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 5 steps: β-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 WT strain was the reason why it didnt produce any adipic acid.We then demonstrated that in T.fusca B6,the maximal titer of adipic acid on 50 g/L glucose was 2.23 g/L with 0.045 g/g-glucose yield and 0.22 g/L adipic acid was produced from 19.38 g/L 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 Study: T.fusca B6 was the first reported microorganism using its native-occurring pathway to accumulate adipic acid.It had the highest reported yield and titer of adipic acid so far.