Recombinant Macrocyclic Lanthipeptides Incorporating Non-Canonical Amino Acids

Article abstract of DOI:10.1021/jacs.7b04159

Nisin is a complex lanthipeptide that has broad spectrum antibacterial activity. In efforts to broaden the structural diversity of this ribosomally synthesized lantibiotic, we now report the recombinant expression of Nisin variants that incorporate noncan

Full text of DOI:10.1021/jacs.7b04159

Communication
pubs.acs.org/JACS
Recombinant Macrocyclic Lanthipeptides Incorporating Non-
Canonical Amino Acids
Claudio Zambaldo, Xiaozhou Luo, Angad P. Mehta, and Peter G. Schultz*
The Scripps Research Institute, 10550 N Torrey Pines Road, La Jolla, California 92037, United States
S
* Supporting Information
(tRNA/aaRS) can be used to site-specifically insert ncAAs into
Nisin in bacteria.17
ABSTRACT: Nisin is a complex lanthipeptide that has
broad spectrum antibacterial activity. In efforts to broaden
the structural diversity of this ribosomally synthesized
lantibiotic, we now report the recombinant expression of
Nisin variants that incorporate noncanonical amino acids
(ncAAs) at discrete positions. This is achieved by
expressing the nisA structural gene, cyclase (nisC) and
dehydratase (nisB), together with an orthogonal nonsense
suppressor tRNA/aminoacyl-tRNA synthetase pair in
Escherichia coli. A number of ncAAs with novel chemical
reactivity were genetically incorporated into NisA,
including an α-chloroacetamide-containing ncAA that
allowed for the expression of Nisin variants with novel
macrocyclic topologies. This methodology should allow
for the exploration of lanthipeptide variants with new or
enhanced activities.
To date, two examples of ncAA incorporation into
lanthipeptides using nonsense codon suppression have been
reported by van der Donk.18,19 Kuipers also reported the
incorporation of tryptophan analogues into Nisin using an
auxotrophic strain.20 With the goal of creating Nisin variants
through biomimetic cyclization reactions (Figure 2), we built
upon the former system18 and began by recombinantly
expressing wild-type (WT) NisA in bacterial strains in which
we have previously genetically encoded a large number of
ncAAs. The nisA structural gene together with the dehydratase
(nisB) were inserted into a pRSF-1 vector to afford pRSF-NisA.
The cyclase, nisC, was inserted into a pACYC backbone to
afford pACYC-NisC. The Ser codon at position 5 of nisA was
then mutated to the amber stop codon TAG (Ser5TAG), and
the resulting plasmid (pZC1) was coexpressed, along with
pACYC-NisC, and a pULTRA plasmid harboring either a
polyspecific Methanocaldococcus jannaschii tRNATyr/TyrRS or
Methanosarcina barkeri pyrrolysine tRNAPyl/PylRS.21 We then
attempted to substitute a number of ncAAs containing diverse
side chains including keto, azide and acetylene groups for Ser5
(Figure 3A). SDS-PAGE and MS analysis (Figure S1−S3)
confirmed incorporation of pAcF, pAzF and ProcK at position
5 in yields ranging from 3 to 5 mg/L (Figure 3B). Although
promising, in our hands the expression system afforded
incomplete dehydration of Ser and Thr residues, giving rise
to a distribution of products.
To solve this issue, we capitalized on the recent finding that
the dehydratase NisB requires glutamylated tRNA (tRNAGlu) in
order to acylate the Ser/Thr side chains of NisA with glutamate
prior to dehydration.22 We reasoned that overexpressing
tRNAGlu could improve the dehydration efficiency. To test
this notion, we constructed a plasmid (pZC2) bearing
Escherichia coli tRNAGlu/GluRS downstream of a proK
constitutive promoter encoded on pACYC-NisC. Coexpression
of pZC2 with pRSF-NisA WT, showed a substantial increase in
dehydration efficiency (WT mNisA = 7299 Da), confirming
our hypothesis (Figure S4, S5).
