title: Insertion Mutation in HMG-CoA Lyase Increases the Production Yield of MPA throughAgrobacterium tumefaciens-Mediated Transformation.
abstract: Mycophenolic acid (MPA) is an antibiotic produced byPenicillium brevicompactum. MPA has antifungal, antineoplastic, and immunosuppressive functions, among others. β-Hydroxy-β-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 theP. brevicompactumHMG-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 intoP. 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 ofP. brevicompactum.
authors: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, P.R. China.
authors: Yuguo
authors: Y
authors: Dong
authors: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, P.R. China.
authors: Jian
authors: J
authors: Zhang
authors: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, P.R. China.
authors: Rui
authors: R
authors: Xu
authors: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, P.R. China.
authors: Xinxin
authors: X
authors: Lv
authors: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, P.R. China.
authors: Lihua
authors: L
authors: Wang
authors: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, P.R. China.
authors: Aiyou
authors: A
authors: Sun
authors: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, P.R. China.
authors: Dongzhi
authors: D
authors: Wei
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics*; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D016254:Mutagenesis, Insertional; D009173:Mycophenolic Acid / Q000378:metabolism*; D007652:Oxo-Acid-Lyases / Q000235:genetics* / Q000378:metabolism*; D010407:Penicillium / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D014170:Transformation, Genetic
chemical_list: D005656:Fungal Proteins; D007652:Oxo-Acid-Lyases; C021410:3-hydroxy-3-methylglutaryl-coenzyme A lyase; D009173:Mycophenolic Acid
keywords: Agrobacterium tumefaciens; HMG-CoA lyase; Penicillium brevicompactum; directional transformation; mycophenolic acid