MICROBIAL POLYCULTURES AND METHODS OF USE THEREOF
20170121748 ยท 2017-05-04
Assignee
Inventors
Cpc classification
C12N9/0071
CHEMISTRY; METALLURGY
C12Y101/01219
CHEMISTRY; METALLURGY
C12Y602/01014
CHEMISTRY; METALLURGY
C12N9/1037
CHEMISTRY; METALLURGY
C12Y203/01074
CHEMISTRY; METALLURGY
C12P19/58
CHEMISTRY; METALLURGY
C12Y114/11009
CHEMISTRY; METALLURGY
C12P17/06
CHEMISTRY; METALLURGY
International classification
C12P19/58
CHEMISTRY; METALLURGY
C12N9/00
CHEMISTRY; METALLURGY
C12P17/06
CHEMISTRY; METALLURGY
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.
Claims
1. A method of producing a product compound in a microbial polyculture; wherein, optionally, the microbial polyculture comprises a TAL module cell comprising an exogenous gene encoding for a tyrosine ammonia lyase (TAL); wherein, optionally, the microbial polyculture comprises 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), and wherein, optionally, the C5 module cell further comprises an exogenous gene encoding for malonyl-CoA synthetase (MatB) and an exogenous gene encoding for putative dicarboxylate carrier protein (MatC); wherein, optionally, the microbial polyculture further comprises a p168 module cell comprising an exogenous gene encoding for a flavanone 3-hydroxylase (F3H), an exogenous gene encoding for a dihydroflavonol 4-reductase (DFR), and an exogenous gene encoding for a leucoanthocyanidin reductase (LAR); and wherein, optionally, the microbial polyculture further comprises 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); 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 microbial polyculture comprises the TAL module cell and 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, further comprising: when the microbial polyculture comprises the TAL module cell, creating the TAL module cell by introducing an exogenous gene encoding for a tyrosine ammonia lyase (TAL) into a host cell for the TAL module cell; when the microbial polyculture comprises the C5 module cell, creating the C5 module cell by introducing 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, optionally, introducing an exogenous gene encoding for malonyl-CoA synthetase (MatB) and an exogenous gene encoding for putative dicarboxylate carrier protein (MatC), into a host cell for the C5 module cell; when the microbial polyculture comprises the p168 module cell, creating the p168 module cell by introducing an exogenous gene encoding for a flavanone 3-hydroxylase (F3H), an exogenous gene encoding for a dihydroflavonol 4-reductase (DFR), and an exogenous gene encoding for a leucoanthocyanidin reductase (LAR) into a host cell for the p168 module cell; and when the microbial polyculture comprises the TAL module cell, creating the Antho module cell by introducing an exogenous gene encoding for an anthocyanidin synthase (ANS), and an exogenous gene encoding for a 3-glucosyl transferase (3GT) into a host cell for the Antho module cell.
3. The method 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), Rhodobacter capsulatus TAL, Rice TAL, Parsley TAL, Tomato TAL, Arabidopsis TAL, or a combination thereof; the exogenous gene encoding for the 4-coumaroyl-CoA ligase (4CL) is a gene encoding for Arabidopsis thaliana 4-coumaroyl-CoA ligase (At4CL), Petroselinum crispum 4-coumaroyl-CoA ligase (Pc4CL), Vitis vinifera 4-coumaroyl-CoA ligase (Vv4CL), or a combination thereof; the exogenous gene encoding for the chalcone synthase (CHS) is a gene encoding for Petuniahybrida chalcone synthase (PhCHS), Citrus maxima chalcone synthase (CmCHS), or a combination thereof; the exogenous gene encoding for the chalcone isomerase (CHI) is a gene encoding for Medicago sativa chalcone isomerase (MsCHI), Citrus maxima chalcone isomerase (CmCHI), or a combination thereof; the exogenous gene encoding for the malonyl-CoA synthetase (MatB) is a gene encoding for Rhizobium trifolii malonyl-CoA synthetase (RtMatB); the exogenous gene encoding for the dicarboxylate carrier protein (MatC) is a gene encoding for Rhizobium trifolii putative dicarboxylate carrier protein (RtMatC); the exogenous gene encoding for the flavanone 3-hydroxylase (F3H) is a gene encoding for Camellia sinensis flavanone 3-hydroxylase (CsF3H), Malus domestica flavanone 3-hydroxylase (MdF3H), Petroselinum crispum flavanone 3-hydroxylase (PcF3H), or a combination thereof; the exogenous gene encoding for the dihydroflavonol 4-reductase (DFR) is a gene encoding for Anthrium andraeanum dihydroflavonol 4-reductase (AaDFR), Camellia sinensis dihydroflavonol 4-reductase (CsDFR), Fragariaananassa dihydroflavonol 4-reductase (FaDFR), or a combination thereof; the exogenous gene encoding for the leucoanthocyanidin reductase (LAR) is a gene encoding for Camellia sinensis leucoanthocyanidin reductase (CsLAR), Desmodium uncinatum leucoanthocyanidin reductase (DuLAR), or a combination thereof; the exogenous gene encoding for the anthocyanidin synthase (ANS) is a gene encoding for Petuniahybrida anthocyanidin synthase (PhANS), Malus domestica ANS, Pyrus communis ANS, Prunus avium ANS, Fragariaananassa ANS, Vitis vinifera ANS, Ipomoea purpurea anthocyanidin synthase (ANS), Camellia sinensis ANS, Citrus sinensis anthocyanidin synthase (ANS), Vaccinium ashei ANS, Populus trichocarpa ANS, or a combination thereof; and the exogenous gene encoding for the 3-glucosyl transferase (3GT) is a gene encoding for Arabidopsis thaliana 3-glucosyl transferase (At3GT), Fragariaananassa 3GT, Vitis vinifera 3GT, Forsynthia 3GT, Eggplant 3GT, Gentian 3GT, Petuniahybrida 3GT, or a combination thereof.
4. The method 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).
5. The method 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 Petuniahybrida 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).
6. The method of claim 1, wherein: the exogenous gene encoding for the flavanone 3-hydroxylase (F3H) is a gene encoding for Camellia sinensis flavanone 3-hydroxylase (CsF3H); the exogenous gene encoding for the dihydroflavonol 4-reductase (DFR) is a gene encoding for Fragariaananassa dihydroflavonol 4-reductase (FaDFR); and the exogenous gene encoding for the leucoanthocyanidin reductase (LAR) is a gene encoding for Desmodium uncinatum leucoanthocyanidin reductase (DuLAR).
7. The method of claim 1, wherein the exogenous gene encoding for the anthocyanidin synthase (ANS) is a gene encoding for Petuniahybrida anthocyanidin synthase (PhANS).
8. The method of claim 1, wherein the exogenous gene encoding for the 3-glucosyl transferase (3GT) is a gene encoding for Arabidopsis thaliana 3-glucosyl transferase (At3GT).
9. The method of claim 1, wherein the phenylpropanoic acid is p-coumaric acid, caffeic acid, cinnamic acid, ferulic acid or a combination thereof.
10. The method of claim 1, wherein the flavanone is naringenin, eriodictyol, pinocembrin, or a combination thereof.
11. 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.
12. The method of claim 11, wherein the flavone is apigenin, luteolin, chrysin, or a combination thereof.
13. The method of claim 11, wherein the flavan-3-ol is afzelechin, catechin, or a combination thereof.
14. The method of claim 11, 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-5methoxyflavan, or a combination thereof.
15. The method of claim 11, wherein the flavonol is kaempferol, quercetin, or a combination thereof.
16. The method of claim 11, wherein the anthocyanin is pelargonidin, cyanidin, delphinidin, peonidin, malvidin, or a combination thereof.
17. The method of claim 1, wherein the anthocyanidin-3-O-glucoside is cyanidin-3-O-glucoside, pelargonidin-3-O-glucoside, delphinidin-3-O-glucoside, peonidin-3-O-glucoside, malvidin-3-O-glucoside, or a combination thereof.
18. The method of claim 1, wherein the phenylpropanoic acid is p-coumaric acid and the flavonoid is (+)-afzelechtin.
19. The method of claim 1, wherein the phenylpropanoic acid is caffeic acid and the flavonoid is (+)-catechin.
20. The method of claim 1, wherein the phenylpropanoic acid is cinnamic acid and flavonoid is 3,5,7-trihydroxyflavan.
21. 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.
22. The method of claim 2, 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 BL21star(DE3)sucCfumC; the host cell for the p168 module cell is E. coli BL21star(DE3); and the host cell for the Antho module cell is E. coli BL21star(DE3).
23. A microbial polyculture, comprising: optionally, a TAL module cell comprising an exogenous gene encoding for a tyrosine ammonia lyase (TAL); optionally, 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), and wherein, optionally, the C5 module cell further comprises an exogenous gene encoding for malonyl-CoA synthetase (MatB) and an exogenous gene encoding for putative dicarboxylate carrier protein (MatC); optionally, a p168 module cell comprising an exogenous gene encoding for a flavanone 3-hydroxylase (F3H), an exogenous gene encoding for a dihydroflavonol 4-reductase (DFR), and an exogenous gene encoding for a leucoanthocyanidin reductase (LAR); and optionally, 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); with a proviso that: the microbial polyculture comprises the TAL module cell and 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.
24. The microbial polyculture of claim 23, wherein: the exogenous gene encoding for the tyrosine ammonia lyase (TAL) is a gene encoding for Rhodotorula glutinis tyrosine ammonia lyase (RgTAL), Rhodobacter capsulatus TAL, Rice TAL, Parsley TAL, Tomato TAL, Arabidopsis TAL, or a combination thereof; the exogenous gene encoding for the 4-coumaroyl-CoA ligase (4CL) is a gene encoding for Arabidopsis thaliana 4-coumaroyl-CoA ligase (At4CL), Petroselinum crispum 4-coumaroyl-CoA ligase (Pc4CL), Vitis vinifera 4-coumaroyl-CoA ligase (Vv4CL), or a combination thereof; the exogenous gene encoding for the chalcone synthase (CHS) is a gene encoding for Petuniahybrida chalcone synthase (PhCHS), Citrus maxima chalcone synthase (CmCHS), or a combination thereof; the exogenous gene encoding for the chalcone isomerase (CHI) is a gene encoding for Medicago sativa chalcone isomerase (MsCHI), Citrus maxima chalcone isomerase (CmCHI), or a combination thereof; the exogenous gene encoding for the malonyl-CoA synthetase (MatB) is a gene encoding for Rhizobium trifolii malonyl-CoA synthetase (RtMatB); the exogenous gene encoding for the dicarboxylate carrier protein (MatC) is a gene encoding for Rhizobium trifolii putative dicarboxylate carrier protein (RtMatC); the exogenous gene encoding for the flavanone 3-hydroxylase (F3H) is a gene encoding for Camellia sinesis flavanone 3-hydroxylase (CsF3H), Malus domestica flavanone 3-hydroxylase (MdF3H), Petroselinum crispum flavanone 3-hydroxylase (PcF3H), or a combination thereof; the exogenous gene encoding for the dihydroflavonol 4-reductase (DFR) is a gene encoding for Anthrium andraeanum dihydroflavonol 4-reductase (AaDFR), Camellia sinensis dihydroflavonol 4-reductase (CsDFR), Fragariaananassa dihydroflavonol 4-reductase (FaDFR), or a combination thereof; the exogenous gene encoding for the leucoanthocyanidin reductase (LAR) is a gene encoding for Camellia sinensis leucoanthocyanidin reductase (CsLAR), Desmodium uncinatum leucoanthocyanidin reductase (DuLAR), or a combination thereof; the exogenous gene encoding for the anthocyanidin synthase (ANS) is a gene encoding for Petuniahybrida anthocyanidin synthase (PhANS), Malus domestica ANS, Pyrus communis ANS, Prunus avium ANS, Fragariaananassa ANS, Vitis vinifera ANS, Ipomoea purpurea anthocyanidin synthase (ANS), Camellia sinensis ANS, Citrus sinensis anthocyanidin synthase (ANS), Vaccinium ashei ANS, Populus trichocarpa ANS, or a combination thereof; and the exogenous gene encoding for the 3-glucosyl transferase (3GT) is a gene encoding for Arabidopsis thaliana 3-glucosyl transferase (At3GT), Fragariaananassa 3GT, Vitis vinifera 3GT, Forsynthia 3GT, Eggplant 3GT, Gentian 3GT, Petuniahybrida 3GT, or a combination thereof.
25. The microbial polyculture of claim 23, wherein the exogenous gene encoding for the tyrosine ammonia lyase (TAL) is a gene encoding for Rhodotorula glutinis tyrosine ammonia lyase (RgTAL).
26. The microbial polyculture of claim 23, 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 Petuniahybrida 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).
27. The microbial polyculture of claim 23, wherein: the exogenous gene encoding for the flavanone 3-hydroxylase (F3H) is a gene encoding for Camellia sinensis flavanone 3-hydroxylase (CsF3H); the exogenous gene encoding for the dihydroflavonol 4-reductase (DFR) is a gene encoding for Fragariaananassa dihydroflavonol 4-reductase (FaDFR); and the exogenous gene encoding for the leucoanthocyanidin reductase (LAR) is a gene encoding for Desmodium uncinatum leucoanthocyanidin reductase (DuLAR).
28. The microbial polyculture of claim 23, wherein the exogenous gene encoding for the anthocyanidin synthase (ANS) is a gene encoding for Petuniahybrida anthocyanidin synthase (PhANS).
29. The microbial polyculture of claim 23, wherein the exogenous gene encoding for the 3-glucosyl transferase (3GT) is a gene encoding for Arabidopsis thaliana 3-glucosyl transferase (At3 GT).
30. The microbial polyculture of claim 23, wherein: a host cell for the TAL module cell is E. coli rpoA14(DE3); a host cell for the C5 module cell is E. coli BL21star(DE3)sucCfumC; a host cell for the p168 module cell is E. coli BL21star(DE3); and a host cell for the Antho module cell is E. coli BL21star(DE3).
31. A method of producing a phenylpropanoic acid in a TAL module cell, wherein the TAL module cell is a microbial cell comprising an exogenous gene encoding for a tyrosine ammonia lyase (TAL); the method comprising: providing a substrate to the TAL module cell, wherein the substrate comprises glucose, glycerol, or a combination thereof; culturing the TAL module cell under conditions permitting synthesis of the phenylpropanoic acid by the TAL module cell; and isolating the phenylpropanoic acid synthesized by the TAL module cell.
32. The method of claim 31, further comprising creating the TAL module cell by introducing an exogenous gene encoding for a tyrosine ammonia lyase (TAL) into a host cell for the TAL module cell.
33. The method of claim 31, wherein: the exogenous gene encoding for the tyrosine ammonia lyase (TAL) is a gene encoding for Rhodotorula glutinis tyrosine ammonia lyase (RgTAL), Rhodobacter capsulatus TAL, Rice TAL, Parsley TAL, Tomato TAL, Arabidopsis TAL, or a combination thereof.
34. The method of claim 31, wherein the phenylpropanoic acid is p-coumaric acid, caffeic acid, cinnamic acid, ferulic acid or a combination thereof.
35. The method of claim 32, wherein the host cell for the TAL module cell is E. coli rpoA14(DE3).
36. A TAL module cell, wherein the TAL module cell is a microbial cell comprising an exogenous gene encoding for a tyrosine ammonia lyase (TAL).
37. The TAL module cell of claim 36, wherein the exogenous gene encoding for the tyrosine ammonia lyase (TAL) is a gene encoding for Rhodotorula glutinis tyrosine ammonia lyase (RgTAL), Rhodobacter capsulatus TAL, Rice TAL, Parsley TAL, Tomato TAL, Arabidopsis TAL, or a combination thereof.
38. The TAL module cell of claim 36, wherein a host cell for the TAL module cell is E. coli rpoA14(DE3).
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] These and other features, aspects, and advantages of the present invention will become better understood when the following detailed description is read with reference to the accompanying drawings in which like characters represent like parts throughout the drawings, wherein:
[0023]
[0024]
[0025]
[0026]
[0027]
[0028]
[0029]
[0030]
[0031]
DETAILED DESCRIPTION OF THE INVENTION
[0032] We developed and optimized polycultures for the efficient production of various compound types, including flavonoids. Flavonoids are high-value molecules with promising potential for pharmaceutical applications resulting from interesting bioactivity (Refs. 39, 46, 47, and 56). In the case of flavan-3-ols, a subclass of flavonoid molecules, high-titer production has been achieved from both the malonyl-CoA requiring upstream module (phenylpropanoic acids to flavanones) (Ref 61) and the NADPH requiring downstream module (flavanones to flavan-3-ols) (Ref 67). However, when the complete pathway is expressed in monoculture, reported titers for flavan-3-ols from phenylpropanoic acids are greater than three orders of magnitude lower than the independent modules (Ref. 39). This observation motivated the choice to attempt co-culture production of flavan-3-ols in E. coli.
[0033] To accomplish this task, careful experimental optimization of carbon source, induction temperature, induction point, inoculation ratio, and strain choice was used to map the production landscape. The experimental optimization was coupled with extensive empirical modeling techniques that were applied to predict conditions for optimal production. Searching the solution space surrounding the predicted optimum resulted in a 65% improvement in flavan-3-ol titer to 40.70.1 mg/L from p-coumaric acid, representing a 970-fold improvement over previous literature reports.
[0034] Some of our novel polycultures are also capable of the de novo production of flavan-3-ols and anthocyanidin-3-O-glucosides in microbial hosts,
[0035] In one embodiment, the invention relates to a method of producing a product compound in a microbial polyculture;
wherein, optionally, the microbial polyculture includes a TAL module cell including an exogenous gene encoding for a tyrosine ammonia lyase (TAL);
wherein, optionally, the microbial polyculture includes a C5 module cell including 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 wherein, optionally, the C5 module cell further includes an exogenous gene encoding for malonyl-CoA synthetase (MatB) and an exogenous gene encoding for putative dicarboxylate carrier protein (MatC);
wherein, optionally, the microbial polyculture further includes a p168 module cell including an exogenous gene encoding for a flavanone 3-hydroxylase (F3H), an exogenous gene encoding for a dihydroflavonol 4-reductase (DFR), and an exogenous gene encoding for a leucoanthocyanidin reductase (LAR); and
wherein, optionally, the microbial polyculture further includes an Antho module cell including an exogenous gene encoding for an anthocyanidin synthase (ANS) and an exogenous gene encoding for a 3-glucosyl transferase (3GT);
the method including: [0036] providing a substrate to the microbial polyculture; [0037] culturing the microbial polyculture under conditions permitting synthesis of the product compound by the microbial polyculture; and [0038] isolating the product compound synthesized by the microbial polyculture;
with a proviso that: [0039] the microbial polyculture includes the TAL module cell and the C5 module cell, the substrate is glucose, glycerol, or a combination thereof, and the product compound is a flavanone; or [0040] the microbial polyculture includes the C5 module cell and the p168 module cell, the substrate is phenylpropanoic acid, and the product compound is a flavonoid; wherein, when the C5 module cell includes 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 [0041] the microbial polyculture includes 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 [0042] the microbial polyculture includes 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 [0043] the microbial polyculture includes 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 includes 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 [0044] the microbial polyculture includes 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.
[0045] Thus, the microbial polycultures of the invention may be any one of the following polycultures: (1) the TAL module cell and the C5 module cell; (2) the C5 module cell and the p168 module cell; (3) the p168 module cell and the Antho module cell; (4) the TAL module cell, the C5 module cell, and the p168 module cell; (5) the C5 module cell, the p168 module cell, and the Antho module cell; or (6) the TAL module cell, the C5 module cell, the p168 module cell, and the Antho module cell. Use of the singular term cell when referring to each module (i.e., TAL module, C5 module, p168 module, and Antho module) is meant to encompass both a single cell of the specified module and a plurality of cells of the specified module.
[0046] The TAL module cell includes an exogenous gene encoding for a tyrosine ammonia lyase (TAL). In some embodiments, the exogenous gene encoding for the tyrosine ammonia lyase (TAL) is a gene encoding for Rhodotorula glutinis tyrosine ammonia lyase (RgTAL). In some embodiments, the exogenous gene encoding for the tyrosine ammonia lyase (TAL) is a gene encoding for Rhodotorula glutinis tyrosine ammonia lyase (RgTAL), Rhodobacter capsulatus TAL, Rice TAL, Parsley TAL, Tomato TAL, Arabidopsis TAL, or a combination thereof.
[0047] The C5 module cell includes 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). In addition to these three geness, the C5 module cell may optionally include an exogenous gene encoding for malonyl-CoA synthetase (MatB) and an exogenous gene encoding for putative dicarboxylate carrier protein (MatC).
[0048] In some embodiments, the exogenous gene encoding for the 4-coumaroyl-CoA ligase (4CL) is a gene encoding for Arabidopsis thaliana 4-coumaroyl-CoA ligase (At4CL), Petrosehnum crispum 4-coumaroyl-CoA ligase (Pc4CL), Vitis vinifera 4-coumaroyl-CoA ligase (Vv4CL), or a combination thereof. In some embodiments, the exogenous gene encoding for the chalcone synthase (CHS) is a gene encoding for Petuniahybrida chalcone synthase (PhCHS), Citrus maxima chalcone synthase (CmCHS), or a combination thereof. In some embodiments, the exogenous gene encoding for the chalcone isomerase (CHI) is a gene encoding for Medicago sativa chalcone isomerase (MsCHI), Citrus maxima chalcone isomerase (CmCHI), or a combination thereof. In some embodiments, the exogenous gene encoding for the malonyl-CoA synthetase (MatB) is a gene encoding for Rhizobium trifolii malonyl-CoA synthetase (RtMatB). In some embodiments, the exogenous gene encoding for the dicarboxylate carrier protein (MatC) is a gene encoding for Rhizobium trifolii putative dicarboxylate carrier protein (RtMatC).
[0049] The p168 module cell includes an exogenous gene encoding for a flavanone 3-hydroxylase (F3H), an exogenous gene encoding for a dihydroflavonol 4-reductase (DFR), and an exogenous gene encoding for a leucoanthocyanidin reductase (LAR).
[0050] In some embodiments, the exogenous gene encoding for the flavanone 3-hydroxylase (F3H) is a gene encoding for Camellia sinensis flavanone 3-hydroxylase (CsF3H), Malus domestica flavanone 3-hydroxylase (MdF3H), Petroselinum crispum flavanone 3-hydroxylase (PcF3H), or a combination thereof. In some embodiments, the exogenous gene encoding for the dihydroflavonol 4-reductase (DFR) is a gene encoding for Anthrium andraeanum dihydroflavonol 4-reductase (AaDFR), Camellia sinensis dihydroflavonol 4-reductase (CsDFR), Fragariaananassa dihydroflavonol 4-reductase (FaDFR), or a combination thereof. In some embodiments, the exogenous gene encoding for the leucoanthocyanidin reductase (LAR) is a gene encoding for Camellia sinensis leucoanthocyanidin reductase (CsLAR), Desmodium uncinatum leucoanthocyanidin reductase (DuLAR), or a combination thereof.
[0051] The Antho module cell includes an exogenous gene encoding for an anthocyanidin synthase (ANS) and an exogenous gene encoding for a 3-glucosyl transferase (3GT). In some embodiments, the exogenous gene encoding for the anthocyanidin synthase (ANS) is a gene encoding for Petuniahybrida anthocyanidin synthase (PhANS). In some embodiments, the exogenous gene encoding for the anthocyanidin synthase (ANS) is a gene encoding for Petuniahybrida anthocyanidin synthase (PhANS), Malus domestica ANS, Pyrus communis ANS, Prunus avium ANS, Fragariaananassa ANS, Vitis vinifera ANS, Ipomoea purpurea anthocyanidin synthase (ANS), Camellia sinensis ANS, Citrus sinensis anthocyanidin synthase (ANS), Vaccinium ashei ANS, Populus trichocarpa ANS, or a combination thereof. In some embodiments, the exogenous gene encoding for the 3-glucosyl transferase (3GT) is a gene encoding for Arabidopsis thaliana 3-glucosyl transferase (At3GT). In some embodiments, the exogenous gene encoding for the 3-glucosyl transferase (3GT) is a gene encoding for Arabidopsis thaliana 3-glucosyl transferase (At3GT), Fragariaananassa 3GT, Vitis vinifera 3GT, Forsynthia 3GT, Eggplant 3GT, Gentian 3GT, Petuniahybrida 3GT, or a combination thereof.
