Production of three oligosaccharide-metabolizing enzymes by reconstituted <italic>Pichia pastoris</italic> and its effect on honey bees

<p indent="0mm">Due to self-incompatibility, <italic>Camellia</italic>’s pollination efficiency is low. When honey bees are introduced for pollination, the raffinose oligosaccharides in the nectar induce bee poisoning. The purpose of this study is to reconstruct <italic>Pichia pastoris</italic> to express three key enzymes of galactose metabolism (GALK, GALT, GALE) in <italic>Andrena camellia</italic> to explore their effect on the detoxification ability of oligosaccharides in honeybees. The pPIC9K-GALK/GALT/GALE expression vector was constructed using plasmid recombination technology, which was then electrotransformed into <italic>Pichia pastoris</italic> GS115 and induced to produce the recombinant enzyme. Protein expression was verified by Western blot. In <italic>in vitro</italic> experiments, we fed honey bees sugar water supplemented with the recombinant enzymes. We then evaluated the metabolic efficiency of the enzymes in galactose, the survival rate, and the median survival of the bees. The results showed that the efficient expression of GALK, GALT, and GALE in <italic>P. pastoris</italic> was successfully achieved. In <sc><italic>in vitro</italic> </sc>digestion experiments, the combination of three enzymes could significantly reduce the content of galactose (<italic>P</italic>​&lt;0.05), and we detected Leloir pathway metabolites (such as UDP-glucose). The feeding experiment showed that the median survival of the worker bees supplemented with three enzymes was 34.43% longer than that of the control group (<italic>P</italic>​&lt;0.05). GALK, GALT, and GALE expressed by <italic>Pichia pastoris</italic> can effectively metabolize galactose in <italic>Camellia oleifera</italic> oligosaccharides, significantly alleviating the symptoms of bee poisoning. This study provides technical support for improving honey bee metabolism using the bioengineering method, which is of great significance for promoting the sustainable development of the <italic>Camellia oleifera</italic> industry.