Microbial ergothioneine biosynthesis

Abstract

Disclosed are methods for ergothioneine biosynthesis. More particularly, the present disclosure relates to methods for microbial ergothioneine biosynthesis.

19 . An engineered host cell for the production of ergothioneine, wherein the host cell comprises:
 a nucleotide sequence encoding a heterologous EgtB comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 2,   a nucleotide sequence encoding a heterologous EgtC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 4,   a nucleotide sequence encoding a heterologous EgtD comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 6, and   a nucleotide sequence encoding a heterologous EgtE comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 8;   wherein the heterologous oxidoreductase (EgtB), the heterologous amidohydrolase (EgtC), the heterologous histidine methyltransferase (EgtD), and the heterologous pyridoxal 5-phosphate binding protein (EgtE) are the only heterologous proteins involved in the production of ergothioneine.   
     
     
         20 . The engineered host cell of  claim 19 , wherein the heterologous EgtB comprises the amino acid sequence of SEQ ID NO:2. 
     
     
         21 . The engineered host cell of  claim 19 , wherein the heterologous EgtC comprises the amino acid sequence of SEQ ID NO:4. 
     
     
         22 . The engineered host cell of  claim 19 , wherein the heterologous EgtD comprises the amino acid sequence of SEQ ID NO:6. 
     
     
         23 . The engineered host cell of  claim 19 , wherein the heterologous EgtE comprises the amino acid sequence of SEQ ID NO:8. 
     
     
         24 . The engineered host cell of  claim 19 , wherein the heterologous EgtB comprises the amino acid sequence of SEQ ID NO:2, the heterologous EgtC comprises the amino acid sequence of SEQ ID NO:4, the heterologous EgtD comprises the amino acid sequence of SEQ ID NO:6, and the heterologous EgtE comprises the amino acid sequence of SEQ ID NO:8. 
     
     
         25 . The engineered host cell of  claim 19 , wherein the host cell is selected from the group consisting of a bacterial cell and a yeast cell. 
     
     
         26 . The engineered host cell of  claim 25 , wherein the host cell is an Escherichia coli cell. 
     
     
         27 . The engineered host cell of  claim 25 , wherein the host cell is selected from the group consisting of a Saccharomyces cell and a Pichia cell. 
     
     
         28 . The engineered host cell of  claim 27 , wherein the host cell is a Saccharomyces cerevisiae cell. 
     
     
         29 . The engineered host cell of  claim 27 , wherein the host cell is a Pichia pastoris cell. 
     
     
         30 . The engineered host cell of  claim 19 , wherein the nucleotide sequence encoding a heterologous EgtB, the nucleotide sequence encoding a heterologous EgtC, the nucleotide sequence encoding a heterologous EgtD, and the nucleotide sequence encoding a heterologous EgtE are on a single vector. 
     
     
         31 . The engineered host cell of  claim 19 , wherein the nucleotide sequence encoding a heterologous EgtB, the nucleotide sequence encoding a heterologous EgtC, the nucleotide sequence encoding a heterologous EgtD, and the nucleotide sequence encoding a heterologous EgtE are on more than one vectors. 
     
     
         32 . A method for producing ergothioneine, the method comprising:
 culturing the host cell of  claim 19 ,   inducing the host cell to express the heterologous EgtB, the heterologous EgtC, the heterologous EgtD and the heterologous EgtE; and   collecting the ergothioneine.   
     
     
         33 . The method of  claim 32 , wherein a substrate selected from the group consisting of histidine, methionine, cysteine, γ-glutamyl cysteine and combinations thereof is added to the culture. 
     
     
         34 . The method of  claim 32 , wherein iron (II) is added to the culture. 
     
     
         35 . The method of  claim 32 , wherein the host cell is an  E. coli  cell. 
     
     
         36 . The method of  claim 32 , wherein the host cell is a  Saccharomyces cerevisiae  cell. 
     
     
         37 . The method of  claim 32 , wherein the host cell is a  Pichia pastoris  cell. 
     
     
         38 . The method of  claim 32 , wherein the heterologous EgtB comprises the amino acid sequence of SEQ ID NO: 2, the heterologous EgtC comprises the amino acid sequence of SEQ ID NO: 4, the heterologous EgtD comprises the amino acid sequence of SEQ ID NO: 6, and the heterologous EgtE comprises the amino acid sequence of SEQ ID NO: 8.