Engineering Extracellular Expression Systems in Escherichia coli Based on Transcriptome Analysis and Cell Growth State

Abstract

Escherichia coli extracellular expression systems have a number of advantages over other systems, such as lower pyrogen levels and a simple purification process. Various approaches, such as the generation of leaky mutants via chromosomal engineering, have been explored for this expression system. However, extracellular protein yields in leaky mutants are relatively low compared to that in intracellular expression systems and therefore need to be improved. In this work, we describe the construction, characterization, and mechanism of enhanced extracellular expression in Escherichia coli. On the basis of the localizations, functions, and transcription levels of cell envelope proteins, we systematically elucidated the effects of multiple gene deletions on cell growth and extracellular expression using modified CRISPR/Cas9-based genome editing and a FlAsH labeling assay. High extracellular yields of heterologous proteins of different sizes were obtained by screening multiple gene mutations. The enhancement of extracellular secretion was associated with the derepression of translation and translocation. This work utilized universal methods in the design of extracellular expression systems for genes not directly associated with protein synthesis that were used to generate strains with higher protein expression capability. We anticipate that extracellular expression systems may help to shed light on the poorly understood aspects of these secretion processes as well as to further assist in the construction of engineered prokaryotic cells for efficient extracellular production of heterologous proteins.

Strategies to Enhance Periplasmic Recombinant Protein Production Yields in Escherichia coli

“…Furthermore, periplasmic protein production can also lead to the “spontaneous” release of the protein into the extracellular milieu ( Kastenhofer et al, 2021 ). The release of a protein into the extracellular milieu can also be promoted by actively making the outer membrane more permeable or by fusing the protein to a fusion partner that is naturally secreted into the extracellular milieu ( Hsiung et al, 1989 ; Zhang et al, 2006 ; Natarajan et al, 2017 ; Gao et al, 2018 )). Extracellular production of a (fusion) protein can even further facilitate protein isolation ( Tripathi, 2016 ).…”

Regulation of Cell Membrane Permeability Enhanced the Non-Classical Secretion of γ-Cyclodextrin Glycosyltransferase in Bacillus subtilis

“…Calcein AM and PI were used for double fluorescence staining. Calcein AM can easily penetrate the viable cell membrane and be hydrolyzed by endogenous esterase in cells, which results in the emission of a strong green fluorescence, while PI, blocked by an intact cell membrane, can only stain cells whose cell membrane integrity has been damaged, resulting in the emission of red fluorescence . Fluorescence micrographs of the double-stained cells are shown in Figure .…”

“…The integrity of cell membranes was detected by a fluorescence microscope using the Calcein/PI Cell Viability/Cytotoxicity Assay Kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions. Specific steps referred to Gao et al with some modifications . In brief, 100 μL of broth containing B.…”

“…Calcein AM can easily penetrate the viable cell membrane and be hydrolyzed by endogenous esterase in cells, which results in the emission of a strong green fluorescence, while PI, blocked by an intact cell membrane, can only stain cells whose cell membrane integrity has been damaged, resulting in the emission of red fluorescence. 27 Fluorescence micrographs of the doublestained cells are shown in Figure 4. In the first 24 h, the bacteria were still in active growth so that red fluorescence can barely be seen in the fluorescence microscope.…”

Developing Gram-negative bacteria for the secretion of heterologous proteins

“…In one study, a ΔlppΔmrcB E. coli strain was able to secrete human parathyroid hormone 1–84 coupled with thioredoxin as a fusion partner at titers of up to 680 mg/L [36]. A recent study by Gao et al used transcriptomics to identify highly expressed lipoproteins that were likely to make the membrane leakier when deleted [95]. They measured secretion of IFN-α and polypeptides of varying sizes to evaluate size limitations.…”