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.