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Title 

Leucine zipper-mediated targeting of multi-enzyme cascade reactions to inclusion bodies in Escherichia coli for enhanced production of 1-butanol

 

대장균에서 부탄올 생산을 위한 루신 지퍼 기반의 다중효소 연쇄반응

Authors 

Gui Hwan HanWonjae SeongY FuPaul Kyung Seok YoonSeong Keun KimSoo Jin YeomDae-Hee LeeSeung Goo Lee

Publisher 

Elsevier

Issue Date 

2017

Citation 

Metabolic Engineering

Keywords 

1-ButanolCellulose binding domainEnzyme cascadeInclusion bodyLeucine zipperMetabolon

Abstract 

Metabolons in nature have evolved to facilitate more efficient catalysis of multistep reactions through the co-localization of functionally related enzymes to cellular organelles or membrane structures. To mimic the natural metabolon architecture, we present a novel artificial metabolon that was created by targeting multi-enzyme cascade reactions onto inclusion body (IB) in Escherichia coli. The utility of this system was examined by co-localizing four heterologous enzymes of the 1-butanol pathway onto an IB that was formed in E. coli through overexpression of the cellulose binding domain (CBD) of Cellulomonas fimi exoglucanase. To target the 1-butanol pathway enzymes to the CBD IB, we utilized a peptide-peptide interaction between leucine zipper (LZ) peptides. We genetically fused the LZ peptide to the N-termini of four heterologous genes involved in the synthetic 1-butanol pathway, whereas an antiparallel LZ peptide was fused to the CBD gene. The in vivo activity of the CBD IB-based metabolon was examined through the determination of 1-butanol synthesis using E. coli transformed with two plasmids containing the LZ-fused CBD and LZ-fused 1-butanol pathway genes, respectively. In vivo synthesis of 1-butanol using the engineered E. coli yielded 1.98?g/L of 1-butanol from glucose, representing a 1.5-fold increase over that obtained from E. coli expressing the LZ-fused 1-butanol pathway genes alone. In an attempt to examine the in vitro 1-butanol productivity, we reconstituted CBD IB-based metabolon using CBD IB and individual enzymes of 1-butanol pathway. The 1-butanol productivity of in vitro reconstituted CBD IB-based metabolon using acetoacetyl-CoA as the starting material was 2.29?mg/L/h, 7.9-fold higher than that obtained from metabolon-free enzymes of 1-butanol pathway. Therefore, this novel CBD-based artificial metabolon may prove useful in metabolic engineering both in vivo and in vitro for the efficient production of desired products.

ISSN 

1096-7176

Link 

http://dx.doi.org/10.1016/j.ymben.2016.12.012

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2019-05-02


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