Heterologous Production of Torularhodin, the Monocyclic Carotenoid with a Terminal Carboxyl Group, in Escherichia coli

Torularhodin is the monocyclic C40 carotenoid with the β-ring and a terminal carboxyl group at the acyclic part, with long conjugated double bonds, only synthesized in fungi called red (oleaginous) yeasts, e.g., the genera <i>Rhodotorula</i> and <i>Sporobolomyces</i>. This unique red pigment with strong antioxidant properties is promising for use in food additives, nutritional supplements, and cosmetics. We aimed to produce torularhodin in <i>Escherichia coli</i> through the identification of the biosynthesis genes needed for its heterologous production, while no genes oxidizing torulene to torularhodin had been reported. The <i>Rhodotorula toruloides crtI</i> (<i>CAR1</i>) and <i>crtYB</i> (<i>CAR2</i>) genes, which were chemically synthesized, proved to lead to the complete conversion of phytoene into torulene when they were introduced into an <i>E. coli</i> cell that carried the <i>Pantoea ananatis crtE</i> and <i>Haematococcus pluvialis IDI</i> genes. We found that the <i>Planococcus maritimus</i> genes coding for C30 carotenoid terminal oxidase (<i>crtP</i>/<i>crtNb</i>/<i>cruO</i>) and aldehyde dehydrogenase (<i>aldH</i>/<i>crtNc</i>), through their introduction into the <i>E. coli</i> transformant synthesizing torulene, mediated the efficient oxidations of torulene to torularhodin, and resulted in the production of torularhodin as the dominant carotenoid. This is the first report of torularhodin production in a heterologous host. We also identified the <i>aldH</i>/<i>crtNc</i> gene in <i>R. toruloides</i>.