Recombinant Production of Biliverdin IXβ and δ Isomers in the T7 Promoter Compatible Nissle
Date
2021-12-08Journal
Frontiers in MicrobiologyPublisher
Frontiers Media S.A.Type
Article
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The ability to obtain purified biliverdin IX (BVIX) isomers other than the commercially available BVIXα is limited due to the low yields obtained by the chemical coupled oxidation of heme. Chemical oxidation requires toxic chemicals, has very poor BVIX yields (<0.05%), and is not conducive to scalable production. Alternative approaches utilizing recombinant E. coli BL21 expressing a cyanobacterial heme oxygenase have been employed for the production BVIXα, but yields are limited by the rate of endogenous heme biosynthesis. Furthermore, the emerging roles of BVIXβ and BVIXδ in biology and their lack of commercial availability has led to a need for an efficient and scalable method with the flexibility to produce all three physiologically relevant BVIX isomers. Herein, we have taken advantage of an optimized non-pathogenic E. coli Nissle (EcN(T7)) strain that encodes an endogenous heme transporter and an integrated T7 polymerase gene. Protein production of the Pseudomonas aeruginosa BVIXβ and BVIXδ selective heme oxygenase (HemO) or its BVIXα producing mutant (HemOα) in the EcN(T7) strain provides a scalable method to obtain all three isomers, that is not limited by the rate of endogenous heme biosynthesis, due to the natural ability of EcN(T7) to transport extracellular heme. Additionally, we have optimized our previous LC-MS/MS protocol for semi-preparative separation and validation of the BVIX isomers. Utilizing this new methodology for scalable production and separation we have increased the yields of the BVIXβ and -δ isomers >300-fold when compared to the chemical oxidation of heme.Rights/Terms
Copyright © 2021 Robinson, Frankenberg-Dinkel, Xue and Wilks.Keyword
E. coli Nissle (EcN(T7))HemO
Pseudomonas aeruginosa
biliverdin IXα
biliverdin IXβ
biliverdin IXδ
biliverdin isomer
heme
Identifier to cite or link to this item
http://hdl.handle.net/10713/17458ae974a485f413a2113503eed53cd6c53
10.3389/fmicb.2021.787609
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