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

Thomas Bayer,Henrik Terholsen,Saskia Buchwald,Ina Menyes,Aileen Becker,Uwe T Bornscheuer,Kathleen Balke,Steven C Almo,Suvi Santala,In Jung Kim

doi:10.3390/catal11080953

Thomas Bayer, Henrik Terholsen + Show 8 more

Open Access

https://doi.org/10.3390/catal11080953

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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.

Highlights

Great progress has been achieved in tailoring microorganisms for the biosynthesis ofnatural chemicals including but not restricted to: fatty acids and derivatives [1,2,3], aromatic and phenolic compounds [4,5,6] and secondary metabolites such as terpenoids [7,8]
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
One example is AlkJ, an alcohol dehydrogenase (ADH) from Pseudomonas putida (P. putida) being capable of oxidizing a variety of primary alcohols [15,16,17]. Due to their high reactivity and cytotoxicity, aldehydes are rapidly metabolized by endogenous enzymes and do not accumulate in many microorganisms including hosts such as Escherichia coli (E. coli) [9,18,19]

Summary

Introduction

Great progress has been achieved in tailoring microorganisms for the biosynthesis of (non-)natural chemicals including but not restricted to: fatty acids and derivatives [1,2,3], aromatic and phenolic compounds [4,5,6] and secondary metabolites such as terpenoids [7,8]. One example is AlkJ, an alcohol dehydrogenase (ADH) from Pseudomonas putida (P. putida) being capable of oxidizing a variety of primary alcohols [15,16,17] Due to their high reactivity and cytotoxicity, aldehydes are rapidly metabolized by endogenous enzymes and do not accumulate in many microorganisms including (heterologous) hosts such as Escherichia coli (E. coli) [9,18,19]. The groups rapidly metabolized by endogenous enzymes and do not accumulate in many microorganisms including (heterologous) hosts such as Escherichia coli (E. coli) [9,18,19] To address this issue, the groups of Atsumi and Prather constructed E. coli platform st2raofin17s by targeted gene knock-outs that reduced the reduction of aliphatic and aromatic aldehydes to the corresponding primary alcohols [20,21]. The combination of the oxidation step (a,b) and LuxAB (highlighted in blue) represents a new cascade producing CAs

Results

General Information

LuxAB-Based HT Screening of Enzymes In Vivo