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-11306331-B2
Publication Date
2022-04-19
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 disclosure describes biotechnology processes for producing a hydroxycinnamic acid of general formula I by contacting a recombinant host cell genetically modified to express a heterologous polypeptide having tyrosine ammonia lyase activity. The recombinant host cell is used in a medium comprising a fermentable carbon substrate and/or a compound of general formula II to produce the hydroxycinnamic acid of general formula I.
The heterologous polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 3, or has at least 80% to at least 85% sequence identity to SEQ ID NO: 2 or SEQ ID NO: 3. The produced hydroxycinnamic acid of general formula I has defined substitution patterns for R1, R2, R3, and R4, where R2 is hydroxyl and R1/R3/R4 are each hydrogen, or R1 is methoxyl or hydroxyl with R3 and R4 being hydrogen.
The disclosure further covers producing the hydroxycinnamic acid of general formula I by contacting a compound of general formula II with a polypeptide to produce the hydroxycinnamic acid, followed by measuring the concentration of the hydroxycinnamic acid or recovering the hydroxycinnamic acid. In recombinant-host embodiments, the disclosure emphasizes controlling host metabolism by using recombinant host cells lacking or having inactivated phenolic acid decarboxylase activity to prevent degradation or byproducts, while maintaining TAL activity for transforming tyrosine derivatives into hydroxycinnamic acids including p-coumaric acid, caffeic acid, and ferulic acid.
Claims Coverage
The partial content includes three independent claims. Each independent claim is directed to producing a hydroxycinnamic acid of general formula I with defined substitution patterns using tyrosine ammonia lyase activity from SEQ ID NO: 2/SEQ ID NO: 3 polypeptides or sequence-identity variants, with one claim additionally limited to recombinant host cell embodiments and host type.
Recombinant host producing hydroxycinnamic acid from general formula II using TAL polypeptide
Contacting a recombinant host cell genetically modified to express a heterologous polypeptide with tyrosine ammonia lyase activity in a medium comprising a fermentable carbon substrate and/or a compound of general formula II to produce a hydroxycinnamic acid of general formula I, wherein the heterologous polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 2 or 3 or has at least 85% sequence identity to SEQ ID NO: 2 or 3, and wherein the recombinant host cell is a bacterium or yeast.
Contacting general formula II with TAL polypeptide and measuring or recovering product
Contacting a compound of general formula II with a polypeptide having tyrosine ammonia lyase activity to produce a hydroxycinnamic acid of general formula I, wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 2 or 3 or has at least 80% sequence identity to SEQ ID NO: 2 or 3, followed by measuring the concentration of the hydroxycinnamic acid or recovering the hydroxycinnamic acid.
Recombinant host fermentation with TAL polypeptide and measuring or recovering product
Contacting a recombinant host cell genetically modified to express a heterologous polypeptide with tyrosine ammonia lyase activity in a fermentation medium comprising a fermentable carbon substrate and/or a compound of general formula II to produce a hydroxycinnamic acid of general formula I, wherein the heterologous polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 2 or 3 or has at least 80% sequence identity to SEQ ID NO: 2 or 3, followed by measuring the concentration of the hydroxycinnamic acid or recovering the hydroxycinnamic acid.
Across the independent claims, coverage centers on producing hydroxycinnamic acid of general formula I by using tyrosine ammonia lyase activity from SEQ ID NO: 2/SEQ ID NO: 3 polypeptides or sequence-identity variants, either by contact of general formula II with a TAL polypeptide followed by measurement and/or recovery, or by fermentation with recombinant host cells expressing the heterologous TAL polypeptide.
Stated Advantages
Produces hydroxycinnamic acids including p-coumaric acid, caffeic acid, and ferulic acid using TAL polypeptides with tyrosine specificity as described in the disclosure.
Reduces degradation and byproducts by using recombinant host cells lacking or having inactivated phenolic acid decarboxylase activity.
Supports enhanced pHCA production and increased yields as described in experimental data in engineered hosts.
Documented Applications
Production of p-coumaric acid from tyrosine derivatives using TAL polypeptides, including embodiments where phenolic acid decarboxylase pathways are disrupted.
Production of caffeic acid and ferulic acid by TAL activity on tyrosine derivatives such as L-dopa and 3-O-methyldopa.
Use of recombinant host cells expressing heterologous TAL polypeptides for producing hydroxycinnamic acids of general formula I, with host examples including bacteria and yeast.
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