[0052] In some embodiments, 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 Petuniahybrida 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).
[0053] In some embodiments, the exogenous gene encoding for the flavanone 3-hydroxylase (F3H) is a gene encoding for Camellia sinensis flavanone 3-hydroxylase (CsF3H); the exogenous gene encoding for the dihydroflavonol 4-reductase (DFR) is a gene encoding for Fragariaananassa dihydroflavonol 4-reductase (FaDFR); and the exogenous gene encoding for the leucoanthocyanidin reductase (LAR) is a gene encoding for Desmodium uncinatum leucoanthocyanidin reductase (DuLAR).
[0054] The polypeptides encoded by the exogenous genes in the cells of the polyculture have various known functions. TAL converts tyrosine and phenylalanine to the corresponding phenylpropanoic acids, coumaric acid, and cinnamic acid. 4CL converts phenylpropanoic acids such as coumaric acid, cinnamic acid, ferulic acid, and caffeic acid to their CoA derivatives. CHS performs condensation of phenylpropanoic-CoA derivatives such as coumaroyl-CoA, cinnamoyl-CoA, caffeoyl-CoA, feruloyl-CoA with 3 moles of malonyl CoA and performs Claisen condensation to form chalcones. CHI performs isomerisation of chalcones to flavanones. Math converts the intracellular malonate to malonyl-CoA. MatC transports malonate across the cell membrane. F3H is a dioxygenase that hydroxylates flavanones such as naringenin and eriodictyol to the corresponding dihydroxyflavanones. DFR is a reductase reducing dixydroxyflavanones to the corresponding leucoanthocyanidins. LAR is a reductase that converts leucoanthocyanidins to flavan-3-ols. ANS is a dioxygenase that converts flavan-3-ols and leucoanthocyanidins to anthocyanidins. 3GT is a glycosyltransferase that adds a glucose group to the 3 OH group of anthocyanidins converting them to the corresponding anthocyanin 3-O-glucoside.
[0055] In some embodiments, the exogenous gene encoding for the tyrosine ammonia lyase (TAL) is a gene that encodes a polypeptide with at least 85%, 90%, or 95% amino acid sequence identity to any one of the TAL amino acid sequences identified herein.
[0056] In some embodiments, the exogenous gene encoding for the 4-coumaroyl-CoA ligase (4CL) is a gene that encodes a polypeptide with at least 85%, 90%, or 95% amino acid sequence identity to any one of the 4CL amino acid sequences identified herein.
[0057] In some embodiments, the exogenous gene encoding for the chalcone synthase (CHS) is a gene that encodes a polypeptide with at least 85%, 90%, or 95% amino acid sequence identity to any one of the CHS amino acid sequences identified herein.
[0058] In some embodiments, the exogenous gene encoding for the chalcone isomerase (CHI) is a gene that encodes a polypeptide with at least 85%, 90%, or 95% amino acid sequence identity to any one of the CHI amino acid sequences identified herein.
[0059] In some embodiments, the exogenous gene encoding for the malonyl-CoA synthetase (MatB) is a gene that encodes a polypeptide with at least 85%, 90%, or 95% amino acid sequence identity to any one of the MatB amino acid sequences identified herein.
[0060] In some embodiments, the exogenous gene encoding for the dicarboxylate carrier protein (MatC) is a gene that encodes a polypeptide with at least 85%, 90%, or 95% amino acid sequence identity to any one of the MatC amino acid sequences identified herein.
[0061] In some embodiments, the exogenous gene encoding for the flavanone 3-hydroxylase (F3H) is a gene that encodes a polypeptide with at least 85%, 90%, or 95% amino acid sequence identity to any one of the F3H amino acid sequences identified herein.
[0062] In some embodiments, the exogenous gene encoding for the dihydroflavonol 4-reductase (DFR) is a gene that encodes a polypeptide with at least 85%, 90%, or 95% amino acid sequence identity to any one of the DFR amino acid sequences identified herein.
[0063] In some embodiments, the exogenous gene encoding for the leucoanthocyanidin reductase (LAR) is a gene that encodes a polypeptide with at least 85%, 90%, or 95% amino acid sequence identity to any one of the LAR amino acid sequences identified herein.
[0064] In some embodiments, the exogenous gene encoding for the anthocyanidin synthase (ANS) is a gene that encodes a polypeptide with at least 85%, 90%, or 95% amino acid sequence identity to any one of the ANS amino acid sequences identified herein.
[0065] In some embodiments, the exogenous gene encoding for the 3-glucosyl transferase (3GT) is a gene that encodes a polypeptide with at least 85%, 90%, or 95% amino acid sequence identity to any one of the 3GT amino acid sequences identified herein.
[0066] In all of the above instances of encoded polypeptides with at least 85%, 90%, or 95% amino acid sequence identity to a specified polypeptide, the function of the encoded polypeptide is the same as the function of the specified polypeptide. Those of skill in the art could readily determine amino acid sequences of such encoded polypeptides. Preservation of the function of the encoded polypeptide would be routine to a person of skill in the art with the benefit of the available information about functional domains of the specified polypeptides. Such information regarding domains includes disclosures in the references listed below, which are incorporated by reference in their entirety herein.
TAL: Crystal structure of phenylalanine ammonia lyase: multiple helix dipoles implicated in catalysis. Calabrese J C, Jordan D B, Boodhoo A, Sariaslani S, Vannelli T., Biochemistry. 2004 Sep. 14, 43(36):11403-16.
4CL: Identification of the substrate specificity-conferring amino acid residues of 4-coumarate:coenzyme A ligase allows the rational design of mutant enzymes with new catalytic properties, Stuible H P, Kombrink E., J Biol Chem. 2001 Jul. 20, 276(29):26893-7.
4CL: The substrate specificity-determining amino acid code of 4-coumarate:CoA ligase, Schneider K, Hvel K, Witzel K, Hamberger B, Schomburg D, Kombrink E, Stuible H P, Proc Natl Acad Sci USA. 2003 Jul. 8, 100(14):8601-6.
CHS: Structure of chalcone synthase and the molecular basis of plant polyketide biosynthesis, Ferrer J L, Jez J M, Bowman M E, Dixon R A, Noel J P., Nat Struct Biol. 1999 August, 6(8):775-84.
CHS: Dissection of malonyl-coenzyme A decarboxylation from polyketide formation in the reaction mechanism of a plant polyketide synthase, Jez J M, Ferrer J L, Bowman M E, Dixon R A, Noel J P., Biochemistry. 2000 Feb. 8, 39(5):890-902.
CHI: Structure and mechanism of the evolutionarily unique plant enzyme chalcone isomerase, Jez J M, Bowman M E, Dixon R A, Noel J P, Nat Struct Biol. 2000 September, 7(9):786-91.
[0067] DFR: Crystal structure of grape dihydroflavonol 4-reductase, a key enzyme in flavonoid biosynthesis, Petit P, Granier T, d'Estaintot B L, Manigand C, Bathany K, Schmitter J M, Lauvergeat V, Hamdi S, Gallois B., J Mol Biol. 2007 May 18, 368(5):1345-57.
LAR: Crystal structure and catalytic mechanism of leucoanthocyanidin reductase from Vitis vinifera., Maug C, Granier T, d'Estaintot B L, Gargouri M, Manigand C, Schmitter J M, Chaudire J, Gallois B., J Mol Biol., 2010 Apr. 9, 397(4):1079-91.
ANS: Structure and mechanism of anthocyanidin synthase from Arabidopsis thaliana, Wilmouth R C, Turnbull J J, Welford R W, Clifton U, Prescott A G, Schofield C J, Structure. 2002 January, 10(1):93-103.
3GT: Structural basis for acceptor-substrate recognition of UDP-glucose: anthocyanidin 3-O-glucosyltransferase from Clitoria ternatea, Hiromoto T, Honjo E, Noda N, Tamada T, Kazuma K, Suzuki M, Blaber M, Kuroki R., Protein Sci. 2015 March, 24(3):395-407. doi: 10.1002/pro.2630, PMID: 25556637.
3GT: Crystal structure of UDP-glucose:anthocyanidin 3-O-glucosyltransferase from Clitoria ternatea, Hiromoto T, Honjo E, Tamada T, Noda N, Kazuma K, Suzuki M, Kuroki R, J Synchrotron Radiat., 2013 November, 20(Pt 6):894-8.
[0068] In some embodiments, the method further includes: (a) when the microbial polyculture includes the TAL module cell, creating the TAL module cell by introducing an exogenous gene encoding for a tyrosine ammonia lyase (TAL) into a host cell for the TAL module cell; (b) when the microbial polyculture includes the C5 module cell, creating the C5 module cell by introducing 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, optionally, introducing an exogenous gene encoding for malonyl-CoA synthetase (MatB) and an exogenous gene encoding for putative dicarboxylate carrier protein (MatC), into a host cell for the C5 module cell; (c) when the microbial polyculture includes the p168 module cell, creating the p168 module cell by introducing an exogenous gene encoding for a flavanone 3-hydroxylase (F3H), an exogenous gene encoding for a dihydroflavonol 4-reductase (DFR), and an exogenous gene encoding for a leucoanthocyanidin reductase (LAR) into a host cell for the p168 module cell; and (d) when the microbial polyculture includes the TAL module cell, creating the Antho module cell by introducing an exogenous gene encoding for an anthocyanidin synthase (ANS), and an exogenous gene encoding for a 3-glucosyl transferase (3GT) into a host cell for the Antho module cell.
[0069] In some embodiments, the host cells are E. coli cells. In one embodiment, the host cell for the TAL module cell is E. coli rpoA14(DE3). In one embodiment, the host cell for the C5 module cell is E. coli BL21star(DE3)sucCfumC. In one embodiment, the host cell for the p168 module cell is E. coli BL21star(DE3). In one embodiment, the host cell for the Antho module cell is E. coli BL21star(DE3).
[0070] The substrate of the TAL module cell is glucose, glycerol, or a combination thereof. The TAL module cell's product compound is a phenylpropanoic acid.
[0071] The substrate of the C5 module cell is a phenylpropanoic acid. When the C5 module cell includes two additional genes, a gene for malonyl-CoA synthetase (MatB) and a gene for putative dicarboxylate carrier protein (MatC), the substrate of the C5 module cell is a phenylpropanoic acid, malonate, or a combination thereof. The C5 module cell's product compound is a flavanone.
[0072] The substrate of the p168 module cell is a flavanone. The p168 module cell's product compound is a flavonoid.
[0073] The substrate of the Antho module cell is a flavonoid. The Antho module cell's product compound is an anthocyanidin-3-O-glucoside.
[0074] The product compound is synthesized within a specified cell of the polyculture and is isolated from that cell, from the media, or from both the cell and the media.
[0075] In some embodiments, the phenylpropanoic acid is p-coumaric acid, caffeic acid, cinnamic acid, ferulic acid, or a combination thereof.
[0076] In some embodiments, the flavanone is naringenin, eriodictyol, pinocembrin, or a combination thereof.
[0077] In some embodiments, the flavonoid is a flavone, a flavan-3-ol, a flavan-4-ol, a flavonol, an anthocyanin, or a combination thereof.
[0078] In some embodiments, the flavone is luteolin, apigenin, chrysin, or a combination thereof.
[0079] In some embodiments, the flavan-3-ol is afzelechin, catechin (e.g., (+)-catechin), or a combination thereof.
[0080] In some embodiments, 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-5methoxyflavan, or a combination thereof.
[0081] In some embodiments, the flavonol is kaempferol, quercetin, or a combination thereof.
[0082] In some embodiments, the anthocyanin is pelargonidin, cyanidin, delphinidin, malvidin, peonidin, or a combination thereof.
[0083] In some embodiments, the anthocyanidin-3-O-glucoside is pelargonidin-3-O-glucoside, cyanidin-3-O-glucoside, delphinidin-3-O-glucoside, malvidin-3-O-glucoside, peonidin-3-O-glucoside, or a combination thereof.
[0084] In some embodiments, the phenylpropanoic acid is p-coumaric acid and the flavonoid is (+)-afzelechin. In some embodiments, the phenylpropanoic acid is caffeic acid and the flavonoid is (+)-catechin. In some embodiments, the phenylpropanoic acid is cinnamic acid and the flavonoid is 3,5,7-trihydroxyflavan. In some embodiments, the substrate is glucose and the flavonoid is pelargonidin-3-O-glucoside.
[0085] In some embodiments, the substrate is glucose and the flavonoid is cyanidin-3-O-glucoside. In some embodiments, the substrate is glucose and the flavonoid is delphinidin-3-O-glucoside. In some embodiments, the substrate is glucose and the flavonoid is peonidin-3-O-glucoside. In some embodiments, the substrate is glucose and the flavonoid is malvidin-3-O-glucoside. In some embodiments, the substrate is glycerol and the flavonoid is cyanidin-3-O-glucoside. In some embodiments, the substrate is glycerol and the flavonoid is pelargonidin-3-O-glucoside. In some embodiments, the substrate is glycerol and the flavonoid is delphinidin-3-O-glucoside. In some embodiments, the substrate is glycerol and the flavonoid is peonidin-3-O-glucoside. In some embodiments, the substrate is glycerol and the flavonoid is malvidin-3-O-glucoside.
[0086] In some embodiments, 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. In some embodiments, the carbon source is any suitable pentose or hexose sugar.
[0087] The conditions permitting synthesis of the flavonoid compound may include an induction point, an induction temperature, and an inoculation ratio.
[0088] Thus, in one embodiment, the induction temperature is from about 10 C. to about 42 C. In one embodiment, the induction temperature of about 30 C. In one embodiment, the induction point is from about 0 hours to about 24 hours. In one embodiment, the induction point is at about 5.5 hours. In another embodiment, the induction point is at about 5 hours.
[0089] When each module cell is used as a plurality of cells, the inoculation ratio of the C5 module cell to the p168 module cell (C5:p168) is a ratio of from about 1:99 to about 99:1. In one embodiment, the inoculation ratio of the C5 module cell to the p168 module cell (C5:p168) is a ratio of about 8:2.
[0090] In one embodiment, the inoculation ratio of the TAL module cell to the C5 module cell to the p168 module cell to the Antho module cell (TAL:C5:p168:Antho) is a ratio of about 1-97:1-97:1-97:1-97. In one embodiment, the inoculation ratio of the TAL module cell to the C5 module cell to the p168 module cell to the Antho module cell (TAL:C5:p168:Antho) is a ratio of about 8:8:2:7.
[0091] In one embodiment, the inoculation ratio of the TAL module cell to the C5 module cell (TAL:C5) is a ratio of about 1-99:1-99. In one embodiment, the inoculation ratio of the TAL module cell to the C5 module cell (TAL:C5) is a ratio of about 8:8.
[0092] In one embodiment, the inoculation ratio of the p168 module cell to the Antho module cell (p168:Antho) is a ratio of about 1-99:1-99. In one embodiment, the inoculation ratio of the p168 module cell to the Antho module cell (p168:Antho) is a ratio of about 2:7.
[0093] In one embodiment, the inoculation ratio of the TAL module cell to the C5 module cell to the p168 module cell (TAL:C5:p168) is a ratio of about 1-98:1-98:1-98. In one embodiment, the inoculation ratio of the TAL module cell to the C5 module cell to the p168 module cell (TAL:C5:p168) is a ratio of about 8:8:2.
[0094] In one embodiment, the inoculation ratio of the C5 module cell to the p168 module cell to the Antho module cell (C5:p168:Antho) is a ratio of about 1-98:1-98:1-98. In one embodiment, the inoculation ratio of the C5 module cell to the p168 module cell to the Antho module cell (C5:p168:Antho) is a ratio of about 8:2:7.
[0095] The inoculation ratios are either volumentric ratios or ratios of the numbers of cells. When using volumetric ratios, cell concentrations are initially starting around 107 total cells/mL and increase to around 109 total cells/mL. These total cell counts can be split between the two or more strains in the polyculture.
[0096] The present invention is also directed to microbial polycultures of the above described methods. Thus, the invention includes a microbial polyculture that includes:
optionally, a TAL module cell including an exogenous gene encoding for a tyrosine ammonia lyase (TAL);
optionally, a C5 module cell including 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 wherein, optionally, the C5 module cell further includes an exogenous gene encoding for malonyl-CoA synthetase (MatB) and an exogenous gene encoding for putative dicarboxylate carrier protein (MatC);
optionally, a p168 module cell including an exogenous gene encoding for a flavanone 3-hydroxylase (F3H), an exogenous gene encoding for a dihydroflavonol 4-reductase (DFR), and an exogenous gene encoding for a leucoanthocyanidin reductase (LAR); and
optionally, an Antho module cell including an exogenous gene encoding for an anthocyanidin synthase (ANS), and an exogenous gene encoding for a 3-glucosyl transferase (3GT);
with a proviso that: [0097] the microbial polyculture includes the TAL module cell and the C5 module cell; or [0098] the microbial polyculture includes the C5 module cell and the p168 module cell; or [0099] the microbial polyculture includes the p168 module cell and the Antho module cell; or [0100] the microbial polyculture includes the TAL module cell, the C5 module cell, and the p168 module cell; or [0101] the microbial polyculture includes the C5 module cell, the p168 module cell, and the Antho module cell; or [0102] the microbial polyculture includes the TAL module cell, the C5 module cell, the p168 module cell, and the Antho module cell.
[0103] The invention is also directed to a method of producing a phenylpropanoic acid in a TAL module cell, wherein the TAL module cell is a microbial cell including an exogenous gene encoding for a tyrosine ammonia lyase (TAL); the method including: providing a substrate to the TAL module cell, wherein the substrate includes glucose, glycerol, or a combination thereof; culturing the TAL module cell under conditions permitting synthesis of the phenylpropanoic acid by the TAL module cell; and isolating the phenylpropanoic acid synthesized by the TAL module cell. In one embodiment, the method further includes creating the TAL module cell by introducing an exogenous gene encoding for a tyrosine ammonia lyase (TAL) into a host cell for the TAL module cell.
[0104] In one embodiment, the exogenous gene encoding for the tyrosine ammonia lyase (TAL) is a gene encoding for Rhodotorula glutinis tyrosine ammonia lyase (RgTAL). In some embodiments, the exogenous gene encoding for the tyrosine ammonia lyase (TAL) is a gene encoding for Rhodotorula glutinis tyrosine ammonia lyase (RgTAL), Rhodobacter capsulatus TAL, Rice TAL, Parsley TAL, Tomato TAL, Arabidopsis TAL, or a combination thereof.
[0105] In some embodiments, the phenylpropanoic acid is p-coumaric acid, caffeic acid, cinnamic acid, ferulic acid, or a combination thereof, all of which could derive from glucose substrate.
[0106] In some embodiments, the host cell for the TAL module cell is E. coli rpoA14(DE3).
[0107] The invention is also directed to a TAL module cell, wherein the TAL module cell is a microbial cell including an exogenous gene encoding for a tyrosine ammonia lyase (TAL). In one embodiment, the exogenous gene encoding for the tyrosine ammonia lyase (TAL) is a gene encoding for Rhodotorula glutinis tyrosine ammonia lyase (RgTAL). In one embodiment, a host cell for the TAL module cell is E. coli, for example, E. coli rpoA14(DE3).
[0108] In general, the terms and phrases used herein have their art-recognized meaning, which can be found by reference to standard texts, journal references and contexts known to those skilled in the art. The following definitions are provided to clarify their specific use in the context of the invention.
[0109] The following nucleic acid name abbreviations are used herein: C or c for cytosine, G or g for guanine, A or a for adenine, T or t for Thymine, and U or u for uracil.
[0110] The following amino acid name abbreviations are used herein: A or Ala for Alanine; M or Met for Methionine; C or Cys for Cysteine; D or Asp for Aspartic Acid; E or Glu for Glutamic Acid; F or Phe for Phenylalanine; G or Gly for Glycine; H or His for Histidine; I or Ile for Isoleucine; K or Lys for Lysine; L or Leu for Leucine; N or Asn for Asparagine; P or Pro for Proline; Q or Glu for Glutamine; R or Arg for Arginine; S or Ser for Serine; T or Thr for Threonine; V or Val for Valine; W or Trp for Tryptophan; and Y or Tyr for Tyrosine.
[0111] The terms microbe and microbial refer to a microscopic living organism, which may be single-celled or multicellular. Microbe, as used herein, includes bacteria, unicellular eukaryotes, archaea, and protozoa. An example of a microbe used in the inventions described herein is E. coli.
[0112] The term isolating the product compound, as used herein, encompases any method that increases purity of the product compound.
[0113] When a reference is made to a gene that encodes for a specified polypeptide, such gene has the meaning of any nucleic acid sequence that encodes for the amino acid sequence of the specified polypeptide. Those of skill in the art could readily determine all possible nucleic acid sequences encoding for the specified polypeptide.
[0114] The term induction point, as used herein, refers to the time point, after the culture has been initiated, at which the inducer is added to the medium.
[0115] The term induction temperature, as used herein, refers to the temperature at which the culture is left to grow after the inducer has been added into the medium.
[0116] The following specific non-limiting examples are illustrative of the invention. Examples 1-14 describe studies that are also described in more detail in Jones, J. A. et al. Experimental and computational optimization of an Escherichia coli co-culture for the efficient production of flavonoids. Metab. Eng. 35, 55-63 (2016) (Ref 24), entire disclosure of which, together with accompanying supplementary data available online at http://dx.doi.org/10.1016/j.ymben.2016.01.006, is incorporated by reference in its entirety.
Example 1
C5 Module and p168 Module PolycultureBacterial Strains, Vectors, and Media
[0117] E. coli DH5 was used to propagate all plasmids, while the BL21star(DE3), BL21star(DE3)sucCfumC, or BL21star(DE3)pgippc was used as the hosts for flavonoid production. The ePathBrick vector, pETM6, was used as the basis for all plasmid construction and pathway expression. Luria Broth (LB) Lennox modification (Sigma) and Andrew's Magic Medium (AMM) (Ref 45) were used where noted. Sequences of all plasmid constructs are available through Addgene.org and are incorporated by reference herein.
Example 2
C5 Module and p168 Module PolycultureFlavonoid Pathways and ePathOptimize Library Construction
[0118] Genes involved in the 12 candidate upstream flavanone production pathways were obtained from previously published literature from the Koffas lab. Vv4CL, Pc4CL, CmCHS, PhCHS, CmCHI, and MsCHI were obtained in ePathBrick vector pETM6 (Refs. 40 and 62), while At4CL was acquired through PCR amplification (ACCUZYME 2 mix, Bioline) of plasmid #3 DNA using primers 1 and 2 (Table 2) (Ref. 53). The ePathBrick destination vector, pETM6, and At4CL PCR amplicon were digested with restriction enzymes NdeI/XhoI (FastDigest, Thermo Scientific) and gel purified (E.Z.N.A. MicroElute Gel Extraction Kit, Omega Bio-tek). Digested At4CL PCR product was ligated with digested pETM6 backbone to create plasmid 2, Table 1. Constructs were then transformed into chemically competent DH5 for verification and plasmid propagation. Colonies were screened via restriction digest and further verified with Sanger sequencing (GENEWIZ, Inc.) using the sequencing primers 3 and 4 in Table 2. Site directed mutagenesis was then preformed using standard protocols to silently remove the NheI restriction site from At4CL using primers 5 and 6 (Table 2). Complete candidate pathways were constructed in monocistronic form using standard ePathBrick methods (Ref. 62) resulting in plasmids 10-27, Table 1. Occasionally the restriction site ApaI was used to replace SalI when the pathway genes either contained internal SalI restriction sites or to optimize the insert:backbone ratio for improved ligation efficiency. Plasmids p148 and p168 containing complete downstream modules were not modified from previous reports (Ref 67).
