Recombinant host cell for biosynthetic production
A cell may include heterologous polynucleotides encoding a multienzyme complex involved in the metabolic pathway of phenylpropanoids and biosynthesis of a vanilloid or a hydroxybenzaldehyde precursor thereof, which multienzyme complex comprises enzymes for the biosynthesis of coumaric acid and a crotonase.
1. A yeast cell comprising heterologous polynucleotides encoding a multienzyme complex, wherein the multienzyme complex is involved in the metabolic pathway of phenylpropanoids and biosynthesis of a vanilloid or a hydroxybenzaldehyde precursor thereof, and wherein the multienzyme complex comprises: a) a crotonase, b) a CoA ligase, c) a 3-monooxygenase; and d) a methyltransferase; wherein a) is enoyl-CoA hydratase (ECH), b) is 4-coumarate-CoA ligase (4CL), c) is hydroxybenzoic acid hydroxylase (HBH), or phenolhydroxylase (PheA) and flavinreductase (FLARED); and d) is O-methyltransferase.
2. The yeast cell according to claim 1 , which multienzyme complex comprises
a) phenylalanine ammonia lyase (PAL), cinnamic acid hydroxylase (C4H), cytochrome P450 reductase (CPR), 4 coumarate-CoA ligase (4CL), enoyl-CoA hydratase (ECH), hydroxybenzoic acid hydroxylase (HBH), or phenolhydroxylase (PheA) and flavinreductase (FLARED), and a O-methyltransferase;
b) tyrosine ammonia lyase (TAL), 4-coumarate-CoA ligase (4CL), enoyl-CoA hydratase (ECH), hydroxybenzoic acid hydroxylase (HBH), or phenolhydroxylase (PheA) and flavinreductase (FLARED), and a O-methyltransferase; or
c) phenylalanine/tyrosine ammonia lyase (PAL TAL), cinnamic acid hydroxylase (C4H), cytochrome P450 reductase (CPR), 4-coumarate-CoA ligase (4CL), enoyl-CoA hydratase (ECH), hydroxybenzoic acid hydroxylase (HBH), or phenolhydroxylase (PheA) and flavinreductase (FLARED), and a O-methyltransferase.
3. The yeast cell according to claim 1 , wherein enoyl-CoA hydratase (ECH) is any of
a) an ECH responsible for the chain reduction reaction on p-coumaroylCoA and/or feruloylCoA;
b) an ECH converting p-coumaroylCoA to 4-hydroxybenzaldehyde; or
c) an ECH converting feruloylCoA to vanillin.
4. The yeast cell according to claim 1 , which further comprises
a) a heterologous polynucleotide encoding a carboxyreductase (CAR), optionally together with a polynucleotide encoding a phosphopantetheinyl transferase (PPTase); and/or
b) a heterologous polynucleotide encoding an alcohol oxidase.
5. The yeast cell according to claim 1 , wherein the yeast cell is of a genera selected from the group consisting of Saccharomyces, Pichia, Hansenula, Schizosaccharomyces, Kluyveromyces, Yarrowia and Candida.
6. The yeast cell according to claim 1 , wherein the yeast cell is a DNA repair deficient cell or a production cell comprising a cluster of polynucleotides assembled in a DNA repair deficient cell.
7. The yeast cell according to claim 1 , wherein polynucleotides encoding a series of enzymes are expressed from a single polycistronic operon, or wherein polynucleotides encoding a series of enzymes are expressed from separate promoters.
8. The yeast cell according to claim 1 , wherein the polynucleotides are stably integrated into the cell genome.
9. The yeast cell according to claim 1 , wherein the polynucleotides originate from at least two different species.
10. The yeast cell according to claim 1 , wherein at least one of the enzymes is a chimeric enzyme.
11. The yeast cell according to claim 10 , wherein the chimeric enzyme is
a) encoded by a nucleotide sequence that is composed of fragments of different polynucleotides, which fragments are assembled to a chimeric nucleotide sequence; and/or
b) encoded by a nucleotide sequence that is obtained by insertion, deletion and/or substitution of one or more nucleotides in a parent polynucleotide.
