Protein secretion in Lactobacillus plantarum

Lactic acid bacteria (LAB) are important micro-organisms in the food and beverages industry, and have for a long time been used in the production of a wide range of foods for humans. Nowadays, LAB also receive increasing attention for applications in probiotic products because of their beneficial effects on human health. Recently, LAB attracted increasing interest as cell factories for the production of enzymes and other proteins, and particularly as mucosal delivery vehicles of therapeutic proteins, vaccines, etc. in probiotic strains for applications in veterinary and human medicine. In this project we propose an approach that is based on the use of Lactobacillus plantarum as the cell factory for the production of proteins as well as a carrier of proteins that are of interest for food and therapeutic applications. The development of a versatile expression tool box for protein secretion and bacterial surface display in lactobacilli using the concept of S-layer proteins is proposed. Combinatorial library construction of different types of signal sequences, anchor-encoding DNA fragments, promoters of the S-layer proteins from selected Lactobacillus spp.and several synthetic promoters will be developed. We have selected two proteins of great interest for food and therapeutic applications to illustrate the usefulness of our system: a -glucosidase from the thermophilic and halophilic bacterium Halothermothrix orenii (HoBGLA) and a coxsackievirus-adenovirus receptor (CAR). HoBGLA, which also possesses high ß-galactosidase activity, is a promising candidate for lactose hydrolysis and formation of galacto-oligosaccharides (GOS), which are prebiotic compounds with health-promoting effects. An economical, sustainable and intelligent use of biocatalysts can be through immobilisation, where the enzyme after extraction from the fermentation process is bound onto a suitable food-grade carrier. Our proposed approach, where the enzyme of interest is displayed on the cell surface of a food-grade organism, should result in the availability of safe, stable food-grade biocatalysts that can be used in different production processes relevant for food industry in a more efficient and sustainable way. Coxsackievirus-adenovirus receptor (CAR) is used by most human adenoviruses. Adenovirus infections, which can cause diarrhea, usually affect children or young adults. In an envisioned therapeutic application, CAR-displaying bacteria may serve as receptor traps which, upon introduction into the intestinal tract, may bind and neutralize adenoviruses and (at least partially) prevent them from infecting their host cells. CAR-displaying bacteria may be introduced as non-living bacteria from outside or may be allowed to colonize the intestinal tract and generate a constant supply of receptor traps for prolonged times. In the second part of this proposed research, the Sec translocon mediated protein secretion in L. plantarum will be investigated to provide a better understanding of the bottleneck for protein production in L. plantarum and help to explain the discrepancy between the protein yields of cytoplasmic and secretory proteins. The optimal window of transcriptional strengths, that does not overload the secretory system in L. plantarum, will be determined and potentially limiting components will be identified. 1

Lactic acid bacteria (LAB) are important micro-organisms in the food and beverages industry, and have for a long time been used in the production of a wide range of foods for humans. Nowadays, LAB also receive increasing attention for applications in probiotic products because of their beneficial effects on human health. Recently, LAB attracted increasing interest as cell factories for the production of enzymes and other proteins, and particularly as mucosal delivery vehicles of therapeutic proteins, vaccines, etc. in probiotic strains for applications in veterinary and human medicine. In this project we propose an approach that is based on the use of Lactobacillus plantarum as the cell factory for the production of proteins as well as a carrier of proteins that are of interest for food and therapeutic applications. For this purpose, a versatile expression tool box for protein secretion and bacterial surface display in lactobacilli using the concept of S-layer proteins was constructed. Combinatorial library construction of different types of signal sequences, anchor-encoding DNA fragments, promoters of the S-layer proteins from selected Lactobacillus spp. was developed. An economical, sustainable and intelligent use of biocatalysts can be through immobilisation, where the enzyme after extraction from the fermentation process is bound onto a suitable food-grade carrier. Our proposed approach, where the enzyme of interest is displayed on the cell surface of a food-grade organism, should result in the availability of safe, stable food-grade biocatalysts that can be used in different production processes relevant for food industry in a more efficient and sustainable way.