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. 2021 Aug 4;20(1):155.
doi: 10.1186/s12934-021-01647-7.

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

Affiliations

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

Jae Ho Shin et al. Microb Cell Fact. .

Abstract

Background: Adipic 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.

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 µg/L of adipic acid.

Conclusions: In 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.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Reverse β-oxidation pathway for adipic acid biosynthesis from TCA metabolites. a Synthetic pathway designed to enable adipic acid biosynthesis from succinyl-CoA and acetyl-CoA. Five enzymatic steps, including condensation, reduction, dehydration, second reduction, and hydrolysis of a thioester bond, are carried out by paaJ, paaH, paaF, ter, and tesB gene products encoding the E. coli PaaJ thiolase, PaaH 3-hydroxyacyl-CoA dehydrogenase, PaaF 2,3-dehydroadipyl-CoA hydratase, Ter trans-2-enoyl-CoA reductase, and TesB thioesterase. PcaF corresponds to a putative acetyl-CoA:acetyltransferase. b Vector design to investigate pathway intermediate compounds
Fig. 2
Fig. 2
Biosynthesis of adipic acid pathway intermediates from glucose. Shake-flask cultivation profile of C. glutamicum harboring (a) pZ8-paaJ-tesB and (b) pZ8-paaH-tesB-paaJ. OD600 (filled circles), residual sugar (empty circles). N = 3; error bars = standard deviation. c Representative GC/MS ion (m/z = 363) extracted chromatogram of MSTFA-derivatized cultivation broth from C. glutamicum transformed with pZ8-paaH-tesB-paaJ. The peak at retention time 13.3 min corresponds to MSTFA-derivatized 3-hydroxyadipic acid. (d) m/z fragmentation pattern of the peak corresponding to MSTFA-derivatized 3-hydroxyadipic acid from (c)
Fig. 3
Fig. 3
Shake-flask cultivation profile of C. glutamicum harboring pZ8-paaH-tesB-paaJF. a Cultivation profile including OD600 (filled circle) and residual sugar (empty circle). N = 3; error bars = standard deviation. b Ion (m/z = 143.03498) extracted chromatogram obtained by LC/MS of trans-2-hexenedioic acid (blue), cultivation broth of C. glutamicum harboring pZ8-paaH-tesB-paaJF (grey), and a control vector (black)
Fig. 4
Fig. 4
Shake-flask cultivation profile of C. glutamicum harboring pZ8-paaH-ter-paaJ-paaF-tesB. a Cultivation parameters include OD600 (filled circles) and residual sugar (empty circles). N = 3; error bars = standard deviation. b Ion (m/z = 363) extracted chromatogram obtained by GC/MS of the cultivation broth (t = 55 h) of C. glutamicum harboring pZ8-paaH-ter-paaJ-paaF-tesB (black line) and an empty vector (grey line). The peak corresponding to 3-hydroxyadipate is indicated with a black arrow. c Cultivation profile of C. glutamicum harboring pZ8-Ptac showing OD600 (filled circles) and residual sugar (empty circles). N = 3; error bars = standard deviation. (d) Ion (m/z = 145.0506) extracted chromatogram obtained by LC/MS of adipic acid standard (purple), cultivation broth of C. glutamicum harboring pZ8-paaH-ter-paaJ-paaF-tesB (green), pZ8-paaH-tesB-paaJF (grey), and an empty vector (black)

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