12. The yeast cell according to claim 1 , wherein the vanilloid is selected from the group consisting of vanillin, vanillic acid, ethyl-vanillin, vanillyl alcohol and vanillin-glycoside.
13. The yeast cell according to claim 1 , wherein the hydroxybenzaldehyde precursor is selected from the group consisting of protocatechuic aldehyde, protocatechuic acid, protocatechuic alcohol, 4-hydroxybenzaldehyde, 4-hydroxybenzoic acid, 4-hydroxybenzyl alcohol, cinnamic acid, coumaric acid, caffeic acid and ferulic acid.
14. A method of engineering a yeast cell according to claim 1 by introducing heterologous polynucleotides encoding a multienzyme complex involved in the metabolic pathway of phenylpropanoids and biosynthesis of a vanilloid or a hydroxybenzaldehyde precursor thereof, into the cell genome, comprising
a) providing the polynucleotides encoding i) a crotonase, ii) a CoA ligase, ii) a-3-monooxygenase; and iv) a methyltransferase, wherein i) is enoyl-CoA hydratase (ECH), ii) is 4-coumarate-CoA ligase (4CL), iii) is hydroxybenzoic acid hydroxylase (HBH), or phenolhydroxylase (PheA) and flavinreductase (FLARED); and iv) is a O-methyltransferase;
b) assembling the polynucleotides into a cluster and integrating said cluster into the cell genome; and
c) optionally engineering a yeast production cell, wherein said cluster is stably integrated in the production cell genome.
15. A method, comprising:
producing a vanilloid or a hydroxybenzaldehyde precursor thereof by heterologous biosynthesis from a yeast cell according to claim 1 .
16. A method of heterologous biosynthesis of a vanilloid or a hydroxybenzaldehyde precursor thereof, by conversion of a precursor compound employing a multienzyme complex, comprising
a) providing a yeast cell according to claim 1 ;
b) cultivating said cell in a cell culture in the presence of the precursor compound;
c) accumulating a vanilloid or a hydroxybenzaldehyde precursor thereof;
and
d) separating said vanilloid or hydroxybenzaldehyde precursor thereof from the cell culture medium.
17. The method of claim 16 , wherein said product is a
i. vanilloid selected from the group consisting of vanillin, vanillic acid, ethyl-vanillin, vanillyl alcohol and vanillin-glycoside; or
ii. a hydroxybenzaldehyde precursor selected from the group consisting of protocatechuic aldehyde, protocatechuic acid, protocatechuic alcohol, 4-hydroxybenzaldehyde, 4-hydroxybenzoic acid, 4-hydroxybenzyl alcohol, cinnamic acid, coumaric acid, caffeic acid and ferulic acid.
18. The yeast cell of claim 1 , wherein the enzymes for the biosynthesis of coumaric acid are selected from the group consisting of: a phenylalanine ammonia lyase (PAL), a tyrosine ammonia lyase (TAL), and a phenylalanine/tyrosine ammonia lyase (PAL/TAL).
19. The yeast cell of claim 18 , wherein the multienzyme complex further comprises one or more enzymes to convert an aromatic amino acid into coumaric acid.
20. The yeast cell of claim 1 , wherein the crotonase is suitable for performing a chain reduction reaction on feruloylCoA or coumaroylCoA.
21. The yeast cell of claim 18 , wherein the crotonase is suitable for performing a chain reduction reaction on feruloylCoA or coumaroylCoA.
22. The yeast cell of claim 19 , wherein the crotonase is suitable for performing a chain reduction reaction on feruloylCoA or coumaroylCoA.
23. The yeast cell of claim 5 , wherein the cell is Saccharomyces cerevisiae.