Scalable production of biliverdin IXα by Escherichia coli

Background: Biliverdin IX alpha is produced when heme undergoes reductive ring cleavage at the alpha-methene bridge catalyzed by heme oxygenase. It is subsequently reduced by biliverdin reductase to bilirubin IXa which is a potent endogenous antioxidant. Biliverdin IX alpha, through interaction with biliverdin reductase, also initiates signaling pathways leading to anti-inflammatory responses and suppression of cellular pro-inflammatory events. The use of biliverdin IXa as a cytoprotective therapeutic has been suggested, but its clinical development and use is currently limited by insufficient quantity, uncertain purity, and derivation from mammalian materials. To address these limitations, methods to produce, recover and purify biliverdin IXa from bacterial cultures of Escherichia coli were investigated and developed. Results: Recombinant E. coli strains BL21(HO1) and BL21(mHO1) expressing cyanobacterial heme oxygenase gene ho1 and a sequence modified version (mho1) optimized for E. coli expression, respectively, were constructed and shown to produce biliverdin IXa in batch and fed-batch bioreactor cultures. Strain BL21(mHO1) produced roughly twice the amount of biliverdin IXa than did strain BL21(HO1). Lactose either alone or in combination with glycerol supported consistent biliverdin IXa production by strain BL21(mHO1) (up to an average of 23. 5mg L-1 culture) in fed-batch mode and production by strain BL21 (HO1) in batch-mode was scalable to 100L bioreactor culture volumes. Synthesis of the modified ho1 gene protein product was determined, and identity of the enzyme reaction product as biliverdin IXa was confirmed by spectroscopic and chromatographic analyses and its ability to serve as a substrate for human biliverdin reductase A. Conclusions: Methods for the scalable production, recovery, and purification of biliverdin IXa by E. coli were developed based on expression of a cyanobacterial ho1 gene. The purity of the produced biliverdin IXa and its ability to serve as substrate for human biliverdin reductase A suggest its potential as a clinically useful therapeutic.