Construction method of double immobilized recombinant yeast engineering bacteria, engineering bacteria, catalyst and application in synthesis of rebaudioside

A construction method of double immobilized recombinant yeast engineering bacteria, comprising the steps of:

• 1) constructing a multi-enzyme complex immobilization module:
• linking a fragment 1 and a fragment 2 to a plasmid Episomal Saccharomyces cerevisiae-URA3 gene (PESC-URA) vector to obtain a recombinant PESC-URA vector;
• linking a fragment 3 to a plasmid RS424 (PRS424) vector to obtain a recombinant plasmid RS424 vector, wherein the fragment 1 is a surface display scaffold protein expression cassette comprised by a signal peptide, a yeast cell wall anchor protein, and an adhesion domain Cohesion;
• the fragment 2 is a uridine piphosphate glucose (UDPG) in-situ regeneration system expression cassette comprised by a signal peptide, a yeast cell wall anchor protein, and sucrose synthase;
• and the fragment 3 is a multi-enzyme complex expression cassette comprised by promoters, a signal peptide, and an anchor domain Dockerin carrying enzyme subunits;
• 2) constructing a bacterial vector-free self-immobilization module:
• integrating a fragment 4 into a yeast genome, wherein the fragment 4 is a vector-free self-immobilization expression cassette comprised by a promoter and a flocculation gene;
• and
• 3) transformation:
• transferring the recombinant PESC-URA vector and the recombinant PRS424 vector into yeast cells to complete the construction of double immobilized recombinant yeast engineering bacteria.