Exploring functionality of the reverse β-oxidation pathway in Corynebacterium glutamicum for production of adipic acid

Background: Adipic acid, a six-carbon platform chemical mainly used in nylon production, can be produced via reverse beta-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 beta-oxidation pathway in C. glutamicum and assessed its functionality for adipic acid biosynthesis. Results: To 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 mu g/L of adipic acid. Conclusions: In the present study, we engineered the reverse beta-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 beta-ketoadipyl-CoA thiolase were determined as potential target for further improvement of the pathway.

Automated summary

In this study, the reverse beta-oxidation pathway was successfully implemented in Corynebacterium glutamicum for production of adipic acid. Despite a small quantity, the presence of adipic acid in the cultivation broth indicated expression and functionality of the synthetic genes. Furthermore, 2,3-dehydroadipyl-CoA hydratase and beta-ketoadipyl-CoA thiolase were identified as potential targets for further improvement of the pathway.