Insertion Mutation in HMG-CoA Lyase Increases the Production Yield of MPA through Agrobacterium tumefaciens-Mediated
Yuguo Dong1, Jian Zhang1, Rui Xu1
1State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, P.R. China.
Abstract:
Mycophenolic acid (MPA) is an antibiotic produced by Penicillium 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 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.
Insights
Genetic modification of *Penicillium brevicompactum* enhanced mycophenolic acid (MPA) production. Inhibiting HMG-CoA lyase increased MPA yield by 71% using *Agrobacterium tumefaciens*-mediated transformation.
Area of Science:
- Microbiology
- Biotechnology
- Metabolic Engineering
Background:
- Mycophenolic acid (MPA), an antibiotic from *Penicillium brevicompactum*, exhibits antifungal, antineoplastic, and immunosuppressive properties.
- β-Hydroxy-β-methylglutaryl-CoA (HMG-CoA) lyase plays a crucial role in a metabolic bypass pathway, influencing MPA biosynthesis.
- Inhibiting HMG-CoA lyase activity can redirect metabolic flux towards increased MPA production by minimizing by-product generation.
Purpose of the Study:
- To clone the HMG-CoA lyase gene from *P. brevicompactum*.
- To enhance MPA production in *P. brevicompactum* through genetic manipulation of HMG-CoA lyase.
- To evaluate *Agrobacterium tumefaciens*-mediated transformation (ATMT) as a tool for genetic modification in *P. brevicompactum*.
Main Methods:
- Cloning of the *P. brevicompactum* HMG-CoA lyase gene using thermal asymmetric interlaced polymerase chain reaction and gene walking.
- Insertion of a mutated HMG-CoA lyase gene into *P. brevicompactum* via ATMT.
- Confirmation of gene insertion using hygromycin screening, PCR, Southern blot analysis, and enzyme assays.
Main Results:
- Successful cloning and insertion of the HMG-CoA lyase gene into *P. brevicompactum*.
- Transformants achieved a maximum MPA production of 2.94 g/l, representing a 71% increase compared to the wild-type strain (ATCC 16024).
- Enzyme assays confirmed the inhibitory effect on HMG-CoA lyase activity.
Conclusions:
- Targeted inhibition of HMG-CoA lyase significantly boosts MPA biosynthesis in *P. brevicompactum*.
- ATMT is demonstrated as an effective and practical method for the genetic engineering of *P. brevicompactum* for enhanced metabolite production.