he lanthipeptides are a subclass of a large family of
Tribosomally synthesized and post-translationally modified
macrocyclic peptides (RiPPs).1 Members that possess anti-
bacterial activity are known as lantibiotics,2 and are defined by
characteristic lanthionine or methyllantionine thioether bridges
that impart backbone rigidity. The most studied member of the
lanthipeptides, Nisin A, depicted as its prepeptide or mNisA
(Figure 1), has been used as a food preservative for the past 50
years.3 It features multiple dehydrated amino acids arising from
the activity of a dehydratase (NisB) on serine (Ser) and
threonine (Thr) residues, and five thioether cross-links (rings
A, B, C and D/E) formed by a cyclase (NisC) that catalyzes
thia-Michael reactions.4 Nisin exerts its antimicrobial activity
through its N-terminal domain that recognizes and sequesters
the pyrophosphate moiety of Lipid II, and the C-terminal
domain that inserts into the outer membrane.5,6 This dual
mode of action, directed toward nonproteinaceous bacterial
targets, has slowed the development of resistant strains.7
However, its poor aqueous solubility and pH sensitivity have
delayed its use as a human therapeutic.8
Given this optimized system, we next explored the possibility
of taking advantage of electrophilic ncAAs and the innate
reactivity of cysteine residues present in NisA, to generate Nisin
variants with altered ring structures. We began by incorporating
mildly reactive ncAAs23 in place of Ser/Thr residues at
lanthionine ring junctions, starting with the ring A Ser3TAG
To further explore the potential utility of this complex
natural product, various efforts have been undertaken to
generate variants of Nisin and other lantibiotics,9 including the
use of solid phase synthesis,10−12 biomimetic approaches,13 the
in vitro biosynthesis of lanthionine-containing peptides14,15 and
an in vitro mutasynthetic approach.16 To expand the number of
amino acids building blocks that can be biosynthetically
incorporated into lantipeptides, we now report that orthogonal
nonsense suppressor tRNA/aminoacyl-tRNA synthetase pairs
Received: April 24, 2017
© XXXX American Chemical Society
A
DOI: 10.1021/jacs.7b04159
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Journal of the American Chemical Society
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Figure 1. Wild-type mNisA. Highlighted thioether rings and key residues for this study (residues 30−34 omitted for simplicity).
Figure 2. Altered ring structures of Nisin created by ncAA incorporation.
mutant (pZC3). Fluoro-pAcF (Figure 3A) was incorporated
into NisA at residue 3 in a yield of 2 mg/L. MS analysis showed
a mass (7418 Da) corresponding to the loss of fluoride (Figure
S6). This result, although encouraging, was somewhat cryptic
due to the similar molecular weight of fluorine and water.
Because water acts as a leaving group during precursor peptide
maturation, an extra post-translational dehydration would result
in a product with a similar mass as that of the desired, cyclized
peptide. This led us to reconsider the ncAA to be used.
PAGE (Figure 4A) and mass analysis revealed cyclization of
thioether rings A, B and C, corresponding to the Ser3TAG,
Thr8TAG and Thr13TAG mutations, respectively, in yields of
1.5 to 3 mg/L (Figure 4B, C and D, Figure S7−S9). Efforts to
create intertwined thioether ring D/E (Thr23TAG and
Thr25TAG) were troublesome, giving rise to a mixture of
desired and truncated products at Lys22.
Our next goal was to further characterize the newly formed
bond in the Ser3 to 1 mutant. To simplify analysis, we decided
to generate a monocyclic Nisin. To this end, we constructed
two plasmids derived from the common vector, NisA Ser3TAG
(pZC3). In the first construct (pZC4), we sequentially mutated
all cysteine residues to Ala (Cys7, 11, 19, 26, 28Ala). The
second construct (pZC5) retained Cys7 and all the remaining
Cys were mutated to Ala (Cys11, 19, 26, 28Ala). Each
construct was individually cotransformed with the accessory
plasmids (pULTRA, pZC2), expressed in the presence of 1,
and the mature peptides were isolated. MS analysis confirmed
cyclization only for the peptide bearing reactive Cys7 (Figure
S11). In contrast, the fully mutated Cys to Ala construct
showed a MS spectrum for a peptide carrying the unreacted
chloroacetamide. Interestingly, this noncyclized peptide showed
We reasoned that a chloroacetamide ncAA24 (Figure 3A, 1)
would be the best compromise in terms of reactivity and leaving
group MW for mass analysis. Gratifyingly, when 1 was
incorporated into ring A of mNisA (Ser3TAG), MS analysis
showed a mass of 7433 Da [M/z]+1, consistent with a fully
dehydrated and cyclized peptide containing a novel ring A
thioether architecture. We next investigated whether this
approach could be extended to other ring junctions as a
general strategy toward lanthipeptides diversification. To this
end, the remaining dehydratable residues (Ser/Thr) involved in
ring cyclization were individually point mutated to TAG and
the resulting constructs cotransformed with pULTRA and
pZC2. Upon expression and isolation of the prepeptides, SDS-
B
DOI: 10.1021/jacs.7b04159
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Journal of the American Chemical Society
Communication
Figure 3. (A) ncAAs used in this study. (B) Biorthogonal chemical handles incorporated into Ser5TAG NisA.