TABLE-US-00001 TABLE 1 List of Strains and Plasmids Number Strain or vector Relevant properties Reference S1 Escherichia coli F.sup., 80d lacZM15, (lacZYA-argF)U169, Novagen DH5 recA1, endA1, hsdR17(rk.sup., mk.sup.+), phoA, supE44.sup., thi.sup.1, gyrA96, relA1 S2 E. coli BL21 Star F.sup.ompT gal dcm rne131 lon hsdS.sub.B (r.sub.B.sup.m.sub.B.sup.) Invitrogen (DE3) (DE3) S3 BLpgippc BL21Star(DE3)pgi::FRTppc::FRT- (a) KanR-FRT S4 BLsumCfumC BL21Star(DE3)fumC::FRTsucC::FRT (b) 1 pETM6 ePathBrick expression vector, ColE1 ori, (c) AmpR 2 pETM6-At4CL #1 with 4CL-1 from A. thaliana This Study 3 pC-At4cl-Vvsts pCDFDuet with 4CL-1 from A. thaliana, STS (d) from V. vinifera 4 pETM6-Pc4CL #1 with 4CL-2 from P. crispum (c) 5 pETM6-Vv4CL #1 with 4CL from V. vinifera (e) 6 pETM6-PhCHS #1 with CHS from P. hybrida (c) 7 pETM6-CmCHS #1 with CHS from C. maxima (e) 8 pETM6-MsCHI #1 with CHI from M. sativa (c) 9 pETM6-CmCHI #1 with CHI from C. maxima (e) 10 pETM6-At4CL- #1 with At4CL and PhCHS, monocistronic This Study PhCHS form 11 pETM6-At4CL- #1 with At4CL and CmCHS, monocistronic This Study CmCHS form 12 pETM6-Pc4CL- #1 with Pc4CL and PhCHS, monocistronic This Study PhCHS form 13 pETM6-Pc4CL- #1 with Pc4CL and CmCHS, monocistronic This Study CmCHS form 14 pETM6-Vv4CL- #1 with Vv4CL and PhCHS, monocistronic This Study PhCHS form 15 pETM6-Vv4CL- #1 with Vv4CL and CmCHS, monocistronic This Study CmCHS form 16 pETM6-At4CL- #1 with At4CL, PhCHS, and MsCHI, This Study PhCHS-MsCHI monocistronic form 17 pETM6-At4CL- #1 with At4CL, PhCHS, and CmCHI, This Study PhCHS-CmCHI monocistronic form 18 pETM6-At4CL- #1 with At4CL, CmCHS, and MsCHI, This Study CmCHS-MsCHI monocistronic form 19 pETM6-At4CL- #1 with At4CL, CmCHS, and CmCHI, This Study CmCHS-CmCHI monocistronic form 20 pETM6-Pc4CL- #1 with Pc4CL, PhCHS, and MsCHI, This Study PhCHS-MsCHI monocistronic form 21 pETM6-Pc4CL- #1 with Pc4CL, PhCHS, and CmCHI, This Study PhCHS-CmCHI monocistronic form 22 pETM6-Pc4CL- #1 with Pc4CL, CmCHS, and MsCHI, This Study CmCHS-MsCHI monocistronic form 23 pETM6-Pc4CL- #1 with Pc4CL, CmCHS, and CmCHI, This Study CmCHS-CmCHI monocistronic form 24 pETM6-Vv4CL- #1 with Vv4CL, PhCHS, and MsCHI, This Study PhCHS-MsCHI monocistronic form 25 pETM6-Vv4CL- #1 with Vv4CL, PhCHS, and CmCHI, This Study PhCHS-CmCHI monocistronic form 26 pETM6-Vv4CL- #1 with Vv4CL, CmCHS, and MsCHI, This Study CmCHS-MsCHI monocistronic form 27 pETM6-Vv4CL- #1 with Vv4CL, CmCHS, and CmCHI, This Study CmCHS-CmCHI monocistronic form 28 p148 #1 with CsF3H.sup.syn-AaDFR.sup.syn-DuLAR.sup.syn, (f) monocistronic form 29 p168 #1 with CsF3H.sup.syn-FaDFR.sup.syn-DuLAR.sup.syn, (f) monocistronic form 30 pETM6-mCherry #1 with mCherry fluoroscent reporter (c) 31 pETM6-C4- #30 Modified with mutated C4 T7 promoter (g) mCherry sequence 32 pETM6-G6- #30 Modified with mutated G6 T7 promoter (g) mCherry sequence 33 pETM6-H9- #30 Modified with mutated H9 T7 promoter (g) mCherry sequence 34 pETM6-H10- #30 Modified with mutated H10 T7 (g) mCherry promoter sequence 35 pFlavo.sup.opt or C5 #17 with C4 mutant T7 promoter controlling This Study mutant CmCHI References cited in Table 1: (a) Chemler J A, Fowler Z L, McHugh K P, Koffas MAG (2010) Improving NADPH availability for natural product biosynthesis in Escherichia coli by metabolic engineering. Metab Eng 12(2): 96-104. (b) Xu P, Ranganathan S, Fowler Z L, Maranas C D, Koffas M a G (2011) Genome-scale metabolic network modeling results in minimal interventions that cooperatively force carbon flux towards malonyl-CoA. Metab Eng 13(5): 578-87. (c) Xu P, Vansiri A, Bhan N, Koffas MAG (2012) ePathBrick: A Synthetic Biology Platform for Engineering Metabolic Pathways in E. coli. ACS Synth Biol 1(7): 256-66. (d) Lim C G, Fowler Z L, Hueller T, Schaffer S, Koffas MAG (2011) High-yield resveratrol production in engineered Escherichia coli. Appl Environ Microbiol 77(10): 3451-60. (e) Cress B F, et al. (2015) CRISPathBrick: Modular Combinatorial Assembly of Type II-A CRISPR Arrays for dCas9-Mediated Multiplex Transcriptional Repression in E. coli. ACS Synth Biol 4(9): 987-1000. (f) Zhao S, et al. (2015) Improvement of catechin production in Escherichia coli through combinatorial metabolic engineering. Metab Eng 28: 43-53. (g) Jones J A, et al. (2015) ePathOptimize: A Combinatorial Approach for Transcriptional Balancing of Metabolic Pathways. Sci Rep 5: 11301.
TABLE-US-00002 TABLE2 ListofPlasmids Number PrimerID PrimerSequence(5->3) 1 At4CL_FWDwithNdeI GCGCCGCATATGGCGCCACAAGA (SEQIDNO:1) ACAAG 2 At4CL_REVwithXhoI GCGCGGCTCGAGTCACAATCCATT (SEQIDNO:2) TGCT 3 Seq_T7_FWD TAATACGACTCACTATAGGG (SEQIDNO:3) 4 Seq_T7Term_REV GCTAGTTATTGCTCAGCGG (SEQIDNO:4) 5 SDM_At4CL_NheI_FWD GAATGACGGAAGCAGGTCCAGTG (SEQIDNO:5) CTCGCAATGTCGTTAGGTTTTGCA AAG 6 SDM_At4CL_NheI_REV CTTTGCAAAACCTAACGACATTGC (SEQIDNO:6) GAGCACTGGACCTGCTTCCGTCAT TC
[0119] The upstream pathway genes were cloned in monocistronic form with randomized promoter strengths using previously published methods (Ref. 50). Multiple transformations were oftentimes completed to ensure sufficient library sampling and retention. The final plasmid library, pETM6-xxAt4CL-xxPhCHS-xxCmCHI, was transformed into BL21star(DE3)sucCfumC for screening. The xx feature represents the inclusion of a single random mutant T7 promoter from the five-member ePathOptimize library.
Example 3
C5 Module and p168 Module PolycultureSmall-Scale Cultivation Protocol
[0120] Single colonies of each strain were inoculated separately into 25 mL of AMM in a 125 mL non-baffled shake flask with ampicillin (80 g/mL) and grown overnight at 37 C. After 14 hours, the overnight cultures were mixed volumetrically to the indicated inoculation ratios and were inoculated at 2% (40 uL) into 2 mL of AMM and allowed to grow at 37 C. before induction with 1 mM IPTG. Upon induction, the cultures were transferred to the appropriate induction temperature and grown for 48 hours. All small-scale screening was completed in polypropylene 48-well plates (5 mL, VWR). Except where noted, the cultures were grown in AMM with 20 g/L Glycerol, 100 mg/L of substrate was added at induction, and 30 C. was used as the induction temperature.
Example 4
C5 Module and p168 Module PolycultureBioreactor Fermentation Protocol
[0121] Fed-batch style fermentation was performed using a DASGIP parallel bioreactor at an initial working volume of 500 mL of AMM with 20 g/L glycerol as a carbon source. Overnight cultures were prepared identically to the small-scale protocol presented above. The bioreactor was inoculated at an initial ratio of 7:3 (C5:p168) at 2% of final volume. The pH and DO of the fermentation broth was maintained at 7.2 and 50 percent saturation through addition of 6M sodium hydroxide and application of stirring cascade control, respectively. The feed solution [250 g/L glycerol, 4 g/L casamino acids, 7 g/L (NH.sub.4).sub.2HPO.sub.4, and 80 g/mL ampicillin] and 2MOPS mix (Ref 50) was fed at 2 mL per hour from 5-15 hours and 4 mL per hour from 15-26 hours. The fermentation was induced with IPTG to a final concentration of 1 mM after 7 hours of growth (OD.sub.600=7.1) and the system was cooled to 30 C. The substrate, p-coumaric acid, was added in 50 mg/L aliquots at 1, 4, and 7 hours post induction. Samples were taken periodically for measurement of OD.sub.600 and metabolite analysis.
Example 5
C5 Module and p168 Module PolycultureMetabolite Analysis
[0122] Fermentation broth was mixed with an equal volume of absolute ethanol and vortexed for 10 seconds prior to centrifugation (10 min, 20,000g). The supernatant (25 L) was used for HPLC analysis carried out using Agilent 1200 series HPLC equipped with a ZORBAX SB-18 column (5 m, 4.6150 mm) and a diode array detector. The mobile phase was acetonitrile (solvent A) and water (solvent B) (both contain 0.1% formic acid) at a flow rate of 1 mL/min. HPLC program was as follows: 10 to 40% A (0-10 min) and 40 to 60% A (10-15 min). Absorbance at 280 nm was monitored in all cases. Titer of products was determined using authentic standards while (+)-afzelechin was quantified using the (+)-catechin calibration curve. All experiments were performed in duplicate. Error bars represent 1 standard deviation of biological duplicate. Significance of data was determined using a two-tailed unpaired t-test with a 95 percent confidence interval.
Example 6
C5 Module and p168 Module PolycultureEmpirical Modeling Methods
[0123] Experimental conditions were modeled using empirical modeling methods, which are described in detail in Jones, J. A. et al. Experimental and computational optimization of an Escherichia coli co-culture for the efficient production of flavonoids. Metab. Eng. 35, 55-63 (2016) (Ref 24).
Example 7
C5 Module and p168 Module Polyculture
[0124] The production of flavan-3-ols from phenylpropanoic acid precursors proceeded through six enzymatic steps: 4-coumaroyl-CoA ligase, 4CL; chalcone synthase, CHS; chalcone isomerase, CHI; flavanone 3-hydroxylase, F3H; dihydroflavonol 4-reductase, DFR; leucoanthocyanidin reductase, LAR; (
Example 8
C5 Module and p168 Module PolycultureIndependent Optimization of Upstream and Downstream Modules
[0125] The ability to tailor the genetic optimization of each strain in a co-culture system for improved flux towards necessary co-factors and substrates through the pathway of interest and away from unwanted side products is a major advantage over monoculture methods. We began our modular optimization by focusing on the upstream strain containing 4CL, CHS, and CHI. Building on previous efforts to optimize malonyl-CoA availability, BL21star(DE3)sucCfumC was chosen as the host strain for this upstream module (Ref. 61). We then chose homologs for each of the three enzymes from different plant sources, resulting in 12 combinations of potential upstream pathways. Upon screening for functional conversion of two phenylpropanoic acid precursors to their corresponding flavanones, several high-titer homolog combinations were discovered (
[0126] Using the recently published ePathOptimize technique for modulating the transcriptional landscape (Ref 50), the promoter strengths of each gene in the upstream module were randomized to one of five mutant T7 promoters of various strength. The library members were then screened for conversion of p-coumaric acid to naringenin in vivo (
[0127] Optimization of the downstream pathway has been previously explored through screening of 18 homolog gene combinations resulting in two combinations that exhibit efficient conversion of both naringenin and eriodictyol substrates across a wide range of substrate concentrations (67 Zhao et al., 2015). To confirm the findings of this previous study, both the p148 and p168 constructs were tested using a cultivation protocol and substrate concentration realistic to the levels expected in the current study. Similar titers and trends were obtained with p168 slightly out-performing p148, leading towards the choice of p168 for the downstream module in the co-culture optimization. Further optimization of plasmid p168 was not performed due to limiting fluxes through the upstream module. With independent genetic optimization of the upstream and downstream modules completed, the lead candidates for each module were then screened for strain compatibility in co-culture.
Example 9
C5 Module and p168 Module PolycultureDetermination of Co-Culture Compatibility
[0128] Strain compatibility is a significant factor in any co-culture system. The strains must be able to efficiently grow in the same media, have the same antibiotic selection, and must not produce toxic compounds that significantly harm the other members of the microbial community. Many of these criteria can be easily addressed by using strains of similar background, but module specific mutations towards improving intercellular conditions for the pathway of interest can impact cellular compatibility in co-culture. Furthermore, pathway metabolites that connect the individual members of the co-culture must be readily transferred across the cell membrane from the producer to the consumer.
[0129] Two strains from each the upstream and downstream module were tested for their cross compatibility in co-culture. For the upstream strain, the transcriptionally optimized pFlavo.sup.opt mutant and the consensus control plasmid (#17, Table 1) were used in strain BL21star(DE3)sucCfumC, while for the downstream module a single plasmid, p168, was tested in two host strains: wild type BL21star(DE3) and BL21star(DE3)pgippc. We have noticed a significant decrease in cell growth for the pgippc strain background and hypothesized that this would affect strain performance in co-culture. Four co-culture combinations were tested across various initial inoculation cell ratios (
Example 10
C5 Module and p168 Module PolycultureDetermination of Important Optimization Parameters
[0130] To begin fermentation optimization of the co-culture system, we identified two key parameters predicted to result in high sensitivity: induction point and inoculation ratio. Both the upstream and downstream modules contain pET expression cassettes controlled by the T7-lac system, and therefore protein production is inducible with the addition of Isopropyl -D-1-thiogalactopyranoside (IPTG). A wide variety of optimum induction points have been presented in the primary literature for pET-based systems indicating that the optimum induction point is linked to division of cellular resources and is more complex than purely affecting protein production levels (Refs. 34 and 48). Due to this complexity, the optimum induction point is specific to the particular system and set of cultivation conditions and must be determined experimentally.
[0131] The initial inoculation ratio of upstream to downstream cells in the fermentation is another important parameter that adds to the complexity of co-culture systems. Variation of this ratio allows for changes to be made in population dynamics, accounting for differences in population growth rate and specific activity of the strains in co-culture. Interestingly, when various induction points were crossed with multiple inoculation ratios, we saw an orthogonal response in product titer from the two parameters (
Example 11
C5 Module and p168 Module PolycultureEffect of Carbon Source
[0132] Previous literature reports and early experimental evidence (data not shown) fueled the decision to use the Andrew's Magic Medium (AMM) with 20 g/L of glucose as the initial production media for individual strain optimization and preliminary co-culture experiments. In an attempt to reduce the production costs at the industrial scale, and because of the increased interest to utilize glycerol for industrial fermentations (Refs. 42 and 55), we varied the proportion of glucose to glycerol in the culture media. In addition to economic incentives, the preference for glycerol over other carbon sources has been reported for different microbial strains due to strain-specific differences in gene expression and metabolite profiles upon growth on glycerol (Ref. 36). With all media having 20 g/L total carbon source, five carbon source ratios were tested ranging from glucose only to glycerol only (
Example 12
C5 Module and p168 Module PolycultureInduction Temperature Optimization
[0133] Fermentation temperature can affect cellular growth dynamics, enzyme folding, and specific enzyme activity (Ref. 44). These effects have not been well documented on the systems level, such that optimum fermentation temperature could be predicted for any given system a priori. We therefore decided to test co-culture production at induction temperatures of 10, 20, or 30 C. The co-culture was grown at 37 C. prior to induction at which the temperature was then dropped to the specified induction temperature after induction. Previous efforts have maintained an induction temperature of 30 C. A significant decrease in optimal titer was observed in the 10 C. case with the 20 and 30 C. cases showing similar maximum achieved titers (
Example 13
C5 Module and p168 Module PolycultureSystem Modeling for Prediction of Optimum Operating Conditions
[0134] The aforementioned observations suggested that the titer achieved by the system could be improved by selecting optimized experimental conditions. To identify potential conditions that could result in an optimal titer, an empirical modeling approach was utilized (Refs. 35 and 43). Due to the trends observed from preliminary data showing the dependence of titer on induction point, inoculation ratio, carbon source, and induction temperature, we constructed an empirical scaled-Gaussian model, which uses these four experimental variables as inputs and computes the titer. This model contains 21 parameters that were fitted using 72 experimental data points. In particular, titer was measured at each combination of the following: induction point3, 4, 5, 6 hours; inoculation ratio (upstream:downstream)49:1, 9:1, 1:1; carbon source (glucose:glycerol)1:0, 1:1, 0:1; induction temperature20, 30 C. The model demonstrates a close fit with the training data, and follows the general trend of additional data that were not used for model fitting. The optimal point of the model function was determined computationally, and was used to direct future experiments in search of optimal operating conditions to maximize titer. Interestingly, the optimal point of the model function was found to be at operating conditions not tested previously, and within a gap between previously tested experimental points. Specifically, the optimal conditions predicted by the model were: induction point of 5.5 hours; inoculation ratio of 7:3 (upstream:downstream); carbon source ratio of 0:1 (glucose:glycerol); and induction temperature of 25 C.
[0135] Experiments were subsequently performed at conditions in the region of the model-predicted optimum. These experiments resulted in a maximum titer of 40.70.1 mg/L, a 65% increase over the highest titer measured prior to computational optimization. This maximal titer was achieved experimentally at an induction point of 6 hours; inoculation ratio of 8:2 (upstream:downstream); carbon source ratio of 0:1 (glucose:glycerol); and induction temperature of 30 C. This point was within the set of experimental points we tested based on proximity with the model-predicted optimum, but the point differs slightly from the model-predicted optimum. This is not surprising, as a scaled-Gaussian model was used for fitting the data and computing the optimum, whereas the behavior of the true system is likely more complex than can be fully captured by such an empirical model. That being said, using a scaled-Gaussian model represented a good trade-off between model complexity and quality of fit for the available data, and the model was ultimately successful in guiding experiments to achieve substantially higher titers. This suggests that relatively simple empirical models can be effective tools for informing titer optimization efforts.
Example 14
C5 Module and p168 Module PolycultureBioreactor Scale-Up: Proof of Principle
[0136] To demonstrate the stability and scalability of our co-culture system, we showed scale-up of the fermentation from a 2 mL culture in a 48-well plate directly to a bioreactor with a 500 mL working volume. Utilizing near optimum conditions from previous small-scale optimization experiments, the bioreactor demonstrated slightly lower (34 vs. 41 mg/L) product titers than that of the optimized small-scale system. We predict this is due to a shift in the production landscape as a result of scale-up but believe that global trends due to induction point, inoculation ratio, media composition, and induction temperature will remain constant for the system. The additional control gained through the use of bioreactors also results in additional complexity from a pathway optimization standpoint. To that end, the complete fermentation optimization of our co-culture system is beyond the scope of this work but represents a promising direction for future optimization studies.
[0137] The ability to harness the power of multiple strains in co-culture allows for a division of metabolic burden across the population, as well as the ability to genetically optimize each module individually for specific co-factor and precursor requirements. Through exploitation of these advantages and empirical modeling techniques, we were able to improve production of flavonoids to 40.70.1 mg/L, a 970-fold improvement over previous monoculture efforts.
Example 15
TAL Module, C5 Module, p168 Module, and Antho Module PolycultureBacterial Strains, Vectors, and Media
[0138] E. coli DH5 was used to propagate all plasmids, while BL21star(DE3), BL21star(DE3)sucCfumC, rpoA14(DE3), or QH4 was used as the hosts for flavonoid production. The expression vectors, pETM6 or pXPA, were the basis for all plasmid construction and pathway expression. Luria Broth (LB) Lennox modification (Sigma) and Andrew's Magic Media (AMM) (Ref. 15) were used where noted. Sequences of all plasmid constructs are available through addgene.org and are incorporated by reference herein.
Example 16
TAL Module, C5 Module, p168 Module, and Antho Module PolyculturePlasmid Construction
[0139] Many preexisting flavonoid modules were used directly or slightly modified for this work. All plasmids used are summarized in Table 3 and all plasmid modifications are described below. Site directed mutagenesis was performed to silently remove an internal NdeI restriction site from the open reading from of Rhodotorula glutinis Tyrosine Ammonia Lyase (RgTAL.sup.syn) on pTrc-RgTAL.sup.syn (Ref 16) using standard methods and primers 13-14, Table 4. The mutagenized RgTAL.sup.syn was PCR amplified from pTrc-RgTAL.sup.syn using primers 11-12, Table 4. The resulting PCR product was digested (FastDigest, Thermo Scientific) with NdeI and SpeI, gel purified (E.Z.N.A MicroElute Gel Extraction Kit, Omega Bio-tek), and ligated with pETM6 backbone also digested with NdeI and SpeI and gel extracted corresponding to standard methods to create pETM6-RgTAL.sup.syn, (#10, Table 3). The corresponding plasmid was sequence verified (GENEWIZ, Inc.) and used together with pETM6-HpaBC (#12, Table 3) (Ref. 17) to create pETM6-RgTALsyn-HpaB-HpaC via standard ePathBrick cloning protocols (Ref. 18).
[0140] To create the constitutive expression plasmid, pXy1A, we replaced the T7-lac feature on pETM6 with the P.sub.xy1A promoter from Bacillus megaterium found on the commercial vector, pMM1522 (Mobitec). To this end, a gBlock (Integrated DNA Technologies, sequence provided in Table 5) was synthesized containing the MCS of pETM6 under the control of the P.sub.xy1A promoter sequence, flanked by AvrII and SpeI restriction sites on the 5 and 3 ends, respectively. The P.sub.xy1A fragment was then cloned into pETM6 and sequence verified. Two constitutive TAL expression plasmids were obtained by sub-cloning RgTAL.sup.syn from pETM6-RgTAL.sup.syn into pXy1A and pXPA-eGFP (P.sub.GAP promoter) at restriction sites NdeI and SpeI using standard methods.
