From the Producer Microorganisms to the Lasso Scaffold - Lasso Peptides: Bacterial Strategies to Make and Maintain Bioactive Entangled Scaffolds - page 14

Table 2.3 Conditions for heterologous expression of lasso peptides. The order of the peptides
follows the alphabetical order inside sub-classes as defined in Table 2.1 . The type I lasso peptide
is highlighted in grey

EĂŴĞ KƉƟŴĂŮŴĞĚŜƵŴŽŶĚŜƟŽŶƐĨŽƋŽƉƟŴĂŮ

ƉƋŽĚƵĐƟŽŶ

ZŜĞŮĚ;ŴŐͬ>ĐƵŮƚƵƋĞͿ

^ǀŜĐĞƵĐŜŶ 'ZD ΡĚ͕ϮϬΣ ϬΚ

ƐƚĞDŽŜŶϬ DΕ ϬĚ͕ϮϳΣ ŶĚ

ƐƚĞDŽŜŶϮ DΕ ϬĚ͕ϮϳΣ ŶĚ

ƐƚĞDŽŜŶϮ DΕ ϬĚ͕ϮϳΣ ŶĚ

ƵƋŚŜNJŜŶ DΕ ϬĚ͕ϮϳΣ ŶĚ

ĂƵŮŽŶŽĚŜŶ/ DΕ ϮĚ͕ϮϬΣ Ϯ͘Κ

ĂƵŮŽŶŽĚŜŶ// DΕ ϮĚ͕ϮϬΣ Ϯ͘Ϯ

ĂƵŮŽŶŽĚŜŶ/// DΕ ϮĚ͕ϮϬΣ Ϭ͘Ρ

ĂƉŜƐƚƋƵŜŶ DϮϬ ϮĚ͕ϮϳΣ Ϭ͘Ϯ

ĂƵŮŽƐĞŐŶŜŶ/ DΕ ϮĚ͕ϮϬΣ Ϭ͘Ϯ

ĂƵŮŽƐĞŐŶŜŶ// DΕ ϮĚ͕ϮϬΣ Ϭ͘ϬΡ

ĂƵŮŽƐĞŐŶŜŶ/// DΕ ϮĚ͕ϮϬΣ Ϭ͘Ϭ

ZŚŽĚĂŶŽĚŜŶ DΕ ϮĚ͕ϮϬΣ ŶĚ

ZƵĎƋŜǀŜŶŽĚŜŶ DΕ ϬĚ͕ϮϳΣ Ϭ͘Ρ

^ƉŚŜŶŐŽŶŽĚŜŶ/ DΕ ϮĚ͕ϮϬΣ Ϭ͘Ε

^ƉŚŜŶŐŽŶŽĚŜŶ// DΕ ϮĚ͕ϮϬΣ ŶĚ

^ƉŚŜŶŐŽƉLJDŽŜŶ/ DΕ ϮĚ͕ϮϬΣ Ϯ͘Κ

^ƉŚŜŶŐŽƉLJDŽŜŶ// DΕ ϮĚ͕ϮϬΣ Ϭ͘Κ

^LJĂŶŽĚŜŶ/ DΕ ϮĚ͕ϮϬΣ Ρ͘Ϯ

ƵĐŜŶŽĚŜŶ DΕ ϮĚ͕ϮϬΣ ŶĚ

moter from MccJ25 system was used. The utility of the latter was demonstrated for
caulosegnin I, II and III whose production was increased 16-, 24-, 50-fold compared
to the original cluster, respectively (Hegemann et al. 2013a ). In cases of clusters
with multiple A genes, such as those of caulosegnins and caulonodins (Hegemann
et al. 2013a ; b ), single precursor constructs resulted in higher production of the
corresponding peptides. Usually, the yields of peptides from multiple precursors in
one cluster were not equal. For caulosegnin II and III, it was demonstrated that hy-
brid precursor genes with the leader peptide from caulosegnin I encoded improved
their production. Caulosegnin I showed the greatest yield among the three peptides,
which might be attributed to its longer leader sequence. E. coli culture conditions
play essential roles for expression of proteobacterial lasso peptides. Minimal media
are required and frequently used, such as M9 and M20 with vitamin supplements.
Generally, very little or no peptide production can be observed in rich LB medium
as exemplified by capistruin (Knappe et al. 2008 ). With regards to the growth tem-
perature, some peptides require 37 °C for 1 day for optimal production, while the
others prefer 20 °C for 3 days. A summary of the conditions for heterologous pro-
duction of lasso peptides is given in Table 2.3 .
Worth of note, MccJ25 is one exception of proteobacterial lasso peptides,
which is produced from the natural gene cluster in the native host with high yields
(1-5 mg/L culture). Nevertheless, Link and coworkers engineered MccJ25 gene
clusters on the pQE60 vector in an attempt to further increase MccJ25 titer (Pan
et al. 2010 ). In these constructs, mcjA was placed under the control of its own pro-
moter or a strong T5 promoter, whereas the mcjBCD operon was either behind the