Biotechnological potential and initial characterization of two novel sesquiterpene synthases from Basidiomycota Coniophora puteana for heterologous production of δ-cadinol

Abstract

Background Terpene synthases are versatile catalysts in all domains of life, catalyzing the formation of an enormous variety of different terpenoid secondary metabolites. Due to their diverse bioactive properties, terpenoids are of great interest as innovative ingredients in pharmaceutical and cosmetic applications. Recent advances in genome sequencing have led to the discovery of numerous terpene synthases, in particular in Basidiomycota like the wood rotting fungus Coniophora puteana, which further enhances the scope for the manufacture of terpenes for industrial purposes. Results In this study we describe the identification of two novel (+)-δ-cadinol synthases from C. puteana, Copu5 and Copu9. The sesquiterpene (+)-δ-cadinol was previously shown to exhibit cytotoxic activity therefore having an application as possible, new, and sustainably sourced anti-tumor agent. In an Escherichia coli strain, optimized for sesquiterpene production, titers of 225 mg l−1 and 395 mg l−1, respectively, could be achieved. Remarkably, both enzymes share the same product profile thereby representing the first two terpene synthases from Basidiomycota with identical product profiles. We solved the crystal structure of Copu9 in its closed conformation, for the first time providing molecular details of sesquiterpene synthase from Basidiomycota. Based on the Copu9 structure, we conducted structure-based mutagenesis of amino acid residues lining the active site, thereby altering the product profile. Interestingly, the mutagenesis study also revealed that despite the conserved product profiles of Copu5 and Copu9 different conformational changes may accompany the catalytic cycle of the two enzymes. This observation suggests that the involvement of tertiary structure elements in the reaction mechanism(s) employed by terpene synthases may be more complex than commonly expected. Conclusion The presented product selectivity and titers of Copu5 and Copu9 may pave the way towards a sustainable, biotechnological production of the potentially new bioactive (+)-δ-cadinol. Furthermore, Copu5 and Copu9 may serve as model systems for further mechanistic studies of terpenoid catalysis. Graphical Abstract

Structural Understanding of Fungal Terpene Synthases for the Formation of Linear or Cyclic Terpene Products

“…Its structure factors and atomic coordinates have been deposited in the Protein Data Bank with accession number 8GY0. The overall structure is a typical class I TPS where the α-domain contains two conserved metal-ion coordination motifs ( 84 DEXXD 88 and 219 NSE 227 ) as well as the 302 WXXXXXRY 309 motif known to be close to the active site via salt bridge formation upon substrate binding . As TPSs are known to be inactive in the absence of Mg 2+ ions, − structural determination/analysis and molecular modeling were carried out by using the structure of Ap.LS* obtained in the presence of Mg 2+ .…”

Characterization and Crystal Structures of a Cubebol-Producing Sesquiterpene Synthase from Antrodia cinnamomea

“…The crystal structure of AcCop4 was determined by the molecular replacement method using Phaser in the PHENIX package . The coordinates of Copu9 (PDB code: 7OFL) were used as the search model. The initial structure was refined with iterative cycles of simulated annealing, energy minimization, and manual rebuilding using PHENIX refinement and COOT .…”

“…Structural Comparison of AcCop4 with Its Homologs. Based on a DALI analysis, AcCop4 shares a high structural similarity with a group of class I terpenoid cyclase members with very low sequence identity, including Copu9 (PDB code: 7OFL, Z-score: 42.1, 36% identity), 25 selinadiene synthase (PDB code: 4OKM, Z-score: 34.6, 20% identity), 31 epiisozizaene synthase (PDB code: 3KB9, Z-score: 33.7, 20% identity), 32 and pentalenene synthase (PDB code: 6WKD, Zscore: 32.3, 23% identity). 33 Except for the α-helical class I terpenoid cyclase fold, AcCop4 and Copu9 possess an additional unique two-stranded β-sheet.…”

Catalytic Mechanism and Heterologous Biosynthesis Application of Sesquiterpene Synthases

“…It suggested that the helix-turn-helix motif may be a hot spot for product-shifting mutations and thus serve as a guide for improving related class I products. 55 W279 is on the C helix of the CS and toward the bottom of the active site cleft. The larger volume of residue at position 279 extended the channel that allowed water access to the active site, increasing the ratio of alcohol to hydrocarbon products.…”

“…Similarly, mutations of the corresponding glycine and cysteine (G186 and C187) residues in COS2, both of which are located in the conserved “kink” region between the α-helices G1 and G2, altered the product profile from (+)-δ-cadinol to germacrene D-4-ol. It suggested that the helix-turn-helix motif may be a hot spot for product-shifting mutations and thus serve as a guide for improving related class I products …”