TABLE-US-00003 TABLE 3 Strains and plasmids used in this study. Cited reference numbers correspond to the numbered references provided in the Bibliography. Number Strain or vector Relevant properties Reference S1 Escherichia coli F.sup., 80d lacZM15, (lacZYA- Novagen DH5 argF)U169, recA1, endA1, hsdR17(rk.sup., mk.sup.+), phoA, supE44.sup., thi.sup.1, gyrA96, relA1 S2 E. coli BL21 Star F.sup.ompT gal dcm rne131 lon hsdS.sub.B Invitrogen (DE3) (r.sub.B.sup.m.sub.B.sup.) (DE3) S3 BLpgippc BL21Star(DE3)pgi::FRTppc::FRT- 30 KanR-FRT S4 BLsumCfumC BL21Star(DE3)fumC::FRTsucC::FRT 31 S5 rpoA14(DE3) E. coli K12 pheA tyrR lacZ::P.sub.LtetO-1- 16 tyrA.sup.fbraroG.sup.fbrtyrR::P.sub.LtetO-1- tyrA.sup.fbraroG.sup.fbr hisH(L82R) pHACM- rpoA14, (DE3) S6 QH4 E. coli ATCC 31884/pheLA-tyrA 21 1 pETM6 ePathBrick expression vector, ColE1 18 ori, AmpR 2 p168 #1 with CsF3H.sup.syn-FaDFR.sup.syn- 32 DuLAR.sup.syn, monocistronic form 3 pETM6-mCherry #1 with mCherry fluoroscent reporter 18 4 pFlavo.sup.opt or C5 #17 with C4 mutant T7 promoter 9 mutant controlling CmCHI 5 pTrc-RgTAL.sup.syn pTrcHis2B carrying codon- 16 optimized R. glutinis TAL 6 pCS-TPTA From pCS27, P.sub.LlacO1; tyrA.sup.fbr-ppsA- 22 tktA-aroG.sup.fbr 7 pZE-TH2 From pZE12, dual operons, P.sub.LlacO1; 21 RgTAL and EcHpaBC 8 pCA1 pTrcHis2B carrying codon-optimized 23 R. glutinis TAL 9 pCA3 pCDFDuet-1 carrying codon- 23 optimized R. glutinis TAL with a trc promoter 10 pETM6-RgTAL.sup.syn #1 with RgTALsyn This Study 11 pETM6-RgTALsyn- #1 with RgTALsyn, HpaB, and HpaC This Study HpaBC in monocistronic form 12 pETM6-HpaBC #1 with HpaB and HpaC in 17 monocistronic form 13 pXylA #1 with constitutive PxylA promoter This Study 14 pXPA-fapO-eGFP pGAP promoter, rrnB terminator and 33 ePathBrick feature carrying one copy of fapO and eGFP 15 pXylA-RgTAL.sup.syn #13 carrying RgTAL.sup.syn This Study 16 pXPA-fapO- #14 carrying RgTAL.sup.syn This Study RgTAL.sup.syn 17 pMM1522 Amp.sup.R (E. coli), Tet.sup.R (B. meg), Mobitec pBR322 ori, P.sub.xylA 18 pETM6-At3GT #1 with 3GT from A. thaliana This Study 19 pETM6-PhANS #1 with ANS from P. hybrida This Study 20 pETM6-At3GT- #1 with At3GT and PhANS, This Study PhANS monocistronic
TABLE-US-00004 TABLE4 Primersusedinthisstudy. PrimerID PrimerName Sequence(5->3) 1 ANS_XbaI_F CCCTCTAGAAATAATTTTGTTT (SEQIDNO:7) AACTTTAAGAAGGAGATATAC ATATGGTGAATGCAGTAGTTA C 2 ANS_XhoI_R CGATCTCGAGCTATTTAGATTC (SEQIDNO:8) TTCAGCAGCAAC 3 3GT_NdeI_F GCATCATATGACCAAACCCTC (SEQIDNO:9) CGACC 4 3GT_XhoI_R CGATCTCGAGTCAAATAATGT (SEQIDNO:10) TTACAACTGCATCC 5 pETM6_ALL_inserts_ CCATCGGTGATGTCGGCGATA (SEQIDNO:11) flank_F TAGG 6 pETM6_ALL_inserts_ GTCGAGGTGCCGTAAAGCACT (SEQIDNO:12) flank_R AAATCG 7 ANS_mid_seq_F CCATCTGGCCTAAAAATCCTA (SEQIDNO:13) CTGACTACAC 8 ANS_mid_seq_R CCTCTTTGAAGACTTTGTGTTC (SEQIDNO:14) AACAGCG 9 3GT_mid_seq_F GCTTCATCAAATGGGTCTTGCT (SEQIDNO:15) TTGC 10 3GT_mid_seq_R GGTGTCATGACCGTACCAAAG (SEQIDNO:16) CTAATG 11 RgTALsyn_FWD_w/NdeI GCGGCGCATATGGCGCCTCGC (SEQIDNO:17) CCGACTTC 12 RgTALsyn_REV_w/SpeI GCGGCGACTAGTTTATGCCAG (SEQIDNO:18) CATCTTCAGCAGAACATTG 13 SDM_RgTALsyn_FWD GCACTGCACGACGCGCACATG (SEQIDNO:19) TTGAGCCTGTTGAGC 14 SDM_RgTALsyn_REV GCTCAACAGGCTCAACATGTG (SEQIDNO:20) CGCGTCGTGCAGTGC 15 pXylA_FOR GCAAGCATGCGAAATGCA (SEQIDNO:21) 16 pXylA_REV GAGTTTCGTTCGAGATCGC (SEQIDNO:22)
TABLE-US-00005 TABLE5 gBlockSequenceforcloningpXylA(SEQIDNO:23) GCAAGCATGCGAAATGCACCTAGGAAAAAAAACATTGAAATAAACATTTA TTTTGTATATGATGAGATAAAGTTAGTTTATTGGATAAACAAACTAACTC AATTAAGATAGTTGATGGATAAACTTGTTCACTTAAATCAAAGGGGGAAA TGTACACATATGGCAGATCTCAATTGGATATCGGCCGGCCACGCGATCGC TGACGTCGGTACCCTCGAGTCTGGTAAAGAAACCGCTGCTGCGAAATTTG AACGCCAGCACATGGACTCGTCTACTAGTCGCAGCTTAATTAAGCGATCT CGAACGAAACTC
[0141] Petuniahybrida anthocyanidin synthase (PhANS) was amplified with primers 1 and 2 using plasmid pMAL-PhANS (unpublished) as a template, and Arabidopsis thaliana anthocyanidin 3-O-glucosyltransferase (At3GT) was amplified with primers 3 and 4 using plasmid pMAL-At3GT (unpublished) as a template. Following restriction digestion of PCR amplicon PhANS (XbaI/XhoI), PCR amplicon At3GT (NdeI/XhoI), and vector pETM6 (XbaI/XhoI for PhANS and NdeI/XhoI for At3GT), digested products were gel purified and ligated (Rapid DNA Ligation Kit, Thermo Scientific) to construct plasmids pETM6-PhANS and pETM6-At3GT. Constructs were transformed into DH5 and confirmed by Sanger sequencing with primers 5-10. Using the ePathBrick sub-cloning procedure (Ref. 18), At3GT and PhANS were then assembled into monocistronic configuration by ligation of restriction digestion fragments from plasmid pETM6-At3GT (NheI/SalI) and pETM6-PhANS (AvrII/SalI), yielding plasmid pETM6-At3GT-m-PhANS.
Example 17
TAL Module, C5 Module, p168 Module, and Antho Module PolycultureFermentation Protocol
[0142] The small scale cultivation protocol was adapted from (Ref. 9) with only minor modification. Except where noted, the cultures were grown in AMM with 20 g/L glucose as the primary carbon source. The cultures were first grown at 37 C. and transitioned to 30 C. upon induction with 1 mM IPTG. In the case of the phenylpropanoic acid production strains, 125 mL non-baffled shake flasks containing 25 mL of media were used to confirm small scale screening studies, allow for more frequent sampling, and limit evaporation effects on final titer.
Example 18
TAL Module, C5 Module, p168 Module, and Antho Module PolycultureMetabolite Analysis
[0143] Analysis methods were slightly adapted from Ref 9. A 25 L injection was used for all polyculture fermentations. Analysis of phenylpropanoic acid titers in monoculture required a 10-fold dilution of culture broth and a 5 L injection volume to reach the linear region for UV detection. Absorbance at 280 nm was monitored in all cases except for anthocyanidin-3-glucosides where 518 nm was used. Product titers were determined using authentic standards, while (+)-afzelechin was quantified using the (+)-catechin standard curve in accordance with previous literature, because (+)-afzelechin is not commercially available. All experiments were performed in at least biological duplicate, with key high-titer conditions reproduced in biological and experimental triplicate. Error bars represent 1 standard deviation from the mean. Significance of data was determined using a two-tailed unpaired t-test with a 95 percent confidence interval.
Example 19
TAL Module, C5 Module, p168 Module, and Antho Module PolycultureResults
[0144] Expanding upon previous co-culture efforts, the development of two additional bioconversion modules has been accomplished to realize the de novo production of both flavan-3-ols and anthocyanidin-3-glucosides for the first time outside of plants.
Example 20
TAL Module, C5 Module, p168 Module, and Antho Module PolycultureDevelopment of TAL Module
[0145] Significant efforts have been focused on improving the de novo production of phenylpropanoic acids in E. coli. Efforts from both the Stephanopoulos and Yan labs have enabled the near gram-scale production of both p-coumaric and caffeic acid. The development of the tyrosine overproducing E. coli strain rpoA14(DE3) represents a major milestone for the de novo production of phenylpropanoic acids (Refs. 16, 19), while the discovery and optimization of the native E. coli non-P450 hydroxylase enabled, for the first time, efficient production of caffeic acid through the ortho-hydroxylation of p-coumaric acid (Refs. 17, 20-22). Building off of these efforts, we set out to develop a phenylpropanoic acid production module that was compatible with our previously described C5 and p168 modules to enable the de novo production of flavan-3-ols in vivo.
TABLE-US-00006 TABLE 6 Twenty-eight potential phenylpropanoic acid production modules. Q in the strain name indicates strain QH4, while R in strain name indicates strain rpoA14(DE3) Name Plasmids Q1 pZE-TH2, pCS-TPTA Q2 pZE-TH2 Q3 pETM6-RgTAL.sup.syn, pCS-TPTA Q4 pETM6-RgTAL.sup.syn Q5 pCA1, pCS-TPTA Q6 pCA1 Q7 pCA3, pCS-TPTA Q8 pCA3 Q9 pETM6-RgTAL.sup.syn-HpaBC, pCS-TPTA Q10 pETM6-RgTAL.sup.syn-HpaBC Q11 pXPA-RgTAL.sup.syn Q12 pXPA-RgTAL.sup.syn, pCS-TPTA Q13 pXylA-RgTAL.sup.syn Q14 pXylA-RgTAL.sup.syn, pCS-TPTA R1 pZE-TH2, pCS-TPTA R2 pZE-TH2 R3 pETM6-RgTAL.sup.syn, pCS-TPTA R4 pETM6-RgTAL.sup.syn R5 pCA1, pCS-TPTA R6 pCA1 R7 pCA3, pCS-TPTA R8 pCA3 R9 pETM6-RgTAL.sup.syn-HpaBC, pCS-TPTA R10 pETM6-RgTAL.sup.syn-HpaBC R11 pXPA-RgTAL.sup.syn R12 pXPA-RgTAL.sup.syn, pCS-TPTA R13 pXylA-RgTAL.sup.syn R14 pXylA-RgTAL.sup.syn, pCS-TPTA
[0146] To accomplish this task, we collected the most efficient plasmids and strains from the recent literature (Refs. 16, 21, 23) and along with several plasmids constructed in the Koffas' lab, built 28 strain-plasmid combinations for screening of phenylpropanoic acid production, Table 6. Twenty of the 28 strains were designed for p-coumaric acid production (TAL overexpression), while the remaining 8 were targeted for caffeic acid production (TAL and HpaBC overexpression). The effect of the endogenous gene supplementation plasmid, pCS-TPTA, was also tested but did not show significant titer improvements for any of the tested combinations,
Example 21
TAL Module, C5 Module, p168 Module, and Antho Module PolycultureOptimization of Phenylpropanoic Acid Production
[0147] Three p-coumaric acid (R4, R11, R13) and one caffeic acid (R2) production strains from the initial screen were subjected to further optimization to determine the full potential of these modules in monoculture. Through course optimization of induction point, inducer concentration, production temperature, and carbon source, the highest titer production to date was realized for both p-coumaric and caffeic acid at 2.510.03 and 1.030.02 g/L, respectively (
Example 22
TAL Module, C5 Module, p168 Module, and Antho Module PolycultureProduction of Flavan-3-Ols De Novo
[0148] Combining the previously published co-culture system for the efficient production of flavan-3-ols from phenylpropanoic acids with the recently developed phenylpropanoic acid production module enables the production of flavan-3-ols from glucose. Highlighting the drop-in modularity of polyculture systems we conserved the previously optimized ratio of C5:p168 of 8:2 (Ref 9) and varied only the proportion of the TAL module over several induction points in the range of the predicted optimum from previous work. Using this simple optimization strategy, we were able to demonstrate the de novo production of afzelechin for the first time in a microbial host (
Example 23
TAL Module, C5 Module, p168 Module, and Antho Module PolycultureProduction of Anthocyanidin-3-Glucosides De Novo
[0149] Our previous successes using polycultures for the production of flavonoids has urged the further application of this technology to expand what is currently possible in vivo. Previous efforts in the Koffas' lab have developed strains capable of high titer anthocyanidin-3-glucoside production from flavan-3-ols, but efforts to further extend the pathway towards the phenylpropanoic acid precursors have not been successful. Building off of these efforts, we cloned the previously characterized ANS and 3GT enzymes into a synthetic monocistronic operon in the ePathBrick plasmid pETM6. Transforming this plasmid into our baseline host BL21star(DE3) resulted in our Antho module to be combined with the previously described TAL, C5, and p168 modules for the de novo production of anthocyanidin-3-glucosides in vivo. In a similar fashion as before, the previously determined optimum ratio 8:8:2 (TAL:C5:p168) was conserved with the fraction of the new module being varied to result in the first account of a functional synthetic four strain polyculture. This microbial consortium enabled, for the first time outside of plants, the production of the anthocyanidin-3-glucoside, callistephin, from glucose,
[0150] Adding two additional enzyme overexpressions, matBC, to the previously published C5 module, further highlights the flexibility of the polyculture platform for rapid expansion and modification. These enzymes enable the uptake of externally supplemented sodium malonate and subsequent activation to malonyl-CoA, a key and limiting substrate for the chalcone synthase enzyme. Significantly (p-value<0.05) higher production of callistephin from glucose was achieved across a wide range of inoculation ratios, while conserving the optimum fermentation conditions from previous experiments.
[0151] In summary, the rapid success of these polycultures to realize the de novo production of various late-pathway flavonoid metabolites demonstrates the power of these techniques over traditional monoculture metabolic engineering efforts. Additionally, the ease at which these pathways were re-optimized through conservation of the previously optimized inoculation ratio further highlights the benefits of polyculture modularity over that of traditional monoculture techniques (Ref. 26). In traditional monoculture techniques, extending the current heterologous overexpression pathway would require additional genes to be cloned and expressed in the previously optimized strain, consequently un-optimizing the strain from both a genetic and fermentation perspective. Genetic re-optimization is a difficult task. Oftentimes, it is impossible to regain the fluxes previously achieved, due to increased metabolic burden or natural precursor and co-factor requirements, limiting the overall titer, yield and productivity of the process. Polycultures, however, enable the genetic optimization of each module to be conserved only requiring minor fermentation optimization to adjust the inoculation ratio of the new strain. The simplicity of this optimization and the smooth trends observed in corresponding production landscapes support the hypothesis that these cultures are stable through the production phase of the fermentation.
[0152] In conclusion, we have demonstrated the development of a high-titer phenylpropanoic acid module and a plan to demonstrate its true potential through bioreactor scale-up. Utilizing this module along with the previously published modules (C5 and p168), we demonstrate the de novo production of flavan-3-ols for the first time outside of the native plant hosts. Further expanding on this polyculture theme, we incorporated a fourth module (Antho) containing the genes ANS and 3GT. Using all four modules, we were able to demonstrate the production of the anthocyindin-3-glucoside, callistephin, from glucose. This feat was possible due to the modularity of the polyculture scaffold conserving the genetic optimization of each module only requiring basic fermentation optimization to achieve peak production. Finally, we outline the path forward for expanding upon this polyculture work. These plans include potential additional modules, expansion into the terpenoid and alkaloid pathways, and methods to address the stability of the individual strain populations with time. In summary, co-culture and polyculture techniques have demonstrated their potential to rapidly expand what is deemed to be possible with metabolic engineering, but this power comes with additional complexities that must be addressed from a systematic approach to achieve the highest titer, yield, and productivities possible.
Example 24
[0153]
