Modified Escherichia coli having an increased expression of a CysP transporter, an ATP sulfurylase, an APS kinase and a PAP phosphatase
Inventors
Jendresen, Christian Bille • Nielsen, Alex Toftgaard
Assignees
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Publication Number
US-10954536-B2
Publication Date
2021-03-23
Expiration Date
Abstract
The present invention generally relates to the field of biotechnology as it applies to the production of aryl sulfates using recombinant host cells. More particularly, the present invention pertains to recombinant host cells comprising (e.g., expressing) a polypeptide having aryl sulfotransferase activity, wherein said recombinant host cells have been modified to have an increased uptake of sulfate compared to identical host cells that does not carry said modification. Further provided are processes for the production of aryl sulfates, such as zosteric acid, employing such recombinant host cells.
Core Innovation
The invention relates to an Escherichia coli bacterium comprising a heterologous polypeptide having an aryl sulfotransferase activity. The bacterium is modified to have increased protein expression of a CysP transporter, an ATP sulfurylase, an APS kinase, and a PAP phosphatase, and is capable of enhanced sulfation of a phenolic compound compared to an identical Escherichia coli bacterium that does not carry the modifications.
The heterologous aryl sulfotransferase polypeptide comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 1, 6, 7, 8 or 13. The CysP transporter comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 27, the ATP sulfurylase comprises polypeptides having at least 95% sequence identity with SEQ ID NO: 28 and SEQ ID NO: 29, the APS kinase comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 32, and the PAP phosphatase comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 37.
The invention is described as increasing PAPS supply and decreasing PAP to improve sulfation, including overexpression or mutation/operon modifications of cysD/cysN/cysC and cysQ. The document also describes production of sulfated phenolic compounds by contacting media with the engineered bacterium, using phenolic compounds, phenolic compound precursors, or fermentable carbon substrates converted to phenolic compounds.
Claims Coverage
The provided independent claim centers on one inventive feature set: an engineered Escherichia coli bacterium with a heterologous aryl sulfotransferase and increased expression of CysP transporter, ATP sulfurylase, APS kinase, and PAP phosphatase, defined by sequence identity to specified SEQ ID NOs and capable of enhanced sulfation of a phenolic compound. Dependent claims further define the sequence selections and a production approach using phenolic inputs or precursors.
Engineered Escherichia coli with heterologous aryl sulfotransferase and increased sulfation-related expression
An Escherichia coli bacterium comprising a heterologous polypeptide having an aryl sulfotransferase activity, wherein the bacterium has been modified to have increased protein expression of a CysP transporter, an ATP sulfurylase, an APS kinase and a PAP phosphatase compared to an identical bacterium that does not carry said modifications, and wherein the bacterium is capable of enhanced sulfation of a phenolic compound compared to the identical bacterium.
Aryl sulfotransferase sequence identity at least 95% with specified SEQ ID NOs
The heterologous polypeptide having an aryl sulfotransferase activity comprises an amino acid sequence which has at least 95% sequence identity with SEQ ID NO: 1, 6, 7, 8 or 13.
CysP transporter sequence identity at least 95% with SEQ ID NO: 27
The CysP transporter comprises an amino acid sequence which has at least 95% sequence identity with SEQ ID NO: 27.
ATP sulfurylase defined by SEQ ID NO: 28 and 29
The ATP sulfurylase comprises i) a polypeptide comprising an amino acid sequence which has at least 95% sequence identity with SEQ ID NO: 28, and ii) a polypeptide comprising an amino acid sequence which has at least 95% sequence identity with SEQ ID NO: 29.
APS kinase sequence identity at least 95% with SEQ ID NO: 32
The APS kinase comprises an amino acid sequence which has at least 95% sequence identity with SEQ ID NO: 32.
PAP phosphatase sequence identity at least 95% with SEQ ID NO: 37
The PAP phosphatase comprises an amino acid sequence which has at least 95% sequence identity with SEQ ID NO: 37.
Sulfated phenolic compound production by contacting with bacterium
A process produces a sulfated phenolic compound by contacting a bacterium with a phenolic compound, a fermentable carbon substrate that the bacterium converts to a phenolic compound, or a phenolic compound precursor that the bacterium converts to the phenolic compound.
The claim coverage centers on engineered Escherichia coli that co-expresses a heterologous aryl sulfotransferase and increased PAPS-pathway components, with sequence identity constraints tied to specified SEQ ID NOs. It also covers production of sulfated phenolic compounds by contacting media with the bacterium using phenolic inputs and/or phenolic precursors.
Stated Advantages
Enhanced sulfation of a phenolic compound compared to an identical Escherichia coli bacterium that does not have the increased expression modifications.
Documented Applications
Production of sulfated phenolic compounds by contacting a medium with the engineered bacterium using phenolic compound inputs, phenolic precursor inputs, or fermentable carbon substrates that the bacterium converts to phenolic compounds.
Production of sulfated phenolic products such as zosteric acid.
Sulfation of phenolic compounds such as p-coumaric acid and resveratrol.
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