Vajente, M. | bioRxiv Science

bioRxiv2025

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. 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 outperformed the gold standard, E. coli BL21(DE3) in producing 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. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/688765v2_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@1a6584corg.highwire.dtl.DTLVardef@1ef094dorg.highwire.dtl.DTLVardef@4bb4c3org.highwire.dtl.DTLVardef@489c85_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2024

Using Cupriavidus necator H16 to provide a roadmap for increasing electroporation efficiency in non-model bacteria

Bacteria are a treasure trove of metabolic reactions, but most industrial biotechnology applications rely on a limited set of established host organisms. In contrast, adopting non-model bacteria for the production of various chemicals of interest is often hampered by their limited genetic amenability coupled with their low transformation efficiency. In this study, we propose a series of steps that can be taken to increase electroporation efficiency in non-model bacteria. As a test strain, we use Cupriavidus necator H16, a lithoautotrophic bacterium that has been engineered to produce a wide range of products from CO2 and hydrogen. However, its low electroporation efficiency hinders the high-throughput genetic modifications required to develop C. necator into an industrially relevant host organism. First, we propose a species-independent technique based on natively methylated DNA and Golden Gate assembly to increase one-pot cloning and electroporation efficiency by 70-fold. Second, bioinformatic tools were used to predict defense systems and develop a restriction avoidance strategy that was used to introduce suicide plasmids by electroporation to obtain a domesticated strain. The results are discussed in the context of metabolic engineering of non-model bacteria. TABLE OF CONTENT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=161 SRC="FIGDIR/small/596136v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@dfc76aorg.highwire.dtl.DTLVardef@19d130forg.highwire.dtl.DTLVardef@14e6ed3org.highwire.dtl.DTLVardef@4e0024_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