TABLE-US-00007 Sequences RtMatB-Rhizobiumtrifolii Nucleicacidsequence(SEQIDNO:24) GTGAGCAACCATCTTTTCGACGCCATGCGGGCCGCCGCGCCCGGTAACGCACCATTC ATCCGGATCGATAACACGCGCACATGGACCTATGACGACGCCGTCGCTCTTTCCGGC CGCATTGCCGGCGCGATGGACACGCTCGGCATTCGCCCCGGCGACCGCGTTGCGGT GCAGGTCGAGAAAAGTGCCGAGGCATTGATCCTCTATCTCGCCTGTCTTCGAAGCGG CGCCGTTTACCTGCCGCTCAACACCGCCTATACGCTGGCTGAGCTCGATTATTTTATC GGCGATGCGGAGCCGCGTTTGGTGGTTGTTGCATCGTCGGCTCGAGCGGGCGTGGA GACAATCGCCAAGCCCCGCGGTGCGATCGTCGAAACTCTCGACGCTGATGGCAGCG GCTCGTTGCTGGATCTCGCCCGCGATGAGCCGGCTGACTTTGTCGATGCCTCGCGCT CCGCCGATGATCTGGCTGCGATCCTCTACACCTCGGGAACGACGGGACGCTCCAAG GGGGCGATGCTCACGCATGGGAACCTGCTCTCGAACGCCCTGACCTTGCGAGATTTT TGGCGCGTCACCGCCGGCGATCGACTGATCCATGCCTTGCCGATCTTCCACACGCAT GGGCTGTTCGTCGCCACGAACGTCACTTTACTCGCCGGCGCCTCGATGTTCCTGCTG TCGAAGTTCGACCCGGAGGAGATCCTGTCGCTGATGCCGCAGGCAACGATGCTGAT GGGCGTGCCGACCTTCTACGTGCGCCTCCTGCAAAGCCCGCGCCTCGACAAGCAAG CAGTCGCCAACATCCGCCTCTTCATTTCCGGTTCGGCTCCACTGCTTGCAGAAACAC ATACCGAGTTCCAGGCACGTACCGGTCACGCCATTCTCGAGCGCTACGGCATGACG GAAACCAATATGAACACGTCCAACCCTTATGAGGGGAAACGGATTGCCGGAACGGT CGGCTTCCCGCTGCCTGATGTGACGGTGCGCGTCACCGATCCCGCCACCGGGCTCGC GCTGCCGCCTGAAGAAACAGGCATGATCGAGATCAAGGGGCCGAACGTTTTCAAGG GCTATTGGCGCATGCCCGAAAAAACCGCGGCCGAATTCACCGCCGACGGTTTCTTCA TCAGCGGCGATCTCGGCAAGATCGACCGGGACGGTTATGTCCACATCGTCGGCCGT GGCAAGGATCTGGTGATTTCCGGTGGATACAACATCTATCCGAAAGAGGTGGAGGG CGAGATCGACCAGATCGAGGGTGTGGTTGAGAGCGCTGTGATCGGCGTGCCGCATC CCGATTTCGGAGAAGGCGTGACCGCCGTCGTCGTGCGCAAACCCGGCGCTGTCCTCG ATGAAAAGGCCATCGTCAGCGCCCTCCAGGACCGGCTCGCGCGCTACAAACAACCC AAGCGCATCATCTTTGCCGAAGACTTGCCGCGCAACACGATGGGCAAGGTTCAGAA AAACATCCTGCGGCAGCAATACGCCGATCTTTACACCAGGACGTAA RtMatB-Rhizobiumtrifolii Aminoacidsequence(SEQIDNO:25) MSNHLFDAMRAAAPGNAPFIRIDNTRTWTYDDAVALSGRIAGAMDTLGIRPGDRVAVQ VEKSAEALILYLACLRSGAVYLPLNTAYTLAELDYFIGDAEPRLVVVASSARAGVETIAK PRGAIVETLDADGSGSLLDLARDEPADFVDASRSADDLAAILYTSGTTGRSKGAMLTHG NLLSNALTLRDFWRVTAGDRLIHALPIFHTHGLFVATNVTLLAGASMFLLSKFDPEEILS LMPQATMLMGVPTFYVRLLQSPRLDKQAVANIRLFISGSAPLLAETHTEFQARTGHAILE RYGMTETNMNTSNPYEGKRIAGTVGFPLPDVTVRVTDPATGLALPPEETGMIEIKGPNV FKGYWRMPEKTAAEFTADGFFISGDLGKIDRDGYVHIVGRGKDLVISGGYNIYPKEVEG EIDQIEGVVESAVIGVPHPDFGEGVTAVVVRKPGAVLDEKAIVSALQDRLARYKQPKRII FAEDLPRNTMGKVQKNILRQQYADLYTRT RtMatC-Rhizobiumtrifolii Nucleicacidsequence(SEQIDNO:26) ATGGGCATCGAACTGCTGAGTATTGGTCTGCTGATTGCTATGTTTATTATTGCTACGA TTCAACCGATTAACATGGGTGCTCTGGCATTCGCAGGCGCTTTTGTGCTGGGTAGCA TGATTATCGGCATGAAAACCAACGAAATTTTCGCAGGCTTTCCGTCTGACCTGTTTCT GACCCTGGTGGCGGTTACGTACCTGTTTGCGATTGCCCAGATCAATGGCACCATCGA CTGGCTGGTTGAATGCGCGGTGCGTCTGGTTCGTGGCCGCATTGGTCTGATCCCGTG GGTGATGTTCCTGGTTGCGGCCATTATCACCGGTTTTGGTGCACTGGGTCCGGCAGC TGTTGCAATTCTGGCACCGGTCGCACTGAGCTTCGCAGTGCAATATCGCATTCATCC GGTTATGATGGGTCTGATGGTCATCCACGGCGCACAGGCTGGCGGTTTTTCACCGAT TTCGATCTACGGCGGTATTACCAACCAAATCGTGGCAAAAGCAGGTCTGCCGTTCGC ACCGACGAGTCTGTTTCTGAGCAGCTTTTTCTTTAATCTGGCAATTGCTGTCCTGGTG TTCTTTGTGTTTGGCGGTGCACGTGTTATGAAACACGATCCGGCTTCTCTGGGTCCGC TGCCGGAACTGCATCCGGAAGGCGTGAGCGCGTCTATTCGTGGTCATGGCGGCACC CCGGCAAAACCGATCCGCGAACATGCGTATGGCACCGCAGCAGACACGGCAACCAC GCTGCGTCTGAACAATGAACGCATTACCACGCTGATCGGTCTGACCGCACTGGGTAT TGGTGCACTGGTTTTCAAATTTAACGTCGGTCTGGTGGCTATGACCGTGGCAGTGGT TCTGGCACTGCTGAGCCCGAAAACGCAGAAAGCAGCTATTGATAAAGTCAGTTGGT CCACCGTGCTGCTGATCGCGGGTATTATCACGTATGTTGGCGTCATGGAAAAAGCGG GCACCGTTGACTACGTCGCCAATGGTATTAGTTCCCTGGGTATGCCGCTGCTGGTCG CGCTGCTGCTGTGTTTCACCGGCGCCATCGTGTCCGCGTTTGCCTCATCGACGGCACT GCTGGGTGCTATTATCCCGCTGGCCGTTCCGTTCCTGCTGCAGGGCCATATTAGTGC AATCGGTGTCGTGGCGGCCATTGCTATCTCCACCACGATTGTGGATACCAGCCCGTT TTCTACGAACGGCGCGCTGGTTGTCGCAAATGCTCCGGATGACTCACGTGAACAGGT TCTGCGCCAACTGCTGATCTATTCGGCCCTGATTGCTATTATTGGTCCGATTGTCGCC TGGCTGGTTTTCGTTGTGCCGGGTCTGGTCTAA RtMatC-Rhizobiumtrifolii Aminoacidsequence(SEQIDNO:27) MGIELLSIGLLIAMFIIATIQPINMGALAFAGAFVLGSMIIGMKTNEIFAGFPSDLFLTLVA VTYLFAIAQINGTIDWLVECAVRLVRGRIGLIPWVMFLVAAIITGFGALGPAAVAILAPV ALSFAVQYRIHPVMMGLMVIHGAQAGGFSPISIYGGITNQIVAKAGLPFAPTSLFLSSFFF NLAIAVLVFFVFGGARVMKHDPASLGPLPELHPEGVSASIRGHGGTPAKPIREHAYGTA ADTATTLRLNNERITTLIGLTALGIGALVFKFNVGLVAMTVAVVLALLSPKTQKAAIDK VSWSTVLLIAGIITYVGVMEKAGTVDYVANGISSLGMPLLVALLLCFTGAIVSAFASSTA LLGAIIPLAVPFLLQGHISAIGVVAAIAISTTIVDTSPFSTNGALVVANAPDDSREQVLRQL LIYSALIAIIGPIVAWLVFVVPGLV RgTALsyn-Rhodotorulaglutinis Nucleicacidsequence(SEQIDNO:28) atggcgcctcgcccgacttcgcaaagccaggcccgcacttgcccgacgacgcaggttacccaagttgatatcgttgag aaaatgttggcggctcctactgatagcacgctggagctggacggttatagcctgaatctgggtgatgtcgtgagcgct gcgcgtaagggtcgtcctgtccgtgtcaaagatagcgatgaaatccgcagcaaaatcgacaagagcgttgaattcctg cgcagccaactgagcatgtcggtttacggtgtgacgaccggattggcggctccgcggacacgcgcacggaggacgcaa ttagcctgcaaaaggcgttgctggaacaccagctgtgtggtgtgttgccgagcagatcgacagattcgcttgggtcgt ggtctggagaatagcctgccgttggaagtcgttcgcggtgcaatgaccattcgtgtgaattcgctgacccgtggccat agcgctgttcgtctggttgttctggaagcactgacgaactttctgaaccacggtattaccccgattgttccgctgcgc ggtacgatctccgcgagcggcgatctgtctccactgtcgtacattgcagcggcgattagcggtcacccggatagcaaa gttcacgtggtccatgaaggcaaagagaagatcctgtacgcgcgcgaagcgatggcgctgtttaacctggagccggtg gttttgggtccgaaggagggcctgggtctggtgaatggtacggcagtctccgcgagcatggcaacgctggcactgcac gacgcgcatatgttgagcctgttgagccaatcgctgaccgcgatgaccgtggaggcgatggtcggtcacgcgggcaga tccatccattcctgcacgatgttacgcgtccgcacccgacgcaaatcgaggtcgcgggtaacattcgcaaactgctgg agggctcgcgcttcgcggtccaccacgaggaagaggttaaggtcaaggatgatgaaggcattttgcgtcaggatcgtt atccgttgcgcacgagcccgcaatggttgggtccgctggtgtccgacctgattcacgctcatgccgtcttgacgatcg aagcgggtcaaagcaccaccgataacccactgatcgatgttgagaataagaccagccatcacggtggcaactttcaag cggcagcggttgccaacacgatggaaaagacccgtctgggcttggcccaaatcggtaaactgaatttcacccagctga cggagatgctgaacgcgggcatgaatcgtggcttgccgagctgcctggcggctgaagacccatccctgagctatcatt gcaaaggtctggacattgcggcggctgcatatacgagcgaactgggccacctggctaacccggtcaccacccacgtcc aaccggctgaaatggcaaaccaggcggtgaatagcttggcgttgattagcgcacgtcgtaccacggaatctaacgacg ttctgtccctgctgctggcaacgcacctgtactgcgtgctgcaggcgatcgacctgcgtgcgattgagttcgagttca agaaacagtttggtcctgccattgttagcctgatcgaccaacactttggtagcgcgatgacgggtagcaatctgcgtg atgagctggttgaaaaggtcaataagactctggccaagcgtttggagcaaaccaatagctacgatctggttccgcgct ggcacgacgcttttagcttcgctgcaggcactgttgtcgaggttctgtccagcacgagcctgagcttggcggccgtga acgcatggaaggttgcggcagccgagagcgcgatctccttgacgcgccaggtccgtgaaacgttttggtccgctgcaa gcacctccagcccggcgttgtcttacttgagcccgcgcacgcagatcctgtacgcatttgtgcgtgaggaactgggtg tcaaagcccgccgtggtgacgtcttcttgggtaaacaagaagttaccatcggcagcaacgttagcaagatttacgaag ccatcaagagcggccgtatcaacaatgttctgctgaagatgctggcataa RgTALsyn-Rhodotorulaglutinis Aminoacidsequence(SEQIDNO:29) MAPRPTSQSQARTCPTTQVTQVDIVEKMLAAPTDSTLELDGYSLNLGDVVSAARKGRP VRVKDSDEIRSKIDKSVEFLRSQLSMSVYGVTTGFGGSADTRTEDAISLQKALLEHQLCG VLPSSFDSFRLGRGLENSLPLEVVRGAMTIRVNSLTRGHSAVRLVVLEALTNFLNHGITPI VPLRGTISASGDLSPLSYIAAAISGHPDSKVHVVHEGKEKILYAREAMALFNLEPVVLGP KEGLGLVNGTAVSASMATLALHDAHMLSLLSQSLTAMTVEAMVGHAGSFHPFLHDVT RPHPTQIEVAGNIRKLLEGSRFAVHHEEEVKVKDDEGILRQDRYPLRTSPQWLGPLVSDL IHAHAVLTIEAGQSTTDNPLIDVENKTSHEIGGNFQAAAVANTMEKTRLGLAQIGKLNFT QLTEMLNAGMNRGLPSCLAAEDPSLSYHCKGLDIAAAAYTSELGHLANPVTTHVQPAE MANQAVNSLALISARRTTESNDVLSLLLATHLYCVLQAIDLRAIEFEFKKQFGPAIVSLID QHFGSAMTGSNLRDELVEKVNKTLAKRLEQTNSYDLVPRWHDAFSFAAGTVVEVLSST SLSLAAVNAWKVAAAESAISLTRQVRETFWSAASTSSPALSYLSPRTQILYAFVREELGV KARRGDVFLGKQEVTIGSNVSKIYEAIKSGRINNVLLKMLA At4CL-Arabidopsisthaliana Nucleicacidsequence(SEQIDNO:30) atggcgccacaagaacaagcagtttctcaggtgatggagaaacagagcaacaacaacaacagtgacgtcattttccgatcaaagttaccgg atatttacatcccgaaccacctatctctccacgactacatcttccaaaacatctccgaattcgccactaagccttgcctaatcaacggaccaacc ggccacgtgtacacttactccgacgtccacgtcatctcccgccaaatcgccgccaattttcacaaactcggcgttaaccaaaacgacgtcgt catgctcctcctcccaaactgtcccgaattcgtcctctctttcctcgccgcctccttccgcggcgcaaccgccaccgccgcaaaccctttcttc actccggcggagatagctaaacaagccaaagcctccaacaccaaactcataatcaccgaagctcgttacgtcgacaaaatcaaaccacttc aaaacgacgacggagtagtcatcgtctgcatcgacgacaacgaatccgtgccaatccctgaaggctgcctccgcttcaccgagttgactca gtcgacaaccgaggcatcagaagtcatcgactcggtggagatttcaccggacgacgtggtggcactaccttactcctctggcacgacggg attaccaaaaggagtgatgctgactcacaagggactagtcacgagcgttgctcagcaagtcgacggcgagaacccgaatctttatttccaca gcgatgacgtcatactctgtgttttgcccatgtttcatatctacgctttgaactcgatcatgttgtgtggtcttagagttggtgcggcgattctgata atgccgaagtttgagatcaatctgctattggagctgatccagaggtgtaaagtgacggtggctccgatggttccgccgattgtgttggccattg cgaagtatcggagacggagaagtatgatttgagctcgataagagtggtgaaatctggtgctgctcctcttggtaaagaacttgaagatgccg ttaatgccaagtttcctaatgccaaactcggtcagggatacggaatgacggaagcaggtccagtgctcgcaatgtcgttaggttttgcaaagg aaccttttccggttaagtcaggagcttgtggtactgttgtaagaaatgctgagatgaaaatagttgatccagacaccggagattctctttcgagg aatcaacccggtgagatttgtattcgtggtcaccagatcatgaaaggttacctcaacaatccggcagctacagcagagaccattgataaaga cggttggcttcatactggagatattggattgatcgatgacgatgacgagcttttcatcgttgatcgattgaaagaacttatcaagtataaaggtttt caggtagctccggctgagctagaggctttgctcatcggtcatcctgacattactgatgttgctgttgtcgcaatgaaagaagaagcagctggt gaagttcctgttgcatttgtggtgaaatcgaaggattcggagttatcagaagatgatgtgaagcaattcgtgtcgaaacaggttgtgttttacaa gagaatcaacaaagtgttcttcactgaatccattcctaaagctccatcagggaagatattgaggaaagatctgagggcaaaactagcaaatg gattgtga At4CL-Arabidopsisthaliana Aminoacidsequence (SEQIDNO:31) MAPQEQAVSQVMEKQSNNNNSDVIFRSKLPDIYIPNHLSLHDYIFQNISEFATKPCLINGP TGHVYTYSDVHVISRQIAANFHKLGVNQNDVVMLLLPNCPEFVLSFLAASFRGATATAA NPFFTPAEIAKQAKASNTKLIITEARYVDKIKPLQNDDGVVIVCIDDNESVPIPEGCLRFTE LTQSTTEASEVIDSVEISPDDVVALPYSSGTTGLPKGVMLTHKGLVTSVAQQVDGENPNL YFHSDDVILCVLPMFHIYALNSIMLCGLRVGAAILIMPKFEINLLLELIQRCKVTVAPMVP PIVLAIAKSSETEKYDLSSIRVVKSGAAPLGKELEDAVNAKFPNAKLGQGYGMTEAGPV LAMSLGFAKEPFPVKSGACGTVVRNAEMKIVDPDTGDSLSRNQPGEICIRGHQIMKGYL NNPAATAETIDKDGWLHTGDIGLIDDDDELFIVDRLKELIKYKGFQVAPAELEALLIGHP DITDVAVVAMKEEAAGEVPVAFVVKSKDSELSEDDVKQFVSKQVVFYKRINKVFFTESI PKAPSGKILRKDLRAKLANGL Pc4CL-Petroselinumcrispum Nucleicacidsequence (SEQIDNO:32) atgggagactgtgtagcacccaaagaagaccttattttccgatcgaaactccctgatatttacatcccgaaacaccttccgttacatacttattgt ttcgaaaacatctcgaaagttggcgacaagtcctgtttaataaatggcgctacaggcgaaacgttcacttattcccaagttgagctcctttccag gaaagttgcatcagggttaaacaaactcggcattcaacagggcgataccatcatgcttttgctccctaactcccctgagtattttttcgctttctta ggcgcatcgtatcgtggtgcaatttctactatggccaatccgtttttcacttctgctgaggtgatcaaacagctcaaagcatcccaagctaagct cataattacgcaagcttgttacgtagacaaagtgaaagactacgcagcagagaaaaatatacagatcatttgcatcgatgatgctcctcagga ttgtttacatttctccaaacttatggaagctgatgaatcagaaatgcctgaggttgtgatcaattcagacgatgtcgtcgcgttaccttactcatcg ggtactacaggactaccgaaaggtgttatgttgacacacaaaggacttgttactagcgtggcacaacaagttgatggagacaatccgaattta tatatgcatagcgaggatgtgatgatctgcatattgcctttgtttcatatttattcgcttaacgcggtgttgtgctgtggactcagagcaggggtga cgatcttgattatgcagaaatttgatattgtgccatttttggaactgatacagaaatataaagttacaattggaccgtttgtgccaccaattgtgttg gcaattgcgaaaagtccagtggtggataaatatgacttgtcgtcggtgaggacggttatgtctggagctgctccgttagggaaggagcttga agatgctgttagagctaagtttcctaatgccaaacttggtcagggatatggaatgacagaggcagggccagttttagcaatgtgcctggcgttt gcaaaggaaccatacgagatcaaatcgggtgcctgtggaactgttgtgaggaatgctgaaatgaaaattgtggatcctgagaccaacgcct ctcttccacgaaaccaacgcggagagatttgcattcgaggtgaccaaattatgaaaggctacctcaatgatcctgaatcaacaaggacaaca atagacgaagaaggctggttgcacacaggagatataggcttcattgacgacgatgatgagctatttattgttgatagacttaaggaaataatca aatacaaaggatccaggttgcccctgctgaacttgaagctctgctacttactcatcctaccatttccgatgctgcagttgttcccatgatagatg agaaagcaggagaggtgcctgtggatttgttgtgagaacaaacggificaccaccactgaggaagaaatcaagcaattcgtctcgaaaca ggtggtgttctacaagagaatatttcgtgtattttttgttgatgcaattccgaaatcaccatctggaaagattcttcgaaaggacttgagagcaaa aatagcatccggtgatcttcccaaataa Pc4CL-Petroselinumcrispum Aminoacidsequence(SEQIDNO:33) MGDCVAPKEDLIFRSKLPDIYIPKHLPLHTYCFENISKVGDKSCLINGATGETFTYSQVEL LSRKVASGLNKLGIQQGDTIMLLLPNSPEYFFAFLGASYRGAISTMANPFFTSAEVIKQLK ASQAKLIITQACYVDKVKDYAAEKNIQIICIDDAPQDCLHFSKLMEADESEMPEVVINSD DVVALPYSSGTTGLPKGVMLTHKGLVTSVAQQVDGDNPNLYMHSEDVMICILPLFHIYS LNAVLCCGLRAGVTILIMQKFDIVPFLELIQKYKVTIGPFVPPIVLAIAKSPVVDKYDLSS VRTVMSGAAPLGKELEDAVRAKFPNAKLGQGYGMTEAGPVLAMCLAFAKEPYEIKSG ACGTVVRNAEMKIVDPETNASLPRNQRGEICIRGDQIMKGYLNDPESTRTTIDEEGWLH TGDIGFIDDDDELFIVDRLKEIIKYKGFQVAPAELEALLLTHPTISDAAVVPMIDEKAGEV PVAFVVRTNGFTTTEEEIKQFVSKQVVFYKRIFRVFFVDAIPKSPSGKILRKDLRARIASG DLPK Vv4CL-Vitisvinifera Nucleicacidsequence(SEQIDNO:34) atgattagtattgaaacgcaaaacccggatgttagcaacctggacacctcgcactctattccgaaaatggcaaaccgtattgatgaccatgtgt ttcgttctaaactgccggaaattccgatcagtaaccatctgccgctgcacacgtattgcttcgaaaattactcgcagtttgcagaccgtccgtgt ctgattgttggctcgacgaacaaaacctatagatcgctgaaacccatctgatctctcgcaaagtgggcgcaggttttgctcacctgggtctga aacagggcgatgtggttatgattctgctgcaaaattgcgcggaatttgccttcagctttctgggtgcgtctatggttggcgccgtcaccacgac cgcaaacccgttctacacgtccgcggaaatcttcaaacagctgaacgcatcaaaagctaaaatcgtcgtgacccaggcgcaatatgtggat aaactgcgcgactacccggatggtcaagttgccaaaattggcgaaggtttcacggtcattaccatcgatgacccgccggaaaactgtatgca ttttagtgttgtctccgaagcgaacgaaagcgaactgccggaagtctcaattaattcggatgacccggtggccctgccgtttagctctggtac gaccggcctgccgaaaggcgtggttctgacgcacaaatcactgatcacctcggtcgcccagcaagtggatggtgaaaacccgaatctgca tctgaccccggatgacgtcgtgctgtgcgtgctgccgctgttccacatttatagcctgaactctgttctgctgtgtagtctgcgtgcaggtgcag cagtgctgctgatgcagaaatttgaaattggtaccctgctggaactgatccaacgttaccgcgtgagcgttgcagctgttgtcccgccgctgg ttctggcactggctaaaaatccgatggtggaatcgtttgatctgagttccatccgtgtggttctgagcggtgcagcaccgctgggcaaagaac tggaagcagctctgcgttcccgcgttccgcaggcagtcctgggccaaggttatggcatgacggaagcaggcccggtgctgtcaatgtgcct gggtttcgctaaacagccgtttccgacgaaatcaggttcgtgtggcaccgtcgtgcgtaacgcggaactgaaagttgtggatccggaaacc ggttgctccctgggccgtaatcagccgggtgaaatttgtatccgcggccagcaaattatgaaaggttatctgaatgatccggaagcgacggc ctctaccattgacgttgatggctggctgcataccggtgacatcggctacgtggatgacgatgaagaagtgttcattgttgatcgcgtcaaaga actgatcaaattcaaaggtMcaggttccgccggcagaactggaagctctgctggtgtctcacccgtccattgccgatgcggccgtggttcc gcaaaaagacgatgttgctggcgaagtcccggtggcgttcgtcgtgcgttctaacggttttgaactgaccgaagaagcagtgaaagaattca tcagtaaacaggttgtcttttataaacgcctgcataaagtgtactttgttcacgcgattccgaaaagcccgtctggcaaaatcctgcgtaaagat ctgcgcgcgaaactggccgaaaaaaccccggaaccgaac Vv4CL-Vitisvinifera Aminoacidsequence(SEQIDNO:35) MISIETQNPDVSNLDTSHSIPKMANRIDDHVFRSKLPEIPISNHLPLHTYCFENYSQFADRP CLIVGSTNKTYSFAETHLISRKVGAGFAHLGLKQGDVVMILLQNCAEFAFSFLGASMVG AVTTTANPFYTSAEIFKQLNASKAKIVVTQAQYVDKLRDYPDGQVAKIGEGFTVITIDDP PENCMHFSVVSEANESELPEVSINSDDPVALPFSSGTTGLPKGVVLTHKSLITSVAQQVD GENPNLHLTPDDVVLCVLPLFHIYSLNSVLLCSLRAGAAVLLMQKFEIGTLLELIQRYRV SVAAVVPPLVLALAKNPMVESFDLSSIRVVLSGAAPLGKELEAALRSRVPQAVLGQGYG MTEAGPVLSMCLGFAKQPFPTKSGSCGTVVRNAELKVVDPETGCSLGRNQPGEICIRGQ QIMKGYLNDPEATASTIDVDGWLHTGDIGYVDDDEEVFIVDRVKELIKFKGFQVPPAEL EALLVSHPSIADAAVVPQKDDVAGEVPVAFVVRSNGFELTEEAVKEFISKQVVFYKRLH KVYFVHAIPKSPSGKILRKDLRAKLAEKTPEPN PhCHS-PetuniaXhybrida Nucleicacidsequence(SEQIDNO:36) atggtgacagtcgaggagtatcgtaaggcacaacgtgctgaaggtccagccactgtcatggccattggaacagccacaccttcaaactgtg ttgatcaaagcacttaccctgatttttattttcgtatcactaacagtgagcacaagactgatcttaaggagaaatttaagcgcatgtgtgaaaaat caatgattaagaaaaggtacatgcacttaacagaggaaatcttgaaagagaatcctagtatgtgtgaatacatggcaccttctcttgatgctag gcaagacatagtggtggttgaagtgcccaaacttggcaaagaggcagctcaaaaggccatcaaggaatggggccagcccaagtccaaaa ttacccatttggtcttttgcacaaccagtggtgtggacatgcctgggtgtgactatcaactcactaagctacttgggcttcgtccatcggtcaag aggcttatgatgtaccaacaaggttgctttgctggtggcacggttcttcggttagccaaggacttggctgaaaacaacaagggcgctcgagt ccttgttgtttgttcagaaatcaccgcggtcactttccgtgggccaaatgatactcatttggatagtttagttggccaagcactttttggtgatggg gcaggcgcgatcattataggttctgatccaattccaggggtcgaaaggcctttgttcgagctcgtttcagcagcccaaactcttctcccagata gccatggtgctattgatggccatctccgtgaagttgggcttacattccacttactcaaagatgttcctgggctgatctcaaaaaatattgagaag agccttgaggaagcattcaaacctttgggcatttctgattggaactctctattctggattgctcatccaggtgggcctgcaattttggaccaagtt gaaataaagttgggcctaaagcccgagaaacttaaggctacaaggaatgtgttaagtaactatggtaacatgtcaagtgcttgtgtactgtttat tttggatgaaatgagaaaggcctcagccaaagaaggtttaggaactactggtgaagggcttgagtggggtgttctttttggatttgggcctgg gctaacagttgagactgttgtcctccacagtgttgctacttaa PhCHS-PetuniaXhybrida Aminoacidsequence(SEQIDNO:37) MVTVEEYRKAQRAEGPATVMAIGTATPSNCVDQSTYPDFYFRITNSEHKTDLKEKFKR MCEKSMIKKRYMHLTEEILKENPSMCEYMAPSLDARQDIVVVEVPKLGKEAAQKAIKE WGQPKSKITHLVFCTTSGVDMPGCDYQLTKLLGLRPSVKRLMMYQQGCFAGGTVLRL AKDLAENNKGARVLVVCSEITAVTFRGPNDTHLDSLVGQALFGDGAGAIIIGSDPIPGVE RPLFELVSAAQTLLPDSHGAIDGHLREVGLTFHLLKDVPGLISKNIEKSLEEAFKPLGISD WNSLFWIAHPGGPAILDQVEIKLGLKPEKLKATRNVLSNYGNMSSACVLFILDEMRKAS AKEGLGTTGEGLEWGVLFGFGPGLTVETVVLHSVAT CmCHS-Citrusmaxima Nucleicacidsequence(SEQIDNO:38) atggctacggtccaagaaatccgcaacgctcaacgcgcagatggtccggcgacggtcctggcaatcggcacggcaaccccggctcatag cgtgaaccaggcagattatccggactattactttcgtattaccaaatctgaacacatgacggaactgaaagaaaaattcaaacgtatgtgcgat aaaagtatgattaaaaaacgctacatgtacctgaccgaagaaatcctgaaagaaaacccgaatatgtgtgcctacatggcaccgagcctgg atgcgcgccaggacattgtggttgtcgaagttccgaaactgggtaaagaagcggccaccaaagccatcaaagaatggggccaaccgaaa