Recombinant microbial system for directed evolution of glycocins and method of preparation thereof

A method for constructing a recombinant microbial system the method comprising:

• a. mutating the nucleotide sequence of a pRSFDuet-1vector by site directed mutagenesis using a primer having sequence as set forth in SEQ ID NO:
• 12 to obtain a vector 1, wherein the vector 1 is pRSFDuet-1SapI vector;
• b. amplifying and sub cloning a polynucleotide sequence having the sequence selected from the group consisting of SEQ ID NO:
• 63, SEQ ID NO:
• 64 and SEQ ID NO:
• 65 encoding a glycosyltransferase using primers in the vector 1 of step (a) to obtain a vector 2;
• c. amplifying and sub cloning an acceptor substrate gene having a polynucleotide sequence selected from the group consisting of SEQ ID NO:
• 60, SEQ ID NO:
• 61 and SEQ ID NO:
• 62 using primers in the vector 2 of step (b) to obtain a vector 3;
• d. amplifying and subcloning a nucleotide sequence encoding a fusion tag MC from a vector 4 using primers in the vector 3 of step (c) to obtain a vector 5;
• e. extending the fusion tag MC in the vector 5 of step (d) using primers to obtain a vector 6;
• f. amplifying and sub cloning an acceptor substrate gene having a polynucleotide sequence selected from the group consisting of SEQ ID NO:
• 60, SEQ ID NO:
• 61 and SEQ ID NO:
• 62 using primers in NcoI and SapI sites of the vector 6 of step (e) to generate a vector comprising a gene cassette A, the gene cassette A having a polynucleotide sequence as set forth in a sequence selected from the group consisting of SEQ ID NO:
• 1, SEQ ID NO:
• 2, SEQ ID NO:
• 3, SEQ ID NO:
• 4 and SEQ ID NO:
• 5;
• and
• g. transforming the vector comprising the gene cassette A of step (f) in an E. coli to obtain the recombinant microbial system.