Functional Genetic Elements for Controlling Gene Expression in Cupriavidus necator H16.

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

doi:10.1128/aem.00878-18

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 P phaC , with the majority of promoters displaying up to a 9-fold higher activity. Positively (AraC/P araBAD -l-arabinose and RhaRS/P rhaBAD -l-rhamnose) and negatively (AcuR/P acuRI -acrylate and CymR/P cmt -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. necatorIMPORTANCE This report provides tools for robust and predictable control of gene expression in the model lithoautotroph C. necator H16. To address a current need, we designed, built, and tested promoters and RBSs for controlling gene expression in C. necator H16. To answer a question on how existing and newly developed inducible systems compare, two positively (AraC/P araBAD -l-arabinose and RhaRS/P rhaBAD -l-rhamnose) and two negatively (AcuR/P acuRI -acrylate and CymR/P cmt -cumate) regulated inducible systems were quantitatively evaluated and their induction kinetics analyzed. To establish if gene expression can be further improved, the effect of genetic elements, such as mRNA stem-loop structure and A/U-rich sequence, on gene expression was evaluated. Using isoprene production as an example, the study investigated if and to what extent chemical compound yield correlates to the level of gene expression of product-synthesizing enzyme.

Highlights

A robust and predictable control of gene expression plays an important role in synthetic biology and biotechnology applications
To enable measurement of their strengths, promoters were transcriptionally fused to a gene encoding enhanced yellow fluorescence protein and a strong ribosome binding sites (RBSs), a 27-nucleotide upstream sequence of the bacteriophage T7 gene 10 (T7g10) [35], was placed directly upstream of the reporter gene
A variety of promoter strengths was achieved by combining core promoter sequences of previously characterized promoters from C. necator, E. coli, and bacteriophages T4 and T5 [3, 36,37,38] with upstream and downstream insulation sequences (Fig. 1A)

Summary

Introduction

A robust and predictable control of gene expression plays an important role in synthetic biology and biotechnology applications. Cupriavidus necator H16 is a Gram-negative betaproteobacterium, formerly known as Ralstonia eutropha, Alcaligenes eutrophus, Wautersia eutropha, and Hydrogenomonas eutropha It has been extensively studied for its capacity to store large amounts of organic carbon in the form of poly-3-hydroxybutyrate (PHB) that can be utilized as a source of bioplastics [1, 2]. Several studies demonstrated that use of C. necator allows achievement of a high cell density, more than 200 g/liter of biomass, without accumulating growth inhibitory organic acids and seemingly precluding formation of inclusion bodies in recombinant protein production [9, 10, 20] These beneficial characteristics imply that C. necator H16 can be used as an alternative expression host

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