Biosynthetic functionalization of nonribosomal peptides

David L. Niquille, Ines B. Folger, Sophie Basler, and Donald Hilvert*

Communication

Biosynthetic Functionalization of Nonribosomal Peptides

†

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sı Supporting Information

ABSTRACT: Nonribosomal peptides (NRPs) are a therapeutically important class of secondary metabolites that are produced by

modular synthetases in assembly-line fashion. We previously showed that a single Trp-to-Ser mutation in the initial Phe-loading

adenylation domain of tyrocidine synthetase completely switches the speciﬁcity toward clickable analogues. Here we report that this

minimally invasive strategy enables eﬃcient functionalization of the bioactive NRP on the pathway level. In a reconstituted

tyrocidine synthetase, the W227S point mutation permitted selective incorporation of Phe analogues with alkyne, halogen, and

benzoyl substituents by the initiation module. The respective W2742S mutation in module 4 similarly permits eﬃcient incorporation

of these functionalized substrate analogues at position 4, expanding this strategy to elongation modules. Eﬃcient incorporation of an

alkyne handle at position 1 or 4 of tyrocidine A allowed site-selective one-step ﬂuorescent labeling of the corresponding tyrocidine

analogues by Cu(I)-catalyzed alkyne−azide cycloaddition. By combining synthetic biology with bioorthogonal chemistry, this

approach holds great potential for NRP isolation and molecular target elucidation as well as combinatorial optimization of NRP

therapeutics.

enetic code expansion with engineered aminoacyl-tRNA

synthetases (aaRSs) has enabled myriad novel chem-

Phe, was activated as eﬃciently as native L-Phe by the variant

(Table S1). Although the speciﬁc activity decreased substan-

tially in some cases, it is generally not rate-limiting in

tyrocidine biosynthesis.

istries in polypeptides, including site-speciﬁc incorporation of

bioorthogonal functional groups. These chemical handles

have since become indispensable tools for studying and

To test the incorporation of these bioorthogonal function-

alities into the antibiotic tyrocidine A (1), we reconstituted the

full nonribosomal synthetase encompassing the initiation

module TycA (123 kDa), trimodular TycB (405 kDa), and

modifying peptides and proteins. Similarly, bioorthogonal

functionality in nonribosomal peptides (NRPs) would greatly

facilitate chemical derivatization of this therapeutically

important class of secondary metabolites. Unlike ribosomal

polypeptides, however, NRPs are produced by a series of

multidomain modules that control amino acid loading,

hexamodular TycC (724 kDa) in vitro (Figure 1).₁The

corresponding genes were cloned by Gibson assembly from

the natural producer Brevibacillus parabrevis into IPTG-

inducible vectors under the control of the strong P_t_r_cpromoter.

modiﬁcation, and concatenation (Figure 1). The aaRS

equivalents in these modular assembly lines are adenylation

A) domains that activate amino acids and tether them to the

synthetase. For the biosynthesis of NRPs with bioorthogonal

groups, A domains thus need to be repurposed to process the

corresponding functionalized building blocks.

The resulting His -tagged constructs were produced in

(

Escherichia coli HM0079, which additionally harbors the

Bacillus subtilis 4′-phosphopantetheine transferase Sfp for

cofactor loading, and puriﬁed by Ni-NTA aﬃnity chromatog-

raphy (Figure S2 ). When the recombinant synthetase was

In previous work, we showed that a single Trp-to-Ser

mutation in the active site of the Phe-speciﬁc A domains of

GrsA (W239S) and TycA (W227S) switches the substrate

preference from native L-Phe to the clickable analogue O-

incubated in vitro with ATP, Mg , and all of the substrate

amino acids, HPLC analysis revealed e ﬃ cient biosynthesis of 1

−1

with a k_o_b_sof 3.4 ± 0.5 min (Figure S3) and 750 ± 50 total

turnovers. The high turnover allowed facile in vitro production

and puriﬁcation of 1 on a preparative scale. The macrocyclic

peptide was thoroughly characterized by mass spectrometry

(Figure S5 ) and NMR spectroscopy (see the Supporting

Information) and shown to exhibit the expected antibiotic

propargyl-L-Tyr. To map the potential of the enlarged binding

pocket of W227S TycA for accommodating additional

bioorthogonal functionalities, we used a pyrophosphate

exchange assay to determine the steady-state kinetics for the

adenylation of various para-substituted Phe analogues (Figure

). Beyond clickable amino acids, we tested p-bromo-L-Phe

14,15

activity.

and p-iodo-L-Phe bearing halogen substituents for palladium-

catalyzed cross-coupling reactions as well as the photoreactive

Received: January 25, 2021

Published: February 11, 2021

amino acid p-benzoyl-L-Phe for nonspeciﬁc cross-linking to

molecular targets and binding partners. For all of these

substrates, the W227S point mutation in TycA aﬀorded

substantial speciﬁcity switches, in particular for the halo-

genated analogues. Even the bulkiest substrate, p-benzoyl-L-

2021 American Chemical Society

Journal of the American Chemical Society

J. Am. Chem. Soc. 2021, 143, 2736−2740

736

Communication

tested substrate analoguesO-propargyl-L-Tyr, p-bromo-L-

Phe, p-iodo-L-Phe, and p-benzoyl-L-Phewere incorporated

at position 1 to aﬀord tyrocidine analogues 2−5, highlighting

the remarkable tolerance of the nine downstream modules

(

Figure 3A,B and Table S2 ). The propargylated derivative 2,

for example, is produced eﬃciently with a k of 1.1 ± 0.2

min (Figure S3) and 220 ± 70 total turnovers.

