4.7 Article

Silk fibroin production in Escherichia coli is limited by a positive feedback loop between metabolic burden and toxicity stress

期刊

METABOLIC ENGINEERING
卷 77, 期 -, 页码 231-241

出版社

ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ymben.2023.03.011

关键词

13C-MFA; Acetate overflow; Entner-doudoroff pathway; Metabolic burden; Silk fibroin; Toxicity

向作者/读者索取更多资源

This study investigates the metabolic flexibility and limitations in the production of recombinant silk proteins in Escherichia coli. It reveals that metabolic burden leads to metabolic flux redistribution and changes in the Ent-ner-Doudoroff pathway. Additionally, accumulation of acetate is highly toxic to silk-producing strains, resulting in decreased silk protein production. The study proposes three solutions to address these issues and discusses other strategies.
To investigate the metabolic elasticity and production bottlenecks for recombinant silk proteins in Escherichia coli, we performed a comprehensive characterization of one elastin-like peptide strain (ELP) and two silk protein strains (A5 4mer, A5 16mer). Our approach included 13C metabolic flux analysis, genome-scale modeling, transcription analysis, and 13C-assisted media optimization experiments. Three engineered strains maintained their central flux network during growth, while measurable metabolic flux redistributions (such as the Ent-ner-Doudoroff pathway) were detected. Under metabolic burdens, the reduced TCA fluxes forced the engineered strain to rely more on substrate-level phosphorylation for ATP production, which increased acetate overflow. Acetate (as low as 10 mM) in the media was highly toxic to silk-producing strains, which reduced 4mer pro-duction by 43% and 16mer by 84%, respectively. Due to the high toxicity of large-size silk proteins, 16mer ' s productivity was limited, particularly in the minimal medium. Therefore, metabolic burden, overflow acetate, and toxicity of silk proteins may form a vicious positive feedback loop that fractures the metabolic network. Three solutions could be applied: 1) addition of building block supplements (i.e., eight key amino acids: His, Ile, Phe, Pro, Tyr, Lys, Met, Glu) to reduce metabolic burden; 2) disengagement of growth and production; and 3) use of non-glucose based substrate to reduce acetate overflow. Other reported strategies were also discussed in light of decoupling this positive feedback loop.

作者

我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。

评论

主要评分

4.7
评分不足

次要评分

新颖性
-
重要性
-
科学严谨性
-
评价这篇论文

推荐

暂无数据
暂无数据