Characterisation of a 3-hydroxypropionic acid-inducible system from Pseudomonas putida for orthogonal gene expression control in Escherichia coli and Cupriavidus necator

Abstract

3-hydroxypropionic acid (3-HP) is an important platform chemical used as a precursor for production of added-value compounds such as acrylic acid. Metabolically engineered yeast, Escherichia coli, cyanobacteria and other microorganisms have been developed for the biosynthesis of 3-HP. Attempts to overproduce this compound in recombinant Pseudomonas denitrificans revealed that 3-HP is consumed by this microorganism using the catabolic enzymes encoded by genes hpdH, hbdH and mmsA. 3-HP-inducible systems controlling the expression of these genes have been predicted in proteobacteria and actinobacteria. In this study, we identify and characterise 3-HP-inducible promoters and their corresponding LysR-type transcriptional regulators from Pseudomonas putida KT2440. A newly-developed modular reporter system proved possible to demonstrate that PpMmsR/PmmsA and PpHpdR/PhpdH are orthogonal and highly inducible by 3-HP in E. coli (12.3- and 23.3-fold, respectively) and Cupriavidus necator (51.5- and 516.6-fold, respectively). Bioinformatics and mutagenesis analyses revealed a conserved 40-nucleotide sequence in the hpdH promoter, which plays a key role in HpdR-mediated transcription activation. We investigate the kinetics and dynamics of the PpHpdR/PhpdH switchable system in response to 3-HP and show that it is also induced by both enantiomers of 3-hydroxybutyrate. These findings pave the way for use of the 3-HP-inducible system in synthetic biology and biotechnology applications.

Functional Genetic Elements for Controlling Gene Expression in Cupriavidus necator H16

“…Inducible promoters are indispensable tools within the context of synthetic biology to control gene expression. To date, a number of transcription-based inducible systems have been tested in C. necator ( 16 , 27 – 31 ). However, the genetic contexts in which they have been evaluated differ considerably, making it difficult to compare their outputs.…”

“…Reporter systems were designed as described recently for the modular reporter plasmid pEH006. It contains the l -arabinose-inducible system and was demonstrated to be a suitable original vector for the evaluation of inducible systems ( 28 ). Vectors were constructed in such a way that the transcriptional regulator (TR) coding sequences are located in opposite direction of the rfp reporter gene ( Fig.…”

“…A few studies have reported on the characterization of functional genetic elements in C. necator H16. The heterologous inducible promoters P araBAD ( l -arabinose), P xylS ( m -toluic acid), P lac (lactose), P hpdH (3-hydroxypropionic acid), and P rhaBAD ( l -rhamnose) ( 16 , 27 – 29 ), the synthetic anhydrotetracycline- and cumate-inducible promoters ( 30 , 31 ), and several native promoters ( 27 ) have been shown to be suitable to control and drive gene expression in C. necator . Escherichia coli -derived promoters P lacUV5 , P trc , and P trp have been applied to regulate expression of R-specific enoyl coenzyme A (enoyl-CoA) hydratases, allowing modulation of the proportion of the ( R )-3-hydroxyhexanoate monomer in poly[( R )-3-hydroxybutyrate-co-3-hydroxyhexanoate] ( 32 ).…”

A genome-wide approach for identification and characterisation of metabolite-inducible systems

“…Sixteen putative inducible systems resulting from a pool that comprised over 400 TRs were identified in this single bacterium. With exception of the acetoin-and 3-HP-inducible systems 25,47 , we identify and characterise 14 systems from C. necator. Two previously proposed regulators 48,49 , responding to tartrate and sulfonatoacetate, were experimentally validated to be involved in transcription activation of their corresponding metabolic genes in this study.…”

The Transcriptome and Flux Profiling of Crabtree‐Negative Hydroxy Acid‐Producing Strains of Saccharomyces cerevisiae Reveals Changes in the Central Carbon Metabolism

“…Investigations of the transport mechanisms of 3HP from the cytosol to the extracellular space could help to pull flux from malonic semialdehyde and restore growth. Another strategy may be to engineer a malonic semialdehyde sensor to dynamically control flux through the pathway, similar to the previous work carried out on 3HP biosensors …”