Production of Novel Enzymes and Peptides Using a Synthetic Biology Approach in Lactococcus lactis

Abstract

Lactococcus lactis is a lactic acid bacterium that is widely used in food fermentation and it is a good candidate for heterologous gene expression. One exciting possibility is the use of this bacterium to produce novel enzymes or peptides which have some beneficial effects on nutritional characteristics of different foods. Galactooligosaccharides (GOS) are novel prebiotic ingredients that are proposed to be fermented by beneficial bacteria in the large intestine. The production of GOS could be achieved by using β-galactosidase from various sources via transgalactosylation activity. According to the literature, the highest production of GOS was obtained by using a thermophilic β-galactosidase enzyme from Sulfolobus solfataricus, which allowed GOS to be produced at a high-temperature condition. To produce this enzyme efficiently, a synthetic lacSt gene was designed and codon optimized using the preferred codon of L. lactis. The optimized gene was subsequently cloned into a two-component regulatory system. The thermophilic β-galactosidase enzyme was successfully expressed in L. lactis. Permeabilized whole cell enzyme enabled a high yield of 197g/L GOS, including both tri-saccharides and tetra-saccharides, with 400g/L initial lactose at pH 6 and 85 °C. To make it more suitable for food application, the whole cell enzyme was immobilized in chitosan. This facilitated efficient GOS production as well as the easy separation. More than 60% of lactose in a 5% initial lactose solution was converted into GOS at pH 5.5 and 90 °C. Bisin is a broad-spectrum lantibiotic produced by Bifidobacterium longum DJO10A. Attempts have been made for lantibiotic isolation and purification. However, it was found that no production occurred in broth media and only a little was observed in agar media. Therefore, L. lactis was used as an alternative host to express genes which were predicted to be involved in bisin production and immunity bypassing the tightly controlled regulatory system. Genes were codon optimized and then cloned into L. lactis. Although no antimicrobial activity was detected, peptide fragments from lan-gene were successfully expressed, as confirmed by LC-MS. Therefore, L. lactis can be used as a production system and help to identify the proper conditions for bioactive lantibiotic production.