From the Producer Microorganisms to the Lasso Scaffold - Lasso Peptides: Bacterial Strategies to Make and Maintain Bioactive Entangled Scaffolds - page 13

conditions for peptide production. The optimization of these parameters must be
strongly connected to the growth phase of the producing bacteria, which is also a
critical parameter for metabolite production. Depending on the peptides and the
producing strains, lasso peptides are produced either in early exponential phase,
such as capistruin (Knappe et al. 2008 ), or in stationary phase, as observed for
MccJ25 (Chiuchiolo et al. 2001 ). Therefore, duration of the fermentation has to be
determined in accordance with bacterial growth curves measured in parallel with
lasso peptide production.
When culture media and fermentation conditions have been optimized for maxi-
mum lasso peptide production, large-scale fermentation procedures can be applied
in order to obtain appropriate amounts of peptides for further structural studies and
biological assays. Nearly, all known lasso peptides from Actinobacteria were iso-
lated from large-scale fermentations. When this approach fails and insufficient pro-
duction yields are obtained, heterologous production (see Sect. 2.2.2 below) is the
best way for permitting the characterization of novel lasso peptides.

2.2.2 Gene Cluster Engineering and Heterologous Production

It is not uncommon to observe that natural producers produce very low amounts
of lasso peptides, even when the culture conditions have been scrutinized and opti-
mized. Large-scale fermentation was generally required to obtain enough peptides
for structure elucidation, as described in Sect. 2.2.1. The last 5 years have seen a
new era of lasso peptide discovery, which is guided by genome-mining approaches.
As lasso peptide clusters are small and amenable to manipulation, heterologous
expression is an obvious choice. Indeed, heterologous expression in E. coli was
successfully applied for lasso peptides from Proteobacteria since the discovery of
capistruin in 2008 (Knappe et al. 2008 ). This strategy circumvents the inconve-
nience of large-scale fermentation and in many cases is indispensible, as the natural
producers do not produce at all the concerned peptide under laboratory conditions
(e.g. caulosegnin III from Caulobacter segnis (Hegemann et al. 2013a )).
As the first example, Knappe et al. cloned the capistruin gene cluster ( capABCD )
into pET-41a vector in a way that the transcription of the four genes was under the
control of a T7 promoter (Knappe et al. 2008 ). A vector-borne ribosome binding
site (RBS) was in front of capA , while capBCD was transcribed from an intrinsic
RBS from B . thailandensis . The resulting construct was transformed into E. coli
BL21(DE3) for expression and led to the production of capistruin with a yield of
0.2 mg/L culture in the defined M20 medium. Link and coworkers further improved
the yield of capistruin to 1.6 mg/L culture by engineering a new construct, in which
capA was under the control of a tetracycline-inducible promoter and the transcrip-
tion terminator-containing intergenic region between capA and capBCD operon
was replaced by an optimized E . coli RBS sequence (Pan et al. 2011 ). Thus, the
modification of the intergenic region between the precursor gene and the processing
enzyme genes could be the key for successful heterologous expression. For this, ei-
ther the optimized E . coli RBS or a combination of terminator and the mcjBCD pro-