We chose the anthocyanidin "pelargonidin" as our dye of choice. Pelargonidin is a secondary metabolite in plants. We reconstructed the biosynthesis of this metabolite by cloning 7 enzymes under control of a T7 promotor and expressed the operon in BL21 (DE3) using IPTG.

The pathway starts with the amino acid tyrosine, which is naturally produced in plants via Shikimic acid pathway. Our first enzyme is the tyrosine ammonia-lyase (TAL, from rhodobacter sphaeroides), which catalyzes the deamination of tyrosine to p-coumaric acid. Secondly p-coumaric acid is linked to coenzyme A by 4-coumarate-CoA ligase (4CL, from Arabidopsis thaliana) and coumaroyl-CoA is formed. Under consumption of 3 malonyl-CoA molecules Chalcone Synthase (CHS, from Gerbera) builds up an aromatic system. Naringenin chalcone reacts under cellular conditions in an annulation reaction to naringenin without enzymatic catalysis. Unfortunately, only enzymatic catalysis ensures a turnover of 100% in S-configuration. For this reason we used Chalcone isomerase (CHI, from Petunia), which catalyzes the stereospecific reaction to naringenin. Dr. Siedler published a naringenin biosensor in 2014 (FdeR), which we used in our project for measuring the synthesis of naringenin. The fifth enzyme of the pathway is the flavonon-3beta-hydroxylase (F3H, from Arabidopsis thaliana). It catalyzes the hydroxylation of naringenin to (2R,3S)-trans-dihydrokaempferol. In the next step the carbonyl group of (2R,3S)-trans-dihydrokaempferol is reduced and (2R,3S,4S)-cis-leucopelargonidin is formed. Responsible for this reaction is the dihydroflavonol 4-reductase (DFR, from Dianthus caryophyllus). In the last step pelargonidin is formed out of  (2R,3S,4S)-cis-leucopelargonidin. Thereby Anthocyanidin synthase (ANS, from fragaria) dehydroxylates the compound and enlarges the aromatic system. After this step we gained erythroid cell pellets.

We were challenged by the toxicity of naringenin and unwanted side reactions. So we planned to build up a protein scaffold consisting of F3H, DFR and ANS. We aimed at higher fluxes through substrate channeling and at hindering side reactions. In addition we expect lower cellular stress because of low naringenin concentrations. Unfortunately, we had no time left for constructing an operon with scaffold tags.

Figure 1: Anthocyanidin pathway from tyrosine to pelargonidin using seven enzymes

E. Grätzel likes working with the anthocyanin pathway.