Inhibition of E. coli Host RNA Polymerase Allows Efficient Extracellular Recombinant Protein Production by Enhancing Outer Membrane Leakiness,Biotechnology Journal

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Inhibition of E. coli Host RNA Polymerase Allows Efficient Extracellular Recombinant Protein Production by Enhancing Outer Membrane Leakiness
Biotechnology Journal ( IF 3.2 ) Pub Date : 2020-09-11 , DOI: 10.1002/biot.202000274
Jens Kastenhofer ₁ , Lukas Rettenbacher ₁ , Lukas Feuchtenhofer ₂ , Juergen Mairhofer ₂ , Oliver Spadiut ₁

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With the growing interest in continuous cultivation of Escherichia coli, secretion of product to the medium is not only a benefit, but a necessity in future bioprocessing. In this study, it is shown that induced decoupling of growth and heterologous gene expression in the E. coli X‐press strain (derived from BL21(DE3)) facilitates extracellular recombinant protein production. The effect of the process parameters temperature and specific glucose consumption rate (q_S) on growth, productivity, lysis and leakiness, is investigated, to find the parameter space allowing extracellular protein production. Two model proteins are used, Protein A (SpA) and a heavy‐chain single‐domain antibody (VHH), and performance is compared to the industrial standard strain BL21(DE3). It is shown that inducible growth repression in the X‐press strain greatly mitigates the effect of metabolic burden under different process conditions. Furthermore, temperature and q_S are used to control productivity and leakiness. In the X‐press strain, extracellular SpA and VHH titer reach up to 349 and 19.6 mg g⁻¹, respectively, comprising up to 90% of the total soluble product, while keeping cell lysis at a minimum. The findings demonstrate that the X‐press strain constitutes a valuable host for extracellular production of recombinant protein with E. coli.

中文翻译：

大肠杆菌宿主RNA聚合酶的抑制通过增强外膜渗漏允许有效的细胞外重组蛋白生产。

随着人们对大肠杆菌的连续培养的兴趣日益浓厚，将产物分泌到培养基中不仅是一种好处，而且是未来生物处理的必要条件。在这项研究中，表明在大肠杆菌X-press菌株（源自BL21（DE3））中，诱导的生长和异源基因表达脱钩促进了细胞外重组蛋白的产生。工艺参数温度和比葡萄糖消耗率（q _S在生长，生产力，裂解和渗漏方面进行了研究，以找到允许细胞外蛋白质产生的参数空间。使用了两种模型蛋白，蛋白A（SpA）和重链单域抗体（VHH），并将其性能与工业标准菌株BL21（DE3）进行了比较。结果表明，X-press菌株中可诱导的生长抑制大大减轻了不同工艺条件下代谢负担的影响。此外，温度和q _S用于控制生产率和泄漏。在X-press菌株中，细胞外SpA和VHH滴度可达349和19.6 mg g ^-1分别占总可溶性产物的90％，同时将细胞裂解保持在最低水平。这些发现表明，X-press菌株是大肠杆菌胞外生产重组蛋白的重要宿主。

更新日期：2020-09-11

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