Báo cáo khoa học: Biochemical characterization of the minimal polyketide synthase domains in the lovastatin nonaketide synthase LovB

Biochemical characterization of the minimal polyketide synthase domains in the lovastatin nonaketide synthase LovB

Author(s):

Suzanne M. Ma and Yi Tang

Field:

Chemical and Biomolecular Engineering

Document Content:

This study investigates the minimal polyketide synthase (PKS) domains of LovB, a megasynthase involved in lovastatin biosynthesis by the filamentous fungus Aspergillus terreus. LovB synthesizes dihydromonacolin L using nine malonyl-coenzyme A molecules. The research focuses on characterizing the ketosynthase (KS) and malonyl-CoA:acyl carrier protein acyltransferase (MAT) domains, their substrate specificities, and their interactions with acyl carrier protein (ACP) domains. The findings shed light on the mechanisms of iterative PKS, the priming of polyketide synthesis, and potential applications in engineering novel polyketides.

Detailed Table of Contents:

• Keywords: filamentous fungi; ketosynthase; lovastatin; megasynthase; polyketide
• Abstract
• Introduction
• Figure 1. Biosynthetic pathway for lovastatin in A. terreus.
• Domains of PKS
• LovB structure and function
• Figure 2. Domain organization of LovB and the standalone domains studied in this work.
• Results and Discussion
• Expression of minimal PKS domains
• Biochemical characterization of the LovB KS-MAT didomain
• Figure 3. [1,2-14C]malonyl-CoA and [1-14C]acetyl-CoA labeling of the LovB KS-MAT.
• Isolation and cloning of standalone MAT domain
• Figure 4. Identification of a MAT proteolytic fragment.
• Characterization of the standalone MAT protein
• Figure 5. Michaelis-Menten plot of the rate of acylation catalyzed by LovB MAT domain.
• Biochemical characterization of the LovB KS-MAT (S656A) domain
• Figure 6. LovB KS-MAT° mediated malonyl transfer from [14C]malonyl-CoA to different ACP acceptors.
• Conclusion
• Experimental procedures
• Strains
• Cloning of domains
• Site-directed mutagenesis
• Expression and purification of ACP proteins
• Expression and purification of the LovB KS-MAT, LovB MAT, LovB MAT (S208A), LovB MAT (S209A) and LovB KS-MAT (S656A)
• Labeling of enzymes by [14C]-malonyl- and [14C]-acetyl-CoA
• Transacylation activity of enzymes
• Polyketide turnover assay
• Acknowledgements
• References