From the Producer Microorganisms to the Lasso Scaffold - Lasso Peptides: Bacterial Strategies to Make and Maintain Bioactive Entangled Scaffolds - page 15

natural or an arabinose-induced promoter. None of these constructs resulted in sig-
nificant improvement, indicating that Mcc25 natural cluster is already well-tuned
for optimal production. However, the engineered Mcc25 gene cluster allowed facile
cloning to generate variant libraries, and served as a robust platform for MccJ25
engineering in the follow-up studies (Pan and Link 2011 ).
Heterologous expression of lasso peptides from Actinobacteria in E . coli is not
an appropriate choice, as work in our lab showed little success of such strategy
(unpublished data). It is likely due to the differences of codon usage and gene regu-
lation mechanisms between the natural and heterologous host. A more suitable host
that is closer to the production strain should be considered. Our laboratory produced
successfully sviceucin from Streptomyces sviceus in Streptomyces coelicolor for
NMR study (Ducasse et al. 2012a ). However, this strategy did not bring success
for all actinobacterial lasso peptides studied in our laboratory (unpublished data).
It thus appears to us that deciphering the regulation mechanism is the key to access
lasso peptide production. Screening various antibacterial hosts and genetic engi-
neering of the cluster may be required for successful heterologous expression.
Other ways to improve or facilitate production of lasso peptides, which can be
used in addition to heterologous production or independently, are the use of immo-
bilization techniques of the bacterial strains and optimized continuous production
conditions (Scannell et al. 2000 ), or the exploitation of regulation mechanisms in
the native hosts similar strategy being applied for bacteriocins from gram-positive
bacteria (Diep et al. 1995 , 1996 ; Kuipers et al. 1995 ). These approaches are cur-
rently underway in our laboratory.

2.2.3 Purification Procedures

The first lasso peptides to be characterized were isolated from fermentation broths
of wild-type strains of Actinobacteria or Proteobacteria. Since the use of genome
mining approaches, heterologous expression in E. coli for proteobacterial and in S.
coelicolor for actinobacterial lasso peptides, became the essential mode of produc-
tion of the recently isolated representatives. The purification process starts with
centrifugation to separate cell pellets and culture supernatants. Lasso peptides
produced by natural producers from Proteobacteria, such as MccJ25 or capistruin
(Blond et al. 1999 ; Knappe et al. 2008 ), are most often isolated from supernatants
due to their export in the culture media. When expressed heterologously in E. coli
under controlled conditions, they are detected and isolated from cell pellets (e.g.
astexins, caulosegnins, caulonodins, caulosegnins, sphingopyxins etc.) (Hegemann
et al. 2013a , b ; Zimmermann et al. 2013 ). Lasso peptides from Actinobacteria are
mainly isolated from mycelia. Being generally hydrophobic in nature (Table 2.4 ),
lasso peptides from the supernatants are first submitted to solid phase extraction
from the aqueous phase using C8 or C18 reversed phase cartridges. A size exclusion
chromatography step is often introduced, mainly using Sephadex LH20. A second
purification step is applied using high performance liquid chromatography (HPLC)
on C18 reversed-phase columns, in a preparative or semi-preparative scale in order