Polyterpens by enzymatic conversion of isoprene – POLYTERP

In order to fulfill our objectives, competences going from biology to chemistry and to physico-chemistry were needed. In a first step, biomolecular biology tools allowed us to produce several enzymes and proteins in bacteria (E. coli) after gene extraction from the plants. Biochemical and chemical tools were further employed for the in vitro synthesis investigations. Eventually, physico-chemical tools allowed us to characterize the synthesized products but also the different interactions between the different NR constituents.

It was not possible to fulfill all the initial objectives. For instance, the in vitro transformation of isoprene into IPP was unsuccessful. As a consequence, some initial objectives were abandoned (bacterial production of terpenes). On the contrary, some investigation, not initially envisaged, led to interesting developments (better comprehension of protein-lipid interactions in rubber latex). These results were obtained thanks to the initiation of new collaborations and allowed us to start new projects that are ongoing.

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Up to now, 6 articles have been published in highly ranked international journals (Biochimie, PLoS One, BBA, Journal of Chromatography A) and 1 book chapter. Some others will be submitted very soon. Results have also been presented in 4 international congresses and 4 national congresses.

Among the four main families of natural compounds produced by plants and animals, terpene derivatives constitute the largest and most diverse group of functional hydrocarbon-based derivatives including molecules from C5 hemiterpenes to very high molar mass polyisoprenes such as Natural Rubber (NR). They are synthesized in vivo in presence of specific enzymes from a common C5 elementary precursor, isopentenyl pyrophosphate (IPP). Although in vitro biosynthesis of many terpenoids is described, this strategy of synthesis remains restricted to laboratory studies because of the low availability and stability of IPP. We have recently discovered that isoprene (IP) can be recognized by enzymes and used as initial substrate in place of IPP to produce isoprenoids. In particular, it was found that addition of IP to fresh Hevea brasiliensis latex containing active cis-prenyltransferase yields to an over production of very high molar mass 1,4-cis polyisoprene with a structure close to NR.

In this project we plan to develop this approach for the production of various terpenoids. The elementary mechanisms leading to the formation of new NR from IP will be first investigated. Besides the possibility to significantly improve the NR biosynthesis, this strategy will be extended to the preparation of other terpenoid of economical interest. To that aim complementary experiments using labeled IP will be conducted on fresh rubber particles and on specific enzymes involved in terpenoid synthesis (IPP/DMAPP isomerase, prenyl transferase,…). Enzymatic systems will be obtained from plant extracts and through cloning protocols.

It is anticipated that the use of IP, a substrate readily available from oil and the biomass and easy to handle, in place of IPP will open extremely interesting perspectives for the production and valorization of terpenoids and of related compounds. This new synthesis pathway should permit enhancing the productivity of NR producing plants as well as an industrial scale-up of the production of terpenoids of interest using microbial platform technology.