Biofilm-forming bacteria are sources of infections because they are often resistant to antibiotics and chemical removal. Recombinant biofilm-degrading enzymes have the potential to remove biofilms gently, but they can be toxic toward microbial hosts and are therefore difficult to produce in bacteria. Here, we investigated Nicotiana species for the production of such enzymes using the dispersin B-like enzyme Lysobacter gummosus glyco 2 (Lg2) as a model. We first optimized transient Lg2 expression in plant cell packs using different subcellular targeting methods. We found that expression levels were transferable to differentiated plants, facilitating the scale-up of production. Our process yielded 20 mg kg⁻¹ Lg2 in extracts but 0.3 mg kg⁻¹ after purification, limited by losses during depth filtration. Next, we established an experimental biofilm assay to screen enzymes for degrading activity using different Bacillus subtilis strains. We then tested complex and chemically defined growth media for reproducible biofilm formation before converting the assay to an automated high-throughput screening format. Finally, we quantified the biofilm-degrading activity of Lg2 in comparison with commercial enzymes against our experimental biofilms, indicating that crude extracts can be screened directly. This ability will allow us to combine high-throughput expression in plant cell packs with automated activity screening.

形成生物膜的细菌是感染源，因为它们通常对抗生素和化学去除剂具有抗药性。重组生物膜降解酶具有温和去除生物膜的潜力，但它们对微生物宿主有毒，因此难以在细菌中产生。在这里，我们调查了烟草 使用分散素 B 样酶生产此类酶的物种 胶溶菌糖 2 (Lg2) 作为模型。我们首先使用不同的亚细胞靶向方法优化了植物细胞包中的瞬时 Lg2 表达。我们发现表达水平可以转移到分化的植物，促进生产的扩大。我们的过程在提取物中产生了 20 mg kg ^-1 Lg2，但在纯化后产生了0.3 mg kg ^-1，受限于深度过滤过程中的损失。接下来，我们建立了一个实验性生物膜测定法来筛选酶的降解活性，使用不同的枯草芽孢杆菌菌株。然后，我们测试了复杂的化学成分确定的生长培养基，在将测定转换为自动高通量筛选格式之前，可重现生物膜的形成。最后，我们量化了 Lg2 与商业酶对我们的实验生物膜的生物膜降解活性，表明可以直接筛选粗提取物。这种能力将使我们能够将植物细胞组中的高通量表达与自动活性筛选相结合。