Diels–Alder Reactions During the Biosynthesis of Sorbicillinoids

Abstract

The sorbicillinoids are a class of biologically active and structurally diverse fungal polyketides arising from sorbicillin. Through co‐expression of sorA, sorB, sorC, and sorD from Trichoderma reesei QM6a, the biosynthetic pathway to epoxysorbicillinol and dimeric sorbicillinoids, which resemble Diels–Alder‐like and Michael‐addition‐like products, was reconstituted in Aspergillus oryzae NSAR1. Expression and feeding experiments demonstrated the crucial requirement of the flavin‐dependent monooxygenase SorD for the formation of dimeric sorbicillinoids, hybrid sorbicillinoids, and epoxysorbicillinol in vivo. In contrast to prior reports, SorD catalyses neither the oxidation of 2′,3′‐dihydrosorbicillin to sorbicillin nor the oxidation of sorbicillinol to oxosorbicillinol. This is the first report that both the intermolecular Diels–Alder and Michael dimerization reactions, as well as the epoxidation of sorbicillinol are catalysed in vivo by SorD.

Molecular methods unravel the biosynthetic potential of Trichoderma species

“…Hence, TrSorD was the rst avin-dependent enzyme reported to catalyze epoxidation, Diels-Alder and Michael addition reactions, which emphasize the immense chemical, genetic and enzymatic potential of Trichoderma spp. 90 Another knockout of sorA in T. reesei resulted not only in abolition of sorbicillinoid production, but also in the accumulation of a compound that was identied as scytolide 46, which was not previously reported from Trichoderma. 90 Isolation of spirosorbicillinol 43a/b from Trichoderma species was reported previously and its biosynthesis was proposed to be a result of Diels-Alder reaction between sorbicillinol 41a and scytolide 46.…”

“…Expression of all four genes (TrsorA-D) nally led to the production of compounds 42a/b, 44a/b/c and 45a/b. 90 Another interesting nding reported by Derntl et al 89 is that sorbicillinoid production results in reduction of cellular biomass, a nding that was related then to the possible growth limiting effect of sorbicillinoids against T. reesei itself, metabolic burden or due to the intracellular accumulation of sorbicillin and dihydrosorbicillin. 89 However, subsequent investigations by the same group reported the presence of an Xpp1 TF that acts as a switch between primary and secondary metabolism in T. reesei and leads to either accumulation of secondary metabolites or production of cell biomass in T. reesei.…”

“…For example, prediction of the function of a new gene in the sorbicillin BGC sor4/D in T. reesei based merely on a knockout experiment in the native host was later proved inaccurate aer heterologous expression of this gene in A. oryzae (Scheme 3, Section 4.1). 89,90 Therefore the application of more than one metabolic engineering strategy is oen preferable when trying to link Trichoderma metabolites to their biosynthetic gene clusters. In the following sections, we will discuss some selected examples of PKS, terpene and PKS-NRPS hybrid biosynthetic gene clusters that have been successfully linked to their secondary…”

“…Spirosorbicillinols 43a/ b were only produced in the latter strain. 90 Kahlert et al 90 then proposed the sorbicillinoid biosynthetic pathway (Scheme 3) based on the reconstitution of the sor BGC from T. reesei Qm6a (TrsorA-D) in the heterologous host A. oryzae NSAR1. Heterologous expression of TrsorA or TrsorB alone in A. oryzae led to the production of no new compounds.…”

“…This might also explain the production of trichopyrone 47 and related pyrone 48 as side products upon heterologous expression of sorA and sorB in A. oryzae (Scheme 4). 20,90 As in the sorbicillinoid pathway, the hr-PKS TlnA synthesize the triketide 66. The nr-PKS TlnB accepts the triketide 66 and extend it to the tetraketide 67, which is then methylated by the action of TlnC to the tetraketide 68.…”