Last year, we devised Flavorator which preserve food by fragrance component with antibacterial effect and introduced its concept. We chose geraniol and farnesol as the fragrance having an antibacterial effect, and we made E.coli possible produce these fragrance components by recombination. However, the synthetic amount of geraniol and farnesol was lower than expected, and it is not sufficient for practical use.

This year, to improve functionality and feasibility of Flavorator, we focused on the production of farnesol instead of two fragrances for three reasons. 1. Farnesol does not impair flavor of food because it is almost odorless. 2. Farnesol has a high antifungal activity than others. 3. E.coli already possesses synthetic pathway for farnesol so that it is easier to modify for over-expression of farnesol using authentic genes.

Fig.1 Pathway of our design DXP: 1-Deoxy-D-xylulose 5-phosphate. MEP: 2-C-methylerythritol 4-phosphate. CDP-ME: 4-diphosphocytidyl-2-C-methylerythritol. CDP-MEP: 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate. MEC: 2-C-methyl-D-erythritol 2, 4-cyclodiphosphate. HMBPP: (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate. IPP: Isopentenyl pyrophosphate. DMAPP: Dimethylallyl pyrophosphate. GPP: geranyl diphosphate. FPP: farnesyl diphosphate.

To increase farnesol production, we improved farnesol synthetic process by adding three genes to E.coli from the last year. 1, The gene that increases the intermediate product of farnesol synthesis. As 1-deoxy-D-xylulose 5-phosphate (DXP), a key gene for farnesol synthesis, is also used for another compound synthesis, we added a new DXP synthase to increase the intermediate product. 2, The dephosphorylation enzyme gene Last year, a synthesis of farnesol relied on endogenous dephosphorylation enzyme of E. coli, and this could be a bottleneck for farnesol production. This year, we introduced an additional endogenous phosphatase gene (YbjG, PgpB) in E. coli to increase the production of farnesol. 3, A gene to improve resistance against farnesol. As farnesol is an antibacterial fragrance, it is toxic to E.coli itself. So, we introduced the endogenous activator gene of AcrAB-TolC efflux pump, marA to improve resistance against farnesol．

Also, to improve the feasibility of Flavorator in the real situation, we thought that it is important to produce farnesol by non-recombinant E.coli. Therefore we attempted to increase production of farnesol using CRISPER / Cas9 system. E. coli has their pathway to produce the farnesol. However, the gdhA gene, which is not lethal to E.coli, produce a byproduct from farnesol synthetic pathway and decrease the production of farnesol. We tried to knockout gdhA with CRISPER-CAS9.

Fig.2 Pathway of our design We tried to knockout gdhA, gene did not relate to falnesol synthesis, with CRISPER-CAS9 and be accumulate intermediate metabolite. DXP: 1-Deoxy-D-xylulose 5-phosphate. MEP: 2-C-methylerythritol 4-phosphate. CDP-ME: 4-diphosphocytidyl-2-C-methylerythritol. CDP-MEP: 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate. MEC: 2-C-methyl-D-erythritol 2, 4-cyclodiphosphate. HMBPP: (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate. IPP: Isopentenyl pyrophosphate. DMAPP: Dimethylallyl pyrophosphate. GPP: geranyl diphosphate. FPP: farnesyl diphosphate.

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