Synthesis of bilirubin in Escherichia coli

Abstract

Bilirubin is a bilirubin produced by the metabolic decomposition of heme. It is widely present in animals, especially in the liver, where it participates in the metabolism of bilirubin. It has various pharmacological activities and is also one of the main raw materials for preparing artificial bezoar. Bilirubin has significant application value in fields such as medicine, food and cosmetics. Traditionally, bilirubin has mainly been extracted from animal tissues, but this method is costly, has a low yield and poses ethical issues. This article intends to utilize biocatalysis technology to catalyze the synthesis of bilirubin in the whole cells of Escherichia coli as the host. First of all, Construct several heme oxygenases (AtHO, TeHO) and biliverdin reductases from different sources The overexpression vectors pETDuet-1-AtHO、pETDuet-1-TeHO、pETDuet-1-AtHO-RnBvR and pETDuet-1-TeHO-SsBvR (RnBvR, SsRnBvR) Escherichia coli BL21 (DE3) and Nissle 1917 were transformed respectively. Later, whole-cell catalysis showed that in the case of adding heme co-solvents such as DMF, Nissle 1917 (pETDuet-1-AtHO-RnBvR) could more effectively sequentially catalyze the synthesis of bilirubin from substrate heme. It reflects the influence of different backgrounds of the host metabolic network on product synthesis. Further, the Nissle 1917 heme pump coding gene was cloned into the pRSFDuet-1 vector. After co-conversion with pETDuet-1-AtHO-RnBvR for BL21 (DE3), continuous whole-cell catalysis was carried out in a pH 8.0 0.2 mM Tris-HCl buffer for 20 hours. The amount of bilirubin synthesized from the substrate heme conversion was 35% higher than that of Nissle 1917 (pETDuet-1-AtHO-RnBvR), reaching 21 mg/mL. This study provides basic data for reference for the biosynthesis of bilirubin, especially for the subsequent development of synthetic biology techniques.