Development of cellulose-degrading lactic acid bacterium Lactococcus cremoris by genetic engineering

Cellulose is one of the most abundant potential sources of carbon for sustainable microbial production of biochemicals. Lactic acid bacteria that produce a range of valuable metabolites are generally unable to grow on cellulose. Here, we aimed to develop the lactic acid bacteria Lactococcus cremoris for growth on cellulosic substrate. Genes encoding cellulases Cel5I, Cel9A and Cel5H from different cellulolytic bacteria were introduced into L. cremoris NZ9000 strain. The genes were designed for constitutive expression, with the produced cellulases being either secreted or displayed on the cell surface. Four promoters for cellulase gene expression and two noncovalent anchors for surface display of cellulases were evaluated using immunoblotting, confocal microscopy and flow cytometry. With the most effective promoter PepN, the cells secreted milligram amounts of cellulases per liter culture. The highest cellulolytic activity on amorphous (phosphoric-acid swollen cellulose, PASC) and microcrystalline (Avicel) cellulose was observed for the secreted Cel5H and Cel5I cellulases, respectively. The major cellodextrin produced by Cel5H and Cel5I was cellobiose, as determined by high performance anion exchange chromatography. The strains secreting cellulases outperformed the corresponding strains displaying the same cellulase on the surface in growth on PASC as the primary carbon source. The fastest growth was observed for the strain secreting Cel9A, followed by strain secreting Cel5H. By demonstrating growth of lactic acid bacteria expressing heterologous cellulases on PASC as a major carbon source for the first time, this study presents an important step towards the realization of consolidated bioprocesses involving lactic acid bacteria.