Directed Biosynthesis of Mitragynine Stereoisomers.

Mitragyna speciosa ("kratom") is used as a natural remedy for pain and management of opioid dependence. The pharmacological properties of kratom have been linked to a complex mixture of monoterpene indole alkaloids, most notably mitragynine. Here, we report the central biosynthetic steps responsible for the scaffold formation of mitragynine and related corynanthe-type alkaloids. We illuminate the mechanistic basis by which the key stereogenic center of this scaffold is formed. These discoveries were leveraged for the enzymatic production of mitragynine, the C-20 epimer speciogynine, and fluorinated analogues.

Stereochemical characterisation of the non-canonical α-humulene synthase from Acremonium strictum.

The non-canonical fungal α-humulene synthase was investigated through isotopic labelling experiments for its stereochemical course regarding inversion or retention at C-1, the face selectivity at C-11, and the stereoselectivity of the final deprotonation. A new and convenient desymmetrisation strategy was developed to enable a full stereochemical analysis of the catalysed steps to the achiral α-humulene product from stereoselectively labelled farnesyl diphosphate.

Understanding and Engineering the Stereoselectivity of Humulene Synthase.

The non-canonical terpene cyclase AsR6 is responsible for the formation of 2E,6E,9E-humulene during the biosynthesis of the tropolone sesquiterpenoid (TS) xenovulene A. The structures of unliganded AsR6 and of AsR6 in complex with an in crystallo cyclized reaction product and thiolodiphosphate reveal a new farnesyl diphosphate binding motif that comprises a unique binuclear Mg2+ -cluster and an essential K289 residue that is conserved in all humulene synthases involved in TS formation. Structure-based site-directed mutagenesis of AsR6 and its homologue EupR3 identify a single residue, L285/M261, that controls the production of either 2E,6E,9E- or 2Z,6E,9E-humulene. A possible mechanism for the observed stereoselectivity was investigated using different isoprenoid precursors and results demonstrate that M261 has gatekeeping control over product formation.

Synthetic Biology Driven Biosynthesis of Unnatural Tropolone Sesquiterpenoids.

Tropolone sesquiterpenoids (TS) are an intriguing family of biologically active fungal meroterpenoids that arise through a unique intermolecular hetero Diels-Alder (hDA) reaction between humulene and tropolones. Here, we report on the combinatorial biosynthesis of a series of unprecedented analogs of the TS pycnidione 1 and xenovulene A 2. In a systematic synthetic biology driven approach, we recombined genes from three TS biosynthetic gene clusters (pycnidione 1, xenovulene A 2 and eupenifeldin 3) in the fungal host Aspergillus oryzae NSAR1. Rational design of the reconstituted pathways granted control over the number of hDA reactions taking place, the chemical nature of the fused polyketide moiety (tropolono- vs. monobenzo-pyranyl) and the degree of hydroxylation. Formation of unexpected monobenzopyranyl sesquiterpenoids was investigated using isotope-feeding studies to reveal a new and highly unusual oxidative ring contraction rearrangement.

Investigating the biosynthesis of Sch-642305 in the fungus Phomopsis sp. CMU-LMA.

Sch-642305 is an unusual bicyclic 10-membered macrolide produced by the filamentous fungus Phomopsis sp. CMU-LMA for which no biosynthetic evidence exists. Here, we generate a draft genome sequence of the producing organism and discover the biosynthetic gene cluster responsible for formation of Sch-642305. Targeted gene disruptions together with reconstitution of the pathway in the heterologous host Aspergillus oryzae dissect key chemical steps and shed light on a series of oxidoreductions occuring in the pathway.

Three previously unrecognised classes of biosynthetic enzymes revealed during the production of xenovulene A.

Xenovulene A is a complex fungal meroterpenoid, produced by the organism hitherto known as Acremonium strictum IMI 501407, for which limited biosynthetic evidence exists. Here, we generate a draft genome and show that the producing organism is previously unknown and should be renamed as Sarocladium schorii. A biosynthetic gene cluster is discovered which bears resemblance to those involved in the biosynthesis of fungal tropolones, with additional genes of unknown function. Heterologous reconstruction of the entire pathway in Aspergillus oryzae allows the chemical steps of biosynthesis to be dissected. The pathway shows very limited similarity to the biosynthesis of other fungal meroterpenoids. The pathway features: the initial formation of tropolone intermediates; the likely involvement of a hetero Diels-Alder enzyme; a terpene cyclase with no significant sequence homology to any known terpene cyclase and two enzymes catalysing oxidative-ring contractions.