Comparison of two genetic systems for protein production in Cupriavidus necator H16

Many chemical manufacturing routes are being replaced with enzymatic processes to improve sustainability. In biocatalysis, enzyme production is often the main bottleneck. Bacterial 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 chemolithoautotrophic metabolism and high polyhydroxybutyrate (PHB) accumulation. Previous studies have demonstrated 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 investigated the bottlenecks limiting protein production in C. necator. We optimized a T7 RNA polymerase genetic system and quantified the impact of several genetic elements such as T7 promoter, RBS strength and codon usage towards GFP production. Codon usage was the main factor limiting protein production in C. necator. Tuning the RBS strength and selecting a different T7 promoter strongly influenced expression leakiness. We then produced the ene-reductase YqjM from Bacillus subtilis in C. necator, analyzing the performance of the engineered C. necator strain and our previously developed pMVRha expression plasmid. C. necator produced high amounts of soluble protein and one of the strains produced higher amounts of FMN-loaded enzymes compared to E. coli BL21(DE3) in the conditions tested. This result highlights the potential of non-model bacteria to achieve high-yield enzyme production and promote the transition to biocatalysis-driven chemical synthesis.