Unveiling of Swainsonine Biosynthesis via a Multibranched Pathway in Fungi

Abstract

The indolizidine alkaloid swainsonine (SW) is a deadly mycotoxin to livestock that can be produced by different plant-associated fungi, including the endophytic entomopathogenic fungi Metarhizium species. The SW biosynthetic gene cluster has been identified but the genetic mechanism of SW biosynthesis remains obscure. To unveil the SW biosynthetic pathway, we performed gene deletions in M. robertsii, heterologous expression of a core biosynthetic gene, substrate feedings, mass spectrometry, and bioassay analyses in this study. It was unveiled that SW is produced via a multibranched pathway by the hybrid nonribosomal peptide−polyketide synthase (NRPS-PKS) gene cluster in M. robertsii. The precursor pipecolic acid can be converted from lysine by both the SW biosynthetic cluster and the unclustered genes such as lysine cyclodeaminase. The hybrid NRPS-PKS enzyme produces three intermediates with and without domain skipping. Intriguingly, the biosynthetic process is coupled with the cis to trans nonenzymatic epimerization of C1-OH for both hydroxyl-and dihydroxyl-indolizidine intermediates. We also found that SW production was dispensable for fungal colonization of plants and infection of insect hosts. Functional characterization of the SW biosynthetic genes in this study may benefit the safe use of Metarhizium fungi as insect biocontrol agents and the management of livestock pastures from SW contamination by genetic manipulation of the toxin-producing fungi.

Rethinking of the Roles of Endophyte Symbiosis and Mycotoxin in Oxytropis Plants

“…Some progress has been made to help understand PA formation during swainsonine biosynthesis. Continuing from the previous identification of the SWN cluster, Luo et al [61] further studied its function in M. robertsii. The disruption of swnA decreased PA by fivefold and therefore it should play an important role in the production of PA.…”

“…In fact, swainsonine producing strains showed slightly less virulence than the knockout mutant. Inoculation experiments showed that by M. robertsii infection did not produce detectable swainsonine in maize (Zea mays), soybean (Glycine max), and milkvetch (Astragalus sinicus) [61]. Thus, the production of swainsonine is not necessary for colonization of those characterized fungi.…”

“…For comparison, genes in purple indicate all the annotated genes with proposed function and position from the locoweed endophytic fungus A. oxytropis, based on its genome sequencing [63]; genes in green represent the proposed and some verified genes in M. robertsii, and "?" indicates that the function and position of activity of the gene still need further characterization [60,61]. Refer to the text for the name of the genes.…”

“…Refer to the text for the name of the genes. Certain chemical structures and reactions in the schematics were adapted from previous work by Cook et al and Luo et al [60,61].…”

The Chemistry of High‐Oxidation State Securinega Alkaloids

“…For group I and II high‐oxidation state securinega alkaloids, we propose that a direct C4 oxidation would occur in an analogous fashion to the C−H hydroxylation during the swainsonine biosynthesis (Scheme 15). [54,55] Group I high‐oxidation state securinega natural products can undergo further oxidations to group II high‐oxidation state securinega alkaloids. We hypothesize that group III and IV high‐oxidation state securinega alkaloids are all derived from N ‐oxide derivative 163 .…”