Flexizyme-Enabled Benchtop Biosynthesis of Thiopeptides

Article abstract of DOI:10.1021/jacs.8b11521

Thiopeptides are natural antibiotics that are fashioned from short peptides by multiple layers of post-translational modification. Their biosynthesis, in particular the pyridine synthases that form the macrocyclic antibiotic core, has attracted intensive research but is complicated by the challenges of reconstituting multiple-pathway enzymes. By combining select RiPP enzymes with cell free expression and flexizyme-based codon reprogramming, we have developed a benchtop biosynthesis of thiopeptide scaffolds. This strategy side-steps several challenges related to the investigation of thiopeptide enzymes and allows access to analytical quantities of new thiopeptide analogs. We further demonstrate that this strategy can be used to validate the activity of new pyridine synthases without the need to reconstitute the cognate prior pathway enzymes.

Full text of DOI:10.1021/jacs.8b11521

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Communication
Flexizyme-enabled benchtop biosynthesis of thiopeptides.
Steven R. Fleming, Tessa E. Bartges, Alexander A. Vinogradov, Christine L.
Kirkpatrick, Yuki Goto, Hiroaki Suga, Leslie M. Hicks, and Albert A. Bowers
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b11521 • Publication Date (Web): 02 Jan 2019
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Flexizyme-enabled benchtop biosynthesis of thiopeptides.
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Steven R. Fleming, Tessa E. Bartges, Alexander A. Vinogradov, Christine L. Kirkpatrick, Yuki
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Goto, Hiroaki Suga, Leslie M. Hicks, and Albert A. Bowers
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Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, University of North Caro-
lina at Chapel Hill, Chapel Hill, North Carolina 27599, United States.
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Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States.
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Department of Chemistry, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan.
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JST, PRESTO, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
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JST, CREST, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan.
Supporting Information Placeholder
ABSTRACT: Thiopeptides are natural antibiotics that are
fashioned from short peptides by multiple layers of post-
translational modification. Their biosynthesis, in particular
the pyridine synthases that form the macrocyclic antibiotic
core, has attracted intensive research but is complicated by
the challenges of reconstituting multiple pathway enzymes.
By combining select RiPP enzymes with cell free expression
and Flexizyme-based codon reprogramming, we have devel-
oped a benchtop biosynthesis of thiopeptide scaffolds. This
strategy side-steps several challenges related to the investi-
gation of thiopeptide enzymes and allows access to analyti-
cal quantities of new thiopeptide analogs. We further
demonstrate that this strategy can be used to validate the ac-
tivity of new pyridine synthases without the need to recon-
stitute the cognate prior pathway enzymes.
Ribosomally synthesized and post-translationally mod-
ified peptides (RiPPs) are a growing family of peptide-de-
rived natural products that exhibit natural combinatorial bi-
osynthetic logic.1 RiPP biosyntheses initiate from a gene-
encoded precursor peptide, which contains a core region that
Figure 1. Flexizyme-enabled benchtop biosynthesis of thi-
undergoes enzymatic post-translational modification, and a
opeptide scaffolds. a) Biosynthetic gene clusters of thiocil-
leader region, which is typically responsible for recruiting
lins, thiomuracin GZ, and aesturamide. Genes for key en-
and coordinating these enzymes through specific recognition
zymes used in this work are highlighted with asterisks. b)
sequences (RSs). These RSs have affinity for select domains
Proposed hybrid route to the thiocillin core.
of RiPP biosynthetic enzymes, increasing substrate local
concentration to the otherwise promiscuous enzyme active
sites, and allowing the modification of diverse cores.2,3 Nat-
Thiopeptides are one of the most extensive natural ex-
amples of this combinatorial, RS-directed biosynthesis,9,10
ural pathways exhibit leader peptides with multiple RSs and
recruit whole suites of post-translational modifying enzymes
to convert precursor peptides into mature natural products.
