobium trifolii putative dicarboxylate carrier protein (RtMatC).

4. The microbial polyculture of claim 1 , wherein:
the exogenous gene encoding for the flavanone 3β-hydroxylase (F3H) is a gene encoding for Camellia sinensis flavanone 3β-hydroxylase (CsF3H);
the exogenous gene encoding for the dihydroflavonol 4-reductase (DFR) is a gene encoding for Fragaria x ananassa dihydroflavonol 4-reductase (FaDFR); and
the exogenous gene encoding for the leucoanthocyanidin reductase (LAR) is a gene encoding for Desmodium uncinatum leucoanthocyanidin reductase (DuLAR).

5. The microbial polyculture of claim 1 , wherein the exogenous gene encoding for the anthocyanidin synthase (ANS) is a gene encoding for Petunia X hybrida anthocyanidin synthase (PhANS).

6. The microbial polyculture of claim 1 , wherein the exogen

Assignees

• Rensselaer Polytech Inst

Inventors

• Koffas Mattheos
• Jones John Andrew

Classifications

• C12P19/58 Primary

  having an oxygen atom of the saccharide radical directly bound through only acyclic carbon atoms to a non-saccharide heterocyclic ring, e.g. bleomycin, phleomycin · CPC title

• C12N9/0006

  acting on CH-OH groups as donors (1.1) · CPC title

• C12N9/0071

  acting on paired donors with incorporation of molecular oxygen (1.14) · CPC title

• C12N9/0093

  acting on CH or CH2 groups (1.17) · CPC title

• C12N9/1037

  Naringenin-chalcone synthase (2.3.1.74), i.e. chalcone synthase · CPC title