Fungal polyketide synthase product chain-length control by partnering thiohydrolase

Abstract

Fungal highly reducing polyketide synthases (HRPKSs) are an enigmatic group of multidomain enzymes that catalyze the biosynthesis of structurally diverse compounds. This variety stems from their intrinsic programming rules, which permutate the use of tailoring domains and determine the overall number of iterative cycles. From genome sequencing and mining of the producing strain Eupenicillium brefeldianum ATCC 58665, we identified an HRPKS involved in the biosynthesis of an important protein transport-inhibitor Brefeldin A (BFA), followed by reconstitution of its activity in Saccharomyces cerevisiae and in vitro. Bref-PKS demonstrated an NADPH-dependent reductive tailoring specificity that led to the synthesis of four different octaketide products with varying degrees of reduction. Furthermore, contrary to what is expected from the structure of BFA, Bref-PKS is found to be a nonaketide synthase in the absence of an associated thiohydrolase Bref-TH. Such chain-length control by the partner thiohydrolase was found to be present in other HRPKS systems and highlights the importance of including tailoring enzyme activities in predicting fungal HRPKS functions and their products.

Figure 1

Putative biosynthethic pathway for 1. The HRPKS is proposed to synthesize the precisely reduced octaketide precursor, which could then be directly offloaded by the thiohydrolase enzyme followed by a P450-mediated formation of the cyclopentane ring and macrocyclization to afford the 7-deoxy BFA 2 (top scheme). Alternatively, the first ring annulation can also occur on the ACP-tethered intermediate before the thiohydrolase release and lactonization (bottom scheme). The C7-hydroxylation is believed to be the final step in the process to obtain the final structure of 1.

Figure 2

Transcriptional analysis of genes in Contig_286 determines the putative boundary of the bref cluster. (A) Arrangement of genes in Contig_286. (B) RT-PCR analysis on the annotated genes within the contig. The template mRNA was extracted from a Day2 BFA-producing culture of E. brefeldianum in the optimized production media, MEM.

Figure 3

In vitro products of Bref-PKS and Bref-TH are acyclic octaketides with variable degrees of β-reduction. (A) HPLC and EIC trace of the in vitro reaction between Bref-PKS and Bref-TH. (B) Production of compounds 3–6 from the S. cerevisiae-NpgA strain co-expressing Bref-PKS and Bref-TH. Notice the change in the production profile between days 1 and 3. The compounds were purified according to their peak production period. (C) Elucidated structures of compounds 3–6 from the corresponding NMR spectra.

Figure 4

In vitro reactions with Bref-PKS demonstrate the TH-controlled PKS chain length release. (A) EIC spectra of the in vitro reactions showed the variation in the product profiles of Bref-PKS with Bref-TH; Bref-PKS with base hydrolysis; Bref-PKS with Bref-TH H276A; and Bref-PKS with other in trans releasing enzymes CazE and Fma-AT. The reactions consist of 20 μM Bref-PKS, 2 mM mCoA, and 10 mM NADPH with either 20 μM concentration of the releasing enzyme or base hydrolysis (1 M NaOH at 65 °C for 10 min). (B) Proposed structures of 7 and 8.

Figure 5

Fma-PKS produces longer polyenes in the absence of cognate Fma-AT. (A) HPLC profiles of Fma-PKS with Fma-AT; base hydrolysis; or Bref-TH. Fma-PKS produces a hexaketide polyene in the presence of the partner Fma-AT. In its absence or in the presence of the non-cognate Bref-TH, the PKS catalyzes 1 or 2 more extension steps to yield the heptaketide 10 and octaketide 11. The reactions consist of 20 μM Fma-PKS, 2 mM mCoA, and 2 mM NADPH with either 20 μM of the releasing enzyme or base hydrolysis (1 M NaOH at 65 °C for 10 min). (B) Proposed structures of the polyene compounds produced in the in vitro assay.

Figure 6

Summary of the programming rules exhibited by Bref-PKS. From the in vitro studies, we were able to fully reconstitute the complex programming of this model HRPKS. Bref-PKS uses different permutations of the reductive domains at each extension cycle and selectively offloads the correct octaketide products with the partner Bref-TH or the longer nonaketide products with base hydrolysis. Compounds 4 and 6 that resulted from additional enoyl reduction at the final extension are italicized.