Offloading Role of a Discrete Thioesterase in Type II Polyketide Biosynthesis

Abstract

Type II polyketides are a group of secondary metabolites with various biological activities. In nature, biosynthesis of type II polyketides involves multiple enzymatic steps whereby key enzymes, including ketoacyl-synthase (KS_α), chain length factor (KS_β), and acyl carrier protein (ACP), are utilized to elongate the polyketide chain through a repetitive condensation reaction. During each condensation, the biosynthesis intermediates are covalently attached to KS_α or ACP via a thioester bond and are then cleaved to release an elongated polyketide chain for successive postmodification. Despite its critical role in type II polyketide biosynthesis, the enzyme and its corresponding mechanism for type II polyketide chain release through thioester bond breakage have yet to be determined. Here, kinamycin was used as a model compound to investigate the chain release step of type II polyketide biosynthesis. Using a genetic knockout strategy, we confirmed that AlpS is required for the complete biosynthesis of kinamycins. Further in vitro biochemical assays revealed high hydrolytic activity of AlpS toward a thioester bond in an aromatic polyketide-ACP analog, suggesting its distinct role in offloading the polyketide chain from ACP during the kinamycin biosynthesis. Finally, we successfully utilized AlpS to enhance the heterologous production of dehydrorabelomycin in Escherichia coli by nearly 25-fold, which resulted in 0.50 g/liter dehydrorabelomycin in a simple batch-mode shake flask culture. Taken together, our results provide critical knowledge to gain an insightful understanding of the chain-releasing process during type II polyketide synthesis, which, in turn, lays a solid foundation for future new applications in type II polyketide bioproduction.

Keywords: biosynthesis; natural product; offloading; thioesterase; type II polyketides.

FIG 1

Biosynthesis of kinamycin. (A) Kinamycin biosynthetic gene cluster in Streptomyces galtieri Sgt26. The genes related with the biosynthesis of the dehydrorabelomycin are marked with green, and alpS is highlighted in red. (B) The proposed biosynthetic pathway of kinamycin.

FIG 2

HPLC analysis of the knockout and complement experiments of alpS in S. albus J1074. (i) S. albus J1074 (2E9 ΔalpW ΔalpS); (ii) S. albus J1074 (2E9 ΔalpW ΔalpS::alpS); (iii) S. albus J1074 (2E9 ΔalpW) (control). HPLC traces were recorded at 424 nm.

FIG 3

Sequence alignment of AlpS and other type II TEs with the editing function in type I PKSs. The conserved GHSMG motifs are marked by rectangles, and the Ser-His-Asp triad is highlighted in red.

FIG 4

Analysis of the hydrolytic activity of AlpS toward SNAC esters. (A) Hydrolysis of Ac-SNAC by AlpS and propionyl-SNAC by FscTE. (B) Time courses of AlpS (■) and Fsc-TE (●) hydrolysis of propionyl-SNAC. (C) HPLC analysis of hydrolysis Ac-SNAC: (i) Ac-SNAC standard; (ii) Ac-SNAC with boiled AlpS; (iii) Ac-SNAC with AlpS; (iv) anthraquinone-2-carboxylic acid standard. HPLC traces were recorded at 276 nm.

FIG 5

AlpS hydrolysis activity toward Ac-SNAC is dependent on the catalytic triad. HPLC analysis of the hydrolytic activity of AlpS and its catalytic triad mutants. (i) Ac-SNAC standard; (ii) Ac-SNAC with AlpS; (iii) Ac-SNAC with AlpS S89A mutant; (iv) Ac-SNAC with AlpS D202N mutant; (v) Ac-SNAC with AlpS H230A mutant; (vi) anthraquinone-2-carboxylic acid standard. HPLC traces were recorded at 276 nm.

FIG 6

The phylogenetic of the characterized TE proteins (domains) and some discrete thioesterases in type II polyketide biosynthetic clusters. AlpS, MtmZ, X26-TEII (ORF33), GrhD, Lcz34, San2, SimC2, SimC3, SauT, EncL, ZhuC, DpsD, and OxyP are discrete thioesterases found in type II polyketide biosynthetic clusters. TylO, FscTE, RifR, PikAV, EryTII, MonCII, NanE, and NigCII are type II TEs in type I PKSs. GrsT, TycF, BacT, SrfD, and YbtT are type II TEs in NRPSs. DEBS_TE, PICS_TE, NysK_TE, Ampho_TE, and Spn_TE are type I TE domains in type I PKSs. Lch_TE, Bat_TE, Srf_TE, HctF_TE, and CrpD_TE are type I TE domains in NRPSs. The accession numbers of the proteins used in this phylogenetic tree are listed in Table S3.

FIG 7

The influence of AlpS in the E. coli dehydrorabelomycin production. (i) E. coli BAP1/pGro7/pXY-2/pXY-3/pXY-6 without IPTG inducing (control); (ii) E. coli BAP1/pGro7/pXY-2/pXY-3/pXY-6; (iii) E. coli BAP1/pGro7/pKM-1/pXY-3/pXY-6 (the crude extract was diluted 10 times before HPLC analysis). pKM-1contains one copy of alpS. HPLC traces were recorded at 424 nm.

FIG 8

Time courses of the dehydrorabelomycin production by BAP1/pGro7/pKM-1/pXY-3/pXY-6 (■) and BAP1/pGro7/pXY-2/pXY-3/pXY-6 (●). The error bars represent standard deviation of the experimental measurements for three independent experiments.