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Open Access Research article

Scalable production of biliverdin IXα by Escherichia coli

Dong Chen1, Jason D Brown1, Yukie Kawasaki2, Jerry Bommer3 and Jon Y Takemoto12*

Author Affiliations

1 Synthetic Bioproducts Center, 620 North 600 East, Utah State University, North Logan, Utah, 84341, USA

2 Department of Biology, 5305 Old Main Hill, Utah State University, Logan, Utah, 84322, USA

3 Frontier Scientific, Inc, 195 South 700 West, Logan, Utah, 84323, USA

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BMC Biotechnology 2012, 12:89  doi:10.1186/1472-6750-12-89

Published: 23 November 2012

Abstract

Background

Biliverdin IXα is produced when heme undergoes reductive ring cleavage at the α-methene bridge catalyzed by heme oxygenase. It is subsequently reduced by biliverdin reductase to bilirubin IXα which is a potent endogenous antioxidant. Biliverdin IXα, 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 IXα 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 IXα 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 IXα in batch and fed-batch bioreactor cultures. Strain BL21(mHO1) produced roughly twice the amount of biliverdin IXα than did strain BL21(HO1). Lactose either alone or in combination with glycerol supported consistent biliverdin IXα 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 IXα 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 IXα by E. coli were developed based on expression of a cyanobacterial ho1 gene. The purity of the produced biliverdin IXα and its ability to serve as substrate for human biliverdin reductase A suggest its potential as a clinically useful therapeutic.

Keywords:
Biliverdin IXα; Heme oxygenase; Escherichia coli; HO1; Bilirubin; Anti-inflammatory; Biliverdin reductase; Bioreactor