tcaaaaattacgcacctgatcttttgcaccacgtcgggtgtggatatgccgggtgcagactatcagctgaccaaactgctgggtctgcgtccg agcgttaaacgctttatgatgtaccagcaaggctgcttcgcaggcggtacggtcctgcgtctggctaaagatctggcggaaaacaataaag gtgctcgcgttctggtggtttgtagtgaaattaccgctgtcacgtttcgtggtccggcggatacccatctggactccctggttggccaggccct gttcggcgatggtgcagctgcggttatcgtcggcgcagatccggacacgagtgtggaacgtccgctgtatcagctggtttcaacctcgcaa acgattctgccggattccgacggtgcgatcgatggccatctgcgcgaagtgggtctgacctttcacctgctgaaagacgttccgggcctgat ttcaaaaaacatcgaaaaaagcctgtctgaagcctttgcaccggttggtatttcggattggagctctattttctggatcgcacatccgggcggtc cggcaatcctggaccaggtggaaagcaaactgggtctgaaagaagaaaaactgaaagctacccgtcaagtcctgtctgaatacggcaata tgagttccgcgtgtgtgctgttcattctggatgaaatgcgcaaaaaatctgccgaagaagctaaagcgaccacgggcgaaggtctggattgg ggcgtgctgtttggtttcggtccgggtctgaccgtcgaaacggtcgtgctgcacagtgtgccgatcaaagcgggcggtggcggttccggcg gtggtggtagtggtggtggtggctctccgccgccggccctgccgccgaaacgtcgtcgctaa CmCHS-Citrusmaxima Aminoacidsequence(SEQIDNO:39) MATVQEIRNAQRADGPATVLAIGTATPAHSVNQADYPDYYFRITKSEHMTELKEKFKR MCDKSMIKKRYMYLTEEILKENPNMCAYMAPSLDARQDIVVVEVPKLGKEAATKAIKE WGQPKSKITHLIFCTTSGVDMPGADYQLTKLLGLRPSVKRFMMYQQGCFAGGTVLRLA KDLAENNKGARVLVVCSEITAVTFRGPADTHLDSLVGQALFGDGAAAVIVGADPDTSV ERPLYQLVSTSQTILPDSDGAIDGHLREVGLTFHLLKDVPGLISKNIEKSLSEAFAPVGISD WSSIFWIAHPGGPAILDQVESKLGLKEEKLKATRQVLSEYGNMSSACVLFILDEMRKKS AEEAKATTGEGLDWGVLFGFGPGLTVETVVLHSVPIKAGGGGSGGGGSGGGGSPPPAL PPKRRR CmCHI-Citrusmaxima Nucleicacidsequence(SEQIDNO:40) atgaatccgtcgccgtctgttaccgaactgcaagtggaaaatgtcacctttacgccgagtctgcaaccgccgggctctaccaaatcgcatttt ctgggcggtgcaggtgaacgtggcctggaaatcgaaggcaaatttgttaaattcaccgctattggtgtctatctggaagaaaacgccgtgcc gctgctggcaggcaaatggaaaggcaaaaccgccggtgaactgacggaatctgtcgaatttttccgcgatgtggttaccggcccgtttgaa aaattcatgaaagtgaccatgatcctgccgctgacgggtgcgcagtattcagaaaaagttgctgaaaattgcatggcgatttggaaatttttcg gcatctacaccgatgcagaagctaaagcgattgaaaaatttacggaagtgttcaaagacgaaatttttccgccgggcagctctatcctgttca cccaaagttccggttcgctgacgatttcattttcgaaagatggcagcatcccgaaagacggtgtcgcggtgattgaaaacaatctgctgagc gaagccgttctggaatctatgatcggtaaaaacggcgtcagtccggcggccaaaaaatccctggccgaacgtctgtcagcactgctgaatg ttgatccgacaaaatgaaaggcggtggcggctcaggtggcggtggctctggtggcggtggttcaggcgtcaaagaaagtctggtgtga CmCHI-Citrusmaxima Aminoacidsequence(SEQIDNO:41) MNPSPSVTELQVENVTFTPSLQPPGSTKSHFLGGAGERGLEIEGKFVKFTAIGVYLEENA VPLLAGKWKGKTAGELTESVEFFRDVVTGPFEKFMKVTMILPLTGAQYSEKVAENCMA IWKFFGIYTDAEAKAIEKFTEVFKDEIFPPGSSILFTQSSGSLTISFSKDGSIPKDGVAVIE NNLLSEAVLESMIGKNGVSPAAKKSLAERLSALLNVASDKMKGGGGSGGGGSGGGGS GVKESLV MsCHI-Medicagosativa Nucleicacidsequence(SEQIDNO:42) atggctgcatcaatcaccgcaatcactgtggagaaccttgaatacccagcggtggttacctctccggtcaccggcaaatcatatttcctcggt ggcgctggggagagaggattgaccattgaaggaaacttcatcaagttcactgccataggtgtttatttggaagatatagcagtggcttcacta gctgccaaatggaagggtaaatcatctgaagagttacttgagacccttgacttttacagagacatcatctcaggtccctttgaaaagttaattag agggtcaaagattagggaattgagtggtcctgagtactcaaggaaggttatggagaactgtgtggcacacttgaaatcagttggaacttatgg agatgcagaagctgaagctatgcaaaaatttgctgaagctttcaagcctgttaattttccacctggtgcctctgttttctacaggcaatcacctaa tggaatattagggcttagtttctctccggatacaagtataccagaaaaggaggctgcactcatagagaacaaggcagtttcatcagcagtgtt ggagactatgatcggcgagcacgctgtttcccctgatcttaagcgctgtttagctgcaagattacctgcgttgttgaacgagggtgctttcaag attggaaactga MsCHI-Medicagosativa Aminoacidsequence(SEQIDNO:43) MAASITAITVENLEYPAVVTSPVTGKSYFLGGAGERGLTIEGNFIKFTAIGVYLEDIAVA SLAAKWKGKSSEELLETLDFYRDIISGPFEKLIRGSKIRELSGPEYSRKVMENCVAHLKS VGTYGDAEAEAMQKFAEAFKPVNFPPGASVFYRQSPNGILGLSFSPDTSIPEKEAALIEN KAVSSAVLETMIGEHAVSPDLKRCLAARLPALLNEGAFKIGN CsF3H-Camelliasinensis Nucleicacidsequence(SEQIDNO:44) atggcaccgaccaccaccctgaccgcactggcagaagaaaaaagcctgcagcagaaatttgttcgtgatgaagatgaacgtccgaaagtt gcctataatgtgtttagcaatgaaatcccggttattagcctggcaggtattgatgaaattgaaggtcgtcgtagcgaaatttgccgtaaaattgtt gaagcatgtgaaggttggggtgtttttcaggttgttgatcatggtgttgatgcaaatctgattgcagaaatgacccgtctggcacgtgaatttttt gcactgcctccggaagaaaaactgcgttttgatatgagcggtggtaaaaaaggtggttttattgttagcagccatctgcagggtgaagcagtt caggattggcgtgaaattgttacctatttcagctatccgattcgtgcacgtgattatagccgttggcctgataaaccggaaggttggcgtgcag ttaccgaaacctatagcgaaaaactgatggatctggcatgtaaactgctggaagttctgagcgaagcaatgggtctggaaaaagaggcact gaccaaagcatgtgttgatatggatcagaaagtggtgatcaacttctatccgaaatgtccgcagccggatctgaccctgggtctgaaacgtca taccgatccgggtacaattaccctgctgctgcaagatcaggtgggtggtctgcaggcaacccgtgatggtggcaaaacctggattaccgttc agccggttgaaggtgcatttgttgttaatctgggtgatcatggccattatctgagcaatggtcgctttaaaaacgcagatcatcaggcagttgtt aatagcaattgtagccgtctgagcattgcaacctttcagaatccggcaccggaagcaaccgtttatccgctgaaaattcgtgaaggtgaaaaa ccgattctggaagaaccgattacctttgccgatatgtataaacgcaaaatgagcaaagatatcgagctggccaaactgaaaaaactggcgaa agaaaaaaaactgctgcaagaccagcaggatatcgaaaaagcaaaactggaaatcaaaagcaccgatgaaatcttcgccctggttggtgc actgatgcatgttatgcagaaacgtagccgtgcaattcatagcagtgatgaaggtgaagatcaagccggtgatgaagatgaggat CsF3H-Camelliasinensis Aminoacidsequence(SEQIDNO:45) MAPTTTLTALAEEKSLQQKFVRDEDERPKVAYNVFSNEIPVISLAGIDEIEGRRSEICRKIV EACEGWGVFQVVDHGVDANLIAEMTRLAREFFALPPEEKLRFDMSGGKKGGFIVSSHL QGEAVQDWREIVTYFSYPIRARDYSRWPDKPEGWRAVTETYSEKLMDLACKLLEVLSE AMGLEKEALTKACVDMDQKVVINFYPKCPQPDLTLGLKRHTDPGTITLLLQDQVGGLQ ATRDGGKTWITVQPVEGAFVVNLGDHGHYLSNGRFKNADHQAVVNSNCSRLSIATFQN PAPEATVYPLKIREGEKPILEEPITFADMYKRKMSKDIELAKLKKLAKEKKLLQDQQDIE KAKLEIKSTDEIFALVGALMHVMQKRSRAIHSSDEGEDQAGDEDED MdF3H-Malusdomestica Nucleicacidsequence(SEQIDNO:46) atggcaccgcctgcaaccaccctgaccagcattgcacatgaaaaaaccctgcagcagaaatttgttcgtgatgaagatgaacgtccgaaag tggcctataatgaatttagcaacgaaatcccgattattagcctggcaggtattgatgaagttgaaggtcgtcgtgccgaaatctgcaaaaaaat cgttgaagcatgtgaagattggggcatttttcagattgttgatcatggtgttgatgccgaactgattagcgaaatgaccggtctggcaaaagaa ttttttgatctgccgagcgaagaaaaactgcgttttgatatgagcggtggtaaaaaaggtggttttattgttagcagccatctgcagggtgaagc agttcaggattggcgtgaaattgttacctattttctgtatccgattcgccaccgtgattatagccgttggcctgataaaccggaagcatggcgtg aagttaccaaaaaatacagtgatgaactgatgggtctggcatgtaaactgctgggtgttctgagcgaagcaatgggcctggataccgaagc actgaccaaagcatgtgttgatatggatcagaaagtggtggttaacttctatccgaaatgtccgcagccggatctgaccctgggtctgaaacg tcataccgatccgggtacaattaccctgctgctgcaagatcaggttggcggtctgcaggcaacccgtgatgatggtaaaacctggattaccg ttcagccggttgaaggtgcatttgttgttaatctgggtgatcatggccattttctgagcaatggtcgctttaaaaacgcagatcatcaggcagttg ttaatagcaatagcagccgtctgagcattgcaacctttcagaatccggcacaggatgcaattgtttatccgctgagcgttcgtgaaggtgaaa aaccgattctggaagcaccgattacctataccgagatgtataaaaaaaaaatgagcaaagatctggaactggcacgcctgaaaaaactggc caaagaacagcagctgcaggatctggaaaaagcaaaagttgaaaccaaaccggcagatgatatctttgccctggttggtgcactgatgcat gttatgcagaaacgtagccgtgcaattcatagcagtgatgaaggtgaagatcaagccggtgatgaagatgaggat MdF3H-Malusdomestica Aminoacidsequence(SEQIDNO:47) MAPPATTLTSIAHEKTLQQKFVRDEDERPKVAYNEFSNEIPIISLAGIDEVEGRRAEICKKI VEACEDWGIFQIVDHGVDAELISEMTGLAKEFFDLPSEEKLRFDMSGGKKGGFIVSSHLQ GEAVQDWREIVTYFLYPIRHRDYSRWPDKPEAWREVTKKYSDELMGLACKLLGVLSEA MGLDTEALTKACVDMDQKVVVNFYPKCPQPDLTLGLKRHTDPGTITLLLQDQVGGLQ ATRDDGKTWITVQPVEGAFVVNLGDHGHFLSNGRFKNADHQAVVNSNSSRLSIATFQN PAQDAIVYPLSVREGEKPILEAPITYTEMYKKKMSKDLELARLKKLAKEQQLQDLEKAK VETKPADDIFALVGALMHVMQKRSRAIHSSDEGEDQAGDEDED PcF3H-Petroselinumcrispum Nucleicacidsequence(SEQIDNO:48) atggcaccgagcaccctgaccgcactggcacaagaaaaaaccctgaatagcaaatttgtgcgcgacgaagatgaacgtccgaaaattgca tataacaaattcagcgacgaaatcccggttattagcctggcaggtattgatgatgatagcgttgataaacgtagccagatttgccgtaaaattgt tgaagcatgtgaagattggggcatttttcaggttgttgatcatggcattgatatcgatctgattagcgaaatgacccgtctggcacgtcagtttttt gcactgcctgcagaagaaaaactgcgttttgatatgaccggtggtaaaaaaggtggttttattgttagcagccatctgcagggtgaagcagtt caggattggcgtgaaattgttacctatttcagctatccgattcaggcacgtgattatagccgttggcctgataaaccggaaggttggcgtagca ttaccgaaatgtatagtgatgaactgatggcactggcatgtaaactgctggaagttctgagcgaagcaatgggtctggaaaaagagggtctg accaaagcatgtgttgatatggatcagaaagtgatcgtgaactactatccgaaatgtccgcagccgaatctgaccctgggtctgaaacgtcat accgatccgggtacaattaccctgctgctgcaggatcaggttggtggtctgcaggcgacccgtgatggtggcaaaacctggattaccgttca gccggttgaaggtgcatttgttgttaatctgggtgatcatggtcactatctgagcaatggtcgctttaaaaacgcagatcatcaggcagttgtta atagcaatagcagccgtatgagcattgcaacctttcagaatccggcaccgaatgcaaccgtttatccgctgaaaattcgtgaaggtgaaaaa gccgttatggaagaaccgattacctttgccgagatgtataaacgtaaaatgagccgtgatattgaaatggccaccctgaaaaaactggccaa agaaaaagttctgcaggaccaagaagtggaaaaagcaaaactgcagatgaccccgaaaagcgcagatgaaatttttgccctggttggtgc actgatgcatgttatgcagaaacgtagccgtgcaattcatagcagtgatgaaggtgaagatcaagccggtgatgaagatgaggat PcF3H-Petroselinumcrispum Aminoacidsequence(SEQIDNO:49) MAPSTLTALAQEKTLNSKFVRDEDERPKIAYNKFSDEIPVISLAGIDDDSVDKRSQICRK IVEACEDWGIFQVVDHGIDIDLISEMTRLARQFFALPAEEKLRFDMTGGKKGGFIVSSHL QGEAVQDWREIVTYFSYPIQARDYSRWPDKPEGWRSITEMYSDELMALACKLLEVLSE AMGLEKEGLTKACVDMDQKVIVNYYPKCPQPNLTLGLKRHTDPGTITLLLQDQVGGLQ ATRDGGKTWITVQPVEGAFVVNLGDHGHYLSNGRFKNADHQAVVNSNSSRMSIATFQ NPAPNATVYPLKIREGEKAVMEEPITFAEMYKRKMSRDIEMATLKKLAKEKVLQDQEV EKAKLQMTPKSADEIFALVGALMHVMQKRSRAIHSSDEGEDQAGDEDED AaDFR-Anthuriumandraeanum Nucleicacidsequence(SEQIDNO:50) atgatgcataaaggcaccgtttgtgttaccggtgcagcaggttttgttggtagctggctgattatgcgtctgctggaacagggttatagcgttaa agcaaccgttcgtgatccgagcaatatgaaaaaagttaaacatctgctggatctgcctggtgcagcaaatcgtctgaccctgtggaaagcag atctggttgatgaaggtagattgatgaaccgattcagggttgtaccggtgtttttcatgttgcaaccccgatggattttgaaagcaaagatccg gaaagcgaaatgattaaaccgaccattgaaggtatgctgaatgttctgcgtagctgtgcccgtgcaagcagcaccgttcgtcgtgttgttttta ccagcagcgcaggtacagttagcattcatgaaggtcgtcgtcatctgtatgatgaaaccagttggagtgatgttgatttttgccgtgccaaaaa aatgaccggctggatgtattttgttagcaaaaccctggcagaaaaagcagcatgggattttgcagagaaaaataacatcgacttcatcagcat tattccgaccctggttaatggtccgtttgttatgccgaccatgcctccgagcatgctgagcgcactggcactgattacccgtaatgaaccgcat tatagcattctgaatccggtgcagtttgttcatctggatgatctgtgtaacgcccacatttttctgtttgaatgtccggatgcaaaaggtcgttatat ttgtagcagccatgatgttaccattgcaggtctggcacagattctgcgtcagcgttatccggaatttgatgttccgaccgaatttggtgaaatgg aagtgtttgatatcatcagctatagcagcaaaaaactgacggatctgggtttcgaattcaaatatagcctggaagatatgttcgatggtgcaatt cagagctgtcgtgaaaaaggtctgctgcctccggcaaccaaagaaccgagctatgcaaccgaacagctgattgcaaccggtcaggataat ggtcatcctcctcctgcactgcctccgaaacgtcgtcgt AaDFR-Anthuriumandraeanum Aminoacidsequence(SEQIDNO:51) MMHKGTVCVTGAAGFVGSWLIMIRLLEQGYSVKATVRDPSNMKKVKHLLDLPGAANR LTLWKADLVDEGSFDEPIQGCTGVEHVATPMDFESKDPESEMIKPTIEGMLNVLRSCAR ASSTVRRVVFTSSAGTVSIHEGRRHLYDETSWSDVDFCRAKKMTGWMYFVSKTLAEKA AWDFAEKNNIDFISIIPTLVNGPFVMPTMPPSMLSALALITRNEPHYSILNPVQFVHLDDL CNAHIFLFECPDAKGRYICSSHDVTIAGLAQILRQRYPEEDVPTEFGEMEVEDIISYSSKKL TDLGFEFKYSLEDMFDGAIQSCREKGLLPPATKEPSYATEQLIATGQDNGHPPPALPPKR RR CsDFR-Camelliasinensis Nucleicacidsequence(SEQIDNO:52) atgaaagatagcgttgcaagcgcaaccgcaagcgcaccgggtacagtttgtgttaccggtgcagcaggttttattggtagctggctggttatg cgtctgctggaacgtggttatattgttcgtgcaaccgttcgtgatccggcaaatctgaaaaaagttaaacatctgctggatctgccgaaagcag ataccaatctgaccctgtggaaagccgatctgaatgaagagggtagctttgatgaagcaattgaaggttgtagcggtgtttttcatgttgcaac cccgatggattttgaaagcaaagatccggaaaacgaagtgattaaaccgaccattaacggtgtgctgagcattattcgtagctgtaccaaag caaaaaccgttaaacgtctggtttttaccagcagcgcaggtacagttaatgttcaagaacatcagcagccggtgtttgatgaaaacaattgga gcgatctgcacttcatcaacaaaaaaaaaatgaccggctggatgtattttgtgagcaaaaccctggcagaaaaagcagcatgggaagcagc aaaagaaaacaacattgatttcatcagcattatcccgaccctggttggtggtccgtttattatgccgacctttccgcctagcctgattaccgcact gagcccgattacccgtaatgaaggtcattattccattatcaaacagggccagtttgtgcatctggatgatctgtgtgaaagccacatttttctgta tgaacgtccgcaggcagaaggtcgttatatttgtagcagccatgatgcaaccattcatgatctggccaaactgatgcgtgaaaaatggcctga atataatgttccgaccgaattcaaaggcatcgataaagatctgccggttgttagcttttccagcaaaaaactgattggcatgggcttcgagttca aatatagcctggaagatatgtttcgtggtgccattgatacctgtcgtgaaaaaggtctgctgccgcatagctttgcagaaaatccggttaatgg caacaaagtgcctcctcctgcactgcctccgaaacgtcgtcgt CsDFR-Camelliasinensis Aminoacidsequence(SEQIDNO:53) MKDSVASATASAPGTVCVTGAAGFIGSWLVMRLLERGYIVRATVRDPANLKKVKHLL DLPKADTNLTLWKADLNEEGSFDEAIEGCSGVFHVATPMDFESKDPENEVIKPTINGVLS IIRSCTKAKTVKRLVFTSSAGTVNVQEHQQPVFDENNWSDLHFINKKKMTGWMYFVSK TLAEKAAWEAAKENNIDFISIIPTLVGGPFIMPTFPPSLITALSPITRNEGHYSIIKQGQFVH LDDLCESHIFLYERPQAEGRYICSSHDATIHDLAKLMREKWPEYNVPTEFKGIDKDLPVV SFSSKKLIGMGFEEKYSLEDMERGAIDTCREKGLLPHSFAENPVNGNKVPPPALPPKRRR FaDFR-Fragariaxananassa Nucleicacidsequence(SEQIDNO:54) atgggtctgggtgcagaaagcggtagcgtttgtgttaccggtgcaagcggttttgttggtagctggctggttatgcgtctgctggaacatggtt ataccgttcgtgcaaccgtgcgtgatccggcaaatctgaaaaaagttcgtcatctgctggaactgccgcaggcagcaacccgtctgaccctg tggaaagcagatctggatgttgaaggtagattgatgaagccattaaaggttgtaccggtgtttttcatgttgcaaccccgatggattttgaaag cgaagatccggaaaacgaagttattaaaccgaccattaacggcatgctggatattatgaaagcatgcctgaaagcaaaaaccgttcgtcgtc tggtttttaccagcagtgccggtgcagttgcaattgaagaacatccgaaagaagtgtacagcgaaaataactggtcagatgttgtgttttgccg caaagttaaaatgaccggctggatgtattttgtgagcaaaaccctggcagaacaggcagcatggaaatttgcaaaagaaaacaacatcgac ttcatcaccattattccgaccctggttattggtccgtttctggcaccgagcatgcctccgagcctgattagcggtctgagtccgctgaccggtaa tgaagcacattatggtattatcaaacagtgccagtatgtgcatctggatgatctgtgtcagagccatatttttctgtatgaacatgcaaaagccga gggtcgttatatttgtagcagccatgatgcaaccattcacgatattgcaaaactgctgaacgagaaatacccgaaatacaacgttccgaaaaa attcaaaggcatcgaagaaaacctgaccaacattcactttagcagcaaaaaactgaaagagatgggcttcgaatttaaacacagcctggaa gatatgtttacaggtgccgttgatgcatgtcgtgaaaaaggtctgctgccgctgccgcaagaagaagaaaccgaaaaacgtcgtgcaggtc ctcctcctgcactgcctccgaaacgtcgtcgt FaDFR-Fragariaxananassa Aminoacidsequence(SEQIDNO:55) MGLGAESGSVCVTGASGFVGSWLVMRLLEHGYTVRATVRDPANLKKVRHLLELPQAA TRLTLWKADLDVEGSFDEAIKGCTGVFHVATPMDFESEDPENEVIKPTINGMLDIMKAC LKAKTVRRLVFTSSAGAVAIEEHPKEVYSENNWSDVVFCRKVKMTGWMYFVSKTLAE QAAWKFAKENNIDFITIIPTLVIGPFLAPSMPPSLISGLSPLTGNEAHYGIIKQCQYVHLDD LCQSHIFLYEHAKAEGRYICSSHDATIHDIAKLLNEKYPKYNVPKKFKGIEENLTNIHFSS KKLKEMGFEEKHSLEDMFTGAVDACREKGLLPLPQEEETEKRRAGPPPALPPKRRR CsLAR-Camelliasinensis Nucleicacidsequence(SEQIDNO:56) atggcaatggccatggcaaccaccaccacaaccaccaaaccgatgattggtgcaaaagcagcatgtgttgttggtggcaccggttttgttgc agcaaccctggttaaaatgctgctggaacgtggttatagcgttaataccaccgttcgtgatccggacaacaaaaaaaacattagccatctggt tgcactggaaggtatgggtaatctgaaaatctttcgtgcagatctgaccgatgaacagagctttgatgcaccgattgcaggttgtgatctggttt ttgatgttgccacaccggttaattttgcaagcgaagatccggaaaacgacatgattaaactggcaattcagggtgttctgaatgtgctgaaagc atgtgccaaagcaggcaccgttaaacgtgttattctgaccagcagcgcagcaagcgttaccattaatcagctggatggtacaggtctggttat ggatgaaagccattggagtgatgttgaatttctgacctcagttaaaccgcctacctggggtcatccggttagcaaaaccctggcagaaaaag cagcctggaaatttgcagaagaaaataacctgaatctgattaccgttgttccgaccctgaccgcaggtccgagcctgaccagcgaagttccg aatagcattgaactggccatgagcctgattacgggtaatgaattcctgattgatggtctgaaaggtatgcgtattctgtcaggtagcattagcat tacccatgttgaagatgtttgtggtgcccatatttttgtggccgaaaaagaaagcgcaagcggtcgttatatttgttgtggtgttaatagcagcgt gccggaactggcacgttttctgaataaacgttatccgcagtataatgtgccgaccgattttggtgatctgccgagcaaagcaaaactgattatt agcagcgagaaactgatcaaagaaggatcagatcaaatatggcatcgaagaaatttttgcacacagcgttgcatatctgaaaaccaaagg tctgctgcagaacggtgttaaagaaagcctggtt CsLAR-Camelliasinensis Aminoacidsequence(SEQIDNO:57) MAMAMATTTTTTKPMIGAKAACVVGGTGEVAATLVKMLLERGYSVNTTVRDPDNKK NISHLVALEGMGNLKIFRADLTDEQSFDAPIAGCDLVEDVATPVNEASEDPENDMIKLAI QGVLNVLKACAKAGTVKRVILTSSAASVTINQLDGTGLVMDESHWSDVEFLTSVKPPT WGHPVSKTLAEKAAWKFAEENNLNLITVVPTLTAGPSLTSEVPNSIELAMSLITGNEFLI DGLKGMRILSGSISITHVEDVCGAHIEVAEKESASGRYICCGVNSSVPELARFLNKRYPQ YNVPTDFGDLPSKAKLIISSEKLIKEGFSFKYGIEEIFAHSVAYLKTKGLLQNGVKESLV DuLAR-Desmodiumuncinatum Nucleicacidsequence(SEQIDNO:58) atgaccgttagcggtgcaattccgagcatgaccaaaaatcgtaccctggttgttggtggcaccggttttattggtcagtttattaccaaagcaa gcctgggttttggttatccgacctttctgctggttcgtccgggtccggttagcccgagcaaagcagttattatcaaaacctttcaggataaaggt gccaaagtgatttatggcgtgatcaacgataaagaatgcatggaaaaaattctgaaagagtacgagatcgacgttgttattagcctggtgggt ggtgcacgtctgctggatcagctgaccctgctggaagcaattaaaagcgttaaaaccatcaaacgttttctgccgagcgaatttggccatgat gttgatcgtaccgatccggttgaaccgggtctgaccatgtataaagaaaaacgtctggtgcgtcgtgccgttgaagaatatggtattccgttta ccaatatctgctgcaatagcattgcaagctggccgtattatgataattgtcatccgagccaggttccgcctccgatggatcagtttcagatttatg gtgatggtaacaccaaagcctatttcattgatggcaacgatatcggcaaatttaccatgaaaaccatcgatgatattcgcaccctgaacaaaaa tgttcattttcgtccgagcagcaactgctacagcattaatgaactggcaagcctgtgggagaaaaaaatcggtcgtacactgcctcgttttacc gttaccgcagataaactgctggcacatgcagcagaaaacattattccggaaagcattgttagcagctttacccacgatatctttattaacggttg