Establishing betaxanthin pigment biosynthesis in cyanobacteria

Betalains are water-soluble pigments with two major classes: red-violet betacyanins and yellow-orange betaxanthins. These pigments are increasingly being sought after as natural replacements for synthetic pigments in the food industry. Traditionally, betalains are extracted from cultivated plants but, due to low native pigments, the process is inherently inefficient. Now, as consumer demand increases, scalable and sustainable production routes are needed. To address this challenge, we engineered a heterologous pathway for the production of betaxanthins into cyanobacteria. The pathway consists of the cytochrome P450 CYP76AD1 and the L-DOPA 4,5-dioxygenase DODA1 from Beta vulgaris (beet). Introduction of the two-enzyme betaxanthin pathway in Synechocystis sp. PCC 6803 did not result in detectable betaxanthins. Subsequent metabolic adjustments to the shikimate pathway, using a feedback resistant AroGfbr from E. coli, led to an overaccumulation of the aromatic amino acids phenylalanine, tryptophan, and tyrosine, and the production of 0.11 mg L-1 phenylalanine-betaxanthin. Optimization of the cultivation conditions (i.e., growth in nutrient-rich medium and CO2-enriched air) increased titers almost 150 times and led to the production of phenylalanine-betaxanthin with a final titer of 15.3 mg L-1. Our work establishes a microbial system for photoautotrophic betaxanthin pigment production without the need for exogenous amino acid supplementation.