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Abstract

In times of global warming, photosynthetic microorganisms hold great promise as biotechnological platforms for the environmentally sustainable production of a wide range of commodity chemicals and biofuels. However, little effort has been made to study the capacity of photosynthetic bacteria to produce fine chemicals, some of which are required in larger quantities and thus represent attractive targets for carbon-neutral production processes. One of these compounds is the light-sensitive B-vitamin riboflavin, which is used as a food and feed additive for humans and farm animals. This study describes the first photoautotrophic bioprocess for the over-production of a vitamin compound, riboflavin (B2), which was achieved by metabolic engineering of the cyanobacterium Synechococcus sp. strain PCC 7002. By expressing the riboflavin biosynthesis genes from Bacillus subtilis in Synechococcus sp. PCC 7002 under the control of strong cyanobacterial promoters the supernatant concentrations of riboflavin was increased 211-fold compared to wild-type, reaching a final concentration of riboflavin of ~78 µM over the course of 38 days. The light-sensitivity of riboflavin at wavelengths below 500 nm was overcome by culturing the production strain under red LED light. Additionally, a Synechococcus sp. PC 7002 strain sensitive to the toxic antimetabolite roseoflavin was generated. Selection of this strain on the toxic riboflavin-analog roseoflavin yielded several roseoflavin-resistant riboflavin-overproducing mutants. Integration of the riboflavin biosynthesis genes into such a parent strain further increased the riboflavin levels, thus showing that the selection approach is compatible with genetic engineering for riboflavin production. Additionally, the endogenous riboflavin biosynthesis genes of Synechococcus sp. PCC 7002 were characterized. Overall, this project lays the foundation to build a potent phototrophic riboflavin production strain and demonstrate that cyanobacteria can serve as biotechnological platforms for the sustainable production of vitamins.