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

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 phaC1, P rrsC, P j5, and P g25) 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 BAD 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 BAD 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.

Design and application of genetically-encoded malonyl-CoA biosensors for metabolic engineering of microbial cell factories.

Malonyl-CoA is the basic building block for synthesizing a range of important compounds including fatty acids, phenylpropanoids, flavonoids and non-ribosomal polyketides. Centering around malonyl-CoA, we summarized here the various metabolic engineering strategies employed recently to regulate and control malonyl-CoA metabolism and improve cellular productivity. Effective metabolic engineering of microorganisms requires the introduction of heterologous pathways and dynamically rerouting metabolic flux towards products of interest. Transcriptional factor-based biosensors translate an internal cellular signal to a transcriptional output and drive the expression of the designed genetic/biomolecular circuits to compensate the activity loss of the engineered biosystem. Recent development of genetically-encoded malonyl-CoA sensor has stood out as a classical example to dynamically reprogram cell metabolism for various biotechnological applications. Here, we reviewed the design principles of constructing a transcriptional factor-based malonyl-CoA sensor with superior detection limit, high sensitivity and broad dynamic range. We discussed various synthetic biology strategies to remove pathway bottleneck and how genetically-encoded metabolite sensor could be deployed to improve pathway efficiency. Particularly, we emphasized that integration of malonyl-CoA sensing capability with biocatalytic function would be critical to engineer efficient microbial cell factory. Biosensors have also advanced beyond its classical function of a sensor actuator for in situ monitoring of intracellular metabolite concentration. Applications of malonyl-CoA biosensors as a sensor-invertor for negative feedback regulation of metabolic flux, a metabolic switch for oscillatory balancing of malonyl-CoA sink pathway and source pathway and a screening tool for engineering more efficient biocatalyst are also presented in this review. We envision the genetically-encoded malonyl-CoA sensor will be an indispensable tool to optimize cell metabolism and cost-competitively manufacture malonyl-CoA-derived compounds.