Summary The recent interest in plant pigment betalains as bioactive compounds and chemopreventive agents has led to the search for a reliable and scalable process to obtain them. The cloning of the novel and efficient enzyme 4,5‐DOPA‐extradiol dioxygenase from Gluconacetobacter diazotrophicus in an expression vector, and the subsequent heterologous expression in Escherichia coli cultures has led to the start‐up of a biotechnological production system of individual pigments. The aim of this study was to search for the optimal conditions for the production of betalamic acid in microbial factories and the scaled‐up obtention of the derived pigments. Four different betaxanthins and two betacyanins were obtained after the addition of non‐transformable amines and amino acids and their condensation with the betalamic acid produced by the dioxygenase. The scaled‐up obtention and purification of betalains improved the yields of the previous methodologies reaching quantities by up to 150 mg of pure compounds.