ccaggtgaactttagcatcgatgaacatagtgatgtggaaatcgatacactgtatccggatgaaaaatttcgtagcctggatgattgctatgaa gattttgttccgatggtgcacgataaaattcatgcaggtaaaagcggtgaaatcaaaatcaaagatggtaaaccgctggttcagaccggcac cattgaagaaattaacaaagacattaaaaccctggtggaaacccagccgaatgaagagatcaaaaaagatatgaaagcactggttgaagcc gttccgattagcgcaatgggtggtgttaaagaaagcctggtt DuLAR-Desmodiumuncinatum Aminoacidsequence(SEQIDNO:59) MTVSGAIPSMTKNRTLVVGGTGFIGQFITKASLGFGYPTFLLVRPGPVSPSKAVIIKTFQ DKGAKVIYGVINDKECMEKILKEYEIDVVISLVGGARLLDQLTLLEAIKSVKTIKRFLPS EFGHDVDRTDPVEPGLTMYKEKRLVRRAVEEYGIPFTNICCNSIASWPYYDNCHPSQVP PPMDQFQIYGDGNTKAYFIDGNDIGKFTMKTIDDIRTLNKNVHFRPSSNCYSINELASLW EKKIGRTLPRFTVTADKLLAHAAENIIPESIVSSFTHDIFINGCQVNFSIDEHSDVEIDTL YPDEKFRSLDDCYEDFVPMVHDKIHAGKSGEIKIKDGKPLVQTGTIEEINKDIKTLVETQ PNEEIKKDMKALVEAVPISAMGGVKESLV PhANS-PetuniaXhybrida Nucleicacidsequence(SEQIDNO:60) atggtgaatgcagtagttacaactccttcaagagttgaaagcttggctaaaagtggaatccaggccatccctaaggagtatgtgaggccaca agaagagttgaatggaatcggaaacatcttcgaggaagagaagaaagatgaagggcctcaagtaccaacaattgatttgaaagaaattgac tccgaggacaaggagattcgcgagaaatgccaccaggagttgaagaaagcagccatggaatggggtgtcatgcaccttgtgaatcatggc atatccgatgagctaatcaatcgtgtcaaggttgctggagagaccttctttgatcaacctgttgaagaaaaggagaagtatgctaatgaccaa gccaatggcaatgtccaaggctacggcagcaagctagcaaatagtgcttgtggtcagcttgagtgggaggattatttcttccattgtgctttcc ctgaagacaagcgcgacttgtccatctggcctaaaaatcctactgactacactccagcaacaagtgaatatgccaagcagatcagggccct agcaacaaagattttgacagtgctttctattgggctggggctggaagaaggaagactagagaaggaagttggaggcatggaggatctgctg cttcaaatgaagattaactactatcccaagtgcccccaaccagaactagcacttggcgtcgaagctcatacagatgtcagcgcactgactttc atcctccacaatatggtgcccggcttgcaactcttctatgaaggccagtgggtaactgctaagtgtgtgcctaattctatcatcatgcacatagg ggacaccattgaaatcctaagcaatggaaagtacaagagcatccttcatagaggggttgtgaataaagagaaagtaaggatctcatgggcc attttctgcgagccacctaaggagaagatcatccttaagcccctacctgagactgtcactgaggctgagccacctcgattcccacctcgcacc tttgcacagcatatggcacacaagctcttcaggaaggatgacaaggatgccgctgttgaacacaaagtcttcaaagaggatgaactggatac tgctgctgaacataaggtcctcaagaaggataatcaggatgctgttgctgagaataaagacatcaaggaggatgaacagtgtggccctgct gagcacaaagatatcaaggaggatggacagggtgccgctgctgagaacaaagtcttcaaggagaataatcaggatgttgctgctgaagaa tctaaatag PhANS-PetuniaXhybrida Aminoacidsequence(SEQIDNO:61) MVNAVVTTPSRVESLAKSGIQAIPKEYVRPQEELNGIGNIFEEEKKDEGPQVPTIDLKEI DSEDKEIREKCHQELKKAAMEWGVMHLVNHGISDELINRVKVAGETFFDQPVEEKEKY ANDQANGNVQGYGSKLANSACGQLEWEDYFFHCAFPEDKRDLSIWPKNPTDYTPATSE YAKQIRALATKILTVLSIGLGLEEGRLEKEVGGMEDLLLQMKINYYPKCPQPELALGVE AHTDVSALTFILHNMVPGLQLFYEGQWVTAKCVPNSIIMHIGDTIEILSNGKYKSILHRG VVNKEKVRISWAIFCEPPKEKIILKPLPETVTEAEPPRFPPRTFAQHMAHKLFRKDDKDA AVEHKVFKEDELDTAAEHKVLKKDNQDAVAENKDIKEDEQCGPAEHKDIKEDGQGAA AENKVFKENNQDVAAEESK At3GT-Arabidopsisthaliana Nucleicacidsequence(SEQIDNO:62) atgaccaaaccctccgacccaaccagagactcccacgtggcagttctcgcttttcctttcggcactcatgcagctcctctcctcaccgtcacg cgccgcctcgcctccgcctctccttccaccgtcttctctttcttcaacaccgcacaatccaactcttcgttattttcctccggtgacgaagcagat cgtccggcgaacatcagagtatacgatattgccgacggtgttccggagggatacgtgtttagcgggagaccacaggaggcgatcgagctg tttcttcaagctgcgccggagaatttccggagagaaatcgcgaaggcggagacggaggttggtacggaagtgaaatgtttgatgactgatg cgttatctggttcgcggctgatatggcgacggagataaatgcgtcgtggattgcgttttggaccgccggagcaaactcactctctgctcatct ctacacagatctcatcagagaaaccatcggtgtcaaagaagtaggtgagcgtatggaggagacaataggggttatctcaggaatggagaa gatcagagtcaaagatacaccagaaggagttgtgtttgggaatttagactctgttttctcaaagatgcttcatcaaatgggtatgctttgcctcg tgccactgctgttttcatcaattcttttgaagatttggatcctacattgacgaataacctcagatcgagatttaaacgatatctgaacatcggtcctc tcgggttattatcttctacattgcaacaactagtgcaagatcctcacggttgtttggcttggatggagaagagatcttctggttctgtggcgtacat tagattggtacggtcatgacaccgcctcctggagagatgcggcgatagcagaagggttggaatcgagtaaagtgccgtttgtttggtcgct taaggagaagagcttggttcagttaccaaaagggtttttggataggacaagagagcaagggatagtggttccatgggcaccgcaagtggaa ctgctgaaacacgaagcaacgggtgtgtttgtgacgcattgtggatggaactcggtgttggagagtgtatcgggtggtgtaccgatgatttgc aggccattttttggggatcagagattgaacggaagagcggtggaggttgtgtgggagattggaatgacgattatcaatggagtatcacgaa agatgggtttgagaagtgtttggataaagttttagttcaagatgatggtaagaagatgaaatgtaatgctaagaaacttaaagaactagcttacg aagctgtctcttctaaaggaaggtcctctgagaatttcagaggattgttggatgcagttgtaaacattatttga At3GT-Arabidopsisthaliana Aminoacidsequence(SEQIDNO:63) MTKPSDPTRDSHVAVLAFPFGTHAAPLLTVTRRLASASPSTVFSFFNTAQSNSSLFSSGD EADRPANIRVYDIADGVPEGYVFSGRPQEAIELFLQAAPENFRREIAKAETEVGTEVKCL MTDAFFWFAADMATEINASWIAFWTAGANSLSAHLYTDLIRETIGVKEVGERMEETIGV ISGMEKIRVKDTPEGVVFGNLDSVFSKMLHQMGLALPRATAVFINSFEDLDPTLTNNLRS RFKRYLNIGPLGLLSSTLQQLVQDPHGCLAWMEKRSSGSVAYISFGTVMTPPPGELAAIA EGLESSKVPFVWSLKEKSLVQLPKGFLDRTREQGIVVPWAPQVELLKHEATGVFVTHCG WNSVLESVSGGVPMICRPFFGDQRLNGRAVEVVWEIGMTIINGVFTKDGFEKCLDKVLV QDDGKKMKCNAKKLKELAYEAVSSKGRSSENFRGLLDAVVNII Fragariaxananassa3GT Aminoacidsequence(SEQIDNO:64) MGSAVAVELVFIPAPGVGHIMSTMEMAKLLINRHQSIATTVLLIHPPYSSSVLTNYIQSLL TNPIQRIRFIQLPQDQETASKLDLKAPFTSFYEFINSHRNYVRNVVSDMLSRPGSVRITGL VVDILCTGMIDVANEFSIPSYAFFTSNAAFLGFKLYMDTLCRNQKQEGIIALSKSDGELRI PSFVKPVPMTVYPAVYQTRDGLDFLTVSIQKFREAKAIMVNTFLELETHAIESFSSYTNFP SVYAVGPVLNLNGVAGKDEDKDVIRWLDGQPPSSVVFLCFGSMGSFEEVQLKEIAYAL ERSGHRFVWSVRRPPSPEQSFKVLPDDYDDPRSILPDGFLERTNGFGKVIGWAPQVSILA HEAVGGFVSHCGWNSVLESICCKVPILAWPMMAEQHLNARMVVEEIKIGLRVETCDGS VRGFVQADGLKKMVKELMEGENGEIVRKRVEGIGEGAKKAMAEGGSSWRTLNELIDE LQCVRNSNGGRFPSSEGDSDKSKGESYVPMDNLSLVSI Vitisvinifera3GT Aminoacidsequence(SEQIDNO:65) MSQTTTNPHVAVLAFPFSTHAAPLLAVVRRLAAAAPHAVFSFFSTSQSNASVFHDSMHT MQCNIKSYDVSDGVPEGYVFAGRPQEDIELFMRAAPEGFRQGMVMAVAETGRPVSCLV ADAFIWFAADMAAEMGVAWLPFWTAGPNSLSTHVYTDEIREKIGVSGIQGREDELLNFI PGMYEVRFRDLQEGIVFGNLNSLFSRMLHRMGQVLPKATAVFINSFEELDDSLTNDLKS KLKTYLNIGPFNLITPPPVVPNTTGCLQWLKERKPTSVVYISFGTVTTPPPAELVALAEAL EASRVPFIWSLRDKARVHLPEGFLEKTRGYGMVVPWAPQAEVLAHEAVGAFVTHCGW NSLWESVAGGVPLICRPFFGDQRLNGRMVEDVLEIGVRIEGGVFTKSGLMSCFDQILSQE KGKKLRENLRALRETADRAVGPKGSSTENFKTLVDLVSKPKDV Forsynthia3GT Aminoacidsequence(SEQIDNO:66) MAIHSHIGVLAFPFGTHAAPLLTLVRRLVLDSSSQGITFSFFNTAKSNCAIFSGQEFDNIKA YDVWDGTHEGEAFTGSNILEAMQLFLAATPGNFEKVMKEAEVKNGMKISCLLSDAFLW FTCDLAEERGIPWVSFWTAASCSLSAHMYTDQIWSLMRSTGTAKTEEKTLSFVPGMTSV RFSDLPEEILSDNLESPLTLMIYKMVQKLSKSTAIVVNSFEEIDPVITNDLKSKFQNFLNIG PSILSSPTLSNGDSGQECLLWLEKQRHASVIYISFGTVITPQPREMAGLAEALETGEFPFL WSLRDNAMKLLPDGFLDRTSKFGMIVSWAPQLKVLENPSVGAFITHCGWNSILESISFG VPMICRPFFGDQNLNSKMVEDVWKIGVRLEGGVFTKNGTIEALHSVMLNETGKAIRENI NKLKRKAQNAVKFDGTSTKNFRALLELIKSPRGI Eggplant3GT Aminoacidsequence(SEQIDNO:67) MTTSQLHIAFLAFPFGTHATPLLTLVQKISPFLPSSTIFSFFNTSSSNSSIFSKVPNQENIKIY NVWDGVKEGNDTPFGLEAIKLFIQSTLLISKITEEAEEETGVKFSCIFSDAFLWCFLVKLP KKMNAPGVAYWTGGSCSLAVHLYTDLIRSNKETSLKIPGFSSTLSINDIPPEVTAEDLEGP MSSMLYNMALNLHKADAVVLNSFQELDRDPLINKDLQKNLQKVFNIGPLVLQSSRKLD ESGCIQWLDKQKEKSVVYLSFGTVTTLPPNEIGSIAEALETKKTPFIWSLRNNGVKNLPK GFLERTKEFGKIVSWAPQLEILAHKSVGVFVTHCGWNSILEGISFGVPMICRPFFGDQKL NSRMVESVWEIGLQIEGGIFTKSGIISALDTFFNEEKGKILRENVEGLKEKALEAVNQMM EVQQKISRF Gentian3GT Aminoacidsequence(SEQIDNO:68) MDQLHVFFFPFLANGHILPTIDMAKLFSSRGVKATLITTHNNSAIFLKAINRSKILGFDISV LTIKFPSAEFGLPEGYETADQARSIDMMDEFFRACILLQEPLEELLKEHRPQALVADLFFY WANDAAAKFGIPRLLFHGSSSFAMIAAESVRRNKPYKNLSSDSDPFVVPDIPDKIILTKSQ VPTPDETEENNTHITEMWKNISESENDCYGVIVNSFYELEPDYVDYCKNVLGRRAWHIG PLSLCNNEGEDVAERGKKSDIDAHECLNWLDSKNPDSVVYVCFGSMANFNAAQLHELA MGLEESGQEFIWVVRTCVDEEDESKWFPDGFEKRVQENNKGLIIKGWAPQVLILEHEAV GAFVSHCGWNSTLEGICGGVAMVTWPLFAEQFYNEKLMTDILRTGVSVGSLQWSRVTT SAVVVKRESISKAVRRLMAEEEGVDIRNRAKALKEKAKKAVEGGGSSYSDLSALLVELS SYPHN Petuniaxhybrida3GT Aminoacidsequence(SEQIDNO:69) MTTSQLHIALLAFPFGSHAAPLLTLVQKLSPFLPSDTIFSFFNTSQSNTSIFSEGSKPDNIKV YNVWDGVTETNGNKPVGLEAIKLFIQATPTNFEKVMKEAEEETGVKFSCIFSDAFLWFS YKLAEKINVPWIAFWTAASGSLSVHLYTDFIRSNDETSLNIPGFSSTLKISDMPPEVMAEN LDLPMPSMLYNMALNLHKAAAVVLNSFEELDPTINKDLKVKLQKVLNIGPLVLQPTSPK KVLDACDERGCIIWLEKQKEESVVYLSFGTVTTLPPNEIVAVAEALEAKKFPFIWSLKDN GIKNLPTGFLERTGQFGKIVSWAPQLEILNHSAVGVFVTHCGWNSILEGISCGVPMICRPF FGDQKLNSRMVESVWQIGLQIEGGSFTKIGTISALDTFFSEEKGKVLRENVKGLKERALE AVKPDGSSSKNFKDLVELVKCHKLT Aminoacidsequence(SEQIDNO:70) MVSSDSVNSRVETLAGSGISTIPKEYIRPKDELVNIGDIFEQEKNNEGPQVPTIDLKEIESD NEKVRAKCREKLKKATVDWGVMHLVNHGISDELMDKVRKAGKAFFDLPIEQKEKYAN DQASGKIQGYGSKLANNASGQLEWEDYFFHCVYPEDKRDLSIWPQTPADYIEATAEYA KQLRELATKVLKVLSLGLGLDEGRLEKEVGGLEELLLQMKINYYPKCPQPELALGVEAH TDVSALTFILHNMVPGLQLFYEGKWVTAKCVPNSIVMHIGDTLEILSNGKYKSILHRGM VNKEKVRISWAVFCEPPKEKIILKPLPETVSEDEPAMFPPRTFAEHIQHKLFRKSQEALLP K PyruscommunisANS Aminoacidsequence(SEQIDNO:71) MVSSDSVNSRVETLAGSGISTIPKEYIRPKDELVNIGDIFEQEKNNEGPQVPTIDLKEIESD NEKVRAKCREELKKAAVDWGVMHLVNHGISDELMDKVRKAGKAFFDLPIEQKEKYAN DQASGKIQGYGSKLANNASGQLEWEDYFFHCVYPEDKRDLSIWPQTPADYIEATAEYA KQLRELATKVLKVLSLGLGLDEGRLEKEVGGLEELLLQMKINYYPKCPQPELALGVEAH TDVSALTFILHNMVPGLQLFYEGKWVTAKCVPNSIVMHIGDTLEILSNGKYKSILHRGM VNKEKVRISWAVFCEPPKEKIILKPLPETVSEDEPAMFPPRTFAEHIQHKLFRKSQEALLP K PrunusaviumANS Aminoacidsequence(SEQIDNO:72) MVSSDSVNSRVETLASSGIATIPKEYIRPKEELINIGDIFEQEKSTDGPQVPTIDLKEIDSEN EKVRERCREELNKAAVDWGVMHLVNHGISDELMDRVRKAGKAFFDLPIEQKEKYAND QASGKIQGYGSKLANNASGQLEWEDYFFHLIFPEDKRDLSIWPQTPADYIEATAEYAKE LRALATKVLRVLSLGLGLEEGRLEKEVGGLEELLLQMKINYYPVCPQPELALGVEAHTD VSALTFILHNMVPGLQLFYEGKWVTAKCVPNSIVMHIGDTIEILSNGKYKSILHRGMVN KEKVRISWAVFCEPPKEKIILKPLPETVSETEPPIFPPRTFAEHIQHKLFRKSQEALLNK FragariaxananassaANS Aminoacidsequence(SEQIDNO:73) MVTAASIGSRVESLASSGISTIPKEYVRPEEELVNIGDIFEDEKSTEGPQVPTIDLKEIDSED IKVREKCREELKKAAIDWGVMHLVNHGISDELMERVKKAGKAFFDLPVEQKEKYAND QASGKIQGYGSKLANNASGQLEWEDYFFHCVYPEDKRDLSIWPQTPSDYIVATSEYAKE LRGLTTKILSILSLGLGLEEGRLEKEVGGLEELLLQMKINYYPKCPQPELALGVEAHTDIS ALTFILHNMVPGLQLFYGGKWVTAKCVPNSVVMHIGDTLEILSNGKYKSILHRGLVNKE KVRISWAVFCEPPKEKIILKPLPETVSEEEPAIFPPRTFFEHIQHKLFRQSQEALVSTKESAA LKSTKESALKSTKEAALISTN VitisviniferaANS Aminoacidsequence(SEQIDNO:74) MVTSVAPRVESLSSSGIQSIPKEYIRPQEELTSIGNVFEEEKKDEGPQVPTIDLKDIESEDE VVRERCREELKKAAMEWGVMHLVNHGISDDLINRVKVAGETFFNLPMEEKEKYANDQ ASGKIAGYGSKLANNASGQLEWEDYFFHLIFPEDKRDMTIWPKTPSDYVPATCEYSVKL RSLATKILSVLSLGLGLEEGRLEKEVGGMEELLLQKKINYYPKCPQPELALGVEAHTDVS ALTFILHNMVPGLQLFYEGKWVTAKCVPNSIIMHIGDTIEILSNGKYKSILHRGLVNKEK VRISWAVFCEPPKEKIILKPLPETVSETEPPLFPPRTFSQHIQHKLFRKTQEALLSK Ipomoeapurpureaanthocyanidinsynthase(ANS) Aminoacidsequence(SEQIDNO:75) MLSTITATVPSRVERLAGSGIERIPKEYIRPEEERRSIGDIFEEEKIAGGPQVPTVDLKGINS EDLEVREKCREELRKAAVDWGVMHLVNHGIPEELTGRVKAAGEGFFGQPIEEKEKYAN DQAAGNVQGYGSKLANNASGQLEWEDYFFHCIFPEDKTDLSIWPKTPSDYIDATREYAK QLRALATKVLAVLSLGLGLEEGRLEKEVGGMEELLLQMKINYYPKCPQPELALGVEAH TDVSALTFILHNMVPGLQLFYGGKWVTAKCVPNSIIMHVGDTVEILSNGKYKSILHRGV VNREKVRVSWAVFCEPPKDKILLQPLPETVSEAEPPRFPPRTFAQHIKHKLFRQSDQEAA HTPKPDNDDDHQSN CamelliasinensisANS Aminoacidsequence(SEQIDNO:76) MTTVAAPRVQSLATSGIESIPKEYVRPKEELTGIGNIFEEEKNEEGPQVPTIDLKDIDSEVE EVRERCREALKKAAVDWGVMHLVNHGIADDVRERVKVAGEGFFEQPVEEKEKYANDP DNGNLQGYGSKLANNACGQFEWEDYFFHLAYPEDKCDMSIWPKTPTDYIPATVEYAKQ LRALATKTLSILSLGLGLEENKLEKEVGGKEELLLQMKINYYPKCPQPELALGVEAHTDL SAVSFILPSMVPGLQLFYEGKWITAKCVPNSIIMLIGDTVEILSNGKYKSILHRGLVNKEK VRISWAVFCEPPKEKIILKPLPETVSEAEPPLEPPRTFAQHIQHKLFRKSQELGSK Citrussinensisanthocyanidinsynthase(ANS) Aminoacidsequence(SEQIDNO:77) MVTPTARRVESLARSGIQAIPKEYVRPKEELMGIGNIFEEEEKDEGPQVPTIDLKEIDSED RVEREKCREELKKAAMDWGVMHLVNHGISDDLTERVKRAGQAFFDQPVEEKEKYANE QASGKIQGYGSKLANNASGQLEWEDYFFHLIYPEDKRDMSIWPKTPSDYTEATSEYARQ LRSLATKILAVLSLGLGLEEGRLEKEVGGLEELLLQMKINYYPKCPQPELALGVEAHTD VSALTFILHNMVPGLQLFYKDKWVTAKCVPNSIILHIGDTIEILSNGEYKSILHRGLVNKE KVRISWAVFCEPPKDKIILKPLPETVSEQKPAMFPPRTFQQHIEHKLFRRTQDALLSDEE VacciniumasheiANS Aminoacidsequence(SEQIDNO:78) MVSTMVAAPSRVESLASSGIQSIPKEYVRPKEELTSIGNIFEEEKKHEGPQVPTIDLEDLVS EDKEARERCHEALKKAATEWGVMHLVNHGVPEELMDRVRVAGEGFENQPVEEKEKY ANDHDTGNSGKIQGYGSKLANNASGQLEWEDYFFHTVYPEDKRDMKIWPKNPSDYIPA TSEYANHLRALTTKVLSALSVCLGLEEDRLEKEVGGKDELVIQMKINYYPKCPQPELAL GVEAHTDVSALTFILHNMVPGLQLFYEGKWITAKCVPNSIIMHIGDTVEILSNGKYKSIL HRGLVNKEKVRISWAAFCEPPKEKIILKPLPETVSETEPARYPPRTFSQHIEHKLFRKTQA LNGA PopulustrichocarpaANS Aminoacidsequence(SEQIDNO:79) MMVTSSFVVPRVESLASSGIQSIPKEYIRPQEELSSIRDVFEEEKKVEGPQVPTIDLKEMES EDKVVREKCREELVKAATEWGVMHLVNHGIPDDLIDRVKKAGQAFFDLPIEEKEKHAN DQASGNVQGYGSKLANNASGQLEWEDYFFHLIFPEDKRDFSIWPKTPSDYTEVTSEYAR QLRSLATKILSVLSLGLGLEEGRLEKEVGGLEELLLQMKINYYPKCPQPDLALGVEAHSD VSALTFILHNMVPGLQLLYEGKWITAKCVPNSIIMHIGDTVEILSNGKYKSIIHRGLVNKE KVRISWAVFCEPPKAKIILKPLAEIVTEAEPPLFPPRTFSQHIEHKLFRKTQDSLLPRKAN RhodobactercapsulatusTAL Aminoacidsequence(SEQIDNO:80) MLDATIGRKRMTLQSQTAKDCLALDGALTLVQCEAIATHRSRISVTPALRERCARAHAR LEHAIAEQRHIYGITTGEGPLANRLIGADQGAELQQNLIYHLATGVGPKLSWAEARALM LARLNSILQGASGASPETIDRIVAVLNAGFAPEVPAQGTVGASGDLTPLAHMVLALQGR GRMIDPSGRVQEAGAVMDRLCGGPLTLAARDGLALVNGTSAMTAIAALTGVEAARAID AALRHSAVLMEVLSGHAEAWHPAFAELRPHPGQLRATERLAQALDGAGRVCRTLTAA RRLTAADLRPEDHPAQDAYSLRVVPQLVGAVWDTLDWHDRVVTCELNSVTDNPIFPEG CAVPALHGGNFMGVHVALASDALNAALVTLAGLVERQIARLTDEKLNKGLPAFLHGG QAGLQSGFMGAQVTATALLAEMRANATPVSVQSLSTNGANQDVVSMGTIAARRARAQ LLPLSQIQAILALALAQAMDLLDDPEGQAGWSLTARDLRDRIRAVSPGLRADRPLAGHIE AVAQGLRHPSAAADPPA RiceTAL Aminoacidsequence(SEQIDNO:81) MAGNGPINKEDPLNWGAAAAEMAGSHLDEVKRMVAQFREPLVKIQGATLRVGQVAA VAQAKDAARVAVELDEEARPRVKASSEWILTCIAHGGDIYGVTTGFGGTSHRRTKDGP ALQVELLRYLNAGIFGTGSDGHTLPSETVRAAMLVRINTLLQGYSGIRFEILEAITKLLNT GVTPCLPLRGTITASGDLVPLSYIAGLITGRPNAQAISPDGRKVDAAEAFKLAGIEGGFFT LNPKEGLAIVNGTSVGSALAATVMFDANILAVLSEVLSAVFCEVMNGKPEYTDHLTHKL KHHPGSIDAAAIMEHILAGSSFMSHAKKVNEMDPLLKPKQDRYALRTSPQWLGPQIQVI RAATKSIEREVNSVNDNPVIDVHRGKALHGGNFQGTPIGVSMDNARLAIANIGKLMFAQ FSELVNEFYNNGLTSNLAGSRNPSLDYGFKGTEIAMASYSSELQYLANPITNHVQSAEQH NQDVNSLGLVSARKTLEAVDILKLMTSTYIVALCQAVDLRHLEENIKSSVKNCVTQVAK KVLTMNPTGDLSSARFSEKNLLTAIDREAVFSYADDPCSANYPLMQKLRAVLVEHALTS GDRRARGLRVLQDHQVRGGAPLCAAPGDRGRPRRRRQRTAPVANRIVESRSFPLYRFV REELGCVFLTGEKLKSPGEECNKVFLGISQGKLIDPMLDCLKEWNGEPLPIN ParsleyTAL Aminoacidsequence(SEQIDNO:82) FLNAGIFGNGSDNTLPHSATRAAMLVRINTLLQGYSGIRFEILEAITKFLNQNITPCLPLRG TITASGDLVPLSYIAGLLTGRPNSKAVGPTGVILSPEEAFKLAGVEGGFFELQPKEGLALV NGTAVGSGMASMVLFEANILAVLAEVMSAIFAEVMQGKPEFTDHLTHKLKHHPGQIEA AAIMEHILDGSAYVKAAQKLHEMDPLQKPKQDRYALRTSPQWLGPQIEVIRSSTKMIER EINSVNDNPLIDVSRNKAIHGGNFQGTPIGVSMDNTRLAIAAIGKLMFAQFSELVNDFYN NGLPSNLSGGRNPSLDYGFKGAEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNSLG LISSRKTSEAVEILKLMSTTFLVGLCQAIDLRHLEENLKSTVKNTVSSVAKRVLTMGVNG ELHPSRFCEKDLLRVVDREYIFAYIDDPCSATYPLMQKLRQTLVEHALKNGDNERNLST SIFQKIATFEDELKALLPKEVESARAALESGNPAIPNRIEECRSYPLYKFVRKELGTEYLT GEKVTSPGEEFEKVFIAMSKGEIIDPLLECLESWNGAPLPIC TomatoTAL Aminoacidsequence(SEQIDNO:83) MDLCKKSINDPLNWEMAADSLRGSHLDEVKKMVDEFRKPIVKLGGETLSVAQVASIAN VDDKSNGVKVELSESARAGVKASSDWVMDSMSKGTDSYGVTAGFGATSHRRTKNGG ALQKELIRFLNAGVFGNGIESFHTLPHSATRAAMLVRINTLLQGYSGIRFEILEAITKLINS NITPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAVGPNGEKLNAEEAFCVAGISGGFFE LQPKEGLALVNGTAVGSAMASIVLFESNIFAVMSEVLSAIFTEVMNGKPEFTDYLTHKL KHHPGQIEAAAIMEHILDGSSYVKVAQKLHEMDPLQKPKQDRYALRTSPQWLGPQIEVI RAATKMIEREINSVNDNPLIDVSRNKALHGGNFQGTPIGVSMDNTRLALASIGKLMFAQ FSELVNDYYNNGLPSNLTAGRNPSLDYGFKGAEIAMASYCSELQFLANPVTNHVQSAEQ HNQDVNSLGLISARKTAKAVDILKIMSSTYLVALCQAIDLRHLEENLKSVVKNTVSQVA KRTLTMGANGELHPARFSEKELLRVVDREYLFAYADDPCSSNYPLMQKLRQVLVDQA MKNGESEKNVNSSIFQKIGAFEDELIAVLPKEVESVRAVFESGNPLIRNRITECRSYPLYR LVREELGTELLTGEKVRSPGEEIDKVFTAICNGQIIDPLLECLKSWNGAPLPIC ArabidopsisTAL Aminoacidsequence(SEQIDNO:84) MEINGAHKSNGGGVDAMLCGGDIKTKNMVINAEDPLNWGAAAEQMKGSHLDEVKRM VAEFRKPVVNLGGETLTIGQVAAISTIGNSVKVELSETARAGVNASSDWVMESMNKGT DSYGVTTGFGATSHRRTKNGVALQKELIRFLNAGIFGSTKETSHTLPHSATRAAMLVRIN TLLQGFSGIRFEILEAITSFLNNNITPSLPLRGTITASGDLVPLSYIAGLLTGRPNSKATGPN GEALTAEEAFKLAGISSGFFDLQPKEGLALVNGTAVGSGMASMVLFETNVLSVLAEILS AVFAEVMSGKPEFTDHLTHRLKHHPGQIEAAAVMEHILDGSSYMKLAQKLHEMDPLQK PKQDRYALRTSPQWLGPQIEVIRYATKSIEREINSVNDNPLIDVSRNKAIHGGNFQGTPIG VSMDNTRLAIRAIGKLMFAQFSELVNDFYNNGLPSNLTASRNPSLDYGFKGAEIAMASY CSELQYLANPVTSHVQSAEQHNQDVNSLGLISSRKTSEAVDILKLMSTTFLVAICQAVDL RHLEENLRQTVKNTVSQVAKKVLTTGVNGELHPSRFCEKDLLKVVDREQVYTYADDPC SATYPLIQKLRQVIVDHALVNGESEKNAVTSIFHKIGAFEEELKAVLPKEVEAARAAYDN GTSAIPNRIKECRSYPLYRFVREELGTELLTGEKVTSPGEEFDKVFTAICEGKIIDPMMEC LNEWNGAPIPIC
[0154] While only certain features of the invention have been illustrated and described herein, many modifications and changes will occur to those skilled in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as falling within the true spirit of the invention.
