Abstract
BackgroundAcrylic acid (AA) is a widely used commodity chemical derived from non-renewable fossil fuel sources. Alternative microbial-based production methodologies are being developed with the aim of providing “green” acrylic acid. These initiatives will benefit from component sensing tools that facilitate rapid and easy detection of in vivo AA production.ResultsWe developed a novel transcriptional sensor facilitating in vivo detection of acrylic acid (AA). RNAseq analysis of Escherichia coli exposed to sub-lethal doses of acrylic acid identified a selectively responsive promoter (PyhcN) that was cloned upstream of the eGFP gene. In the presence of AA, eGFP expression in E. coli cells harbouring the sensing construct was readily observable by fluorescence read-out. Low concentrations of AA (500 μM) could be detected whilst the closely related lactic and 3-hydroxy propionic acids failed to activate the sensor. We further used the developed AA-biosensor for in vivo FACS-based screening and identification of amidase mutants with improved catalytic properties for deamination of acrylamide to acrylic acid.ConclusionsThe transcriptional AA sensor developed in this study will benefit strain, enzyme and pathway engineering initiatives targeting the efficient formation of bio-acrylic acid.
Highlights
Acrylic acid (AA) is a widely used commodity chemical derived from non-renewable fossil fuel sources
Research focused on endogenous acrylic acid producers, such as the obligate anaerobes Clostridium propionicum and Megasphaera elsdenii that are capable of reducing lactic acid to propionic acid via an acrylyl-CoA intermediate [5]
Identification of AA‐responsive genes by transcriptome analysis Escherichia coli growth curves in the presence of acrylic acid indicated that concentrations > 5 mM were lethal
Summary
We developed a novel transcriptional sensor facilitating in vivo detection of acrylic acid (AA). RNAseq analysis of Escherichia coli exposed to sub-lethal doses of acrylic acid identified a selectively responsive promoter (PyhcN) that was cloned upstream of the eGFP gene. In the presence of AA, eGFP expression in E. coli cells harbouring the sensing construct was readily observable by fluorescence read-out. Low concentrations of AA (500 μM) could be detected whilst the closely related lactic and 3-hydroxy propionic acids failed to activate the sensor. We further used the developed AA-biosensor for in vivo FACS-based screening and identification of amidase mutants with improved catalytic properties for deamination of acrylamide to acrylic acid
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