Biosynthesis Of Adipic Acid Research Articles

Effect of Intensification of the Tricarboxylic Acid Cycle on Biosynthesis of Adipic Acid Through the Inverted Fatty Acid β-Oxidation by Escherichia coli Strains

Effect of Intensification of the Tricarboxylic Acid Cycle on Biosynthesis of Adipic Acid Through the Inverted Fatty Acid β-Oxidation by Escherichia coli Strains

Metabolic engineering of Escherichia coli for adipic acid production

Adipic acid is a high-value-added dicarboxylic acid which is primarily used in the production of nylon-66 for manufacturing polyurethane foam and polyester resins. At present, the biosynthesis of adipic acid is hampered by its low production efficiency. By introducing the key enzymes of adipic acid reverse degradation pathway into a succinic acid overproducing strain Escherichia coli FMME N-2, an engineered E. coli JL00 capable of producing 0.34 g/L adipic acid was constructed. Subsequently, the expression level of the rate-limiting enzyme was optimized and the adipic acid titer in shake-flask fermentation increased to 0.87 g/L. Moreover, the supply of precursors was balanced by a combinatorial strategy consisting of deletion of sucD, over-expression of acs, and mutation of lpd, and the adipic acid titer of the resulting E. coli JL12 increased to 1.51 g/L. Finally, the fermentation process was optimized in a 5 L fermenter. After 72 h fed-batch fermentation, adipic acid titer reached 22.3 g/L with a yield of 0.25 g/g and a productivity of 0.31 g/(L·h). This work may serve as a technical reference for the biosynthesis of various dicarboxylic acids.

The Effect of Glyoxylate Shunt Inactivation on Biosynthesis of Adipic Acid through Inverted Fatty Acid β-Oxidation by Escherichia coli Strains

Using Escherichia coli MG1655 lacIQ, ∆ackA-pta, ∆poxB, ∆ldhA, ∆adhE, ∆fadE, PL-SDφ10-atoB, Ptrc-ideal-4-SDφ10-fadB, PL-SDφ10-tesB, ∆yciA as a core strain, derivatives capable of synthesizing adipic acid from glucose through the inverted fatty acid β-oxidation pathway were obtained. Biosynthesis of the target compound by recombinants was ensured by the primary condensation of acetyl-CoA and succinyl-CoA by 3-oxoacyl-CoA thiolase PaaJ and the catalysis of the final reaction of the cycle by acyl-CoA dehydrogenases FadE and FabI. Deletion in the strains of sucCD genes encoding components of succinyl-CoA synthase did not increase the relative intracellular availability of succinyl-CoA for target biosynthetic reactions and did not lead to an increase in adipic acid accumulation by the recombinants. The secretion of succinic and malic acids by the strains with an impaired tricarboxylic acid cycle remained almost unchanged, indicating the activity in the cells of glyoxylate shunt reactions that compete with the cycle reactions for isocitrate, required for succinyl-CoA formation. When isocitrate lyase, malate synthases A and G, and bifunctional kinase/phosphatase of isocitrate dehydrogenase were inactivated in strains due to deletion of the aceBAK operon genes and glcB, adipic acid synthesis by recombinants increased three-fold and reached 0.33 mM.

Biosynthesis of adipic acid in metabolically engineered Saccharomyces cerevisiae.

Adipic Acid (AA) is a valued platform chemical compound, which can be used as a precursor of nylon-6,6. Due to the generation of an enormous amount of nitric oxide metabolites and the growing depletion of oil resources as a result of AA production from a mixture of cyclohexanol and cyclohexanone, the microbial methods for synthesizing AA have attracted significant attention. Of the several AA-producing pathways, the reverse adipate degradation pathway in Thermobifida fusca (Tfu RADP) is reported to be the most efficient, which has been confirmed in Escherichia coli. In this study, the heterologous Tfu RADP was constructed for producing AA in S. cerevisiae by co-expressing genes of Tfu_0875, Tfu_2399, Tfu_0067, Tfu_1647, Tfu_2576, and Tfu_2576. The AA titer combined with biomass, cofactors and other by-products was all determined after fermentation. During batch fermentation in a shake flask, the maximum AA titer was 3.83 mg/L, while the titer increased to 10.09 mg/L during fed-batch fermentation in a 5-L bioreactor after fermentation modification.

