Details

Autor(en) / Beteiligte

• Lv, Xueqin
• Zhang, Cheng
• Cui, Shixiu
• Xu, Xianhao
• Wang, Lingling
• Li, Jianghua
• Du, Guocheng
• Chen, Jian
• Ledesma-Amaro, Rodrigo
• Liu, Long

Titel

Assembly of pathway enzymes by engineering functional membrane microdomain components for improved N-acetylglucosamine synthesis in Bacillus subtilis

Ist Teil von

• Metabolic engineering, 2020-09, Vol.61, p.96-105

Ort / Verlag

Elsevier Inc

Erscheinungsjahr

2020

Quelle

Elsevier ScienceDirect Journals

Beschreibungen/Notizen

• 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. •The plasma membrane order degree was improved by the engineering of FMM components.•FMM component modification promoted the enzyme assembly to increase the synthesis of GlcNAc.•FMM component modification can obviously improve the cell fitness at the later stage of fermentation.