18-Hydroxydolabella-3,7-diene synthase – a diterpene synthase from Chitinophaga pinensis

Abstract

The product obtained in vitro from a diterpene synthase encoded in the genome of the bacterium Chitinophaga pinensis, an enzyme previously reported to have germacrene A synthase activity during heterologous expression in Escherichia coli, was identified by extensive NMR-spectroscopic methods as 18-hydroxydolabella-3,7-diene. The absolute configuration of this diterpene alcohol and the stereochemical course of the terpene synthase reaction were addressed by isotopic labelling experiments. Heterologous expression of the diterpene synthase in Nicotiana benthamiana resulted in the production of 18-hydroxydolabella-3,7-diene also in planta, while the results from the heterologous expression in E. coli were shown to be reproducible, revealing that the expression of one and the same terpene synthase in different heterologous hosts may yield different terpene products. 1770

Engineering terpenoid production through transient expression in Nicotiana benthamiana

“…Furthermore, the yields of the end products are still in the microgram per litre range in many cases, thus highlighting the need to continue to explore other host systems. The numbers of studies demonstrating the purification of compounds derived from transient expression in plants have increased in recent years, reflecting the increasing popularity of this system (Andersen-Ranberg et al 2016 ; Dickschat et al 2017 ; Geisler et al 2013 ; Huang et al 2017a , b ; Reed et al 2017 ). As described above, transient expression can be achieved at industrial scale, and is currently used for production of pharmaceutical proteins by various companies (Sack et al 2015 ).…”

Bacterial Diterpene Biosynthesis

“…Am echanistically simple DTS was described from Chitinophaga pinensis. [40] Thee nzyme converts GGPP by 1,11-10,14-cyclisation and attack of water at C-15 into 21, possibly in ac oncerted reaction without any cationic intermediates and ac learly defined stereochemical course.T his was shown by incubation experiments with (16-13 C)-and (17-13 C)GGPP,w hich resulted in ac learly defined incorporation of labelling into only one of the two geminal methyl groups of 21 in both cases (Scheme 11). Experiments with enantioselectively deuterated and 13 C-labelled GGPP isotopomers were performed to establish the absolute configuration of 21.…”

“…The gibberellin biosynthetic gene cluster from Sinorhizobium fredii furthermore encodes three cytochrome P450 monooxygenases (CYP117, CYP114, and CYP112), af erredoxin (Fd GA )a cting together with CYP114, and as hort-chain dehydratase-reductase (SDR GA ). [57] It was demonstrated by heterologous expression in Sinorhizobium meliloti that CYP117 catalyses the oxidation of 39 to ent-kaurenoic acid (40). Oxidation with ring contraction to GA 12 aldehyde (41) by CYP114 proceeds with specific extrusion of C-7, as demonstrated by a 13 C-labelling experiment through feeding with (2-13 C)mevalonolactone.…”

“…A mechanistically simple DTS was described from Chitinophaga pinensis . The enzyme converts GGPP by 1,11‐10,14‐cyclisation and attack of water at C‐15 into 21 , possibly in a concerted reaction without any cationic intermediates and a clearly defined stereochemical course.…”

Exploring novel bacterial terpene synthases

“…In a previous study, 262 presumptive bacterial TSs were identified and 27 proteins were functionally characterised in in vivo conditions [37]. Several other bacterial TSs have been characterised and their activities identified with single or multiple substrates [24,28,36,[38][39][40][41]. In this study, presumptive TS homologue protein sequences were clustered by pairwise similarity, and from the resulting neighbour-joining tree (Fig 1) we identified TSs from Gram-positive bacteria, mainly from the order Actinomycetales, as well as Gram-negative bacteria belonging to numerous orders.…”