Processes for the production of hydroxycinnamic acids using polypeptides having tyrosine ammonia lyase activity
Inventors
Jendresen, Christian Bille • Siedler, Solvej • Stahlhut, Steen Gustav • Nielsen, Alex Toftgaard
Assignees
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Publication Number
US-10752923-B2
Publication Date
2020-08-25
Expiration Date
Abstract
The present invention generally relates to the field of biotechnology as it applies to the production of hydroxycinnamic acids using polypeptides having tyrosine ammonia lyase activity. More particularly, the present invention pertains to polypeptides having tyrosine ammonia lyase activity and high substrate specificity towards tyrosine, which makes them particularly suitable in the production of p-coumaric acid and other hydroxycinnamic acids. The present invention thus provides processes for the production of p-coumaric acid and other hydroxycinnamic acids employing these polypeptides as well as recombinant host cells expressing same.
Core Innovation
The invention relates to processes for producing hydroxycinnamic acids of general formula I by contacting a compound of general formula II with a polypeptide having tyrosine ammonia lyase activity. The produced hydroxycinnamic acid is of general formula I with R2 being hydroxyl and R4 being hydrogen, while R1 and R3 are independently hydrogen, hydroxyl, or C1-6-alkoxy. Measurement of the concentration of the hydroxycinnamic acid and/or recovery of the hydroxycinnamic acid is included.
A key aspect is the selection of the tyrosine ammonia lyase polypeptide, including a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 and polypeptides having tyrosine ammonia lyase activity with at least 85% or at least 95% sequence identity to SEQ ID NO: 1. The described concept includes defining TAL polypeptide variants by tolerated substitutions, deletions, and insertions, and optionally including specificity regions.
The invention further covers production using genetically modified recombinant host cells that express a heterologous tyrosine ammonia lyase polypeptide. The host-cell methods include providing a medium comprising a fermentable carbon substrate and/or a compound of general formula II, producing the hydroxycinnamic acid of general formula I, and measuring concentration and/or recovering the product. The document also addresses reducing degradative or byproduct pathways by inactivating phenolic acid decarboxylase (PAD), and reports embodiments across multiple recombinant host systems.
Claims Coverage
The document contains three independent claims covering hydroxycinnamic acid production using (i) contacting a compound of general formula II with a TAL polypeptide, (ii) using genetically modified recombinant host cells expressing a heterologous TAL, and (iii) further restricting polypeptide sequence identity, R-group substitution patterns, and host type (bacterium or yeast). Across the independent claims, the main inventive features focus on TAL polypeptides defined by SEQ ID NO: 1 identity and tyrosine ammonia lyase activity, conversion from general formula II substrates to general formula I products, and measurement or recovery of the hydroxycinnamic acid, with optional host-cell PAD activity reduction constraints in the host-cell claims.
Tyrosine ammonia lyase polypeptide contacting general formula II to produce general formula I hydroxycinnamic acid
A method producing a hydroxycinnamic acid of general formula I by contacting a compound of general formula II with a polypeptide having tyrosine ammonia lyase activity, wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1 or has at least 85% sequence identity to SEQ ID NO: 1, and wherein hydroxycinnamic acid of general formula I is measured and/or recovered.
Hydroxycinnamic acid general formula I substituent constraints (R2 hydroxyl, R4 hydrogen)
The method includes hydroxycinnamic acids of general formula I wherein R1 and R3 are independently hydrogen, hydroxyl, or C1-6-alkoxy, R2 is hydroxyl (—OH), and R4 is hydrogen (—H), with measurement of the concentration and/or recovery of the hydroxycinnamic acid.
Genetically modified recombinant host cell expressing heterologous TAL to produce general formula I hydroxycinnamic acid
A method producing a hydroxycinnamic acid of general formula I by contacting a recombinant host cell genetically modified to express a heterologous polypeptide with a medium comprising a fermentable carbon substrate and/or a compound of general formula II, wherein the heterologous polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1 or has at least 85% sequence identity to SEQ ID NO: 1 and has tyrosine ammonia lyase activity, and wherein hydroxycinnamic acid of general formula I is measured and/or recovered.
Recombinant host-cell PAD activity reduction
In the recombinant host-cell embodiments, the genetically modified recombinant host cell is configured such that it does not express a polypeptide with phenolic acid decarboxylase (PAD) activity.
Higher-sequence-identity TAL polypeptide and restricted hydroxycinnamic acid substitution patterns in bacterium or yeast
A method producing a hydroxycinnamic acid of general formula I by contacting a recombinant host cell (a bacterium or yeast) genetically modified to express a heterologous polypeptide with tyrosine ammonia lyase activity, wherein the heterologous polypeptide comprises SEQ ID NO: 1 or has at least 95% sequence identity to SEQ ID NO: 1, and wherein each of R1, R3, and R4 is hydrogen with R2 hydroxyl, or alternatively R1 is methoxyl (—OCH3), R2 is hydroxyl (—OH), and R3 and R4 are hydrogen (—H).
Overall, the claim set covers producing hydroxycinnamic acids of general formula I from general formula II using tyrosine ammonia lyase polypeptides defined by SEQ ID NO: 1 and sequence-identity thresholds, including embodiments using genetically modified recombinant host cells expressing heterologous TAL. The host-cell claims additionally include constraints related to phenolic acid decarboxylase (PAD) activity exclusion and restrict the TAL polypeptide sequence identity (e.g., at least 95% identity) together with defined R-group substitution patterns and host type (bacterium or yeast).
Stated Advantages
Higher tyrosine specificity relative to phenylalanine activity (low PAL/phenylalanine activity) is described.
Reduces degradation or byproducts by inactivating phenolic acid decarboxylase (PAD) in engineered host cells.
Performance improvements are reported across expression in multiple recombinant host systems.
Documented Applications
Production of hydroxycinnamic acids including p-coumaric acid (pHCA) via deamination of tyrosine derivatives using TAL polypeptides.
Production of caffeic acid from tyrosine derivatives such as L-dopa using TAL activity.
Production of ferulic acid from tyrosine derivatives such as 3-O-methyldopa using TAL activity.
Demonstrated use in recombinant host cells including E. coli, Saccharomyces cerevisiae, Lactococcus lactis, and Bacillus subtilis for TAL-based deamination to hydroxycinnamic acids.
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