Analyzing and Engineering the Product Selectivity of a 2-Methylenebornane Synthase

Abstract

Terpenes constitute the largest class of natural products with more than 70 000 compounds. Many different terpenes find applications in the flavor and fragrance industry or can be used as fine chemicals or drugs. In some bacteria, noncanonical terpenes with 11 carbon atoms are synthesized via a GPP-C2-methyltransferase and the subsequent conversion of 2-methyl-GPP by certain terpene synthases into mainly 2-methylisoborneol and 2methylenebornane. Many other C 11 -terpenes were reported as side products, but they are synthesized only in minor amounts by the bacterial C 11 -terpene biosynthesis pathway. To enable biotechnological synthesis of these largely unexplored natural products, we changed the product selectivity of the 2methylenebornane synthase from Pseudomonas f luorescens by a semirational protein engineering approach. Active site amino acids with impact on the product selectivity were identified and variants with completely altered product spectra could be identified and characterized. The gathered data provide new insights into the structure−function relationship for C 11 -terpene synthases and demonstrate the production of formerly inaccessible noncanonical terpenes.

Microbial Degradation of 2‐Methylisoborneol in Forest Soil**

“…In all cases, the concentrations of 1‐MC were relatively low, way behind the main products 2‐MIB and 2‐MB, and at a maximum of 3 % of total peak area. The only case in which the concentration of 1‐MC was increased in relation to the main products was when the 2‐MB synthase from P. fluorescens was engineered to have its active site modified [25] …”

Methylation of Unactivated Alkenes with Engineered Methyltransferases To Generate Non‐natural Terpenoids

“…Several MTs have been reported to methylate the alkene of isoprene moieties, which is remarkable because alkenes are not particularly good nucleophiles. Currently, however, natural MTs are limited to specific terpenoid substrates, namely complex sterols ( C 30 ) and terpenoids carrying diphosphate moieties, which limits their application for late‐stage derivatization [27–32] . We aimed to utilize MTs for selective methylation in a one‐step reaction with readily available mono‐, sesqui‐, and diterpenoids (Figure 1B).…”

Methylierung nicht‐aktivierter Alkene mit modifizierten Methyltransferasen zur Diversifizierung von Terpenoiden

“…Dies ist bemerkenswert ist, da Alkene keine besonders guten Nukleophile sind. Derzeit sind natürliche MTs jedoch auf bestimmte Terpenoidsubstrate beschränkt, nämlich komplexe Sterole ( C 30 ) oder Terpenoide, die Diphosphatgruppen tragen, was ihre Anwendung für die Derivatisierung in späten Synthesestufen einschränkt [27–32] . Ziel dieser Arbeit ist es, MTs für die selektive Methylierung in einer einstufigen Reaktion mit verfügbaren Mono‐, Sesqui‐ und Diterpenoiden zu nutzen (Abbildung 1B).…”

“…Derzeit sind natürliche MTs jedoch auf bestimmte Terpenoidsubstrate beschränkt, nämlich komplexe Sterole (C 30 ) oder Terpenoide, die Diphosphatgruppen tragen, was ihre Anwendung für die Derivatisierung in späten Synthesestufen einschränkt. [27][28][29][30][31][32] Es ist bekannt, dass die Enzymfamilie der C-24-Sterol-Methyltransferasen (SMTs) verschiedene Sterole für die Methylierung der terminalen Prenylgruppe akzeptiert. [27] Es wird angenommen, dass die Reaktion über einen Carbokation-Übergangszustand abläuft, wodurch verschiedene Produkte erzeugt werden können (Abbildung 2A).…”