Overcoming the plasticity of plant specialized metabolism for selective diterpene production in yeast

Abstract

Plants synthesize numerous specialized metabolites (also termed natural products) to mediate dynamic interactions with their surroundings. The complexity of plant specialized metabolism is the result of an inherent biosynthetic plasticity rooted in the substrate and product promiscuity of the enzymes involved. The pathway of carnosic acid-related diterpenes in rosemary and sage involves promiscuous cytochrome P450s whose combined activity results in a multitude of structurally related compounds. Some of these minor products, such as pisiferic acid and salviol, have established bioactivity, but their limited availability prevents further evaluation. Reconstructing carnosic acid biosynthesis in yeast achieved significant titers of the main compound but could not specifically yield the minor products. Specific production of pisiferic acid and salviol was achieved by restricting the promiscuity of a key enzyme, CYP76AH24, through a single-residue substitution (F112L). Coupled with additional metabolic engineering interventions, overall improvements of 24 and 14-fold for pisiferic acid and salviol, respectively, were obtained. These results provide an example of how synthetic biology can help navigating the complex landscape of plant natural product biosynthesis to achieve heterologous production of useful minor metabolites. In the context of plant adaptation, these findings also suggest a molecular basis for the rapid evolution of terpene biosynthetic pathways.

Orthogonal monoterpenoid biosynthesis in yeast constructed on an isomeric substrate

“…So far, low monoterpene titers have hindered efficient P450 catalyzed oxidation of monoterpenes by a yeast cell factory. We evaluated whether the overall improvements obtained here were sufficient to enable downstream oxidation steps by introducing the sabinene hydroxylase CYP750B1 45 from T. plicata , together with a compatible cytochrome P450 oxidoreductase 38,46 , to establish production of trans -sabin-3-ol. While production of trans -sabin-3-ol was not detectable when only the GPP pathway was used, we were able to synthesize detectable amounts of trans -sabin-3-ol in the NPP-producing MIC2 strain using wild-type Sp SabS.…”

Biochemical characterization of diterpene synthases of Taiwania cryptomerioides expands the known functional space of specialized diterpene metabolism in gymnosperms

“…Ferruginol and abietatriene were observed as major metabolites in Taiwania (Figure b) and are hypothesized as key intermediates en route to taiwaniaquinoids and taiwaniaquinols (Figure ), a group of bioactive abeo‐ abietane diterpenoids with a 6,5,6‐ring structure (Lin et al ., ; Chang et al ., ; Cheng et al ., ; Majetich and Shimkus, ; Guardia et al ., ). In select angiosperms, ferruginol biosynthesis has been demonstrated to proceed through the consecutive activity of a (+)‐CPS and a miltiradiene synthase, followed by non‐enzymatic oxidation of miltiradiene into abietatriene and downstream functional decorations facilitated by P450 enzymes (Guo et al ., ; Zi and Peters, ; Brückner et al ., ; Ignea et al ., ). A similar pathway has been proposed for taiwaniaquinoid biosynthesis (Jana et al ., ; Tapia et al ., ) (Figure ).…”

Finding and analysing the minimum set of driver nodes required to control multilayer networks

“…6 suggest that the specialized metabolism functional class tends to benefit from multicontrol MDSM. This metabolic pathway generates specialized metabolites that are useful for adjusting the cell state to the surroundings and environmental conditions 35 . The secondary metabolism has an inherent plasticity that facilitates its control at different levels.…”