The combination of RS-programmable recruitment and pro-
miscuous enzymes inspires recent efforts at repurposing this
strategy to scaffold new-to-nature hybrid biosynthetic path-
ways. 4-8
and the three class-defining transformations include the for-
mation of azoles, dehydroamino acids, and pyridines from
serine and cysteine residues (Figure 1a). Many of these en-
zymes are remarkably promiscuous and thiopeptide path-
ways have proven capable of generating many variants.11,12
For example, hundreds of mutants of thiocillin, GE37468,
and thiostrepton have been generated by gene replacement
strategies.13-15 However, competition between the pathway
enzymes for functional groups on non-native substrates can
give rise to complex mixtures of products and slower
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processing of mutant substrates or host toxicity can restrict
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positions Ser1 and Ser10 of the thiocillin core. For ease, in-
itial DNA templates were prepared by cloning into plasmid
pMCSG7 which necessarily incorporated an N-terminal se-
quence tag leading to the design of our test substrate (Figure
2a), and transcription-translation reactions were prepared on
2.5 µL analytical scale using NEB PURExpress. 26,27 In the
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production of potential compounds. The in vitro reconstitu-
tion of whole thiopeptide biosynthetic pathways, which has
recently been achieved for thiomuracin, can circumvent
some of these problems, but relies on access to soluble, well-
behaved proteins.16,17 This can be especially challenging
for the tRNA dependent Lantibiotic-type dehydratases. Ad-
ditionally, this strategy still does not overcome enzyme com-
petition. Alternative strategies, such as a chemoenzymatic
approach, could allow rapid access to novel structural vari-
ants and ease characterization of new thiopeptides and thio-
peptide-associated enzymes.18
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We envisioned that carefully chosen RiPP enzymes
might be combined with orthogonal chemical handles to cre-
ate a flexible in vitro platform for the benchtop preparation
of thiopeptides (Figure 1b).19,20 More specifically, in vitro
transcription-translation could be used to express designer
hybrid leader peptide substrates displaying RSs for the cy-
clodehydratase LynD from aesturamide biosynthesis and
pyridine synthase, TclM from thiocillin biosynthesis. Both
enzymes have well-defined RS motifs and broad substrate
promiscuity. Additionally, LynD exhibits excellent selectiv-
ity for Cys conversion to thiazolines while ignoring Ser/Thr
residues.21,22 Alternatively, if oxazoles are of interest, then
PatD, which acts on Ser/Thr/Cys, could be used in place of
LynD. 20 Oxidation of thiazolines to thiazoles might be ef-
fected by the azoline-oxidase, TbtE from thiomuracin bio-
synthesis, which acts in a leader peptide independent man-
ner.16 In place of Lantibiotic dehydratases, we would use
robust Flexizyme technology to introduce the unnatural
amino acid Se-phenylselenocysteine (SecPh), which under-
goes oxidative elimination with H
2
2
O to generate dehydroa-
lanines (Dhas) for the pyridine-forming cycloaddition.23-25
Flexizymes are aptamers developed to condense a wide ar-
ray of amino acid esters with tRNAs of choice (see Supple-
mentary Fig. 6) , allowing codon reprograming in in vitro
transcription/translation systems.26,27 In total, this would
cut the number of enzymes or proteins necessary to prepare
a thiopeptide in vitro from, six, in case of thiocillin
Figure 2. Flexizyme-enabled benchtop biosynthesis of a
thiocillin scaffold. a) Sequence of the designer precursor
peptide, including a pMCSG7-derived sequence, the LynD
RS and TclM RS. The Trp-codon was reprogrammed to in-
corporate SecPh. EICs for SecPh and hexathiazole precur-
sor peptide after LynD/TbtE treatment (b), hexathiazole and
Dha-containing product after oxidative elimination (c), and
fully cyclized thiocillin core (d).
(TbtIJKLMN), to three (LynD, TbtE, and TclM, Figure 1).
Although quantities of peptide made by in vitro transcrip-
tion-translation and Flexizyme reprogramming are small rel-
ative to other technologies, such as amber codon suppres-
sion/in cell expression or solid phase peptide synthesis, the
approach is rapid (~2 hr.), robust, and flexible for peptides
of this size and complexity. Thus, we anticipate that this
strategy might ultimately enable rapid characterization of
new pyridine synthases and associated enzymes and aid elu-
cidation of new thiopeptides.
