Biochemical and genetic insights into asukamycin biosynthesis

Abstract

Asukamycin, a member of the manumycin family metabolites, is an antimicrobial and potential antitumor agent isolated from Streptomyces nodosus subsp. asukaensis. The entire asukamycin biosynthetic gene cluster was cloned, assembled, and expressed heterologously in Streptomyces lividans. Bioinformatic analysis and mutagenesis studies elucidated the biosynthetic pathway at the genetic and biochemical level. Four gene sets, asuA-D, govern the formation and assembly of the asukamycin building blocks: a 3-amino-4-hydroxybenzoic acid core component, a cyclohexane ring, two triene polyketide chains, and a 2-amino-3-hydroxycyclopent-2-enone moiety to form the intermediate protoasukamycin. AsuE1 and AsuE2 catalyze the conversion of protoasukamycin to 4-hydroxyprotoasukamycin, which is epoxidized at C5-C6 by AsuE3 to the final product, asukamycin. Branched acyl CoA starter units, derived from Val, Leu, and Ile, can be incorporated by the actions of the polyketide synthase III (KSIII) AsuC3/C4 as well as the cellular fatty acid synthase FabH to produce the asukamycin congeners A2-A7. In addition, the type II thioesterase AsuC15 limits the cellular level of omega-cyclohexyl fatty acids and likely maintains homeostasis of the cellular membrane.

FIGURE 1.

Structures of asukamycin and related metabolites. Compounds are grouped according to the main A, B, C, and D core units.

FIGURE 2.

Open reading frames identified in the asu cluster. The putative genes were divided into eight groups based on the functional characterization. Group A, 3,4-AHBA biosynthesis and adenylation; Group B, CHC-CoA biosynthesis; Group C, polyketide chain assembly; Group D, C₅N moiety biosynthesis; Group E, oxygenation; Group R, transcriptional regulation. AsuM1, efflux protein. The relative genomic regions and overlapping inserts of three cosmid clones, 2B9, 10D6, and pART1391, are indicated with bold lines.

FIGURE 3.

Assembly of two cosmids with overlapping inserts. a, cosmids 2B9 and 10D6 were linearized by SpeI and XbaI, respectively. Homologous recombination occurred at two overlapping insert and vector regions, indicated by heavy lines. b, the restriction map of 2B9, 10D6, and pART1361. The inserts are shown with solid lines. The EcoRI fragment sizes are indicated (kb). c, comparison of the EcoRI digestion patterns by DNA gel electrophoresis.

FIGURE 4.

HPLC analysis of asukamycin metabolites. a, 20 μl of crude culture extract of the asuB1 mutant. b, the asuC3C4 double mutant. c, the asuC15 mutant. d, S. lividans K4-114/pART1391. e, S. nodosus subsp. asukaensis wild type strain. The related peaks of asukamycins A1–A5 and B1–B4 are indicated. The y axis indicates the absorbance abundance. Panel b was calculated based on a 10× injection amount.

FIGURE 5.

Proposed asukamycin biosynthetic pathway. The primary metabolite substrates are shown in squares. The ACP and aroyl carrier protein are in capsules.