Engineering Lactococcus cremoris strains co-expressing two cellulase genes for growth on cellulose

Lactic acid bacteria are commonly used to convert carbohydrates into valuable products like lactic acid, but they currently rely on carbon sources from food crops, raising sustainability and cost concerns. Lignocellulosic waste is a more sustainable alternative, yet these bacteria cannot naturally break down cellulose to grow on it. Efficient cellulose degradation requires synergetic action of multiple cellulolytic enzymes, each operating through distinct mechanisms.

To design genetic constructs for co-expression of gene pairs encoding different heterologous cellulases (Cel5I, Cel5H or Cel9A) from separate expression cassettes in Lactococcus cremoris, the BglBrick approach was used. The activity of culture supernatants containing co-expressed cellulases was assessed on microcrystalline cellulose and phosphoric acid swollen cellulose (PASC), and compared with that of co-cultures and cultures expressing individual enzymes. Subsequently, growth of selected developed strains on PASC was evaluated.

The A1 strain that co-produced cellulase pair Cel5H-Cel9A achieved the highest activity on PASC and one of the highest activities measured on microcrystalline cellulose. Additionally, the A1 strain showed the highest degradation of PASC during the 16 days of anaerobic cultivation and one of the highest lactic acid production levels.

In this study, co-expression of gene pairs that encode different heterologous cellulases from separate promoters in L. cremoris was achieved for the first time. This approach allows each cellulase to be tuned independently, enabling optimization of their expression levels to maximize synergistic effects and enhance the cellulolytic performance of the engineered bacteria.