Biotechnological applications of type 1 secretion systems

Pourhassan, N Zohreh; Smits, Sander H. J.; Ahn, Jung Hoon; Schmitt, Lutz

doi:10.1016/j.biotechadv.2021.107864

Cited by 13 publications

(21 citation statements)

References 127 publications

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“…[ 4 , 5 ] Recombinant protein secretion is beneficial over intracellular production strategies to meet biotechnological purposes because of various reasons such as simplifying downstream processes, reducing risk of cytosolic degradation, higher quality and solubility of secreted protein, avoidance of inclusion body formation, and reduced costs of production processes. [ 5 , 6 , 7 ]…”

Section: Introductionmentioning

confidence: 99%

“…Attempts to achieve reasonable secretion levels for recombinant protein secretion have been continuously made commonly with limited success, presumably due to the complexity of the multi‐component secretion machineries. [ 6 , 7 , 9 ]…”

Section: Introductionmentioning

confidence: 99%

“…Various examples of successful KnowVolution campaigns have been published, e. g ., with glucose oxidases, phytases, proteases, cellulases, laccases, aryl sulfotransferases, or polymer‐binding peptides. [ 7 , 30 , 31 , 32 , 33 , 34 ]…”

Section: Introductionmentioning

confidence: 99%

“…Escherichia coli ( E. coli ) has been continuously implemented in recombinant protein production [2] and is often still the preferred production host in research laboratories [3] and used in industrial scales [4,5] . Recombinant protein secretion is beneficial over intracellular production strategies to meet biotechnological purposes because of various reasons such as simplifying downstream processes, reducing risk of cytosolic degradation, higher quality and solubility of secreted protein, avoidance of inclusion body formation, and reduced costs of production processes [5–7] …”

Section: Introductionmentioning

confidence: 99%

“…So far, at least eight different secretion systems for Gram‐negative bacteria, including E. coli , have been elucidated that secrete a wide range of transport‐substrates [7,8] . Successful secretion of recombinant proteins by five of these secretion systems has been reported so far, but often with low titers and for a narrow range of substrates.…”

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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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

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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Enzyme Production

Knapp,

Jaeger

2024

Learning Materials in Biosciences

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A step forward to the optimized HlyA type 1 secretion system through directed evolution

Pourhassan,

Cui,

Muckhoff

et al. 2023

Appl Microbiol Biotechnol

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Secretion of proteins into the extracellular space has great advantages for the production of recombinant proteins. Type 1 secretion systems (T1SS) are attractive candidates to be optimized for biotechnological applications, as they have a relatively simple architecture compared to other classes of secretion systems. A paradigm of T1SS is the hemolysin A type 1 secretion system (HlyA T1SS) from Escherichia coli harboring only three membrane proteins, which makes the plasmid-based expression of the system easy. Although for decades the HlyA T1SS has been successfully applied for secretion of a long list of heterologous proteins from different origins as well as peptides, but its utility at commercial scales is still limited mainly due to low secretion titers of the system. To address this drawback, we engineered the inner membrane complex of the system, consisting of HlyB and HlyD proteins, following KnowVolution strategy. The applied KnowVolution campaign in this study provided a novel HlyB variant containing four substitutions (T36L/F216W/S290C/V421I) with up to 2.5-fold improved secretion for two hydrolases, a lipase and a cutinase. Key points • An improvement in protein secretion via the use of T1SS • Reaching almost 400 mg/L of soluble lipase into the supernatant • A step forward to making E. coli cells more competitive for applying as a secretion host Graphical Abstract

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Biotechnological applications of type 1 secretion systems

Cited by 13 publications

References 127 publications

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

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

Enzyme Production

A step forward to the optimized HlyA type 1 secretion system through directed evolution

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