Figure 4. (A) SDS-PAGE mNisA ring variants incorporating 1. Lane 1, ring A; lane 2, ring B; lane 3, ring C. (B−D) QTOF-ESI for mNisA ring
variants incorporating 1: (B) Ser3TAG, ring A. (C) Thr8TAG, ring B. (D) Thr13TAG, ring C. (E) 13C NMR of labeled 1 (red) and 2 (green). (F)
13C NMR mNisA Cys to Ala mutants incorporating labeled 1, noncyclized (red) and cyclized (green) peptides. Asterisks indicate GSH addition
products.
an extra dehydration event that was ablated upon Ser29 to Ala
mutation in the core peptide (Figure S10). This observation is
in accordance with the hypothesis that dehydration and
cyclization events in Nisin biosynthesis are coupled, and that
precise PTM control depends upon conformationally restricted
substrates.25,26
To obtain NMR evidence for the novel thioether linkage, we
synthesized isotopically labeled ncAA (1) bearing a 13C at the
reaction center (2-chloroacetamide position) using chloroacetyl
chloride-2-13C as a starting material. Incorporation of 13C-1
into Nisin mutants should afford chemical shift differences
between cyclized and noncyclized product. As a control
experiment, we recorded the 13C NMR spectrum of labeled
1, and then treated it with excess glutathione (GSH) to form a
thioether bond as a standard. A substantial chemical shift
difference between the starting ncAA-chloroacetamide (42.78
ppm) and the GSH-displaced product 2 (36.09 ppm) was
observed (Figure 4E). We then expressed and HPLC purified
mutants Ala7, Ala11, Ala19, Ala26, Ala28 and Cys7, Ala11,
Ala19, Ala26, Ala28 encoded on pZC4 and pZC5, respectively,
incorporating 13C-(1). The peptide lacking all cysteines showed
13C NMR peaks consistent with the presence of a
C
DOI: 10.1021/jacs.7b04159
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Communication
C.; Willey, J. M.; van der Donk, W. A. Nat. Prod. Rep. 2013, 30 (1),
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chloroacetamide (43.16 ppm) and its GSH adduct. On the
other hand, the peptide harboring reactive Cys7, showed a 13C
resonance (36.84 ppm) consistent with the standard thioether
2, confirming the macrocyclization event (Figure 4F). The
additional thioether resonance (36.30 ppm) is likely due either
to GSH addition to 1, with Cys7 cyclizing to dehydroalanine 5
(Dha5), or GSH addition to Dha5 of the expanded cyclic
peptide. Upon trypsin-mediated cleavage of the leader peptide,
Nisin analogues bearing novel thioeter linkages were tested for
antibacterial activity. Unfortunately, although a halo assay
against Micrococcus luteus indicated that this Nisin ring variants
are devoid of antibacterial activity (whereas WT Nisin retained
activity) (Figure S19), the possibility of constructing non-
natural lanthipeptides with positional precision should allow for
the interrogation of broader chemical space.
In summary, we genetically incorporated diverse ncAAs
containing either biorthogonal handles or mildly reactive
functional groups into Nisin A. NMR and tandem MS
spectroscopy confirmed the formation of a novel ring A
thioether bond for one of the mutants. We are currently
probing various ring topologies with the aim to restore
biologically active conformations. The ability to diversify
lanthipeptide macrocycles at the ribosomal stage may lead to
improvements in their pharmacological properties.
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.7b04159.
■
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Mass spectra (ESI-QTOF, LC MS/MS), NMR data,
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AUTHOR INFORMATION
Corresponding Author
*schultz@scripps.edu
■
ORCID
Xiaozhou Luo: 0000-0001-9808-6890
Peter G. Schultz: 0000-0003-3188-1202
Funding
NIH 5R01 GM062159
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Chem. Rev. 2017, 117 (8), 5457−5520.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Kristen Williams for assistance in manuscript preparation, Dr.
Laura Pasternak for NMR analysis, Dr. Michael J. Bollong and
Dr. Sean A. Reed for helpful discussions.
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D
DOI: 10.1021/jacs.7b04159
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX