Biosynthesis of phenylpropanoids and phenylpropanoid derivatives

1. A method of producing a chalcone compound or a stilbene compound, comprising growing a yeast cell capable of producing a phenylpropanoid or phenylpropanoid derivative compound in a culture medium,

wherein said chalcone compound or stilbene compound is synthesized by the yeast cell, and

wherein the yeast cell comprises a gene encoding a double-bond reductase polypeptide, wherein expression of the gene or activity of the double-bond reductase polypeptide encoded thereby is reduced or eliminated, and wherein the double-bond reductase polypeptide:

(i) comprises SEQ ID NO: 22;

(ii) comprises a polypeptide with at least 90% identity to SEQ ID NO: 22;

(iii) comprises SEQ ID NO: 26;

(iv) comprises a polypeptide with at least 90% identity to SEQ ID NO: 26;

(v) is encoded by a nucleic acid sequence comprising SEQ ID NO: 7;

(vi) is encoded by a nucleic acid sequence comprising a sequence with at least 90% identity to SEQ ID NO: 7;

(vii) is encoded by a nucleic acid sequence comprising SEQ ID NO: 43; or

(viii) is encoded by a nucleic acid sequence comprising a sequence with at least 90% identity to SEQ ID NO: 43.

2. The method of claim 1 , wherein the double-bond reductase polypeptide is S. cerevisiae trans-2-enoyl-CoA reductase TSC13 or S. cerevisiae DFG10.

3. The method of claim 1 , wherein the yeast cell is a recombinant yeast cell and comprises a recombinant gene encoding an enzyme that partially or completely complements the function of the double-bond reductase polypeptide, wherein the recombinant gene:

(a) is any one of SEQ ID NOs: 94-96;

(b) is a nucleotide sequence with at least 90% identity to any one of SEQ ID NOs: 94-96;

(c) encodes any one of SEQ ID NOs: 65-67; or

(d) encodes a polypeptide with at least 90% identity to any one of SEQ ID NOs: 65-67;

and wherein the method further comprises growing the recombinant yeast cell under conditions in which the recombinant gene encoding an anzyme that partially or completelty ocmplements the function of the double-bond reductase polypeptide is expressed.

4. The method of claim 1 , wherein the yeast cell is a recombinant yeast cell and comprises a recombinant gene encoding a polyketide synthase Type III polypeptide, and wherein the method further comprises growing the recombinant yeast cell under conditions in which the recombinant gene encoding the polyketide synthase Type III polypeptide is expressed.

5. The method of claim 4 , wherein the polyketide synthase Type III polypeptide is:

(a) a chalcone synthase polypeptide; or

(b) a stilbene synthase polypeptide.

6. The method of claim 5 , wherein the polyketide synthase Type III polypeptide:

(i) is encoded by a nucleic acid sequence comprising SEQ ID NO: 4;

(ii) is encoded by a nucleic acid sequence comprising a sequence with at least 90% identity to SEQ ID NO: 4;

(iii) comprises SEQ ID NO: 19;

(iv) comprises a polypeptide sequence with at least 90% identity to SEQ ID NO: 19;

(v) is encoded by a nucleic acid sequence comprising SEQ ID NO: 23;

(vi) is encoded by a nucleic acid sequence comprising a sequence with at least 90% identity to SEQ ID NO: 23;

(vii) comprises SEQ ID NO: 24; or

(viii) comprises a polypeptide sequence with at least 90% identity to SEQ ID NO: 24.

7. The method of claim 4 , wherein the recombinant yeast cell further comprises one or more of:

(a) a recombinant gene encoding a phenylalanine ammonia lyase polypeptide;

(b) a recombinant gene encoding a cinnamate 4-hydroxylase polypeptide;

(c) a recombinant gene encoding a 4-coumarate-CoA ligase polypeptide;

(d) a recombinant gene encoding a cytochrome p450 polypeptide; or

(e) a recombinant gene encoding a chalcone isomerase polypeptide.

8. The method of claim 7 , wherein:

(a) the recombinant gene encoding the phenylalanine ammonia lyase polypeptide:

(i) comprises SEQ ID NO: 1;

(ii) comprises a nucleotide sequence with at least 90% identity to SEQ ID NO: 1;

(iii) encodes a polypeptide comprising SEQ ID NO: 16; or

(iv) encodes a polypeptide with at least 90% identity to SEQ ID NO: 16;

(b) the recombinant gene encoding the cinnamate 4-hydroxylase polypeptide:

(i) comprises SEQ ID NO: 2;

(ii) comprises a nucleotide sequence with at least 90% identity to SEQ ID NO: 2;

(iii) encodes a polypeptide comprising SEQ ID NO: 17; or

(iv) encodes a polypeptide with at least 90% identity to SEQ ID NO: 17;

(c) the recombinant gene encoding the 4-coumarate-CoA ligase polypeptide:

(i) comprises SEQ ID NO: 3;

(ii) comprises a nucleotide sequence with at least 90% identity to SEQ ID NO: 3;

(iii) encodes a polypeptide comprising SEQ ID NO: 18; or

(iv) encodes a polypeptide with at least 90% identity to SEQ ID NO: 18;

(d) the recombinant gene encoding the cytochrome p450 polypeptide:

(i) comprises SEQ ID NO: 6;

(ii) comprises a nucleotide sequence with at least 90% identity to SEQ ID NO: 6;

(iii) encodes a polypeptide comprising SEQ ID NO: 21; or

(iv) encodes a polypeptide with at least 90% identity to SEQ ID NO: 21; or

(e) the recombinant gene encoding the chalcone isomerase polypeptide:

(i) comprises any one of SEQ ID NOS: 80-86;

(ii) comprises a nucleotide sequence with at least 90% identity to any one of SEQ ID NOS: 80-86;

(iii) encodes a polypeptide comprising any one of SEQ ID NOS: 87-93; or

(iv) encodes a polypeptide a polypeptide with at least 90% identity to any one of SEQ ID NOS: 87-93.