[0155] Throughout this application, various references are referred to. The disclosures of these publications in their entireties are hereby incorporated by reference as if written herein.
BIBLIOGRAPHY
[0156] 1. Hays, S. G., Patrick, W. G., Ziesack, M., Oxman, N. & Silver, P. A. Better together: engineering and application of microbial symbioses. Curr. Opin. Biotechnol. 36, 40-49 (2015). [0157] 2. Lindemann, S. R. et al. Engineering microbial consortia for controllable outputs. ISME J. (2016). doi:10.1038/ismej.2016.26 [0158] 3. GroBkopf, T. & Soyer, O. S. Synthetic microbial communities. Curr. Opin. Microbiol. 18, 72-77 (2014). [0159] 4. Hatcher, B. G. Coral reef ecosystems: how much greater is the whole than the sum of the parts? Coral Reefs 16, S77-S91 (1997). [0160] 5. Teague, B. & Weiss, R. Synthetic communities, the sum of parts. Science (80-.). 349, 924-925 (2015). [0161] 6. Brown, E. J., Pignatello, J. J., Martinson, M. M. & Crawford, R. L. Pentachlorophenol degradation: a pure bacterial culture and an epilithic microbial consortium. Appl. Environ. Microbiol. 52, 92-97 (1986). [0162] 7. Olson, J. B., Steppe, T. F., Litaker, R. W. & Paerl, H. W. N 2-Fixing Microbial Consortia Associated with the Ice Cover of Lake Bonney, Antarctica. Microb. Ecol. 36, 231-238 (1998). [0163] 8. Rabaey, K., Boon, N., Siciliano, S. D., Verhaege, M. & Verstraete, W. Biofuel Cells Select for Microbial Consortia That Self-Mediate Electron Transfer. Appl. Environ. Microbiol. 70, 5373-5382 (2004). [0164] 9. Jones, J. A. et al. Experimental and computational optimization of an Escherichia coli co-culture for the efficient production of flavonoids. Metab. Eng. 35, 55-63 (2016). [0165] 10. Zhang, H., Pereira, B., Li, Z. & Stephanopoulos, G. Engineering Escherichia coli coculture systems for the production of biochemical products. Proc. Natl. Acad. Sci. U.S.A 112, 8266-8271 (2015). [0166] 11. Zhang, H., Li, Z., Pereira, B. & Stephanopoulos, G. Engineering E. coli-E. coli cocultures for production of muconic acid from glycerol. Microb. Cell Fact. 14, 134 (2015). [0167] 12. Zhou, K., Qiao, K., Edgar, S. & Stephanopoulos, G. Distributing a metabolic pathway among a microbial consortium enhances production of natural products. Nat. Biotechnol. 33, 377-383 (2015). [0168] 13. Willrodt, C., Hoschek, A., Bhler, B., Schmid, A. & Julsing, M. K. Coupling limonene formation and oxyfunctionalization by mixed-culture resting cell fermentation. Biotechnol. Bioeng. 112, 1738-1750 (2015). [0169] 14. Zhang, H. & Wang, X. Modular co-culture engineering, a new approach for metabolic engineering. Metab. Eng. 37, 114-121 (2016). [0170] 15. He, W. et al. Production of chondroitin in metabolically engineered E. coli. Metab. Eng. 27, 92-100 (2015). [0171] 16. Santos, C. N. S., Koffas, M. & Stephanopoulos, G. Optimization of a heterologous pathway for the production of flavonoids from glucose. Metab. Eng. 13, 392-400 (2011). [0172] 17. Jones, J. A., Collins, S. M., Lachance, D. M., Vernacchio, V. R. & Koffas, M. A. G. Optimization of naringenin and p-coumaric acid hydroxylation using the native E. coli hydroxylase complex, HpaBC. Biotechnol. Prog. 32, 21-25 (2016). [0173] 18. Xu, P., Vansiri, A., Bhan, N. & Koffas, M. A. G. ePathBrick: A Synthetic Biology Platform for Engineering Metabolic Pathways in E. coli. ACS Synth. Biol. 1, 256-66 (2012). [0174] 19. Santos, C. N. S. Combinatorial search strategies for the metabolic engineering of microorganisms. (2010). [0175] 20. Lin, Y. & Yan, Y. Biotechnological production of plant-specific hydroxylated phenylpropanoids. Biotechnol. Bioeng. 111, 1895-9 (2014). [0176] 21. Huang, Q., Lin, Y. & Yan, Y. Caffeic acid production enhancement by engineering a phenylalanine over-producing Escherichia coli strain. Biotechnol. Bioeng. 110, 3188-3196 (2013). [0177] 22. Lin, Y. & Yan, Y. Biosynthesis of caffeic acid in Escherichia coli using its endogenous hydroxylase complex. Microb Cell Fact 11, 1-9 (2012). [0178] 23. Zhang, H. & Stephanopoulos, G. Engineering E. coli for caffeic acid biosynthesis from renewable sugars. Appl. Microbiol. Biotechnol. 97, 3333-41 (2013). [0179] 24. Jones, J. A. et al. Experimental and computational optimization of an Escherichia coli co-culture for the efficient production of flavonoids. Metab. Eng. 35, 55-63 (2016). [0180] 25. Kang, S.-Y. et al. Artificial biosynthesis of phenylpropanoic acids in a tyrosine overproducing Escherichia coli strain. Microb. Cell Fact. 11, 153 (2012). [0181] 26. Jones, J. A. & Koffas, M. A. G. Optimizing Metabolic Pathways for the Improved Production of Natural Products. Methods Enzymol. (2016). doi:10.1016/bs.mie.2016.02.010 [0182] 27. Pandey, R. P., Parajuli, P., Koffas, M. A. G. & Sohng, J. K. Microbial production of natural and non-natural flavonoids: Pathway engineering, directed evolution and systems/synthetic biology. Biotechnol. Adv. 1-29 (2016). doi:10.1016/j.biotechadv.2016.02.012 [0183] 28. Yan, Y., Li, Z. & Koffas, M. A. G. High-yield anthocyanin biosynthesis in engineered Escherichia coli. Biotechnol. Bioeng. 100, 126-40 (2008). [0184] 29. Saini, M., Hong Chen, M., Chiang, C.-J. & Chao, Y.-P. Potential production platform of n-butanol in Escherichia coli. Metab. Eng. 27, 76-82 (2015). [0185] 30. Chemler, J. A., Fowler, Z. L., McHugh, K. P. & Koffas, M. A. G. Improving NADPH availability for natural product biosynthesis in Escherichia coli by metabolic engineering. Metab. Eng. 12, 96-104 (2010). [0186] 31. Xu, P., Ranganathan, S., Fowler, Z. L., Maranas, C. D. & Koffas, M. a G. Genome-scale metabolic network modeling results in minimal interventions that cooperatively force carbon flux towards malonyl-CoA. Metab. Eng. 13, 578-87 (2011). [0187] 32. Zhao, S. et al. Improvement of catechin production in Escherichia coli through combinatorial metabolic engineering. Metab. Eng. 28, 43-53 (2015). [0188] 33. Xu, P., Li, L., Zhang, F., Stephanopoulos, G. & Koffas, M. Improving fatty acids production by engineering dynamic pathway regulation and metabolic control. Proc. Natl. Acad. Sci. 111, 11299-11304 (2014). [0189] 34. Andrianantoandro, E., Basu, S., Karig, D. K., Weiss, R., 2006. Synthetic biology: new engineering rules for an emerging discipline. Mol. Syst. Biol. 2, 2006.0028. doi:10.1038/msb4100073 [0190] 35. Bhadouria, A. S., Sorci, M., Gu, M., Belfort, G., Hahn, J., 2014. Optimization of Membrane Separation Processes for Protein Fractionation. Ind. Eng. Chem. Res. 53, 5103-5109. doi:10.1021/ie401303d [0191] 36. Bizzini, A., Zhao, C., Budin-Verneuil, A., Sauvageot, N., Giard, J.-C., Auffray, Y., Hartke, A., 2010. Glycerol is metabolized in a complex and strain-dependent manner in Enterococcus faecalis. J. Bacteriol. 192, 779-85. doi:10.1128/JB.00959-09 [0192] 37. Boock, J. T., Gupta, A., Prather, K. L., 2015. Screening and modular design for metabolic pathway optimization. Curr. Opin. Biotechnol. 36, 189-198. doi:10.1016/j.copbio.2015.08.013 [0193] 38. Brenner, K., You, L., Arnold, F. H., 2008. Engineering microbial consortia: a new frontier in synthetic biology. Trends Biotechnol. 26, 483-9. doi:10.1016/j.tibtech.2008.05.004 [0194] 39. Chemler, J. A., Lock, L. T., Koffas, M. A. G., Tzanakakis, E. S., 2007. Standardized biosynthesis of flavan-3-ols with effects on pancreatic beta-cell insulin secretion. Appl. Microbiol. Biotechnol. 77, 797-807. doi:10.1007/s00253-007-1227-y [0195] 40. Cress, B. F., Toparlak, . D., Guleria, S., Lebovich, M., Stieglitz, J. T., Englaender, J. A., Jones, J. A., Linhardt, R. J., Koffas, M. A. G., 2015a. CRISPathBrick: Modular Combinatorial Assembly of Type II-A CRISPR Arrays for dCas9-Mediated Multiplex Transcriptional Repression in E. coli. ACS Synth. Biol. 4, 987-1000. doi:10.1021/acssynbio.5b00012 [0196] 41. Cress, B. F., Trantas, E. A., Ververidis, F., Linhardt, R. J., Koffas, M. A., 2015b. Sensitive cells: enabling tools for static and dynamic control of microbial metabolic pathways. Curr. Opin. Biotechnol. 36, 205-214. doi:10.1016/j.copbio.2015.09.007 [0197] 42. Da Silva, G. P., Mack, M., Contiero, J., 2009. Glycerol: a promising and abundant carbon source for industrial microbiology. Biotechnol. Adv. 27, 30-9. doi:10.1016/j.biotechadv.2008.07.006 [0198] 43. Dai, W., Word, D. P., Hahn, J., 2014. Modeling and dynamic optimization of fuel-grade ethanol fermentation using fed-batch process. Control Eng. Pract. 22, 231-241. doi:10.1016/j.conengprac.2013.01.005 [0199] 44. Hannig, G., Makrides, S. C., 1998. Strategies for optimizing heterologous protein expression in Escherichia coli. Trends Biotechnol. 16, 54-60. doi:10.1016/S0167-7799(97)01155-4 [0200] 45. He, W., Fu, L., Li, G., Andrew Jones, J., Linhardt, R. J., Koffas, M., 2015. Production of chondroitin in metabolically engineered E. coli. Metab. Eng. 27, 92-100. doi:10.1016/j.ymben.2014.11.003 [0201] 46. Heiss, C., Dejam, A., Kleinbongard, P., Schewe, T., Sies, H., Kelm, M., 2003. Vascular effects of cocoa rich in flavan-3-ols. JAMA 290, 1030-1. doi:10.1001/jama.290.8.1030 [0202] 47. Hooper, L., Kay, C., Abdelhamid, A., Kroon, P. A., Cohn, J. S., Rimm, E. B., Cassidy, A., 2012. Effects of chocolate, cocoa, and flavan-3-ols on cardiovascular health: a systematic review and meta-analysis of randomized trials. Am. J. Clin. Nutr. 95, 740-51. doi:10.3945/ajcn.111.023457 [0203] 48. Jones, J. A., Collins, S. M., Lachance, D. M., Vernacchio, V. R., Koffas, M. A. G., 2015a. Optimization of naringenin and p-coumaric acid hydroxylation using the native E. coli hydroxylase complex, HpaBC. Biotechnol. Prog. n/a-n/a. doi:10.1002/btpr.2185 [0204] 49. Jones, J. A., Toparlak, . D., Koffas, M. A., 2015b. Metabolic pathway balancing and its role in the production of biofuels and chemicals. Curr. Opin. Biotechnol. 33, 52-59. doi:10.1016/j.copbio.2014.11.013 [0205] 50. Jones, J. A., Vernacchio, V. R., Lachance, D. M., Lebovich, M., Fu, L., Shirke, A. N., Schultz, V. L., Cress, B., Linhardt, R. J., Koffas, M. A. G., 2015. ePathOptimize: A Combinatorial Approach for Transcriptional Balancing of Metabolic Pathways. Sci. Rep. 5, 11301. doi:10.1038/srep11301 [0206] 51. Koenig, J. E., Spor, A., Scalfone, N., Fricker, A. D., Stombaugh, J., Knight, R., Angenent, L. T., Ley, R. E., 2011. Succession of microbial consortia in the developing infant gut microbiome. Proc. Natl. Acad. Sci. 108, 4578-4585. doi:10.1073/pnas.1000081107 [0207] 52. L. Gaikwad, G., 2014. Development of Microbial Consortia for the Effective Treatment of Complex Wastewater. J. Bioremediation Biodegrad. 05. doi:10.4172/2155-6199.1000227 [0208] 53. Lim, C. G., Fowler, Z. L., Hueller, T., Schaffer, S., Koffas, M. A. G., 2011. High-yield resveratrol production in engineered Escherichia coli. Appl. Environ. Microbiol. 77, 3451-60. doi:10.1128/AEM.02186-10 [0209] 54. Manz, W., Wagner, M., Amann, R., Schleifer, K.-H., 1994. In situ characterization of the microbial consortia active in two wastewater treatment plants. Water Res. 28, 1715-1723. doi:10.1016/0043-1354(94)90243-7 [0210] 55. Martinez-Gmez, K., Flores, N., Castaeda, H. M., Martinez-Batallar, G., Hernndez-Chvez, G., Ramirez, O. T., Gosset, G., Encarnacin, S., Bolivar, F., 2012. New insights into Escherichia coli metabolism: carbon scavenging, acetate metabolism and carbon recycling responses during growth on glycerol. Microb. Cell Fact. 11, 46. doi:10.1186/1475-2859-11-46 [0211] 56. Monagas, M., Urpi-Sarda, M., Snchez-Patn, F., Llorach, R., Garrido, I., Gmez-Cordovs, C., Andres-Lacueva, C., Bartolom, B., 2010. Insights into the metabolism and microbial biotransformation of dietary flavan-3-ols and the bioactivity of their metabolites. Food Funct. 1, 233. doi:10.1039/c0fo00132e [0212] 57. Paerl, H. W., Pinckney, J. L., 1996. A mini-review of microbial consortia: Their roles in aquatic production and biogeochemical cycling. Microb. Ecol. 31. doi:10.1007/BF00171569 [0213] 58. Roze, L. V., Chanda, A., Linz, J. E., 2011. Compartmentalization and molecular traffic in secondary metabolism: A new understanding of established cellular processes. Fungal Genet. Biol. 48, 35-48. doi:10.1016/j.fgb.2010.05.006 [0214] 59. Saini, M., Hong Chen, M., Chiang, C.-J., Chao, Y.-P., 2015. Potential production platform of n-butanol in Escherichia coli. Metab. Eng. 27, 76-82. doi:10.1016/j.ymben.2014.11.001 [0215] 60. Smid, E. J., Lacroix, C., 2013. Microbe-microbe interactions in mixed culture food fermentations. Curr. Opin. Biotechnol. 24, 148-54. doi:10.1016/j.copbio.2012.11.007 [0216] 61. Xu, P., Ranganathan, S., Fowler, Z. L., Maranas, C. D., Koffas, M. a G., 2011. Genome-scale metabolic network modeling results in minimal interventions that cooperatively force carbon flux towards malonyl-CoA. Metab. Eng. 13, 578-87. doi:10.1016/j.ymben.2011.06.008 [0217] 62. Xu, P., Vansiri, A., Bhan, N., Koffas, M. A. G., 2012. ePathBrick: A Synthetic Biology Platform for Engineering Metabolic Pathways in E. coli. ACS Synth. Biol. 1, 256-66. doi:10.1021/sb300016b [0218] 63. Yadav, V. G., De Mey, M., Giaw Lim, C., Kumaran Ajikumar, P., Stephanopoulos, G., 2012. The future of metabolic engineering and synthetic biology: Towards a systematic practice. Metab. Eng. 14, 233-241. doi:10.1016/j.ymben.2012.02.001 [0219] 64. Young, V. A., Kiefer, A. M., 2014. Kimchi: spicy science for the undergraduate microbiology laboratory. J. Microbiol. Biol. Educ. 15, 297-8. doi:10.1128/jmbe.v15i2.695 [0220] 65. Zhang, H., Li, Z., Pereira, B., Stephanopoulos, G., 2015a. Engineering E. coli-E. coli cocultures for production of muconic acid from glycerol. Microb. Cell Fact. 14, 134. doi:10.1186/s12934-015-0319-0 [0221] 66. Zhang, H., Pereira, B., Li, Z., Stephanopoulos, G., 2015b. Engineering Escherichia coli coculture systems for the production of biochemical products. Proc. Natl. Acad. Sci. U.S.A 112, 8266-8271. doi:10.1073/pnas.1506781112 [0222] 67. Zhao, S., Jones, J. A., Lachance, D. M., Bhan, N., Khalidi, O., Venkataraman, S., Wang, Z., Koffas, M. A. G., 2015. Improvement of catechin production in Escherichia coli through combinatorial metabolic engineering. Metab. Eng. 28, 43-53. doi:10.1016/j.ymben.2014.12.002 [0223] 68. Zhou, K., Qiao, K., Edgar, S., Stephanopoulos, G., 2015. Distributing a metabolic pathway among a microbial consortium enhances production of natural products. Nat. Biotechnol. 33, 377-383. doi:10.1038/nbt.3095