Synthetic biology and metabolic engineering for monoterpenoid biosynthesis in Escherichia coli

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Abstract

Terpenes are one of the three most common metabolites in nature, derived from isoprene units to form the carbon skeleton building block of various terpene classes. Terpenes which are chemically modified, such as by isomerisation or oxidation, are also called terpenoids and are industrially valuable. Thus innovating clean synthetic routes is attractive for producing commercially relevant monoterpenoids, such as those derived from limonene, including perillyl alcohol, carveol and carvone, and menthol with properties such as growth tumour inhibition, anticancer and antibacterial activity, respectively. However, industrial production is limited by the scarce natural resources with low terpenoid concentrations, dependency on environmental conditions and the requirement for several purification phases. In addition, classical industrial chemical synthesis is limited due to poor regiospecific introduction of functional groups including carbonyl or hydroxyl, resulting in racemic mixture formation. Instead, enzymes can be employed as biocatalysts with superior stereo- and regiospecificity and operable in mild conditions. The aim of this project is to develop a green, enzyme-based synthetic carvolactone pathway as proof-of-concept, with the capacity to produce industrial valuable monoterpenoids including carveol, carvone, dihydrocarvone and carvolactone. Thus primarily, enzyme candidates with the potential to catalyse one of the four pathway redox steps, were screened for their solubility in E. coli. Additionally, soluble enzymes were purified to determine the kinetic parameters and optimal conditions with highest activity. GC-analysis of single- or multi enzyme biotransformation with purified enzymes and/or recombinant cells showed that both of them only promoted the forward reaction which yields the desired monoterpenoid product. Following this, the first and second generation lactone operons were assembled, encompassing genes encoding the enzymes characterised in vitro. The regulatory elements of the operon, including synthetic ribosome binding sites and promoters, were characterised based on protein expression levels of the operon and in vitro monoterpenoid production. An in vivo reaction screening was employed and identified the missing functional P450 system that hydroxylate (S)-limonene to (1R, 5S)-carveol, which is the first redox step of the carvolactone pathway. (S)-limonene was synthesised intracellularly by an engineered mevalonate pathway that was co-expressed with the P450 system candidates. Subsequently, the functional P450 system was included in the third generation lactone operon whose assembly was based on the previous two versions, and an alcohol dehydrogenase activity screening that improved the (R)-carvone product yield catalysed by the second redox step. The reported work established proof of concept of a green enzymatic synthetic pathway in vivo, which yields the cyclic ketone, (4R, 7R)-4-isopropenyl-7-methyl-2-oxo-oxepanone lactone, as final product which can be used as a polyester plastic precursor, and industrially valuable flavours as intermediates.

Keyword(s)

Biocatalysis; Biotechnology; Biotransformation; Flavours; Fragrances; Genetic engineering; Metabolic engineering; Monoterpenoid; Synthetic Biology

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