Construction of Lactococcus lactis Expression System via Chromosomal Integration of T7 RNA Polymerase for Levan Production from Fruit Pomace

Abstract

Fruit pomace recycling is critical for green biomanufacturing. Lactococcus lactis, a common host for waste valorization, shows poor protein expression in conventional systems. We constructed T7RNAP-driven inducible and constitutive expression systems in L. lactis NZ9000. The two systems increased target gene transcription by 103-fold and 207-fold, and enzyme activity by 5.24-fold and 10.36-fold, respectively. A signal peptide enabled efficient extracellular secretion of recombinant proteins. The engineered strains synthesized levansucrase and levan, achieving yields of 36.27 g/L and 27.53 g/L in sucrose medium. When using food waste substrates, the levan titer reached 8.84 g/L with molasses, and 3.2, 1.4, 0.5, 0.7, 0.5 g/L with sugar cane bagasse and various fruit peels. This work provides a reliable strategy for strain engineering, pomace reuse and low-cost large-scale levan production.