Plant chloroplasts enable high-yield and cost-effective production of the engineered bacterial enzyme CueO-MacHis as a technical protein via transient expression in Nicotiana benthamiana.

INTRODUCTION: Laccases are versatile oxidoreductases found in bacteria, fungi, and plants., which have a wide range of industrial applications, including the removal of pharmaceutical micropollutants from wastewater. Bacterial laccases are particularly attractive because of their high catalytic activity and stability over broad temperature and pH ranges. One example is the copper efflux oxidase (CueO) from Escherichia coli, which has been fused to the adhesion-promoting peptide Macaque Histatin (MacHis) because immobilized enzymes have proven especially effective for solid-phase wastewater treatment. However, the practical application of this enzyme is constrained by the relatively low production yields of 25-50 mg/L when expressed in recombinant E. coli or Pichia pastoris. METHODS AND RESULTS: Here, we used transient expression in Nicotiana benthamiana to demonstrate that the engineered CueO-MacHis can be produced at substantially higher levels, reaching 250-320 (286 ± 35) mg/kg biomass in the prokaryotic-like environment of chloroplasts in 6-week-old plants, which are typically used for production purposes. Subsequent systematic optimization of the transient expression process based on statistical experimental designs increased the accumulation of recombinant CueO-MacHis approximately threefold to 846 ± 57 mg/kg biomass. Furthermore, we established a simplified and cost-effective purification strategy that leverages the enzyme's temperature and pH stability to clarify extracts before anion exchange chromatography, achieving 76% recovery and 92% purity. Moreover, the plant-derived CueO-MacHis had a specific activity of 7.22 ± 1.55 U/mg, which is in the same range as that reported for other CueO enzymes. DISCUSSION: Our approach paves the way for the large-scale production of CueO-MacHis as a technical enzyme for industrial applications.