In order to validate this strategy, we first had to confirm
that existing Flexizymes could incorporate SecPh into in
vitro translated peptides and that LynD and TbtE could ac-
commodate SecPh at cysteine-adjacent positions. We con-
firmed that the dinitrobenzyl (Dnb) ester-specific Flexizyme
dFx could ligate the Dnb ester of SecPh by means of an in
event, LynD was able to convert all six cysteines in several
test substrates into thiazolines, which were further processed
to thiazoles by TbtE as confirmed by high resolution liquid
chromatography and mass spectrometry (HR LC/MS) (Fig-
ure 2b and Supporting Information). Subsequent treatment
with 1 M H
Dhas (Figure 2c). In the last proof-of-concept step, the ex-
cess H could be quenched by addition of tris(2-carboxy-
2
O
2
efficiently converted both SecPh residues to
2
O
2
vitro microhelix assay (see Supplementary Figure S2).25
Hybrid substrates were prepared by codon reprogramming
the tryptophan codon – although Flexizyme allows a number
of codons to be reprogrammed – due to the scarcity of Trp
residues in thiopeptides, using the AsnE2 tRNA body.28 The
ethyl)phosphine hydrochloride (TCEP) and TclM added in
situ provided complete conversion of the linear substrate
into a cyclic thiopeptide (Figure 2d), confirming that TclM
is compatible with this designed leader strategy.
We next began to explore the requirements of the
designer leader peptide.
The N-termini of natural
loaded AsnE2trp-tRNA was used to incorporate SecPh at
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thiopeptide leader peptides have been implicated in an affin-
RS sequence N-terminal to the complete native TclE leader
peptide was made and subsequently processed to the mature
thiopeptide (Table 1A, entry 10). This last result suggests a
potentially broadly applicable strategy for circumventing re-
constitution of all pathway enzymes in thiopeptide for-
mation: express the full leader as a C-terminal fusion to
LynD RS.
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ity-enhancing interaction with pyridine synthase enzymes.29
Although our studies have shown this interaction dispensa-
ble for pyridine synthase processing, 22 we chose to test the
potential impact of changes to the N-terminus on processing
of designer leader peptide substrates. Thus, we prepared two
new hybrid substrates in which we replaced the original
pMCSG7-derived N-terminus with two different excerpts
from the N-terminus of TclE, the native precursor peptide
for TclM (Table 1A, entries 3 and 4). In a third substrate we
truncated the leader peptide leaving only a short MSSQ tag
before the LynD RS (Table 1A, entry 1). The relative impact
of these changes was assessed by integration of extracted ion
chromatograms (EICs) for the product (Table 1A, entry 2;
Figure 2c,d). Interestingly, the TclE fragment sequences
We next examined allowable changes to the core se-
quence. The TclM-containing thiocillin pathway has been
shown to tolerate a wide array of changes to the core peptide
in vivo; we therefore focused on changes to the core that
were unproductive in those studies (Table 1B, entries 4 and
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).13,30 For example, Val6 of native thiocillin had been re-
5
calcitrant to charged or hydrophilic residues, such as lysine
or aspartic acid. This limitation is a barrier to antibiotic de-
velopment, as Val6 appears to be an ideal position for mod-
ulating the solubility.31 In contrast, hybrid substrates bear-
Table 1. Results of flexizyme-enabled benchtop biosynthe-
a
b
sis with mutant leader peptides (1A) and Cores (1B).
ing a V6D or V6K mutation were readily transformed to the
cyclic thiopeptide in vitro. Additionally, the LynD/TbtE
pair in vitro provided greater product control relative to the
native thiocillin enzymes, TclI, TclJ, and TclN, as exempli-
fied by a C7S mutant (Table 1B, entry 6). In the in vivo
system, a similar mutant gave mixtures of different modifi-
cations and apparent misprocessing. LynD, however, mod-
ifies all cysteines indiscriminately and left the newly intro-
duced serine untouched. We focused considerably more mu-
tagenesis on the C-terminus of the core, because studies have
suggested TclM might be sensitive to C-terminal modifica-
tions, 22,32 and such modifications would be necessary for
linking the current hybrid substrates with mRNA display in
the future (Table 1B, entries 7-13). Deletion of even one
amino acid from the C-terminus was unfavorable for TclM
and/or LynD processing (Table 1B, entries 7-10). In con-
trast, extending the C-terminus (Table 1B, entry 13) did not
significantly impact enzymatic processing. These data sug-
gest that the hybrid strategy is amenable to C-terminal ex-
tensions and new sequences, not previously accessible by in
vivo engineering approaches, although, more extensive in-
vestigations will be needed to understand the limitations.
