Insertion Mutation in HMG-CoA Lyase Increases the Production Yield of MPA through Agrobacterium tumefaciens-Mediated Transformation
2016
Dong, Y., East China University of Science and Technology, Shanghai, China | Zhang, J., East China University of Science and Technology, Shanghai, China | Xu, R., East China University of Science and Technology, Shanghai, China | Lv, X., East China University of Science and Technology, Shanghai, China | Wang, L., East China University of Science and Technology, Shanghai, China | Sun, A., East China University of Science and Technology, Shanghai, China | Wei, D., East China University of Science and Technology, Shanghai, China
Mycophenolic acid (MPA) is an antibiotic produced by Penicillium brevicompactum. MPA has antifungal, antineoplastic, and immunosuppressive functions, among others. beta-Hydroxy-beta- methylglutaryl-CoA (HMG-CoA) lyase is a key enzyme in the bypass metabolic pathway. The inhibitory activity of HMG-CoA lyase increases the MPA biosynthetic flux by reducing the generation of by-products. In this study, we cloned the P. brevicompactum HMG-CoA lyase gene using the thermal asymmetric interlaced polymerase chain reaction and gene walking technology. Agrobacterium tumefaciens-mediated transformation (ATMT) was used to insert a mutated HMG-CoA lyase gene into P. brevicompactum. Successful insertion of the HMG-CoA lyase gene was confirmed by hygromycin screening, PCR, Southern blot analysis, and enzyme content assay. The maximum MPA production by transformants was 2.94 g/l. This was 71% higher than wild-type ATCC 16024. Our results demonstrate that ATMT may be an alternative practical genetic tool for directional transformation of P. brevicompactum.
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