Optimization of heterologous production of Bacillus ligniniphilus L1 laccase in Escherichia coli through statistical design of experiments

Abstract

Laccases are powerful multi-copper oxidoreductases that have wide applicability as "green" biocatalysts in biotechnological, bioremediation, and industrial applications. Sustainable production of large amounts of functional laccases from original sources is limited by low yields, difficulties in purification, slow growth of the organisms, and high cost of production. Harnessing the full potential of these versatile biocatalysts will require the development of efficient heterologous systems that allow high-yield, scalable, and cost-effective production. We previously cloned a temperature- and pH-stable laccase from Bacillus ligniniphilus L1 (L1-lacc) that demonstrated remarkable activity in the oxidation of lignin and delignification for bioethanol production. However, L1-lacc is limited by low enzyme yields in both the source organism and heterologous systems. Here, to improve production yields and lower the cost of production, we optimized the recombinant E. coli BL21 strain for high-level production of L1-lacc. Several culture medium components and fermentation parameters were optimized using one-factor-at-a-time (OFAT) and Plackett-Burman design (PBD) to screen for important factors that were then optimized using response surface methodology (RSM) and an orthogonal design. The optimized medium composition had compound nitrogen (15.6 g/L), glucose (21.5 g/L), K₂HPO₄ (0.15 g/L), MgSO₄ (1 g/L), and NaCl (7.5 g/L), which allowed a 3.3-fold yield improvement while subsequent optimization of eight fermentation parameters achieved further improvements to a final volumetric activity titer of 5.94 U/mL in 24 h. This represents a 7-fold yield increase compared to the initial medium and fermentation conditions. This work presents statistically guided optimization strategies for improving heterologous production of a bacterial laccase that resulted in a high-yielding, cost-efficient production system for an enzyme with promising applications in lignin valorization, biomass processing, and generation of novel composite thermoplastics.

Keywords: Laccase; Medium optimization; Orthogonal array design; Process parameters; Response surface methodology; Statistical optimization.