Numaswitch: an efficient high-titer expression platform to produce peptides and small proteins

Nguyen, Bach-Ngan; Tieves, Florian; Rohr, Thomas; Wobst, Hilke; Schöpf, Felix S.; Solano, José D.; Schneider, Júlia; Stock, Janpeter; Uhde, Andreas; Kalthoff, Thomas; Jaeger, Karl Erich; Schmitt, Lutz; Schwarz, Christian

doi:10.1186/s13568-021-01204-w

Cited by 9 publications

(10 citation statements)

References 24 publications

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“…Another strategy (42) can produce higher peptide yields under fermentation (teriparatide: > 2 g/L) but requires cumbersome cell lysis, inclusion body recovery, and fusion protein cleavage to recover the desired peptide. We have not attempted to optimize export, but our studies using E. coli BL21(DE3) lacking proteases and lowering temperature indicate that peptide secretion abundance can be improved (Fig.…”

Section: Discussionmentioning

confidence: 99%

Export of diverse and bioactive peptides through a type I secretion system

Kim

Parker

Gonzalez-Magaldi

et al. 2023

Preprint

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Microcins are peptide antibiotics secreted by Gram-negative bacteria that inhibit the growth of neighboring microbes. They are exported from the cytosol to the environment in a one-step process through a specific type I secretion system (T1SS). While the rules governing export of natural or non-native substrates have been resolved for T1SSs that secrete large proteins, relatively little is known about substrate requirements for peptides exported through T1SSs that secrete microcins. Here, we investigate the prototypic microcin V T1SS from Escherichia coli and show it can export a remarkably wide range of natural and synthetic peptides. We demonstrate that secretion through this system is not affected by peptide charge or hydrophobicity and appears only constrained by peptide length. A varied range of bioactive peptides, including an antibacterial peptide, a microbial signaling factor, a protease inhibitor, and a human hormone, can all be secreted and elicit their intended biological effect. Secretion through this system is not limited to E. coli, and we demonstrate its function in additional Gram-negative species that can inhabit the gastrointestinal tract. Our findings uncover the highly promiscuous nature of peptide export thorough the microcin V T1SS, which has implications for native cargo capacity and use of Gram-negative bacteria for peptide research and delivery.

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Section: Discussionmentioning

confidence: 99%

Export of diverse and bioactive peptides through a type I secretion system

Kim

Parker

Gonzalez-Magaldi

et al. 2023

Preprint

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“…[ 14 , 15 , 16 , 17 ] It should be noted that an engineered T1SS has recently been developed to allow efficient production of peptides and small proteins. [18] …”

Section: Introductionmentioning

confidence: 99%

“…Despite a long list of successfully secreted heterologous proteins, HlyA1 is still far away from being an universal secretion platform, due to a narrow range of possible substrates as well as low yields of secretion [14–17] . It should be noted that an engineered T1SS has recently been developed to allow efficient production of peptides and small proteins [18] …”

Section: Introductionmentioning

confidence: 99%

“…[14][15][16][17] It should be noted that an engineered T1SS has recently been developed to allow efficient production of peptides and small proteins. [18] The HlyA secretion system forms a tripartite doublemembrane-spanning channel trough association of an inner membrane ATP-binding cassette transporter (HlyB), a membrane fusion protein (HlyD), and an outer membrane protein (TolC). [19] The next component of the HlyA system is HlyC.…”

Section: Introductionmentioning

confidence: 99%

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Optimized Hemolysin Type 1 Secretion System in Escherichia coli by Directed Evolution of the Hly Enhancer Fragment and Including a Terminator Region

Cui

Khosa

et al. 2022

ChemBioChem

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Type 1 secretion systems (T1SS) have a relatively simple architecture compared to other classes of secretion systems and therefore, are attractive to be optimized by protein engineering.Here, we report a KnowVolution campaign for the hemolysin (Hly) enhancer fragment, an untranslated region upstream of the hlyA gene, of the hemolysin T1SS of Escherichia coli to enhance its secretion efficiency. The best performing variant of the Hly enhancer fragment contained five nucleotide mutations at five positions (A30U, A36U, A54G, A81U, and A116U) resulted in a 2-fold increase in the secretion level of a model lipase fused to the secretion carrier HlyA1. Computational analysis suggested that altered affinity to the generated enhancer fragment towards the S1 ribosomal protein contributes to the enhanced secretion levels. Furthermore, we demonstrate that involving a native terminator region along with the generated Hly enhancer fragment increased the secretion levels of the Hly system up to 5-fold.

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“…Especially, pepteins of 30-300 amino acids length, covering such important families as long-chained peptides, disulfide-rich peptides, growth factors, peptide fusions and antibody fragments are of high interest and neither chemical synthesis nor traditional recombinant approaches are a reliable and cost-efficient production platform. To this end, we developed such a production platform called Numaswitch [1]. Here, bifunctional Switchtag proteins were developed combining two essential functions (Figure 1).…”

Section: Introductionmentioning

confidence: 99%