GREENING THE PRODUCTION OF HIGH-VALUE MOLECULES USING RECOMBINANT CYANOBACTERIA AND LIGHT

Cyanobacteria are ancient prokaryotic organisms capable to utilize CO2 and sunlight as their primary sources of carbon and energy. Their unique metabolism, which integrates characteristics of both plants and bacteria, poses them as candidates for sustainable biotechnological applications. Cyanobacteria metabolic pathways and molecular architecture are similar to those of other bacteria (Oliver et al., 2014). Among cyanobacteria, Synechocystis sp. PCC6803 (from now on Synechocystis) has been studied for a long time as model organism to investigate photosynthesis. Synechocystis has been extensively engineered in the last years to express NADPH-dependent oxidoreductases and employed in whole-cell biotransformation to produce several industrially relevant chemicals (Malihan-Yap et al., 2022). In this thesis, Synechocystis has been employed to produce three high-value molecules: bio-indigo, salidroside and L-theanine. All of them are employed in industry and chemically produced through polluted processes, based on the consumption of toxic and hazardous substrates, solvents and catalysts. Bio-indigo was produced employing the stable transgenic strain Syn_mFMO obtained by cloning in wild-type Synechocystis the coding sequence of the Flavine-dependent MonoOxygenase from Methylophaga sp. SK1, mFMO. The expression of such enzymes was shown to affect cells metabolism inducing the production of white granules, observed by transmission electron microscopy, whose identity is compatible with polyhydroxybutyrate (PHB). The quantity of PHB granules increased upon addition of the substrate indole, with consequent production of bio-indigo. The extraction of blue-coloured PHB is also shown from Syn_mFMO cultures. After the optimization of reaction conditions, the yield was 112 mg/L of produced indigo (86% conversion of the furnished substrate). Since indigo was found to be prevalently secreted in the medium, a method for its recovery by employing polyamide nets, directly submerged in the culture, is also presented. Salidroside production in Synechocystis was attempted through the design of a reaction cascade that starts from raspberry ketone and produces tyrosol acetate, which is then hydrolyzed to tyrosol by Syn_CmBVMO. The latter strain was obtained by cloning the coding sequence of the NADPH-dependent Baeyer-Villiger MonoOxygenase from the red alga Cyanidioschyzon merolae (CmBVMO). Since both tyrosol and its acetylated form (tyrosol acetate) are expensive molecules, in this section the optimization of their production and the scaling up from 1mL reaction in vials to 120 mL flat panel photobioreactors are also described. It was demonstrated that, based on the growth method, different rates and yields are obtained. Cultivation in flasks produced 80% conversion to tyrosol acetate in 8 hours, whereas cultivation in tubes produced a 75% conversion of the substrate to tyrosol in 34 hours. The latter molecule is the natural substrate produced in Rhodiola sachalinensis and converted to salidroside by the enzyme UDP Glucuronosyl-Transferase (UGT). The UGT coding sequence has been cloned in Syn_CmBVMO to finally produce a double transgenic strain expressing both CmBVMO and UGT to produce salidroside. The last section describes the attempt to express for the first time in Synechocystsis an ATP-dependent enzyme: the γ-Glutamyl-MethylAmide Synthetase from Methylovorus mays No.9 (MmGMAS). It catalyzes the condensation reaction that produces L-theanine starting from L-glutamate and ethylamine. By testing the strain Syn_MmGMAS in whole-cell biotransformations it was possible to produce L-theanine at the maximum concentration of 1mM in the culture medium. This experiment demonstrated that photosynthetically produced ATP can be employed for catalysis. Moreover, it was observed that the consumption of ATP by the expressed and active enzyme is read as an extra electron demand by the photosystems, that respond by increasing the photosynthetic efficiency.