LuxAB-Based Microbial Cell Factories for the Sensing, Manufacturing and Transformation of Industrial Aldehydes

Abstract

The application of genetically encoded biosensors enables the detection of small molecules in living cells and has facilitated the characterization of enzymes, their directed evolution and the engineering of (natural) metabolic pathways. In this work, the LuxAB biosensor system from Photorhabdus luminescens was implemented in Escherichia coli to monitor the enzymatic production of aldehydes from primary alcohols and carboxylic acid substrates. A simple high-throughput assay utilized the bacterial luciferase—previously reported to only accept aliphatic long-chain aldehydes—to detect structurally diverse aldehydes, including aromatic and monoterpene aldehydes. LuxAB was used to screen the substrate scopes of three prokaryotic oxidoreductases: an alcohol dehydrogenase (Pseudomonas putida), a choline oxidase variant (Arthrobacter chlorophenolicus) and a carboxylic acid reductase (Mycobacterium marinum). Consequently, high-value aldehydes such as cinnamaldehyde, citral and citronellal could be produced in vivo in up to 80% yield. Furthermore, the dual role of LuxAB as sensor and monooxygenase, emitting bioluminescence through the oxidation of aldehydes to the corresponding carboxylates, promises implementation in artificial enzyme cascades for the synthesis of carboxylic acids. These findings advance the bio-based detection, preparation and transformation of industrially important aldehydes in living cells.

Biosensor and chemo-enzymatic one-pot cascade applications to detect and transform PET-derived terephthalic acid in living cells

“…In a previous study, the monooxygenase LuxAB from P. luminescens was expressed in E. coli K-12 MG1655 RARE ( Kunjapur et al., 2014 ), herein referred to as E. coli RARE, and provided a reliable detection tool for aldehydes in living cells in a 96-well microplate format ( Bayer et al., 2021 ), importantly, beyond the previously reported long-chain aliphatic aldehydes ( Colepicolo et al., 1989 ). Furthermore, LuxAB was suitable to sense aldehydes, including aromatic products such as benzaldehyde, cuminaldehyde, and 2-phenylacetaldehyde that were enzymatically produced from carboxylic acid substrates by the co-expression of CAR Mm in the same cell ( Bayer et al., 2021 ). Prompted by the structural relatedness of these aromatic aldehydes to TPA-derived aldehydes, the capabilities of (1) CAR Mm – to reduce one or both carboxylic acid functionalities of TPA to the aldehyde – and (2) LuxAB – to accept aldehyde products formed in situ , thereby yielding bioluminescence – were investigated.…”

“…Therefore, chemically competent E. coli BL21(DE3) cells were transformed with pACYCDuet-1/ car Mm :ppt Ni to co-express CAR Mm and a phosphopantetheinyl transferase from Nocardia iowensis (PPT Ni ) ( Bayer et al., 2021 ). The PPT is required to posttranslationally modify apoCARs to yield the functional holo-CAR enzymes ( Akhtar et al., 2013 ; Finnigan et al., 2017 ; Horvat and Winkler, 2020 ).…”

“…Complementary, genetically encoded biosensor systems have been used for the detection of small molecules and included transcription factors (TFs), riboswitches, or enzyme-coupled sensor devices ( Bayer et al., 2021 ; Dietrich et al., 2010 ; Lehtinen et al., 2017 ; Liu et al., 2015 , 2017 ; Yi et al., 2021 ). To date, only two biosensors have been reported to detect TPA in vivo .…”

“…Most recently, the luciferase LuxAB from Photorhabdus luminescens ( P. luminescens ) was introduced for the detection of structurally diverse aldehydes in Escherichia coli ( E. coli ) ( Bayer et al., 2021 ). In the present work, the carboxylic acid reductase from Mycobacterium marinum (CAR Mm ) was shown to transform TPA into the corresponding aldehydes 4-carboxybenzaldehyde (4-CBAL) and terephthalaldehyde (TAL) in vivo ( Figure 1 ).…”

Oxidase enzymes as sustainable oxidation catalysts

“…Multistep synthesis using AcCO6 has been reported in a sequential amination cascade with aminating enzymes, in both batch [ 33 ] and more recently in continuous flow using a specially designed flow reactor for the enhancement of oxygen-dependent biocatalysts [ 5 , 34 ]. Recently, AcCO6 has also been studied as a catalyst within an in vivo system for the first time [ 35 ]. In this report, a LuxAB biosensor system was implemented in E. coli to monitor the enzymatic production of aldehydes from primary alcohols and carboxylic acid substrates.…”