Establishing heterologous 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 concentrations of native pigments, the process is inherently inefficient. Now, an increase in consumer demand calls for the development of scalable and sustainable betalain production routes. To address this challenge, we introduced a heterologous pathway for the production of betaxanthins into cyanobacteria. The pathway consists of an engineered variant of the cytochrome P450 CYP76AD1 (W13L, F309L) 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 AroG^fbr from E. coli, led to an overaccumulation of the aromatic amino acids phenylalanine, tryptophan, and tyrosine, and the production of low levels of phenylalanine-betaxanthin. Optimization of the cultivation conditions (i.e., growth in nutrient-rich medium and CO₂-enriched air) increased titers approximately 165 times and led to the production of phenylalanine-betaxanthin with a final titer of 18.2 ± 5.1 mg L^-1. Our work establishes a microbial system for photoautotrophic betaxanthin pigment production without the need for exogenous amino acid supplementation.