Method for producing phycocyanobilin using a recombinant Escherichia coli

Patent metadata

Field	Value
Publication number	US-12529084-B2
Application number	US-202418428893-A
Country	US
Kind code	B2
Filing date	Jan 31, 2024
Priority date	Feb 10, 2023
Publication date	Jan 20, 2026
Grant date	Jan 20, 2026

Abstract

Disclosed is a method for producing phycocyanobilin by use of a recombinant Escherichia coli that express heterologous heme oxygenase ho1 and ferredoxin oxidoreductase pcyA derived from Synechocystis sp. PCC6803. According to the present disclosure, heterologous expression of ho1 and pcyA genes leads to conversion of heme to an intermediate biliverdin for phycocyanobilin synthesis, and reduces the accumulation of biliverdin in the process of the phycocyanobilin synthesis. The genome of E. coli is further engineered to overexpress related genes of a metabolic pathway of phycocyanobilin, and a strain of recombinant E. coli with high yield of phycocyanobilin is obtained. The recombinant E. coli strain is cultured for 36 hr in a system using glycerol as a substrate, and the phycocyanobilin yield can reach 147 mg/L.

First claim

What is claimed is:

1 . A strain of recombinant Escherichia coli ( E. coli ) for synthesis of phycocyanobilin, wherein heme oxygenase ho1 and ferredoxin oxidoreductase pcyA derived from Synechocystis sp. PCC6803 are expressed in the strain of the recombinant E. coli.

2 . The recombinant E. coli of claim 1 , wherein the heme oxygenase ho1 and the ferrendoxin oxidoreductase pcyA are expressed using pRSFDuet-1 expression vector.

3 . The recombinant E. coli of claim 1 , wherein nucleotide sequences of genes encoding the heme oxygenase ho1 and the ferredoxin oxidoreductase pcyA are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively.

4 . The recombinant E. coli of claim 1 , wherein the heme oxygenase ho1 and the ferredoxin oxidoreductase pcyA are linked together by short peptide tags RIDD and RIAD.

5 . The recombinant E. coli of claim 1 , wherein at least one of the following improvements is made in the recombinant E. coli: (1) overexpression of endogenous genes hemB, hemC, and hemD; and (2) overexpression of endogenous genes hemE, hemF, hemG, and hemH.

6 . The recombinant E. coli of claim 5 , wherein the genes hemB, hemC, and hemD are integrated at an arsB site of an arsenate transporter gene of the recombinant E. coli.

7 . The recombinant E. coli of claim 5 , wherein the genes hemE, hemF, hemG and hemH are integrated at a position where a heme degrading gene yfeX is located.

8 . The recombinant E. coli of claim 4 , a fusion gene ho1-GGGGS-RIDD-RIAD-GGGGS-pcyA is expressed in E. coli strain BL21 (DE3) using pRSFDuet-1 as the expression vector, wherein endogenous genes hemB, hemC, hemD, hemE, hemF, hemG, and hemH are overexpressed; wherein the nucleotide sequence of the fusion gene ho1-GGGGS-RIDD-RIAD-GGGGS-pcyA is shown in SEQ ID NO:60.

9 . The recombinant E. coli of claim 7 , wherein the genes hemB, hemC and hemD are integrated at an arsB site of an arsenate transporter gene of the recombinant E. coli.

10 . The recombinant E. coli according to claim 8 , wherein the genes hemE, hemF, hemG, and hemH are integrated at a position where a heme degrading gene yfeX is located.

11 . A method for producing phycocyanobilin, wherein the recombinant E. coli of claim 1 is fermented to produce the phycocyanobilin.

12 . The method of claim 11 , wherein the recombinant E. coli is inoculated into a fermentation medium, cultured at 35-37° C. for 2-3 hr, induced with IPTG, and fermented for 24-48 hr.

13 . The method of claim 12 , wherein 0.5 mM IPTG is added and synthesis of phycocyanobilin is induced and carried out at 25° C., 200-220 rpm for 24-48 hr.

14 . The method of claim 12 , wherein the fermentation medium contains KH 2 PO 4 , K 2 HPO 4 ·3H 2 O, (NH 4 ) 2 SO 4 , anhydrous citric acid, MgSO 4 , yeast powder, glycerol, maltodextrin, vitamin B1, and a trace element solution.

15 . The method of claim 14 , wherein the trace element solution contains Fe (III) citrate, ZnCl 2 , MnSO 4 ·H 2 O, CuSO 4 ·5H 2 O, Na 2 MoO 4 ·2H 2 O, CaCl 2 ·2H 2 O, H 3 BO 3 , CoCl 2 ·6H 2 O, and NiSO 4 ·6H 2 O.

Assignees

• Univ Jiangnan

Inventors

• Zhou Jingwen
• Chen Jian
• Wang Yuqi
• Xu Sha
• Zeng Weizhu

Classifications

• C12Y114/14

  with reduced flavin or flavoprotein as one donor, and incorporation of one atom of oxygen (1.14.14) · CPC title

• C12Y103/07005

  Phycocyanobilin:ferredoxin oxidoreductase (1.3.7.5) · CPC title

• C12N2800/101

  for bacteria · CPC title

• C12N15/70

  Vectors or expression systems specially adapted for E. coli · CPC title

• C12N9/0071

  acting on paired donors with incorporation of molecular oxygen (1.14) · CPC title