The Post-Polyketide Synthase Modification Mechanism in Hitachimycin Biosynthesis.

Area of Science:

• Biochemistry
• Molecular Biology
• Natural Product Biosynthesis

Background:

• Hitachimycin is a bicyclic macrolactam antibiotic featuring (S)-β-phenylalanine (β-Phe).
• While initial steps involving β-amino acids and polyketide synthases (PKSs) are understood, the post-PKS modifications forming hitachimycin's unique structure are unknown.

Purpose of the Study:

• To investigate the post-PKS modification mechanism in hitachimycin biosynthesis.
• To identify the specific genes and enzymes responsible for constructing the bicyclic structure.

Main Methods:

• Inactivation of six putative post-PKS modification genes (hitM1-hitM6) in Streptomyces scabrisporus.
• Analysis of accumulated intermediates in gene knockout strains.
• In vitro enzymatic assays using purified enzymes and intermediates.

Main Results:

• Deletion of hitM4 led to accumulation of an all-trans-2,4,6,8,18-pentaene macrolactam, identified as an early post-PKS intermediate.
• Deletion of hitM1 resulted in 10-O-demethyl-10-oxohitachimycin (M1-A).
• Enzymatic studies showed HitM1 (a reductase) and HitM6 (a methyltransferase) are crucial for converting M1-A to hitachimycin.

Conclusions:

• A plausible post-PKS modification pathway for hitachimycin biosynthesis has been proposed.
• HitM4 is involved in an early step, while HitM1 and HitM6 catalyze later modifications, including reduction and methylation, to form the final antibiotic structure.