Ergothioneine-producing engineered bacteria and construction method and use thereof

A construction method of ergothioneine-producing engineered bacteria, comprising:

• 1) integrating a constitutive promoter PrrnD and an ergothioneine-synthesizing gene cluster MnegtABCDE derived from Mycolicibacterium neoaurum into a genome of Escherichia coli BL21 (DE3) as a starting strain,
• wherein the step 1) is as follows:
• subjecting the ergothioneine-synthesizing gene cluster MnegtABCDE derived from the Mycolicibacterium neoaurum to codon optimization for the Escherichia coli BL21 (DE3) to produce an optimized sequence, adding the constitutive promoter PrrnD preceding the optimized sequence to produce a MnegtABCDE gene cluster fragment carrying the constitutive promoter PrrnD, and integrating the MnegtABCDE gene cluster fragment to a motA locus in a genome of Escherichia coli BL21 (DE3), wherein a sgRNA primer for the motA locus is set forth in SEQ ID NO:
• 9;
• the optimized sequence produced after the codon optimization for the Escherichia coli is set forth in SEQ ID NO:
• 1, and the sequence of the constitutive promoter PrrnD is set forth in SEQ ID NO:
• 2;
• and
• 2) mutating glutamate at a position 271 in an ATP phosphoribosyltransferase HisG gene of a strain produced in the step 1) into lysine to produce the ergothioneine-producing engineered bacteria,
• wherein the step 2) is as follows:
• 2-1) with the genome of the Escherichia coli BL21 (DE3) as a template, conducting polymerase chain reaction (PCR) amplification to produce a hisG gene, wherein primers adopted for the PCR amplification are a primer hisG-F shown in SEQ ID NO:
• 12 and a primer hisG-R shown in SEQ ID NO:
• 13;
• 2-2) ligating the hisG gene to a linearized vector pET-24a(+) through a recombination reaction to produce a recombinant vector pET-24a(+)-hisG;
• and conducting whole-plasmid PCR with a primer hisGE271K-F shown in SEQ ID NO:
• 16 and a primer hisGE271K-R shown in SEQ ID NO:
• 17 to produce a recombinant vector pET-24a(+)-hisGE271K;
• 2-3) with the recombinant vector pET-24a(+)-hisGE271K as a template, conducting PCR amplification to produce a hisGE271K gene fragment;
• 2-4) with a pTarget plasmid as a template, constructing a pTargetF-sghisG plasmid targeting the hisG gene;
• 2-5) electrotransforming a pCas plasmid carrying a transporter gene into the strain obtained in the step 1) to produce an engineered bacteria EGT01/pCas, then further preparing to produce an engineered bacteria EGT01/pCas electrocompetent cell, and electrotransforming the hisGE271K gene fragment and the pTargetF-sghisG plasmid simultaneously into the engineered bacteria EGT01/pCas electrocompetent cell to produce Escherichia coli EGT01-hisGE271K/pCas/pTargetF-sghisG;
• and
• 2-6) subjecting the Escherichia coli EGT01-hisGE271K/pCas/pTargetF-sghisG to a screening culture to produce Escherichia coli EGT01-hisGE271K with the pTargetF-sghisG plasmid and the pCas plasmid removed, wherein the Escherichia coli EGT01-hisGE271K is the ergothioneine-producing engineered bacteria.