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

Abstract

BackgroundTerpenes 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.ResultsThree 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.ConclusionThe 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.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-1010-z) contains supplementary material, which is available to authorized users.

The foxtail millet (Setaria italica) terpene synthase gene family

“…Nicotiana benthamiana expression revealed SiTPS15 as an a-bisabolol synthase, SiTPS20 as a b-caryophyllene synthase, SiTPS19 and SiTPS27 as d-cadinene synthases and SiTPS26 as a germacrene D-synthase compared with commercial standards, while SiTPS38 produced germacrene-D-4-ol as verified by comparison with the product of a d-cadinene synthase variant (GhCAD:N403P/L405H) from cotton (Gossypium arboreum)(Yoshikuni et al, 2006) (Figures 4a, e and S5). Co-expression of enzymes not showing activity in N. benthamiana with ZmFPPS in E. coli identified SiTPS21 as a cubebol synthase as validated by comparison with the product profile of cubebol synthase from Coniophora puteana(Mischko et al, 2018) (Figures 4e,f and S5). The primary product of SiTPS25 featured mass ions of m/z 161, 207, 222 and 240 (…”

“…(Table S1) (Wang et al, 2011;Mafu et al, 2018). Additional genes used for producing authentic standards, namely G. arboreum germacrene-D-4-ol/ d-cadinene synthase (NM_001330020) variant N403P/L405H and C. puteana cubebol synthase, Copu3 (XP_007765978), were synthesized for expression in E. coli and cloned into pET-DUET1 according to published literature (Yoshikuni et al, 2006;Mischko et al, 2018).…”

Alternative metabolic pathways and strategies to high-titre terpenoid production inEscherichia coli

“…43 Finally, Haematococcus lacustris idi was used in the highest titre reports for cubebol, bcopaene, and cembratriene-ol production (Table S1 †). 44,45…”

Recent developments of tools for genome and metabolome studies in basidiomycete fungi and their application to natural product research

“…The appeal is obvious, it is a unicellular, well understood and very fastgrowing organism, and robust techniques and tools for its manipulation are available. Examples of its use include, the heterologous expression of sesquiterpene synthases from Coniophora puteana for use in generating β-copaene and cubebol, which were then used to create a heterologous microbial production system, with yields that exceeded all previously published results for these compounds (Mischko et al, 2018). The unspecific peroxygenase MroUPO from the basidiomycete Marasmius rotula was expressed in E. coli alongside computationally guided mutated variants for the purposes of optimising the enzyme (Carro et al, 2019).…”