Improved selectivity of an engineered multi-product terpene synthase

Abstract

Mutation of the sesquiterpene synthase Cop2 was conducted with a high-throughput screen for the cyclization activity using a non-natural substrate. A mutant of Cop2 was identified that contained three amino acid substitutions. This mutant, 17H2, converted the natural substrate FPP into germacrene D-4-ol with 77% selectivity. This selectivity is in contrast to that of the parent enzyme in which germacrene D-4-ol is produced as 29% and α-cadinol is produced as 46% of the product mixture. The mutations were shown to each contribute to this selectivity, and a homology model suggested that the mutations lie near to the active site though would be unlikely to be targeted for mutation by rational methods. Kinetic comparisons show that 17H2 maintains a k cat /K M of 0.62 mM −1 s −1 , which is nearly identical to that of the parent Cop2, which had a k cat /K M of 0.58 mM −1 s −1 .

Cyclopropylmethyldiphosphates are substrates for sesquiterpene synthases: experimental and theoretical results

“…Triene 17 is the homo-analogue of germacrene D (20) and alcohol 18 corresponds to germacrene D-4-ol (21), 15 while the backbone of alcohol 19 is closely related to the sesquiterpene kunzeaol (22). 16 Our previously collected findings on the pronounced promiscuity of presilphiperfolan-8β-ol synthase (BcBOT2) are further confirmed in this study.…”

“…The crude product obtained was subjected to column chromatographic purification (silica gel; n -pentane/Et 2 O = 10 : 1 → 4 : 1 → 2 : 1), which first allowed separation of the triene 17 (24%) from the two alcohols 18 (11%) and 19 (17%) these were present in a ratio of 2 : 3, as indicated by the integrals in the 1 H NMR spectrum. Triene 17 is the homo-analogue of germacrene D ( 20 ) and alcohol 18 corresponds to germacrene D-4-ol ( 21 ), 15 while the backbone of alcohol 19 is closely related to the sesquiterpene kunzeaol ( 22 ). 16…”

Exploring the genomic traits of fungus-feeding bacterial genus Collimonas

“…Thus we characterized CPter91_2617 and CPter291_2730 as mixed mono-, sesqui- and diterpene cyclases, with major product germacrene D-4-ol. The sesquiterpene products suggest that they are functionally related to plant and fungal cadinene/cadinol and germacrene D-4-ol synthases, although sequence homology to these enzymes is low [ 72 , 73 ]. One of the monoterpene products of CPter91_2617 and CPter291_2730, β-pinene, was also observed in the headspace of C. pratensis and showed antibacterial activity, suggesting a role of the Collimonas terpene cyclases in antimicrobial activity.…”

Volatile communication between fungi and bacteria

“…When GPP was applied as a substrate, the production of two monoterpenes identified as β-pinene and β-linalool was observed. The sesquiterpene products suggest that they are functionally related to plant and fungal cadinene/cadinol and germacrene D-4-ol synthases (Yoshikuni et al, 2006, Lauchli et al, 2014. Based on these findings, we suggest that there may be many new terpene products as yet undiscovered hiding in the genomes of bacteria.…”

“…The sesquiterpene products suggest they are functionally related to plant and fungal cadinene/cadinol and germacrene D-4-ol synthases, although sequence homology to these enzymes is low (Yoshikuni et al, 2006, Lauchli et al, 2014. One of the monoterpene products of CPter91_2617 and CPter291_2730, β-pinene, was also observed in the headspace of C. pratensis and …”