Functional analysis of post-PKS enzymes in hitachimycin
biosynthesis
Akimasa MIYANAGA
Department of Chemistry, Tokyo Institute of Technology

Research aims
Hitachimycin, a β-amino acid-containing macrolactam polyketide antibiotic isolated from
Streptomyces scabrisporus, has both antiprotozoal and antitumor activities[1]. The main
structural features of hitachimycin are the β-phenylalanine starter unit of its polyketideskeleton and a bicyclic structure with an internal five-membered carbocycle (Fig. 1). The
bicyclic structure likely results from post-polyketide synthase (post-PKS) modification
reactions. However, the mechanism underlying the formation of such a bicyclic structure
remains unclear. In this study, we used gene-disruption and biochemical analyses toinvestigate putative enzymes that have been proposed to function in the post-PKS
modification reactions of hitachimycin biosynthesis.

Figure 1. β-amino acid-containing macrolactam polyketide antibiotics
Methods
We previously reported the identification of the hitachimycin biosynthetic gene cluster ofS. scabrisporus[2]. Within this cluster, six genes (hitM1, hitM2, hitM3, hitM4, hitM5, andhitM6) have been proposed to play roles in the post-PKS modification reactions ofhitachimycin biosynthesis. While the hitM1 and hitM4 genes encode putativeoxidoreductases, hitM2 and hitM5 encode putative isomerases, hitM3 encodes a putative
cytochrome P450 monooxygenase, and hitM6 encodes a putative methyltransferase. Here,we constructed S. scabrisporus disruption mutants (disruptants) for these six genes (referredto as ∆hitM1 to ∆hitM6 strains) and analyzed the compounds they were producing.
Furthermore, we performed in vitro biochemical analyses of the reaction catalyzed by theHitM4 protein using recombinant HitM4 and the hitachimycin biosynthetic intermediate
produced by the ∆hitM4 strain.
Results
To confirm that the hitM1-6 genes are involved in hitachimycin biosynthesis, we firstexamined whether the ∆hitM1 to ∆hitM6 strains were producing hitachimycin. As expected,
none of the disruptants were producing hitachimycin, showing that these genes are indeed
involved in hitachimycin biosynthesis. Moreover, we found that some of these strains wereaccumulating hitachimycin derivatives. Among them, the ∆hitM1 and ∆hitM6 strains
produced hitachimycin derivatives that contained a bicyclic structure. This observationstrongly suggests that HitM1 and HitM6 catalyze reactions that are downstream of theformation of the bicyclic structure.
Conversely, the ∆hitM4 strain produced a hitachimycin biosynthetic intermediate (referredto as compound 1) that does not contain a bicyclic structure. Therefore, it is likely that
HitM4 enzymatic activity is involved in the formation of the bicyclic structure. To further
investigate the function of HitM4, we expressed recombinant HitM4 in E. coli and purified
the protein to homogeneity. We then optimized the reaction conditions and successfully
detected the conversion of compound 1 by HitM4 in vitro.
Conclusions
In this study, we examined post-PKS modification reactions in the hitachimycin
biosynthetic pathway. Our gene-disruption and biochemical analyses clearly showed that six
enzymes (HitM1 to HitM6) are involved in the post-PKS modification reactions ofhitachimycin biosynthesis. Our data suggest that, among these six enzymes, HitM4 is
responsible for the first step of the post-PKS modification reactions. Although this study
represents significant progress, further analyses are necessary to elucidate the mechanism
underlying the formation of the bicyclic structure of hitachimycin.
References
1) Ōmura, S., Nakagawa, A., Shibata, K, and Sano, H. (1982) The structure of hitachimycin,a novel macrocyclic lactam involving β-phenylalanine. Tetrahedron Lett. 23: 4713-4716.
2) Kudo, F. et al. (2015) Genome mining of the hitachimycin biosynthetic gene cluster:
involvement of a phenylalanine-2,3-aminomutase in biosynthesis. Chembiochem 16:
909-914.