Berberine bridge enzyme-like oxidase-catalysed double bond isomerization acts as the pathway switch in cytochalasin synthesis

Jin-Mei Zhang,Dehai Li,Xuan Liu,Qian Wei,Chuanteng Ma,Yi Zou

doi:10.1038/s41467-021-27931-z

Jin-Mei Zhang, Dehai Li + Show 4 more

Open Access

https://doi.org/10.1038/s41467-021-27931-z

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Abstract

Cytochalasans (CYTs), as well as their polycyclic (pcCYTs) and polymerized (meCYTs) derivatives, constitute one of the largest families of fungal polyketide-nonribosomal peptide (PK-NRP) hybrid natural products. However, the mechanism of chemical conversion from mono-CYTs (moCYTs) to both pcCYTs and meCYTs remains unknown. Here, we show the first successful example of the reconstitution of the CYT core backbone as well as the whole pathway in a heterologous host. Importantly, we also describe the berberine bridge enzyme (BBE)-like oxidase AspoA, which uses Glu538 as a general acid biocatalyst to catalyse an unusual protonation-driven double bond isomerization reaction and acts as a switch to alter the native (for moCYTs) and nonenzymatic (for pcCYTs and meCYTs) pathways to synthesize aspochalasin family compounds. Our results present an unprecedented function of BBE-like enzymes and highly suggest that the isolated pcCYTs and meCYTs are most likely artificially derived products.

Highlights

Cytochalasans (CYTs), as well as their polycyclic and polymerized derivatives, constitute one of the largest families of fungal polyketide-nonribosomal peptide (PK-NRP) hybrid natural products
Based on the abovementioned facts, to investigate the function of each cyt biosynthetic gene clusters (BGCs) gene, and especially to clarify these two common crucial issues in CYT biosynthesis, we turned our attention to aliphatic amino acid-type CYTs
We sequenced the genome of A. flavipes KLA0325, used CcsA as the probe, and found two possible cyt BGCs, which are shown in Fig. 2a. (1) Cluster 1 shares similar gene compositions and organizations with the ccs cluster (Supplementary Fig. 5a), and shares the identical A domain codes of the NRPS module (Supplementary Fig. 5b); cluster 1 should be responsible for the synthesis of L-Phe-type moCYTs. (2) Apart from the four-gene conserved cassette and regulation gene, cluster 2 has three tailoring genes, a cytochrome P450 monooxygenase gene, a short chain dehydrogenase/reductase (SDR) gene and a flavindependent oxidase gene, where aspoA is distinguishable from the flavin-dependent BVMO gene in cluster 1. (3)