Production of α-amylase from gluconate and carbon dioxide by protein synthesis and secretion optimization in Cupriavidus necator H16.

Chemoautotrophic Cupriavidus necator H16 has a strong protein synthesis ability and has been used to produce intracellular protein products. However, studies optimizing its secretion system and the producing extracellular enzyme products (EEPs) are lacking. Here, we focused on investigating the feasibility of synthesizing and secreting EEPs in C. necator H16, using α-amylase as a prototype. α-Amylase expression optimization, genome modification, and secretion system engineering were performed to construct and optimize the α-amylase-producing engineering C. necator H16. Finally, the optimized engineering strain could produce α-amylase, with the α-amylase activity per unit cells reaching up to 5.54 U/OD using gluconate as substrate, which was 29.2-fold compared with that of initial engineering strain. Additionally, when using carbon dioxide as substrate, the α-amylase activity per unit cells of engineered strain reached 4.26 U/OD. Overall, this study demonstrates the feasibility of developing C. necator H16 as a host for autotrophic production of α-amylase.