We last sought to test whether this strategy could be
used to reconstitute new pyridine synthases. Recent work
has suggested that only a fraction of genetically-encoded thi-
opeptides have been isolated.33 Of the >500 predicted thio-
peptide gene clusters, the largest family is comprised of
members that contain a close homolog of LazC, the pre-
dicted pyridine synthase from lactazole biosynthesis.34 Ad-
a
b
EIC area relative to entry 2 as a standard. Checks indicate
a detected EIC. An “X” indicates no EIC detected above the
noise threshold of 1.0E2.
ditionally, while LazC homology is high in this family, the
core peptide diversity is broad, suggesting LazC and its
homologs may exhibit unique substrate promiscuity (see
Supplementary Fig. 5). Despite the preponderance of pre-
dicted LazC homologs, LazC has not yet been reconstituted
in vitro. Therefore, we expressed and purified LazC as its
MBP-fusion and designed three new hybrid sequences as po-
tential substrates (Figure 3b). In one LazC substrate, we in-
tegrated LynD RS directly into the native lactazole leader at
a site with apparent natural homology (termed LacHyb1,
Figure 3a), while in the other two, LynD RS was encoded 15
or 25 residues N-terminal to the core (termed LacHyb2 and
decrease thiopeptide formation and seem to negatively im-
pact LynD processing. Moreover, removal of the pMCSG7-
derived sequence greatly reduces processing by LynD/TbtE
pair. Taken together, these results further confirm that the
N-terminus of TclE is dispensable for TclM processing but
LynD may be sensitive to the location of its cognate RS
within the larger peptide context. To further probe the latter
aspect, we designed a series of leader sequences, in which
spacers were introduced between the LynD RS and TclM RS
(Table 1A, entries 5-9).20 In almost all cases, the substrates
were converted to thiopeptides, suggesting that LynD is
broadly tolerant of diverse sequence space between the RS
and core, although at reduced efficiency. As an extreme ex-
ample of this spacing promiscuity, a substrate with the LynD
3, respectively). Additionally, Trp2 and oxazole-forming
Ser11 were replaced with a serine and thiazole-forming cys-
teine, respectively (see Supplementary Figure 3). Both mu-
tations were previously produced by a gene-replacement
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strategy suggesting that the double mutant may also be a
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In summary, we have developed a new, facile strategy
to access thiopeptide backbones. This approach combines
robust, Flexizyme-assisted incorporation of chemical han-
dles into in vitro transcribed/translated peptides with three
unrelated RiPP enzymes LynD, TbtE, and TclM by using
designer leader peptides. We demonstrate the ability to make
thiocillin variants previously unattainable through natural
biosynthetic processes and use this strategy to reconstitute
the pyridine synthase LazC to make lactazoles for the first
time in vitro. We anticipate this approach will be useful in
making new thiopeptide variants with therapeutic potential,
studying more pyridine synthases and associated enzymes,
and may aid elucidation of new thiopeptide structures. Fi-
nally, we anticipate that this strategy will be compatible with
high throughput screening techniques, such as mRNA dis-
play, which is a current focus in our lab.
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LazC substrate and render the core compatible with Trp-re-
programed SecPh incorporation. Upon treatment with
LynD/TbtE, the four cysteines in each substrate readily un-
derwent conversion to thiazoles and subsequent H
2
2
O oxida-
tion introduced the four Dhas (Figure 3c). Lastly, treatment
with LazC efficiently yielded the new, pyridine-containing
masses, thus confirming activity of LazC as a pyridine syn-
thase (Figure 3d). This result was consistent for all three hy-
brid lactazole leader peptides (Supplementary Fig. 4, traces
S22-24 and S42-44) and suggests that the hybrid in vitro
strategy should work on many other, yet uncharacterized
pyridine synthases and could ultimately allow elucidation of
new thiopeptides.
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ASSOCIATED CONTENT
Experimental details, synthetic schemes, figures available at
http:// pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
abower2@email.unc.edu
ACKNOWLEDGMENT
We thank C. Neumann, B. Li, and H. Onaka for informative
discussions. SRF is recipient of a joint NSF/JSPS EAPSI fel-
lowship.
This work was supported by NIH Grant
GM125005 (AAB).
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