Heterologous expression of 2-methylisoborneol / 2 methylenebornane biosynthesis genes in Escherichia coli yields novel C11-terpenes

Abstract

The structural diversity of terpenoids is limited by the isoprene rule which states that all primary terpene synthase products derive from methyl-branched building blocks with five carbon atoms. With this study we discover a broad spectrum of novel terpenoids with eleven carbon atoms as byproducts of bacterial 2-methylisoborneol or 2-methylenebornane synthases. Both enzymes use 2-methyl-GPP as substrate, which is synthesized from GPP by the action of a methyltransferase. We used E. coli strains that heterologously produce different C11-terpene synthases together with the GPP methyltransferase and the mevalonate pathway enzymes. With this de novo approach, 35 different C11-terpenes could be produced. In addition to eleven known compounds, it was possible to detect 24 novel C11-terpenes which have not yet been described as terpene synthase products. Four of them, 3,4-dimethylcumene, 2-methylborneol and the two diastereomers of 2-methylcitronellol could be identified. Furthermore, we showed that an E. coli strain expressing the GPP-methyltransferase can produce the C16-terpene 6-methylfarnesol which indicates the condensation of 2-methyl-GPP and IPP to 6-methyl-FPP by the E. coli FPP-synthase. Our study demonstrates the broad range of unusual terpenes accessible by expression of GPP-methyltransferases and C11-terpene synthases in E. coli and provides an extended mechanism for C11-terpene synthases.

Reaction mechanism of the farnesyl pyrophosphate C-methyltransferase towards the biosynthesis of pre-sodorifen pyrophosphate by Serratia plymuthica 4Rx13

“…Altogether, the findings of this new type of C -methyltransferases, involved in terpenoid biosynthesis, strongly indicate that substrates for terpenoid biosynthesis are more diverse than previously expected. Such substrate variations greatly increase the structural diversity of terpenoids as shown for some GPP methyltransferases when they were used in synthetic biology to increase terpenoid structures with potentially new/interesting properties 8 , 9 , 41 . It is also interesting to note that the multiple finding of prenyl pyrophosphate methyltransferases highlights a new dimension of substrate promiscuity of the corresponding specialized terpene cyclases in bacteria 8 , 9 , 44 .…”

“…Later on, it was shown in more detail that during the biosynthesis of this volatile by Streptomyces coelicolor , a GPP C -methyltransferase (GPPMT) catalyzed the methylation of GPP to yield a non-canonical acyclic allylic pyrophosphate intermediate (2-methyl GPP), which is the substrate of the methylisoborneol synthase 6 , 7 . This finding led to the use of GPPMT in synthetic biology to increase terpenoid structures with potentially new flavors or biological activities 8 , 9 . Additionally, the C 5 building blocks (IPP and DMAPP) can be mono- or dimethylated enabling the biosynthesis of C 11 , C 12 , C 16 , and C 17 prenyl pyrophosphates 10 .…”

Structural and Molecular Basis of the Catalytic Mechanism of Geranyl Pyrophosphate C6‐Methyltransferase: Creation of an Unprecedented Farnesyl Pyrophosphate C6‐Methyltransferase

“…Recently,t wo groups attempted to produce novel terpenoids by combining terpene cyclases with GPPC2MTs. [28,29] Their results suggest that both BezA and BezA_W210A can also be used to generate novel terpenoids by combining them with promiscuous terpene cyclasesw ith broad substrate specificities.T hus,BezA and BezA_W210A should be useful tools for synthesizing novel terpenoids.N either GPPC6MT nor FPP C6 methyltransferase has been reported to date, other than BezA or BezA_W210A, respectively.…”

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

“…In E. coli, the expression of wild-type and variant 2-methylisoborneol and 2methylenebornane synthases with a GPP methyl transferase and the MVA pathway have allowed the production of several known and unknown avours and fragrances compromising mono-, C11-, sesqui-and C16 terpenoids. 146,147 Another study used an IPP methyl transferase from Streptomyces monomycini to produce a variety of C6 and C7 IPP analogues in E. coli containing the MVA pathway. This has led to the production of several C11, C12, C16 and C17 terpenoids, and new-to-nature methylated carotenoids when co-expressed with b-carotene or zeaxanthin biosynthetic pathways.…”