Biosynthesis of LL‐Z1272β: Discovery of a New Member of NRPS‐like Enzymes for Aryl‐Aldehyde Formation

Abstract

LL-Z1272β (1) is a prenylated aryl-aldehyde produced by several fungi; it also serves as a key pathway intermediate for many fungal meroterpenoids. Despite its importance in the biosynthesis of natural products, the molecular basis for the biosynthesis of 1 has yet to be elucidated. Here we identified the biosynthetic gene cluster for 1 from Stachybotrys bisbyi PYH05-7, and elucidated the biosynthetic route to 1. The biosynthesis involves a polyketide synthase, a prenyltransferase, and a nonribosomal peptide synthetase (NRPS)-like enzyme, which is responsible for the generation of the aldehyde functionality. Interestingly, the NRPS-like enzyme only accepts the farnesylated substrate to catalyze the carboxylate reduction; this represents a new example of a substrate for adenylation domains.

Selective Enzymatic Transformation to Aldehydes in vivo by Fungal Carboxylate Reductase from Neurospora crassa

“…The hit with highest scores was a hypothetical protein with NCBI accession code XP_955820.1. On the sequence level, this protein showed low identities with the original search templates Ni CAR, Mm CAR, Sr CAR and At CAR (17.3%, 17.5%, 18.5% and 22.5%, respectively), and also low identities with CARs that have been published during this study: Mn CAR,9 Tv CAR,10 Ms CAR,11 Nb CAR11 and Sb CAR12 (16.4%, 26.1%, 17.7%, 17.3% and 22.4%, respectively). A codon optimized synthetic gene coding for XP_955820.1 from N. crassa OR74A was expressed with an N ‐terminal TEV cleavable HIS‐tag from the multiple cloning site 2 of the pETDUET1 vector.…”

“…Notably, Nc CAR was expressed very efficiently in E. coli as a soluble protein, in contrast to another fungal CAR from Trametes versicolor under identical conditions 10. Other published fungal CARs ATEG03630 ( An CAR) and StbB ( Sb CAR) have been heterologously expressed in S. cerevisiae 7 and Aspergillus oryzae ,12 respectively. …”

“…Carboxylate reductase (CAR) enzymes exhibit a broad substrate tolerance for the conversion of organic acids to the respective aldehydes 4. However, only few CAR enzyme sequences have been elucidated5, 6, 7, 8, 9, 10, 11, 12 and are available for biocatalysis to date, although Nature provides a great versatility of organisms with the capability to catalyze this reaction. [4,13–18] The reduction of salicylic acid,19 benzoic acid and derivatives20 as well as cinnamic acid and derivatives21 has been observed in the ascomycete Neurospora crassa ( Nc ).…”

Characterization of Type IV Carboxylate Reductases (CARs) for Whole Cell‐Mediated Preparation of 3‐Hydroxytyrosol

“…Among these, only five CARs do not originate from bacteria. These few CARs constitute three of four phylogenetic types of CARs . Further representatives of fungal CARs are not only needed to diversify the basis of phylogenetic analyses and corroborate conclusions from such analyses, but also to eventually assemble a repertoire of new enzymes with different characteristics.…”

“…Biophysical characterization of the type III Nc CAR enzyme indicated a preference for an acidic environment (pH 5.5–6.0) and a broad substrate tolerance . Aspergillus terreus CAR ( At CAR) and Stachybotrys bisbyi CAR ( Sb CAR) belong to the subtype II and are the only CARs so far placed in this same subtype . The first and only CAR of subtype IV was described from Trametes versicolor ( Tv CAR) and was functionally expressed in rather low amounts.…”