A Fluorescent Bioreporter for Acetophenone and 1-Phenylethanol derived from a Specifically Induced Catabolic Operon

Abstract

The β-proteobacterium Aromatoleum aromaticum degrades the aromatic ketone acetophenone, a key intermediate of anaerobic ethylbenzene metabolism, either aerobically or anaerobically via a complex ATP-dependent acetophenone carboxylase and a benzoylacetate-CoA ligase. The genes coding for these enzymes (apcABCDE and bal) are organized in an apparent operon and are expressed in the presence of the substrate acetophenone. To study the conditions under which this operon is expressed in more detail, we constructed a reporter strain by inserting a gene fusion of apcA, the first gene of the apc-bal operon, with the gene for the fluorescent protein mCherry into the chromosome of A. aromaticum. The fusion protein indeed accumulated consistently with the expression pattern of the acetophenone-metabolic enzymes under various growth conditions. After evaluating and quantifying the data by fluorescence microscopy, fluorescence-based flow cytometry and immunoblot analysis, mCherry production was found to be proportional to the applied acetophenone concentrations. The reporter strain allowed quantification of acetophenone within a concentration range of 50 μM (detection limit) to 250 μM after 12 and 24 h. Moreover, production of the Apc-mCherry fusion protein in the reporter strain was highly specific and responded to acetophenone and both enantiomers of 1-phenylethanol, which are easily converted to acetophenone. Other analogous substrates showed either a significantly weaker response or none at all. Therefore, the reporter strain provides a basis for the development of a specific bioreporter system for acetophenone with an application potential reaching from environmental monitoring to petroleum prospecting.

Development of a 2-Nitrobenzoate-Sensing Bioreporter Based on an Inducible Gene Cluster

“…Unlike plasmid-based reporter systems, chromosome-based reporters often encounter issues with relatively poor sensitivity, with the detection limit in the micromolar to millimolar range due to the presence of a single copy reporter gene compared to usual high copy number for reporter plasmids. However, the reporter strain BR prox was able to detect 2NBA with a lower detection limit of 0.5 nM for CFE and 1.0 nM for whole cell ( Figures 2C , 4A ), exhibiting a better sensitivity compared to other reported chromosome-based bioreporters ( Heitzer et al, 1992 ; Hay et al, 2000 ; van der Meer et al, 2004 ; Muhr et al, 2016 ). The higher sensitivity of BR prox strain may be attributed to a strategic disruption of the first functional gene of the pathway i.e., onbA encoding a nitroreductase (that transforms 2NBA to 2-hydroxylaminobenzoate), permitting the presence of a steady level of intracellular 2NBA.…”

“…At the same time, the construction of bioreporter was based on the assumption that the promoter-operator sequence should reside somewhere upstream to the first structural gene onbF ( Figure 1 ), without prior knowledge on the identity and location of the promoter-operator sequence and the role of encoded regulator ( Basu et al, 2016 ). A similar concept had been employed to construct a chromosome-based bioreporter to understand the mechanisms regulating the acetophenone-metabolic genes of an inducible catabolic operon in Aromatoleum aromaticum strain EbN1-SR7, although the constructed bioreporter exhibited broad substrate specificities toward acetophenone, propiophenone, ethylbenzene, 1-phenylethanol and all the monofluoroacetophenone isomers with a comparable sensitivity ( Muhr et al, 2016 ). When subcultured in the absence of antibiotic stress, each day for a month, both the bioreporter strains BR prox and BR dist could retain the EGFP and Kan R genes (data not shown) demonstrating a clear advantage over plasmid-based reporters that are susceptible to frequent loss of reporter gene due to plasmid curing under similar conditions.…”

“…Alternatively, the reporter gene can be inserted into an inducible catabolic operon of host chromosome/megaplasmid where the expression of reporter gene is under the control of an associated promoter-operator-regulator and inducer molecule ( Trögl et al, 2012 ; Xu et al, 2013 ; Hong and Park, 2014 ; Yagur-Kroll et al, 2015 ). However, unlike the plasmid-based construct in E. coli or other heterologous strains, host chromosome-integrated bioreporters ensure a stable construct, that can be easily maintained and circumvents issues associated with analyte transport and compatible sigma factors ( Rysz et al, 2013 ; Muhr et al, 2016 ).…”

Adaptations to a Loss-of-Function Mutation in the Betaproteobacterium Aromatoleum aromaticum: Recruitment of Alternative Enzymes for Anaerobic Phenylalanine Degradation

“…The resulting PCR product was cloned into the suicide vector pK19mobsacB (44) using an XbaI restriction site to get plasmid pK19mobsacB-Δpdh. The vector was transferred into A. aromaticum EbN1 by conjugational plasmid transfer as described previously (30), taking E. coli WM3064 (auxotrophic for DAP) as the donor strain (45). Transconjugants were obtained after incubation of the mixtures on minimal medium agar plates (40, 46) containing 5 mM benzoate as sole carbon source, 10 mM nitrate, and 50 g/ml kanamycin in an anaerobic chamber (N 2 atmosphere) at 28°C for at least 7 days.…”

Biotransformation of 1- and 2-phenylethanol to products of high value via redox reactions

“…Phenolic compounds are of high commercial value owing to their multiple applications. Chiral 1-phenylethanol can be obtained through biotransformation of acetophenone, a substrate which is used in food and cosmetic industries due to its rose-like flavor (Muhr et al, 2016). Optically pure (R )-and (S )-1-phenylethanols are two of the most important chiral building blocks in pharmaceutical and cosmetic industries (Cao et al, 2012).…”