9. The method of claim 1 , wherein the phenylpropanoid compound is cinnamic acid or coumaric acid.

10. The method of claim 1 , wherein the phenylpropanoid derivative compound is a chalcone compound or a stilbenoid compound.

11. The method of claim 10 , wherein the stilbenoid compound is resveratrol.

12. The method of claim 1 , wherein the yeast cell is a Saccharomyces cerevisiae cell, a Schizosaccharomyces pombe cell, a Yarrowia lipolytica cell, a Candida glabrata cell, a Ashbya gossypii cell, a Cyberlindnera jadinii cell, a Pichia pastoris cell, a Kluyveromyces lactis cell, a Hansenula polymorpha cell, a Candida boidinii cell, an Arxula adeninivorans cell, a Xanthophyllomyces dendrorhous cell, or a Candida albicans cell.

13. The method of claim 1 , wherein the chalcone compound or stilbene compound is a compound of formula (III):

or a pharmaceutically acceptable salt thereof, wherein

A is a bond or C═O;

n is an integer 0, 1, 2, 3, or 4;

R is hydrogen when is a double bond, or R and R 5 together with the atoms to which they are attached form a 6-member heterocyclyl when A is C═O and is a single bond;

R 1 is hydrogen or —OR 11 ;

wherein each R 11 is independently hydrogen, C 1 -C 6 alkyl, or glycosyl;

R 2 is hydrogen, —OR 11 , C 1 -C 12 alkyl, or C 2 -C 12 alkenyl, wherein alkyl and alkenyl are optionally substituted with one or more R 7 ;

or R 2 and R 6 together with the atoms to which they are attached form a 5- to 7-member heterocyclyl optionally substituted with one or more R 7 groups;

or R 2 and R 4 together with the atoms to which they are attached form a 5- to 7-member heterocyclyl optionally substituted with one or more R 7 groups;

R 3 is independently selected from nitro, C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 1 -C 6 haloalkyl, C 1 -C 6 hydroxyalkyl, —OR 12 , —N(R 12 ) 2 , —C(O)R 12 , —C(O)OR 12 , —C(O)N(R 12 ) 2 , and —S(O) 2 R 12 , wherein each R 12 is independently hydrogen or C 1 -C 6 alkyl;

R 4 is hydrogen, —OR 11 , C 1 -C 12 alkyl, or C 2 -C 12 alkenyl, wherein alkyl and alkenyl are optionally substituted with one or more R 7 ;

or R 4 and R 2 together with the atoms to which they are attached form a 5- to 7-member heterocyclyl optionally substituted with one or more R 7 groups;

R 5 is hydrogen, —OR 11 , —C(O)OR 10 , or —C(O)N(R 10 ) 2 , wherein each R 10 is independently hydrogen or C 1 -C 6 alkyl; and

R 6 is hydrogen, C 1 -C 6 alkyl, C 2 -C 6 alkenyl, —OR 11 , —N(R 10 ) 2 , —C(O)R 10 , —C(O)OR 10 , or —C(O)N(R 10 ) 2 , wherein alkyl and alkenyl are optionally substituted with one or more R 8 ; or R 6 and R 2 together with the atoms to which they are attached form a 5- to 7-member heterocyclyl optionally substituted with one or more R 8 groups;

each R 7 and R 8 is independently halogen, cyano, nitro, C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 1 -C 6 haloalkyl, C 1 -C 6 hydroxyalkyl, —OR 13 , —SR 13 , —N(R 13 ) 2 , —C(O)R 13 , —C(O)OR 13 , —C(O)N(R 13 ) 2 , or —S(O) 2 R 13 , wherein each R 13 is independently hydrogen or C 1 -C 6 alkyl.

14. The method of claim 1 , further comprising harvesting said chalcone compound or stilbene compound.

15. The method of claim 1 , further comprising isolating said chalcone compound or stilbene compound.

16. The method of claim 1 , wherein the double-bond reductase polypeptide:

(i) comprises SEQ ID NO: 22;

(ii) comprises a polypeptide with at least 90% identity to SEQ ID NO: 22;

(iii) is encoded by a nucleic acid sequence comprising SEQ ID NO: 7; or

(iv) is encoded by a nucleic acid sequence comprising a sequence with at least 90% identity to SEQ ID NO: 7.

17. The method of claim 1 , wherein said chalcone compound or stilbene compound is resveratrol.

18. The method of claim 17 , further comprising isolating the resveratrol.

19. The method of claim 16 , wherein said chalcone compound or stilbene compound is resveratrol.

20. The method of claim 19 , further comprising isolating the resveratrol.