Microbial polycultures and methods of use thereof

1. 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.

2. The method of claim 1 , wherein the phenylpropanoic acid is p-coumaric acid, caffeic acid, cinnamic acid, ferulic acid or a combination thereof.

3. The method of claim 1 , wherein the flavanone is naringenin, eriodictyol, pinocembrin, or a combination thereof.

4. The method of claim 1 , wherein the flavonoid is a flavone, a flavan-3-ol, a flavan-4-ol, a flavonol, an anthocyanin, or a combination thereof.

5. The method of claim 4 , wherein the flavone is apigenin, luteolin, chrysin, or a combination thereof.

6. The method of claim 4 , wherein the flavan-3-ol is afzelechin, catechin, or a combination thereof.

7. The method of claim 4 , wherein the flavan-4-ol is 4,5,7-trihydroxyflavan, 4,5,7,4′-tetrahydroxyflavan, 4,5,7,4′,5′-pentahydroxyflavan, 4,5,7,4′,5′,6′-hexahydroxyflavan, 4,5,7,4′-tetrahydroxy-5′methoxyflavan, or a combination thereof.

8. The method of claim 4 , wherein the flavonol is kaempferol, quercetin, or a combination thereof.

9. The method of claim 4 , wherein the anthocyanin is pelargonidin, cyanidin, delphinidin, peonidin, malvidin, or a combination thereof.

10. The method of claim 1 , wherein the anthocyanidin-3-O-glucoside is cyanidin-3-O-glucoside, elargon din-3-O-glucoside, delphinidin-3-O-glucoside, peonidin-3-O-glucoside, malvidin-3-O-glucoside, or a combination thereof.

11. The method of claim 1 , wherein the phenylpropanoic acid is p-coumaric acid and the flavonoid is (+)-afzelechtin.

12. The method of claim 1 , wherein the phenylpropanoic acid is caffeic acid and the flavonoid is (+)-catechin.

13. The method of claim 1 , wherein the phenylpropanoic acid is cinnamic acid and flavonoid is 3,5,7-trihydroxyflavan.

14. The method of claim 1 , wherein the conditions permitting synthesis of the product compound comprise providing a carbon source to the microbial polyculture, wherein the carbon source is glucose, glycerol, xylose, arabinose, galactose, yeast extract, or a combination thereof.

15. The method of claim 1 , wherein:

the host cell for the TAL module cell is E. coli rpoA14(DE3);

the host cell for the C5 module cell is E. coli BL21starTM(DE3)ΔsucCΔfumC;

the host cell for the p168 module cell is E. coli BL21starTM(DE3); and

the host cell for the Antho module cell is E. coli BL21starTM(DE3).

16. The method according to claim 1 , wherein an inoculation ratio of the TAL module cell to the C5 module cell in the microbial polyculture is about 8:8.