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Abstract
The ribosomally synthesized and post-translationally modified peptide mersacidin is a class II lanthipeptide with good activity against Gram-positive bacteria. The intramolecular lanthionine rings, that give mersacidin its stability and antimicrobial activity, are specific structures with potential applications in synthetic biology. To add the mersacidin modification enzymes to the synthetic biology toolbox, a heterologous expression system for mersacidin in Escherichia coli has recently been developed. While this system was able to produce fully modified mersacidin precursor peptide that could be activated by Bacillus amyloliquefaciens supernatant and showed that mersacidin was activated in an additional proteolytic step after transportation out of the cell, it lacked a mechanism for clean and straightforward leader processing. Here, the protease responsible for activating mersacidin was identified and heterologously produced in E. coli, improving the previously reported heterologous expression system. By screening multiple proteases, the stringency of proteolytic activity directly next to a very small lanthionine ring is demonstrated, and the full two-step proteolytic activation of mersacidin was elucidated. Additionally, the effect of partial leader processing on diffusion and antimicrobial activity is assessed, shedding light on the function of two-step leader processing.
Highlights
The class II lanthipeptide mersacidin is a ribosomally synthesized and post-translationally modified peptide (RiPP; Figure 1; Bierbaum et al, 1995; Arnison et al, 2013)
Produced in E. coli has a higher activity than the PG10 produced ATCC 6633 AprE, despite having a lower yield. This effect could be caused by BH072 AprE cleaving the mersacidin leader more efficiently or by the different expression conditions used for PG10 and BL21(DE3)
The experiments described in this paper show that B. amyloliquefaciens AprE is responsible for activating mersacidin in a second proteolytic step, which occurs after transport and FIGURE 4 | removal of the leader peptide
Summary
The class II lanthipeptide mersacidin is a ribosomally synthesized and post-translationally modified peptide (RiPP; Figure 1; Bierbaum et al, 1995; Arnison et al, 2013). Lanthipeptides contain post-translationally installed intramolecular thioether bridges, which increase resistance to proteolytic degradation and are necessary to give the molecules their rigidity and bioactivity, for example, antimicrobial activity (Repka et al, 2017). The modification enzymes of lanthipeptides and many other RiPPs are guided toward the precursor by a leader peptide sequence (Plat et al, 2013). By combining (parts of) leader sequences of different systems, new molecules can be created that contain modifications from both systems (Burkhart et al, 2017; Wu and van der Donk, 2021). For more in-depth information about the different RiPPs classes and engineering, some excellent reviews are available (Arnison et al, 2013; Montalbán-López et al, 2021).
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