Heterologous gene expression and secretion in Bifidobacterium longum

Strains of the genus Bifidobacterium are used in fermented dairy products where they are supposed to exert health-promoting effects on the consumers. To study Bifidobacterium sp. properties, to improve the strains or to use them for safe delivery of vaccinal or anticarcinogenic polypeptides into the human digestive tract, it is strategically important to develop a system for heterologous expression and secretion. For this purpose, we constructed pBESAF2, a secretion vector based on the alpha-amylase expression and secretion signals (ESS) isolated from Bifidobacterium adolescentis. The signal peptide was analyzed in silico and its cleavage site was experimentally determined by N-terminal sequencing of the mature amylase. To validate this secretion vector, the phytase structural gene from Escherichia coli MC4100 was cloned downstream of the ESS and the resulting plasmid was introduced into Bifidobacterium longum MG1. Recombinant cells expressed phytase (with activity up to 21.1 U) and more than 95% of the phytase activity was found in the culture medium, suggesting that E. coli phytase can be produced and properly secreted and folded when expressed in B. longum. This is the first report of the development of a secretion vector for bifidobacteria and its use in a heterologous protein export in B. longum. This work is the first step towards the production of other proteins of food or therapeutic interest in bifidobacteria.