Engineered strains of Lactococcus lactis envisioning to deliver exogenous β-galactosidases for relieving lactose intolerance symptoms

Lactose intolerance (LI) is a condition affecting many adults. This condition is triggered by the physiological decline of lactase expression in adulthood, which results in a range of gastrointestinal symptoms following the ingestion of lactose-containing foods. Despite the beneficial effect of probiotic and prebiotic preparations, dairy avoidance remains the most widely adopted strategy for treating lactose intolerance. However, intolerant individuals often report a poor life quality associated with dairy avoidance and the occurrence of occasional gastrointestinal symptoms. Lactase replacement strategies based on enzyme supplemented through tablets also exist, but gastric inactivation limits their efficacy. The development of preparations effective in alleviating intolerance symptoms upon dairy products ingestion could result in a significant life quality improvement in intolerant individuals. In recent years, studies highlighted the potential of Lactococcus lactis subspecies lactis for delivering therapeutic molecules in humans. In this work, we constructed a vector for the expression of heterologous β-galactosidases in the NZ9000 L. lactis to employ for treating lactose intolerance. We selected three galactosidases from different microorganisms based on their reported pH and temperature profiles of activity. After this first selection step, we cloned the galactosidases encoding genes under a nisin-inducible promoter and evaluated their activity in different environmental conditions. Among the selected galactosidases, the one encoded by the LacZ gene of L. bulgaricus showed the most promising results. The L. bulgaricus enzyme retained a good activity at 37°C and within a pH range typical of the small intestine. Although affected by intestinal fluids concentrations, this enzyme showed significant activity in this latter. Moreover, the enzyme remained stable for up to 90 minutes in simulated intestinal fluids. These observations allowed us speculating that the galactosidase from L. bulgaricus could perform well during the entire transit throughout the small intestine. Additionally, intestinal fluids seem to trigger the lysis of the transiting L. lactis, promoting the release of the intracellular proteins, including the galactosidase of interest. While these data were promising, some critical aspects need to be evaluated. Firstly, the expression system was designed to allow the extracellular exportation of the galactosidase. However, the activity detectable in the extracellular compartment was limited. The employment of the 5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) instead of lactose as a substrate to assess the enzyme activity has several advantages, but the findings (enzyme activity and activity in different environmental conditions) need to be confirmed in presence of lactose as substrate of the reaction catalyzed by beta-galactosidases. Lastly, the evidence presented here come entirely from in vitro experiments. To corroborate these results, we will carry out additional experiments using lactose as a substrate. We will also optimize the secretion efficiency by employing a modified L. lactis lacking the HtrA protease. Assuming positive results, we will test the effect of this modified L. lactis on a mice model of lactose intolerance and eventually implement a food-grade system for a human application