Improving Stress Resistance Of Lactococcus Lactis By Using Metabolic Engineering Approaches

Lactococcus lactis is widely used in the manufacture of cheese and other fermented dairy products. In addition to its traditional applications, L. lactis has been developed as a Gram-positive gene expression host and extensively used in the field of metabolic engineering. However, low final cell density due to medium acidification hampers the application of L. lactis to produce heterologous proteins. Moreover, the relatively poor oxygen-tolerance of L. lactis also affects its robustness that is desirable for industrial operation. Therefore, engineering lactococcal strain with improved acid stress and oxidative stress resistance will contribute to the development of using L. lactis as a starter culture and cell factory. However, to the author's knowledge, there is no correlative research in our country, and lactic acid bacteria researchers abroad have made little attempt to develop acid stress and oxidative stress resistant strains of L. lactis.L. lactis ssp. cremoris NZ9000 which is incapable of both glutathione (γ-GluCysGly, reduced form GSH) uptake and biosynthesis, and L. lactis ssp. cremoris SK11 which is capable of GSH uptake, were used in this study. By simply introducing heterologous microbial transglutaminase activity into L. lactis NZ9000 and introducing GSH synthetic capability into L. lactis SK11, the acid stress and oxidative stress resistance of both L. lactis strains were greatly increased, resulting in significantly improved growth performances. The possible mechanisms were also investigated. The main results of this dissertation were described as following:1. The leaky expression of the mtg gene in L. lactis NZ9000 significantly enhanced the aerobic growth yield of the host. When grown aerobically in CM medium (non-pH buffer-contained) with the pH controlled at 6.5±0.1, the maximal biomass of strain NZ9000(pFL010) reached 4.73 g·L-1, which was 1.8-fold that of the control, strain NZ9000(pNZ8148). In addition, the efficiency of biomass synthesis relative to glucose consumption (Yx/s) of strain NZ9000(pFL010), 71.7 g of biomass per mol of glucose, was 1.6-fold higher than that of strain NZ9000(pNZ8148). When grown aerobically in CM under non-pH-controlled conditions, the maximal biomass of strain NZ9000(pFL010) reached 4.13 g·L-1, which was 12.1-fold that of strain NZ9000(pNZ8148). Moreover, the Yx/s of strain NZ9000(pFL010) (72.9 g of biomass per mol of glucose) increased 4.4-fold.2. The Leaky expression of the mtg gene in L. lactis NZ9000 exerted great influences on the host cell's physiology. First, strain NZ9000(pFL010) exhibited a thickened cell wall with respect to strain NZ9000(pNZ8148), which contributes to the nisin resistance and the non-inducible effect of nisin on the expression of the mtg gene that was translationally fused to the nisA promoter.