Heterologous biosynthesis of 2-methyl-6-geranylgeranyl benzoquinone and δ-tocotrienol in recombinant Escherichia coli strains

10 Abstract Vitamin E is a group of lipid soluble compounds, consisting of 4 forms () of tocopherols and tocotrienols, each. In nature, all 8 vitamin E compounds are exclusively synthesized by photosynthetic organisms (green plants, some cyanobacteria and some algae), with the exception of recently reported tocopherol synthesis in a non-photosynthetic eukaryote, Plasmodium falciparum. Out of the 8 forms of Vitamin E compounds, tocopherol is ubiquitously found in photosynthetic organisms, while -tocotrienol is a rare form. Unlike -tocopherol, -tocotrienol is being discussed for its possible unique biological functions in humans and animals like anti-cancer effects, neuroprotective and hypocholesterolemic effects apart from its antioxidant activity. Humans and animals cannot produce any of the Vitamin E compounds, and hence, they form an essential dietary component. Chemical synthesis of -tocotrienol (analogue to that found in nature, i.e. single stereoisomer), requires a series of asymmetric synthetic reactions, that usually results in low yields. Even though, all the -tocotrienol found in nature is in its active form, its presence in low amounts (e.g. 15.2 μg of -tocotrienol per gram of fresh weight of palm fruit) makes the extraction process less economical feasible compared to the chemical synthesis route. Additionally, finding a suitable solvent for extraction of -tocotrienol from natural food sources is difficult as it should be environmental friendly and economical for the process technology. Molecular biologists and plant breeding scientists are making efforts to increase the biosynthesis of vitamin E compounds in photosynthetic organism itself. Increasing the vitamin E yield, by approx. 50-100 times in natural sources may result the extraction technology to be more economical than chemical synthesis process. In the current work, a new approach for the biosynthesis of active stereoisomer of tocotrienol and its precursor, 2-methyl-6-geranylgeranyl benzoquinol (MGGBQ) in recombinant non-photosynthetic Escherichia coli (E. coli) strain, has been investigated. -tocotrienols consists of a polar aromatic head group linked to a lipophilic hydrocarbon tail. Wild type E. coli is able to provide the two donor groups, for the biosynthesis of tocotrienol i.e. in form of phydroxyphenyl pyruvate (p-HPP), produced via shikimate pathway and in form of farnesyl pyrophosphate (FPP) and isopentenyl pyrophosphate (IPP) produced via the 1-deoxy-Dxylulose-5-phosphate (DXP) pathway. To realise the heterologous biosynthesis of tocotrienol in recombinant E. coli, four additional genes, encoding for p-hydroxyphenyl-pyruvate dioxygenase (Hpd) from Pseudomonas putida, geranylgeranylpyrophosphate synthase (CrtE) from Pantoea ananatis, homogentisate phytyltransferase (Hpt-Syn) from Synechocystis sp. PCC6803, and tocopherol cyclase (Cyc-At) from Arabidopsis thaliana, were successfully cloned and overexpressed in recombinant E. coli encoded within one expression plasmid. The plasmidencoded strains produced homogentisic acid (HGA) and/or geranylgeranyl pyrophosphate (GGPP) and/or MGGBQ, and/or tocotrienol when tested for in-vivo activity in complex medium. In order to scale up any fermentation process from laboratory/pilot scale to commercial/industrial scale, the heterologous genes should be stable (segregational and structural), during fermentation. Recombinant strains encoded with plasmid(s) have a risk for segregational instability during fermentation, resulting in loss of productivity and hence the plant capacity. Use of antibiotics in the fermentation medium for cultivation of plasmidencoded strains, increases the operating cost of the whole process and is undesired in food related products. To solve the problem of segegrational instability and to reduce the cost of antibiotic during fermentation, homologous recombination techniques were used in the current study to construct plasmid-free recombinant E. coli strains, which could produce