Microbial polycultures and methods of use thereof

A method of producing a product compound in a microbial polyculture;

• wherein the microbial polyculture comprises a first module cell and a least a second module cell, the first and the at least 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 3β-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 (CmCHI) 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 3μ-hydroxylase (CsF3H) of SEQ ID NO:
• 45, Malus domestica flavanone 3μ-hydroxylase (MdF3H) of SEQ ID NO:
• 47, Petroselinum crispum flavanone 3μ-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;
• the method comprising:
• providing a substrate to the microbial polyculture;
• culturing the microbial polyculture under conditions permitting synthesis of the product compound by the microbial polyculture;
• and
• isolating the product compound synthesized by the microbial polyculture;
• with a proviso that:
• the first module cell in the microbial polyculture is the TAL module cell and the second module cell is the C5 module cell, the substrate is glucose, glycerol, or a combination thereof, and the product compound is a flavanone;
• or
• the microbial polyculture comprises the C5 module cell and the p168 module cell, the substrate is a phenylpropanoic acid, and the product compound is a flavonoid;
• wherein, when the C5 module cell comprises an exogenous gene encoding for malonyl-CoA synthetase (MatB) and an exogenous gene encoding for putative dicarboxylate carrier protein (MatC), the substrate is a phenylpropanoic acid, malonate, or a combination thereof and the product is a flavonoid;
• or
• the microbial polyculture comprises the p168 module cell and the Antho module cell, the substrate is a flavanone, and the product compound is an anthocyanidin-3-O-glucoside;
• or
• the microbial polyculture comprises the TAL module cell, the C5 module cell, and the p168 module cell, the substrate is glucose, glycerol, or a combination thereof, and the product compound is a flavonoid;
• or
• the microbial polyculture comprises the C5 module cell, the p168 module cell, and the Antho module cell, the substrate is a phenylpropanoic acid, and the product compound is an anthocyanidin-3-O-glucoside;
• wherein, when the C5 module cell comprises an exogenous gene encoding for malonyl-CoA synthetase (MatB) and an exogenous gene encoding for putative dicarboxylate carrier protein (MatC), the substrate is phenylpropanoic acid, malonate, or a combination thereof and the product is an anthocyanidin-3-O-glucoside;
• or
• the microbial polyculture comprises the TAL module cell, the C5 module cell, the p168 module cell, and the Antho module cell, the substrate is glucose, glycerol, or a combination thereof, and the product compound is an anthocyanidin-3-O-glucoside.