Functional Genetic Elements for Controlling Gene Expression in Cupriavidus necator H16

Area of Science:

• Synthetic biology
• Metabolic engineering
• Microbial biotechnology

Background:

• Robust control of gene expression is crucial for synthetic biology and biotechnology.
• Development and quantitative evaluation of genetic elements like promoters and ribosome binding sites (RBSs) are needed for model organisms.

Purpose of the Study:

• To design, build, and test novel promoters and RBSs for gene expression control in Cupriavidus necator H16.
• To quantitatively evaluate inducible gene expression systems and their impact on product formation.
• To expand the genetic toolbox for C. necator H16.

Main Methods:

• Designed and constructed a series of constitutive promoters with varying strengths.
• Evaluated positively and negatively regulated inducible systems using different inducer concentrations.
• Assessed the impact of RBSs, mRNA stem-loop structures, and A/U-rich sequences on gene expression.
• Correlated gene expression levels with isoprene production yields.

Main Results:

• Developed constitutive promoters with predictable activity over a >700-fold dynamic range, some showing up to 9-fold higher activity than native promoters.
• Achieved >1,000-fold gene expression control using inducible systems (AraC/P-l-arabinose, RhaRS/P-l-rhamnose, AcuR/P-acrylate, CymR/P-cumate).
• Demonstrated a significant correlation between reporter protein synthesis and isoprene yields, with a second-order polynomial relationship observed between RBS activity and isoprene yields.

Conclusions:

• The study provides a suite of robust and predictable genetic tools for controlling gene expression in C. necator H16.
• Quantitative data on regulatory elements and their impact on gene expression and product formation were established.
• The developed genetic elements significantly expand the engineering capabilities for C. necator H16 in biotechnology.