Eine chimäre pilzliche Diterpensynthase der Klade II‐D aus Colletotrichum gloeosporioides produziert Dolasta‐1(15),8‐dien

Abstract

Basierend auf einer Plattform zur Überproduktion von Terpenoiden in Hefe für" Genome-Mining" wurde eine chimäre Diterpensynthase aus dem endophytischen Pilz Colletotrichum gloeosporioides ES026 als (5R,12R,14S)-Dolasta-1(15),8-dien-Synthase charakterisiert. Die absolute Konfiguration wurdeu nabhängig durch die Verwendung enantioselektiv deuterierter Terpenvorstufen bestimmt, wodurchd er vorhergesagte C1-III-IV-Cyclisierungsmodus dieses ersten charakterisierten Enzyms der Klade II-D bestätigt wurde. Isotopenmarkierungsexperimente und die Isolierung des Intermediates (1R)-d-Araneosen stützen den vorgeschlagenen Cyclisierungsmechanismus.

A Branched Diterpene Cascade: The Mechanism of Spinodiene Synthase from Saccharopolyspora spinosa

“…Reprotonation of 3 proceeds with a strict stereochemical course through addition to the Si ‐face, as followed by HSQC. The 1,4‐hydride shift from B to C was confirmed by incubation of (2‐ 2 H)GPP and (3‐ 13 C)IPP with SoS and GGPPS (Figure S45, triplet for C‐7), and the 1,2‐hydride shift to D was demonstrated by the incubation of (2‐ 2 H)DMAPP and (2‐ 13 C)IPP, synthesised from (2‐ 13 C)acetic acid (Scheme S2), with SoS, FPPS, and GGPPS (Figure S46, triplet for C‐10). For the stereochemical investigation of the final deprotonation step, (1 R )‐ and (1 S )‐(1‐ 2 H)IPP were synthesised (Scheme S3, Figure S47) and incubated with isopentenyl diphosphate isomerase (IDI) from Serratia plymuthica , followed by its heat inactivation and elongation of the resulting (1 R )‐ and (1 S )‐(1‐ 2 H)DMAPP with excess unlabelled IPP by FPPS and GGPPS to suppress the incorporation of additional labelled IPP units.…”

“…The biosynthesis of the three SoS products was investigated using all twenty ( 13 C 1 )GGPP isotopomers that were obtained by synthesis or prepared enzymatically from other synthetic 13 C‐labelled precursors such as GPP, FPP, and isopentenyl diphosphate (IPP) using a GGPP synthase (GGPPS) from Streptomyces cyaneofuscatus , and converted by SoS to give a product mixture of singly 13 C‐labelled diterpenes 1 – 3 . 13 C NMR analysis of these samples allowed for an unambiguous assignment of all the carbon atoms in these products to their originating positions in GGPP (Figures S28–S30).…”

“…Theobtained NMR data for the main product 1 (Table S1, Figures S5-S12) suggested the structure of ad iterpene with a6 -7-5 ring system featuring ar are conjugated diene.N OE correlations fixed the relative configuration with an unusual cis stereochemistry at the fused five-membered ring. In terms of ring sizes,s ome similarity of 1 to the dolastane diterpenes [12] can be found, but the ring arrangement is different and 1 represents anew diterpene skeleton. With respect to the origin of its producing enzyme, 1 was named spinodiene A and the TS from S. spinosa characterised as as pinodiene synthase (SoS).…”

An Unusual Skeletal Rearrangement in the Biosynthesis of the Sesquiterpene Trichobrasilenol from Trichoderma

“…The hydride migration from A to B was followed by using (3-13 C,2-2 H)FPP, [24] resulting in a slightly upfield-shifted strong triplet for C3 of 6 in the 13 C NMR spectrum (Scheme 1 D, Figure 3 A). The third hydride migration from B to C was investigated through the same strategy using (2-2 H)GPP [26] and (2-13 C)IPP, [27] which were coupled by FPPS to (2-13 C,6-2 H)FPP. Enzymatic conversion with TaTC6 yielded a product with a direct 13 C À 2 H bond as indicated by a strongly enhanced triplet for C2 in the 13 C-NMR spectrum (Scheme 1 E, Figure 3 B).…”

Eine ungewöhnliche Gerüstumlagerung in der Biosynthese des Sesquiterpens Trichobrasilenol aus Trichoderma

“…Die Hydridwanderung von A zu B wurde mit (3-13 C,2-2 H)FPP verfolgt, [24] wodurch sich im 13 C-NMR-Spektrum ein leicht hochfeldverschobenes starkes Triplett für C3 von 6 ergab (Schema 1 D, Abbildung 3 A). Die dritte Hydridwanderung von B zu C wurde durch dieselbe Strategie unter Verwendung von (2-2 H)GPP [26] und (2-13 C)IPP [27] untersucht, die mit FPPS zu (2-13 C,6-2 H)FPP gekuppelt wurden. Enzy-…”