Production of adipic acid by the native-occurring pathway in Thermobifida fusca B6

Aims: To identify the native-occurring adipate pathway in a previously engineered Thermobifida fusca B6 strain and optimize the adipic acid production of this strain on glucose and corncob. Methods and Results: The native-occurring adipate pathway in T. fusca B6 was identified to be the reverse adipate degradation pathway, including five steps: beta-ketothiolase (Tfu_0875), 3-hydroxyacyl-CoA dehydrogenase (Tfu_2399), 3-hydroxyadipyl-CoA dehydrogenase (Tfu_0067), 5-Carboxy-2-pentenoyl-CoA reductase (Tfu_1647) and succinyl-CoA synthetase (Tfu_2576, Tfu_2577). The cell lysates of T. fusca wild-type strain with the addition of the Tfu_1647 protein produced trace adipic acid, while no adipic acid was produced in the absence of this protein. The above results prove that the low expression of Tfu_1647 in the wild-type strain was the reason why it did not produce any adipic acid. We then demonstrated that in T. fusca B6, the maximal titre of adipic acid on 50 g l(-1) glucose was 2.23 g l(-1) with 0.045 g g(-1) -glucose yield and 0.22 g l(-1) adipic acid was produced from 19.38 g l(-1) milled corncob. Conclusions: The reverse adipate degradation pathway was found to be responsible for the adipate synthesis in T. fusca B6 and Tfu_1647 was the regulatory node on this pathway. Significance and Impact of the Study: Thermobifida fusca B6 was the first reported micro-organism using its native-occurring pathway to accumulate adipic acid. It had the highest reported yield and titre of adipic acid so far.