Expanding the Isoprenoid Building Block Repertoire with an IPP Methyltransferase from Streptomyces monomycini

Abstract

Many synthetic biology approaches aim at expanding the product diversity of enzymes or whole biosynthetic pathways. However, the chemical structure space of natural product forming routes is often restricted by the limited cellular availability of different starting intermediates. Although the terpene biosynthesis pathways are highly modular, their starting intermediates are almost exclusively the C 5 units IPP and DMAPP. To amplify the possibilities of terpene biosynthesis through the modification of its building blocks, we identified and characterized a SAM-dependent methyltransferase converting IPP into a variety of C 6 and C 7 prenyl pyrophosphates. Heterologous expression in Escherichia coli not only extended the intracellular prenyl pyrophosphate spectrum with mono-or dimethylated IPP and DMAPP, but also enabled the biosynthesis of C 11 , C 12 , C 16 , and C 17 prenyl pyrophosphates. We furthermore demonstrated the general high promiscuity of terpenoid biosynthesis pathways toward uncommon building blocks by the E. coli-based production of polymethylated C 41 , C 42 , and C 43 carotenoids. Integration of the IPP methyltransferase in terpene synthesis pathways enables an expansion of the terpenoid structure space beyond the borders predetermined by the isoprene rule which indicates a restricted synthesis by condensation of C 5 units.

Rerouting and Improving Dauc‐8‐en‐11‐ol Synthase from Streptomyces venezuelae to a High Yielding Biocatalyst

“…We and others have recently shown that additional methyl groups can also enzymatically be incorporated into terpenes from methylated IPP derivatives, but in these cases the additional methyl groups were not placed in positions that lead to an altered reactivity of the obtained methylated oligoprenyl diphosphate derivatives. [40,41] Also functional groups such as the keto group in the FPP analogue 16 are tolerated by DcS, similar to recent findings for other terpene synthases, [42][43][44][45] yielding products 33 and 34 with functionalised side chains that allow further chemical transformations. We have also investigated the effect of mutating aromatic amino acid residues that are, according to a homology model, located on the surface of DcS.…”

“…Both the widdrane and the tenuifolane skeletons are difficult to form from natural FPP, as is evident from the low number of known natural products from these classes, but can be reached with the altered reactivity of FPP analogues with non‐natural methylation patterns. We and others have recently shown that additional methyl groups can also enzymatically be incorporated into terpenes from methylated IPP derivatives, but in these cases the additional methyl groups were not placed in positions that lead to an altered reactivity of the obtained methylated oligoprenyl diphosphate derivatives [40,41] . Also functional groups such as the keto group in the FPP analogue 16 are tolerated by DcS, similar to recent findings for other terpene synthases, [42–45] yielding products 33 and 34 with functionalised side chains that allow further chemical transformations.…”

Biotechnological applications of S-adenosyl-methionine-dependent methyltransferases for natural products biosynthesis and diversification

“…The enzymatic activities were verified by overexpressing SodC and SodD genes in Escherichia coli with an elevated amount of FPP, and directly analysing the products formed (Duell et al 2019). In another study, Drummond et al (2019)…”

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

“…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. 148 The second alternative pathway found in nature that is capable of producing terpenoids outside the isoprene rule is the homomevalonate pathway, used by insects to produce C16, C17 or C18 terpenoid hormones (Fig. 4B).…”