Abstract

BackgroundAdipic acid, a six-carbon platform chemical mainly used in nylon production, can be produced via reverse β-oxidation in microbial systems. The advantages posed by Corynebacterium glutamicum as a model cell factory for implementing the pathway include: (1) availability of genetic tools, (2) excretion of succinate and acetate when the TCA cycle becomes overflown, (3) initiation of biosynthesis with succinyl-CoA and acetyl-CoA, and (4) established succinic acid production. Here, we implemented the reverse β-oxidation pathway in C. glutamicum and assessed its functionality for adipic acid biosynthesis.ResultsTo obtain a non-decarboxylative condensation product of acetyl-CoA and succinyl-CoA, and to subsequently remove CoA from the condensation product, we introduced heterologous 3-oxoadipyl-CoA thiolase and acyl-CoA thioesterase into C. glutamicum. No 3-oxoadipic acid could be detected in the cultivation broth, possibly due to its endogenous catabolism. To successfully biosynthesize and secrete 3-hydroxyadipic acid, 3-hydroxyadipyl-CoA dehydrogenase was introduced. Addition of 2,3-dehydroadipyl-CoA hydratase led to biosynthesis and excretion of trans-2-hexenedioic acid. Finally, trans-2-enoyl-CoA reductase was inserted to yield 37 µg/L of adipic acid.ConclusionsIn the present study, we engineered the reverse β-oxidation pathway in C. glutamicum and assessed its potential for producing adipic acid from glucose as starting material. The presence of adipic acid, albeit small amount, in the cultivation broth indicated that the synthetic genes were expressed and functional. Moreover, 2,3-dehydroadipyl-CoA hydratase and β-ketoadipyl-CoA thiolase were determined as potential target for further improvement of the pathway.

Highlights

  • The detrimental impact of petrochemical-based manufacturing on the environment has increased demand for biomass-derived chemicals, fuels, and consumer products [1, 2]

  • Assessment of non‐decarboxylative Claisen condensation of acetyl‐coenzyme A (CoA) and succinyl‐CoA in C. glutamicum by overexpressing paaJ from E. coli The design of synthetic pathway (Fig. 1) is motived by the similarities found in the bacterial phenylalanine catabolism pathways

  • Succinyl-CoA and acetyl-CoA formed by thiolytic cleavage of 3-oxoadipylCoA are assimilated in the tricarboxylic acid (TCA) cycle

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Summary

Introduction

The detrimental impact of petrochemical-based manufacturing on the environment has increased demand for biomass-derived chemicals, fuels, and consumer products [1, 2]. Biomass-based derived compounds include fuels [3], monomers [4], polymers [5], and pharmaceuticals [2], with a few products already exerting a significant impact in the chemical industry [6, 7]. Its industrial production volume amounts to nearly 3 million US tons and is achieved via chemical oxidation of petroleum-derived KA oil (cyclohexanone and cyclohexanol) [10]. A six-carbon platform chemical mainly used in nylon production, can be produced via reverse β-oxidation in microbial systems. We implemented the reverse β-oxidation pathway in C. glutamicum and assessed its functionality for adipic acid biosynthesis

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