Functional genetic elements for controlling gene expression in Cupriavidus necator H16

Swathi Alagesan, Erik K.R. Hanko, Naglis Malys, Muhammad Ehsaan, Klaus Winzer, Nigel P. Minton

Research output: Contribution to journalArticlepeer-review

Abstract

A robust and predictable control of gene expression plays an important role in synthetic biology and biotechnology applications. Development and quantitative evaluation of functional genetic elements, such as constitutive and inducible promoters as well as ribosome binding sites (RBSs), are required. In this study, we designed, built, and tested promoters and RBSs for controlling gene expression in the model lithoautotroph Cupriavidus necator H16. A series of variable-strength, insulated, constitutive promoters exhibiting predictable activity within a >700-fold dynamic range was compared to the native PphaC, with the majority of promoters displaying up to a 9-fold higher activity. Positively (AraC/ParaBAD-L-arabinose and RhaRS/PrhaBAD-Lrhamnose) and negatively (AcuR/PacuRI-acrylate and CymR/Pcmt-cumate) regulated inducible systems were evaluated. By supplying different concentrations of inducers, a >1,000-fold range of gene expression levels was achieved. Application of inducible systems for controlling expression of the isoprene synthase gene ispS led to isoprene yields that exhibited a significant correlation to the reporter protein synthesis levels. The impact of designed RBSs and other genetic elements, such as mRNA stem-loop structure and A/U-rich sequence, on gene expression was also evaluated. A second-order polynomial relationship was observed between the RBS activities and isoprene yields. This report presents quantitative data on regulatory genetic elements and expands the genetic toolbox of C. necator.

Original languageEnglish
Article numbere00878-18
Pages (from-to)1-17
Number of pages17
JournalApplied and environmental microbiology
Volume84
Issue number19
Early online date17 Sept 2018
DOIs
Publication statusPublished - 1 Oct 2018

Keywords

  • Cupriavidus necator H16
  • functional genetic element
  • gene expression
  • isoprene
  • promoter
  • ribosome binding site

Fingerprint

Dive into the research topics of 'Functional genetic elements for controlling gene expression in Cupriavidus necator H16'. Together they form a unique fingerprint.

Cite this