The immunosuppressant brasilicardin: Determination of the biosynthetic gene cluster in the heterologous host Amycolatopsis japonicum†

Nocardia terpenica IFM 0406 is the producer of the immunosuppressants brasilicardins A-D. Brasilicardin is a promising compound because of its unique mode of action and its higher potency and reduced toxicity compared to today's standard drugs. However, production of brasilicardin was so far hampered since N. terpenica IFM 0406 synthesizes brasilicardin in only low amounts and represents a human pathogen (biosafety level 2 BSL2). In order to achieve a safe and high yield production of brasilicardin A (BraA) we heterologously expressed the brasilicardin gene cluster in the nocardioform actinomycete Amycolatopsis japonicum (A. japonicum::bcaAB01), which is fast growing, genetically accessible and closely related to N. terpenica IFM 0406. In Amycolatopsis japonicum::bcaAB01 four brasilicardin congeners, intermediates of the BraA biosynthesis, were produced. Investigation of the genes flanking the previous defined brasilicardin biosynthetic gene cluster revealed two novel genes (bra0, bra12), which are involved in brasilicardin biosynthesis: bra12 encodes a transcriptional activator of the brasilicardin gene cluster. bra0 codes for a dioxygenase involved in methoxylation of brasilicardin. Based on this finding we were able to revise the proposed brasilicardin biosynthesis.