Optimization of a T7 RNA polymerase expression system for high-yield protein production in Cupriavidus necator H16

Many chemical manufacturing routes are being replaced with enzymatic processes to improve sustainability and reduce the use of harmful chemicals. Enzyme production is often the main bottleneck of the process, and proteins are frequently produced using the workhorse E. coli BL21(DE3) and its derivatives. However, other bacteria with beneficial characteristics can also be engineered for this purpose. Cupriavidus necator H16 ( C. necator ), for example, is a Gram-negative bacterium well-known for its lithoautotrophic metabolism and high polyhydroxybutyrate (PHB) accumulation. Previous studies have demonstrated high-yield protein production without inclusion body formation, which is one of the main challenges when producing enzymes in E. coli . Nevertheless, high-yield protein production in C. necator remains an understudied field. Here, we optimized a T7 RNA polymerase genetic system to improve protein production in C. necator . We investigated the impact of codon usage, different inducible promoters, and several genetic elements by expressing the fluorescent reporter protein GFP. Codon usage was the main factor limiting protein production in C. necator . Tuning the RBS strength also strongly reduced leakiness of the promoter. As an application, we compared the performance of our engineered C. necator T7 RNA polymerase-based system to that of an E. coli -based T7 system using the ene-reductase YqjM from Bacillus subtilis . The optimized protein expression system in C. necator outperformed the gold standard, E. coli BL21(DE3), in producing soluble, FMN-loaded enzyme. This result highlights the potential of non-model bacteria to achieve high-yield enzyme production and promote the transition to biocatalysis-driven chemical synthesis.