Biosynthesis of adipic acid by a highly efficient induction-free system in Escherichia coli

Adipic acid is an important dicarboxylic acid, which is an essential building block to synthesize nylon 6-6 fiber. Adipic acid is primarily synthesized from chemical plant, however, this process is associated with a number of environmental concerns including heavy pollution, toxic catalyst and harsh reaction conditions. A decent amount of adipic acid was produced by reconstructing the reversed adipate-degradation pathway (RADP) from Thermobifida fusca in Escherichia coli. However, IPTG was used in the previous study, which was not feasible in the fermentation industry. In this study, strong promoter-5’-UTR complexes (PUTR) were chosen to construct a highly efficient induction-free system to produce adipic acid. First, comparisons of various exogenous 5’-UTR Complexes, as well as a series of E. coli host strains, demonstrated that those genes using E. coli K12 MG1655 as the host strain produced the highest titer of adipic acid. Subsequently, optimizations were applied to enhance the titer of adipate biosynthesizing strains. The highest titer of adipate of 57.6 g L-1 was achieved by fed-batch fermentation. This work offers a better way to enhance the industrial titer of adipate.

One-Pot Biocatalytic Transformation of Adipic Acid to 6-Aminocaproic Acid and 1,6-Hexamethylenediamine Using Carboxylic Acid Reductases and Transaminases

Production of platform chemicals from renewable feedstocks is becoming increasingly important due to concerns on environmental contamination, climate change, and depletion of fossil fuels. Adipic acid (AA), 6-aminocaproic acid (6-ACA) and 1,6-hexamethylenediamine (HMD) are key precursors for nylon synthesis, which are currently produced primarily from petroleum-based feedstocks. In recent years, the biosynthesis of adipic acid from renewable feedstocks has been demonstrated using both bacterial and yeast cells. Here we report the biocatalytic conversion/transformation of AA to 6-ACA and HMD by carboxylic acid reductases (CARs) and transaminases (TAs), which involves two rounds (cascades) of reduction/amination reactions (AA → 6-ACA → HMD). Using purified wild type CARs and TAs supplemented with cofactor regenerating systems for ATP, NADPH, and amine donor, we established a one-pot enzyme cascade catalyzing up to 95% conversion of AA to 6-ACA. To increase the cascade activity for the transformation of 6-ACA to HMD, we determined the crystal structure of the CAR substrate-binding domain in complex with AMP and succinate and engineered three mutant CARs with enhanced activity against 6-ACA. In combination with TAs, the CAR L342E protein showed 50-75% conversion of 6-ACA to HMD. For the transformation of AA to HMD (via 6-ACA), the wild type CAR was combined with the L342E variant and two different TAs resulting in up to 30% conversion to HMD and 70% to 6-ACA. Our results highlight the suitability of CARs and TAs for several rounds of reduction/amination reactions in one-pot cascade systems and their potential for the biobased synthesis of terminal amines.

Biosynthesis of adipic acid via microaerobic hydrogenation of cis,cis-muconic acid by oxygen-sensitive enoate reductase

Adipic acid (AA) is an important dicarboxylic acid used for the manufacture of nylon and polyurethane plastics. In this study, a novel adipic acid biosynthetic pathway was designed by extending the cis,cis-muconic acid (MA) biosynthesis through biohydrogenation. Enoate reductase from Clostridium acetobutylicum (CaER), an oxygen-sensitive reductase, was demonstrated to have in vivo enzyme activity of converting cis,cis-muconic acid to adipic acid under microaerobic condition. Engineered Escherichia coli strains were constructed to express the whole pathway and accumulated 5.8 ± 0.9 mg/L adipic acid from simple carbon sources. Considering the different oxygen demands between cis,cis-muconic acid biosynthesis and its degradation, a co-culture system was constructed. To improve production, T7 promoter instead of lac promoter was used for higher level expression of the key enzyme CaER and the titer of adipic acid increased to 18.3 ± 0.6 mg/L. To decrease the oxygen supply to downstream strains expressing CaER, Vitreoscilla hemoglobin (VHb) was introduced to upstream strains for its ability on oxygen obtaining. This attempt further improved the production of this novel pathway and 27.6 ± 1.3 mg/L adipic acid was accumulated under microaerobic condition.