Abstract
Synthetic biology efforts for the production of valuable chemicals are frequently hindered by the structure and regulation of the native metabolic pathways of the chassis. This is particularly evident in the case of monoterpenoid production in Saccharomyces cerevisiae, where the canonical terpene precursor geranyl diphosphate is tightly coupled to the biosynthesis of isoprenoid compounds essential for yeast viability. Here, we establish a synthetic orthogonal monoterpenoid pathway based on an alternative precursor, neryl diphosphate. We identify structural determinants of isomeric substrate selectivity in monoterpene synthases and engineer five different enzymes to accept the alternative substrate with improved efficiency and specificity. We combine the engineered enzymes with dynamic regulation of metabolic flux to harness the potential of the orthogonal substrate and improve the production of industrially-relevant monoterpenes by several-fold compared to the canonical pathway. This approach highlights the introduction of synthetic metabolism as an effective strategy for high-value compound production.
Highlights
Synthetic biology efforts for the production of valuable chemicals are frequently hindered by the structure and regulation of the native metabolic pathways of the chassis
Monoterpene scaffolds are synthesized by the conversion of the canonical 10-carbon terpene precursor geranyl diphosphate (GPP) by monoterpene synthases, which catalyze the rearrangement of the substrate to generate a plethora of structures[18] (Fig. 1)
One of the factors contributing to this lower efficiency may be the structure of the isoprenoid biosynthesis pathway in yeast, which is optimized for the production of farnesyl diphosphate (FPP)-derived molecules that are essential for growth and viability
Summary
Synthetic biology efforts for the production of valuable chemicals are frequently hindered by the structure and regulation of the native metabolic pathways of the chassis This is evident in the case of monoterpenoid production in Saccharomyces cerevisiae, where the canonical terpene precursor geranyl diphosphate is tightly coupled to the biosynthesis of isoprenoid compounds essential for yeast viability. Erg20p is a bifunctional enzyme that catalyzes two successive steps of the pathway; initially, it condenses DMAPP and IPP to form GPP, and subsequently it catalyzes the condensation of GPP with one more IPP molecule to generate FPP25 (Fig. 1) This facilitates the metabolic economy of the cell[26], as yeast does not naturally produce other GPP-derived compounds. NPP and NPP-derived monoterpene biosynthesis has only been observed in these few plant species and is entirely absent in yeast
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