Identification of sesquiterpene synthases from the Basidiomycota Coniophora puteana for the efficient and highly selective β-copaene and cubebol production in E. coli

Abstract

Background: Terpenes are an important and extremely versatile class of secondary metabolites that are commercially used in the pharmaceutical, food and cosmetics sectors. Genome mining of different fungal collections has revealed the genetic basis for a steadily increasing number of putative terpene synthases without any detailed knowledge about their biochemical properties. The analysis and research of this rich genetic source provides a precious basis for the advancing biotechnological production of an almost endless number of valuable natural metabolites.

Results: Three annotated terpene synthases from the little investigated Basidiomycota Coniophora puteana were studied in this work. For biochemical characterization, the heterologous expression in E. coli was conducted leading to the identification of two sesquiterpene synthases capable of the highly selective generation of β-copaene and cubebol. These compounds are commercially used as food and flavor additives. The new enzymes show the highest reported product selectivity for their main compounds and therefore represent the first exclusive synthases for β-copaene (62% product selectivity) and cubebol (75% product selectivity) generation. In combination with an optimized heterologous microbial production system, we obtained product titers of 215 mg/L β-copaene and 497 mg/L cubebol.

Conclusion: The reported product selectivity and our generated terpene titers exceed all published biotechnological data regarding the production of β-copaene and cubebol. This represents a promising and economic alternative to extraction from natural plant sources and the associated complex product purification.

Keywords: Basidiomycota; Coniophora puteana; Copaene; Cubebol; Fermentation; Heterologous expression; Phylogenetic analysis; Sesquiterpene.

Fig. 1

Design of the synthetic operons for an increased precursor supply and efficient STP production. Enzymatic bottlenecks within the upstream non-mevalonate (MEP) pathway (dxs and idi) as well as the heterologous STPS genes (Copu2 and Copu3) are combined in a single synthetic operon. The integration of an additional geranylgeranyl diphosphate synthase (crtE) allowed to study a potential function as diterpene synthases. A constitutive promoter (P) regulates the pathways

Fig. 2

The biosynthetic generation of β-copaene and cubebol. STP production relies on the precursor supply of the non-mevalonate (MEP) pathway (a). The respective GC–MS chromatograms of E. coli cultures co-expressing either Copu2 or Copu3 and the corresponding MEP bottleneck enzymes reveal new product peaks and distinct product profiles due to the specific cyclization reaction (b). The respective detailed MS spectra allow for a putative compound assignment (c). The asterisks refers to terpenes without any MS spectra match in the NIST database

Fig. 3

Reaction scheme to illustrate the rearrangement from β-copaene to α-copaene. This reaction is observed in the presence of chlorophorm

Fig. 4

Unrooted neighbor-joining phylogram of all 29 reported and experimentally characterized STPSs from Basidiomycota. The protein sequence based analysis includes STPSs from Antrodia cinnamomea [39], Armillaria gallica [40], Boreostereum vibrans [41], Coprinopsis cinereus [35], Fomitopsis pinicola [42], Lignosus rhinocerus [43] Stereum hirsutum [37], Omphalotus olearius [42] as well as Coniophora puteana. The phylogenetic analysis revealed four distinct clades, divided into different cyclization mechanisms and product profiles. Copu1-3 belong to clade I including all other known β-copaene or cubebol synthases from Basidiomycota. For detailed information about the used sequences and accession numbers, see Additional file 1

Fig. 5

Proposed cyclization mechanisms for the generation of the main STPs from Copu2 and Copu3. The reaction starts by ionization and isomerization of farnesyl diphosphate (FPP) creating nerolidyl diphosphate (NPP). The first cyclization is achieved by the formation of the C1–C10 bond. Subsequent C6–C1 bond formation yields a cadinyl cation, which represents a general precursor for various cadalane and cubebane-type STPs [, –47]

Fig. 6

E. coli growth curves and time-dependent concentration of β-copaene and cubebol in controlled fed-batch fermentations. Expression of Copu2 (a) and Copu3 (b) in E. coli resulted in a constant concentration increase, which was finally limited by the stationary phase. The error bars represent the mean values ± standard deviation of technical triplicates