Discovery of a polyketide carboxylate phytotoxin from a polyketide glycoside hybrid by β-glucosidase mediated ester bond hydrolysis

Abstract

Fungal phytotoxins cause significant harm to agricultural production or lead to plant diseases. Discovering new phytotoxins, dissecting their formation mechanism and understanding their action mode are important for controlling the harmful effects of fungal phytopathogens. In this study, a long-term unsolved cluster (polyketide synthase 16, PKS16 cluster) from Fusarium species was thoroughly investigated and a series of new metabolites including both complex α-pyrone-polyketide glycosides and simple polyketide carboxylates were identified from F. proliferatum. The whole pathway reveals an unusual assembly and inactivation process for phytotoxin biosynthesis, with key points as follows: (1) a flavin dependent monooxygenase catalyzes Baeyer-Villiger oxidation on the linear polyketide side chain of α-pyrone-polyketide glycoside 8 to form ester bond compound 1; (2) a β-glucosidase unexpectedly mediates the ester bond hydrolysis of 1 to generate polyketide carboxylate phytotoxin 2; (3) oxidation occurring on the terminal inert carbons of 2 by intracellular oxidase(s) eliminates its phytotoxicity. Our work identifies the chemical basis of the PKS16 cluster in phytotoxicity, shows that polyketide carboxylate is a new structural type of phytotoxin in Fusarium and importantly uncovers a rare ester bond hydrolysis function of β-glucosidase family enzymes.

Fig. 1. Fungal phytotoxins and PKS16 clusters from Fusarium species. (a) The well-known phytotoxins produced by Fusarium sp. (b) PKS16 and its homologue clusters in F. fujikuroi species. The PKS16 cluster in F. fujikuroi IMI 58289 lacks the key tailoring-step genes, while the core hrPKS is truncated in stains C1995 and E282.

Fig. 2. Identification of the homologue pro cluster and confirmation of the corresponding products in F. proliferatum. (a) Organization and proposed gene functions of the pro cluster. (b) LC-MS analysis of the extracts from F. proliferatum and its knockout mutants. (c) Chemical structures of compounds 1–6.

Fig. 3. The hrPKS and α,β-hydrolase collaboratively produce α-pyrone-polyketide precursor 7. (a) Two proposed pathways for the collaboration of hrPKS ProF and α,β-hydrolase ProG. (b) LC-MS analysis of the culture extracts from AN-proF and AN-proFG. (c) The yields of 7 increased nearly fourfold in AN-proFG indicating that ProG plays a supportive role with ProF in the biosynthesis of 7.

Fig. 4. Confirmation of the function of ProE, ProI and ProJ. (a) LC-MS analysis of the culture extracts from AN-proFGIJE and AN-proFGIJ. (b) LC-MS analysis of the culture extracts from proE and proI knockout mutants in F. proliferatum. (c) The in vitro biochemical assays of ProE, ProI and ProJ. (d) The proposed pathway from 7 to 1.

Fig. 5. Investigation of the function of ProL and ProK. (a) LC-MS analysis of the culture extracts from proL and proK knockout mutants in F. proliferatum. (b) LC-MS analysis of the incorporation of H₂¹⁸O into 2. (c) The proposed pathway from 1 to 2 and 9, respectively. β-Glucosidase ProL is involved in the hydrolysis of 1 to form 2. (d) The bioconversion of 1 to 2 by the crude enzymes of AN-proL.

Fig. 6. The root growth inhibition activity tests against Arabidopsis thaliana of compounds 1–9. (a) Phenotypic characteristics of seven-day-old Arabidopsis thaliana grown on Murashige and Skoog basal medium containing 50 μg per mL 1–9 (DMSO: dimethyl sulfoxide). (b) Quantification of the total root length of seven-day-old Arabidopsis thaliana grown on medium containing 50 μg per mL 1–9. In box plots, the center line is the median, box edges delineate first and third quartiles and whiskers show the range of values (n = 3 independent experiments), unpaired two-tailed Student's t-test (***p ≤ 0.001, ****p ≤ 0.0001, ns stands for no significant difference).

Fig. 7. Formation of polyketide carboxylate 2 from polyketide glycoside 1 occurs through β-glucosidase mediated ester bond hydrolysis.