Metabolic engineering of Escherichia coli for efficient biosynthesis of fluorescent phycobiliprotein

Huaxin Chen,Peng Jiang

doi:10.1186/s12934-019-1100-6

Abstract

BackgroundPhycobiliproteins (PBPs) are light-harvesting protein found in cyanobacteria, red algae and the cryptomonads. They have been widely used as fluorescent labels in cytometry and immunofluorescence analysis. A number of PBPs has been produced in metabolically engineered Escherichia coli. However, the recombinant PBPs are incompletely chromophorylated, and the underlying mechanisms are not clear.Results and discussionIn this work, a pathway for SLA-PEB [a fusion protein of streptavidin and allophycocyanin that covalently binds phycoerythrobilin (PEB)] biosynthesis in E. coli was constructed using a single-expression plasmid strategy. Compared with a previous E. coli strain transformed with dual plasmids, the E. coli strain transformed with a single plasmid showed increased plasmid stability and produced SLA-PEB with a higher chromophorylation ratio. To achieve full chromophorylation of SLA-PEB, directed evolution was employed to improve the catalytic performance of lyase CpcS. In addition, the catalytic abilities of heme oxygenases from different cyanobacteria were investigated based on biliverdin IXα and PEB accumulation. Upregulation of the heme biosynthetic pathway genes was also carried out to increase heme availability and PEB biosynthesis in E. coli. Fed-batch fermentation was conducted for the strain V5ALD, which produced recombinant SLA-PEB with a chromophorylation ratio of 96.7%.ConclusionIn addition to reporting the highest chromophorylation ratio of recombinant PBPs to date, this work demonstrated strategies for improving the chromophorylation of recombinant protein, especially biliprotein with heme, or its derivatives as a prosthetic group.

Highlights

The phycobiliproteins (PBPs) are a family of light-harvesting proteins found in cyanobacteria, red algae and the cryptomonads [1]
Increasing the chromophorylation ratio of SLA‐PEB by improving plasmid stability The engineered E. coli strain SLA-V1 was constructed in our previous study to produce SLA-PEB
Inefficient biosynthesis of recombinant PBPs in recombinant E. coli leads to the production of a mixture of holo-PBP and SLA‐PEB production in fed‐batch fermentation We noticed that when E. coli strain producing PBPs were cultured on large-scale, the chromophorylation ratio of recombinant PBPs was remarkably decreased, though its production level was elevated

Summary

Introduction

Phycobiliproteins (PBPs) are light-harvesting protein found in cyanobacteria, red algae and the cryptomonads They have been widely used as fluorescent labels in cytometry and immunofluorescence analysis. The phycobiliproteins (PBPs) are a family of light-harvesting proteins found in cyanobacteria, red algae and the cryptomonads [1] Based on their spectrum properties, PBPs are classified into three main types: phycoerythrin (PE), phycocyanin (PC), and allophycocyanin (APC). These proteins absorb strongly in the visible region of the spectrum because they carry various covalently attached linear tetrapyrrole prosthetic groups (phycobilins) Due to their excellent fluorescent properties and their ability to be covalently linked to various biomolecules, PBPs have been widely used as fluorescent labels in flow cytometry and immunofluorescence analysis [2]. The streptavidin-linked phycobiliprotein can bind to biotinylated proteins, DNA or other biochemical reagents and can be used to probe biological events

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