An Engineered Constitutive Promoter Set with Broad Activity Range for Cupriavidus necator H16

Abstract

Well-characterized promoters with variable strength form the foundation of heterologous pathway optimization. It is also a key element that bolsters the success of microbial engineering and facilitates the development of biological tools like biosensors. In comparison to microbial hosts such as Escherichia coli and Saccharomyces cerevisiae, the promoter repertoire of Cupriavidus necator H16 is highly limited. This limited number of characterized promoters poses a significant challenge during the engineering of C. necator H16 for biomanufacturing and biotechnological applications. In this article, we first examined the architecture and genetic elements of the four most widely used constitutive promoters of C. necator H16 (i.e., P , P, P , and P) and established a narrow 6-fold difference in their promoter activities. Next, using these four promoters as starting points and applying a range of genetic modifications (including point mutation, length alteration, incorporation of regulatory genetic element, promoter hybridization, and configuration alteration), we created a library of 42 constitutive promoters, all of which are functional in C. necator H16. Although these promoters are also functional in E. coli, they show different promoter strength and hierarchical rank of promoter activity. Subsequently, the activity of each promoter was individually characterized, using l-arabinose-inducible P promoter as a benchmark. This study has extended the range of constitutive promoter activities to 137-fold, with some promoter variants exceeding the l-arabinose-inducible range of P promoter. Not only has the work enhanced our flexibility in engineering C. necator H16, it presented novel strategies in adjusting promoter activity in C. necator H16 and highlighted similarities and differences in transcriptional activity between this organism and E. coli.

Adaptive Laboratory Evolution of Cupriavidus necator H16 for Carbon Co-Utilization with Glycerol

“…To gain further insights of the four non-synonymous mutations identified in genome sequencing, the four genes were amplified from v6C6 and cloned for constitutive expression in a P j5 -based constitutive plasmids (Figure 7a, Table S1) [10]. The constructs were transformed into C. necator wild-type and cultivated in MSM with 1.0% ( w / v ) glycerol.…”

“…Since expression of the four mutated genes in v6C6 suggested GlpK may play an important role in glycerol assimilation, the glpK H16 and glpD H16 from gene loci H16_A2507 and H16_A2508 in the wild-type strain were amplified and cloned using pBBR1-based plasmid for expression in C. necator H16 wild-type. All genes were cloned downstream of a constitutive promoter P j5 [A1A3C2] [10] (Figure 7b) and individual effects of GlpK H16 and GlpD H16 expression and in combination (glpKD H16 ) were investigated. Constructs pP j5 [A1A3C2] -glpKD H16 -O1 and pP j5 [A1A3C2] -glpKD H16 -O2 differed in the ribosome binding site (rbs) preceding the glpD H16 gene.…”

“…PCR and gel purifications were performed using NucleoSpin ® Gel and PCR Clean-up (Macherey-Nagel, Düren, Germany). All genes were cloned by replacing the rfp gene in pP j5 [C2] -RFP [10] with the target gene. Standard restrictive digestion and ligation were used for all plasmids except pP j5 [A1A3C2] -glpKD H16 -O2, which was constructed using the NEBuilder ® HiFi DNA Assembly Cloning Kit (New England Biolabs Ltd., Hitchin, UK).…”

“…A promising candidate for glycerol biotransformation is the non-pathogenic Gram-negative bacterium Cupriavidus necator H16. Best known for its natural ability to accumulate polyhydroxybutyrate (PHB) [8], recent literature has evidenced a rapid expansion of C. necator H16 applications [9] and the development of its genetic engineering tools [10]. While C. necator H16 can naturally metabolize glycerol, carbohydrates, lignin derivatives, organic acids, and even carbon dioxide [11], its growth in glycerol is slow compared to favorable carbon sources like gluconate and fructose.…”

Self-Redirection of Metabolic Flux toward Squalene and Ethanol Pathways by Engineered Yeast

“…This can be achieved using strong and well-characterized constitutive promoters capable of producing a higher titer of the enzymes robustly converting the substrate into the final product [26]. Although the repertoire of yeast constitutive promoters is large enough to engineer the medium size metabolic pathway" this repertoire does not possess a large number of strong promoters [27][28][29]. Only a few studies have reported the successful use of weak promoters and low copy plasmids to optimize the expression of the pathway to produce high-end products including, the precursor of Taxol in E. coli [30].…”