Opportunities and challenges for the sustainable production of structurally complex diterpenoids in recombinant microbial systems

Abstract

With over 50.000 identified compounds terpenes are the largest and most structurally diverse group of natural products. They are ubiquitous in bacteria, plants, animals and fungi, conducting several biological functions such as cell wall components or defense mechanisms. Industrial applications entail among others pharmaceuticals, food additives, vitamins, fragrances, fuels and fuel additives. Central building blocks of all terpenes are the isoprenoid compounds isopentenyl diphosphate and dimethylallyl diphosphate. Bacteria like Escherichia coli harbor a native metabolic pathway for these isoprenoids that is quite amenable for genetic engineering. Together with recombinant terpene biosynthesis modules, they are very suitable hosts for heterologous production of high value terpenes. Yet, in contrast to the number of extracted and characterized terpenes, little is known about the specific biosynthetic enzymes that are involved especially in the formation of highly functionalized compounds. Novel approaches discussed in this review include metabolic engineering as well as site-directed mutagenesis to expand the natural terpene landscape. Focusing mainly on the validation of successful integration of engineered biosynthetic pathways into optimized terpene producing Escherichia coli, this review shall give an insight in recent progresses regarding manipulation of mostly diterpene synthases.

Identifying and engineering the ideal microbial terpenoid production host

“…For more detailed information on host engineering, we refer the reader to several excellent reviews that focus either on specific hosts, especially E. coli (Li and Wang 2016; Ward et al 2018) and S. cerevisiae (Paramasivan and Mutturi 2017; Vickers et al 2017; Zhang et al 2017) or on strain engineering for selected targets such as isoprene (Ye et al 2016) mono- (Zebec et al 2016) or diterpenoids (Kemper et al 2017), lycopene (Ma et al 2016a), or fragrance and flavor molecules (Carroll et al 2016).…”

CITRIC: cold-inducible translational readthrough in the chloroplast of Chlamydomonas reinhardtii using a novel temperature-sensitive transfer RNA

“…Inducible systems can be useful in the field of metabolic engineering as they allow the accumulation of cell mass prior to switching on a potentially detrimental enzymatic pathway. For example, some terpenes have cytotoxic effects on the production host (reviewed in [ 41 ]). Constitutive expression of the TPS4 gene, encoding a bifunctional cis -abienol synthase from Abies balsamea (Balsam Fir), in the C. reinhardtii chloroplast produces a slight but reproducible growth defect [ 42 , 43 ].…”

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

“…In this context substrate availability is another issue to be considered: While no GGPP synthase is known in E. coli , this diterpene precursor is produced in the mitochondria of N. benthamiana . The yield of 18-hydroxydolabella-3,7-diene in planta of 26.2 mg per 100 g of fresh leaves is useful for the preparative scale production of the diterpene alcohol that can easily be isolated by extraction and column chromatography, which underpins the potential of plants, besides the recently reviewed microbial hosts for the sustainable production of diterpenes [ 36 ], as expression systems for secondary metabolite genes. The function of the investigated terpene synthase from C. pinensis in its natural context remains elusive, since neither (1 R ,3 E ,7 E ,11 S ,12 S )-18-hydroxydolabella-3,7-diene nor germacrene A or its Cope rearrangement product β-elemene could be detected in laboratory cultures [ 37 ].…”