In Vivo Study of the Sorbicillinoid Gene Cluster in Trichoderma reesei

Abstract

Sorbicillinoids are a diverse group of yellow secondary metabolites that are produced by a range of not closely related ascomycetes, including Penicillium chrysogenum, Acremonium chrysogenum, and Trichoderma reesei. They share a similarity to the name-giving compound sorbicillin, a hexaketide. Previously, a conserved gene cluster containing two polyketide synthases has been identified as the source of sorbicillin, and a model for the biosynthesis of sorbicillin in P. chrysogenum has been proposed. In this study, we deleted the major genes of interest of the cluster in T. reesei, namely sor1, sor3, and sor4. Sor1 is the homolog of P. chrysogenum SorA, which is the first polyketide synthase of the proposed biosynthesis pathway. Sor3 is a flavin adenine dinucleotide (FAD)-dependent monooxygenase, and its homolog in P. chrysogenum, SorC, was shown to oxidize sorbicillin and 2′,3′-dihydrosorbicillin to sorbicillinol and 2′,3′-dihydrosorbicillinol, respectively, in vitro. Sor4 is an FAD/flavin mononucleotide-containing dehydrogenase with an unknown function. We measured the amounts of synthesized sorbicillinoids throughout growth and could verify the roles of Sor1 and Sor3 in vivo in T. reesei. In the absence of Sor4, two compounds annotated to dihydrosorbicillinol accumulate in the supernatant and only small amounts of sorbicillinol are synthesized. Therefore, we suggest extending the current biosynthesis model about Sor4 reducing 2′,3′-dihydrosorbicillin and 2′,3′-dihydrosorbicillinol to sorbicillinol and sorbicillinol, respectively. Sorbicillinol turned out to be the main chemical building block for most sorbicillinoids, including oxosorbicillinol, bisorbicillinol, and bisvertinolon. Further, we detected the sorbicillinol-dependent synthesis of 5-hydroxyvertinolide at early time points, which contradicts previous models for biosynthesis of 5-hydroxyvertinolide. Finally, we investigated whether sorbicillinoids from T. reesei have a growth limiting effect on the fungus itself or on plant pathogenic fungi or on pathogenic bacteria.

Constitutive hyperproduction of sorbicillinoids in Trichoderma reesei ZC121

“…As a well-known industrial strain for the production of cellulases, hemicellulases, and recombinant proteins [ 20 ], T. reesei is also a rich source of secondary metabolites [ 21 , 22 ], a point which often is overlooked. It has been observed that T. reesei forms yellow pigments during growth [ 23 , 24 ], which have been identified as a mixture of sorbicillin, sorbicillinol and sorbicillinoids [ 9 , 25 , 26 ]. More recently, the biosynthesis pathway of sorbicillinoids in P. chrysogenum [ 27 ] and T. reesei [ 25 ] has been proposed, wherein all the related genes are clustered on the genome the same way as most genes involved generally in secondary metabolites production [ 9 , 19 , 25 ].…”

“…It has been observed that T. reesei forms yellow pigments during growth [ 23 , 24 ], which have been identified as a mixture of sorbicillin, sorbicillinol and sorbicillinoids [ 9 , 25 , 26 ]. More recently, the biosynthesis pathway of sorbicillinoids in P. chrysogenum [ 27 ] and T. reesei [ 25 ] has been proposed, wherein all the related genes are clustered on the genome the same way as most genes involved generally in secondary metabolites production [ 9 , 19 , 25 ]. This cluster also contains two transcriptional factors, YPR1 and YRP2, and a transporter [ 9 ].…”

Genome Mining and Comparative Biosynthesis of Meroterpenoids from Two Phylogenetically Distinct Fungi

“…Ap ossible candidate for this step in the atn gene cluster is AtnJ,w hich shows 33 %i dentity to Ops4, af lavin-dependent monooxygenase in oosporein biosynthesis. [18] With compounds 11 a and 11 in hand, we next sought to verify the function of AtnJ in vitro.I ntron-free atnJ was subcloned into pGEX4T-1 and high-level expression was achieved in E. coli. Both 11 and 11 a can be utilized by the atn biosynthetic pathway,s ince feeding of 11 or 11 a to DatnG restored the production of 3 ( Figure S6).…”

Enhancing peptaibols production in the biocontrol fungus Trichoderma longibrachiatum SMF2 by elimination of a putative glucose sensor

“…In addition to tlx1 and tlx2 , the upregulated geneset includes two putative PKS encoding genes, one putative NRPS encoding gene, one putative hybrid PKS/NRPS encoding gene, and one putative terpenoid synthase encoding gene. Of particular note, two genes (SMF2FGGW_102078 and SMF2FGGW_102079) encoding putative PKSs that are close homologues of SOR1 and SOR2, two PKSs responsible for the synthesis of yellow pigment in T. reesei (Derntl et al, ; Derntl, Rassinger, Srebotnik, Mach, & Mach‐Aigner, ), show dramatic decreased expression in the Δ Tlstp1 strain, which is in accordance with the remarkable reduction of yellow pigment in the culture supernatant of Δ Tlstp1 (Figure S3). In addition to these two genes, another two genes encoding a putative PKS and a putative NRPS with unknown products are also downregulated in the absence of Tl STP1.…”