Enzyme clustering can improve catalytic efficiency by facilitating the processing of intermediates. Functional membrane microdomains (FMMs) in bacteria can provide a platform for enzyme clustering. However, the amount of FMMs at the cell basal level is still facing great challenges in multi-enzyme immobilization. Here, using the nutraceutical N-acetylglucosamine (GlcNAc) synthesis in Bacillus subtilis as a model, we engineered FMM components to improve the enzyme assembly in FMMs. First, by overexpression of the SPFH (stomatin-prohibitin-flotillin-HflC/K) domain and YisP protein, an enzyme involved in the synthesis of squalene-derived polyisoprenoid, the membrane order of cells was increased, as verified using di-4-ANEPPDHQ staining. Then, two heterologous enzymes, GlcNAc-6-phosphate N-acetyltransferase (GNA1) and haloacid dehalogenase-like phosphatases (YqaB), required for GlcNAc synthesis were assembled into FMMs, and the GlcNAc titer in flask was increased to 8.30 ± 0.57 g/L, which was almost three times that of the control strains. Notably, FMM component modification can maintain the OD₆₀₀ in stationary phase and reduce cell lysis in the later stage of fermentation. These results reveal that the improved plasma membrane ordering achieved by the engineering FMM components could not only promote the enzyme assembly into FMMs, but also improve the cell fitness.

酶簇可以通过促进中间体的加工来提高催化效率。细菌中的功能性膜微区 (FMM) 可以为酶聚类提供平台。然而，细胞基础水平的 FMM 数量在多酶固定化方面仍面临巨大挑战。在这里，使用枯草芽孢杆菌中的营养品 N-乙酰氨基葡萄糖 (GlcNAc) 合成作为模型，我们设计了 FMM 组件以改进 FMM 中的酶组装。首先，通过 SPFH（stomatin-prohibitin-flotillin-HflC/K）结构域和 YisP 蛋白（一种参与角鲨烯衍生的聚异戊二烯合成的酶）的过表达，细胞的膜顺序增加，如使用 di-4- ANEPPDHQ 染色。然后，将 GlcNAc 合成所需的两种异源酶 GlcNAc-6-磷酸 N-乙酰转移酶 (GNA1) 和卤酸脱卤酶样磷酸酶 (YqaB) 组装成 FMM，烧瓶中的 GlcNAc 滴度增加至 8.30 ± 0.57 g/ L，几乎是对照菌株的三倍。值得注意的是，FMM 组件修改可以保持 OD ₆₀₀稳定期，减少发酵后期细胞裂解。这些结果表明，通过工程 FMM 组件实现的质膜排序改善不仅可以促进酶组装成 FMM，还可以提高细胞适应性。