A construction method of double immobilized
recombinant yeast engineering bacteria,
engineering bacteria, a catalyst and an application in synthesis of rebaudioside. In the present invention,
yeast is used as the
chassis cell, an ordered self-assembled multi-
enzyme cascade surface display system is constructed, and carrier-free immobilization is coupled with
uridine piphosphate glucose (UDPG) in-situ regeneration technology, resulting in a novel
yeast whole cell catalyst with double immobilization of enzymes and
bacteria. The catalyst uses cheap
steviol glycoside (St) and
rebaudioside A (Reb A) as substrates, and can synthesize rebaudioside (Reb D / M) in an efficient and economical one-pot method without additional addition of expensive UDPG. The synthesis process does not require cumbersome operations including
cell disruption,
enzyme separation, purification and immobilization, and avoids
bottleneck problems in the related art including material transmembrane transport resistance,
mass transfer resistance and product
hydrolysis by
intracellular enzyme. The catalyst has
reusability, and can realize continuous and high-intensity rebaudioside
biosynthesis through strain rejuvenation, which provides strong
technical support for its industrial application.