obs

−

Given the importance of the initial Phe residue for

macrocyclization of the linear precursor, the ability of the

terminal thioesterase (TE) domain of TycC to cyclize the

modiﬁed decapeptides is particularly notable. In no case was

the hydrolyzed linear decapeptide observed. Depending on the

substrate analogue, however, the reprogrammed synthetase

does produce some diketopiperazine (DKP) by premature

termination of the growing peptide chain as well as tyrocidines

and 6 by incorporation of L-Phe or L-Tyr instead of the Phe

derivative. Formation of the latter side products correlates with

the adenylation speciﬁcity of W227S TycA (Table S1 ) and

could be suppressed by increasing the concentration of the

functionalized substrate analogue (Figure S4).

Many Phe-activating A domains share the conserved amino

acids that line the substrate binding pocket of the TycA A

domain, including the delimiting Trp residue (Table S4).

These residues constitute a nonribosomal code that is

17,18

diagnostic for substrate speciﬁcity.

The nonribosomal

code for the Phe-speciﬁc A domain of the third TycB module,

for instance, di ﬀ ers from that of TycA at only two of eight

positions (Table S4). To test the incorporation of modiﬁed

amino acids by an elongation module, the point mutation

W2742S, analogous to W227S TycA, was introduced into the

corresponding A domain, and the reprogrammed synthetase

TycA/W2742S TycB/TycC was assembled in vitro. Upon

supplementation with amino acids, ATP, and Mg , this

synthetase eﬃciently produced functionalized tyrocidine

analogues 7−10 with O-propargyl-L-Tyr, p-bromo-L-Phe, p-

iodo-L-Phe, and p -benzoyl-L-Phe, respectively, at position 4

Figure 1. Tyrocidine synthetase assembles the macrocyclic decapep-

tide antibiotic tyrocidine A via 10 modules distributed over three

proteins, TycA, TycB, and TycC. Modules 1 (blue) and 4 (green)

activate L-Phe for incorporation into the NRP and epimerize it via the

associated epimerization (E) domain. A, T, C, and TE refer to the

adenylation, thiolation, condensation, and thioesterase domains,

respectively.

(

Figure 3A,C and Table S3 ). In contrast to the W227S TycA

synthetase, DKP formation was negligible, and only small

amounts of wild-type 1 and the corresponding Tyr-containing

variant 11 were formed, indicating more speciﬁc building block

selection. Indeed, the reprogrammed synthetase incorporated

O-propargyl-L-Tyr with a rate constant of 2.1 ± 0.2 min⁻and

560 ± 70 total turnovers, which approach the values seen for

the wild-type synthetase (TycA/TycB/TycC) with its native

substrate L-Phe (Figure S3 ). The derivatized building blocks

introduced by the reprogrammed A domain are eﬃciently

processed by the upstream C domain and all of the

downstream domains of the tyrocidine NRPS. Unlike position

1, position 4 is not important for tyrocidine cyclization,

which may explain the higher turnover and decreased

formation of side products. Modiﬁcations of Phe1 could

conceivably cause stalling at other points along the assembly

pathway, but given the tolerance of modules 4−10 to the same

modiﬁcations of Phe4, this seems unlikely.

Figure 2. Adenylation kinetics for Phe and four noncanonical

analogues determined with TycA (gray) and W227S TycA (blue)

using a pyrophosphate exchange assay. See Table S1 for details.

For detailed characterization and subsequent chemical

modiﬁcation of the bioactive core, biosynthesis was scaled up

to produce preparative amounts of pure tyrocidine analogues 2

and 7 with an alkyne handle at position 1 or 4, respectively.

Both modiﬁed peptides retained full antimicrobial activity

(Table 1). Harnessing the bioorthogonal handle, 2 and 7 were

then coupled to 6-TAMRA-azide (12) by copper(I)-catalyzed

In vitro reconstitution of tyrocidine synthetase provides a

versatile platform for testing reprogrammed A domains in the

context of a full nonribosomal peptide synthetase (NRPS)

assembly line. To assess incorporation of functionalized amino

acids at position 1 of tyrocidine A, we assembled a

reprogrammed synthetase consisting of the initiation module

W227S TycA and wild-type TycB and TycC. All four of the

9,20

alkyne−azide cycloaddtion

to give the ﬂuorescent

tyrocidine analogues 13 and 14 (Figure 4). While the bulky

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Journal of the American Chemical Society

J. Am. Chem. Soc. 2021, 143, 2736−2740

determined only once. Based on two independent measurements.

Communication

Table 1. Bioactivities of Tyrocidine A (1) and Analogues

MIC/MHC (μM)

strain

E. coli HM0079

B. subtilis JH642

A. baumannii

S. aureus

>100

erythrocytes

Minimal inhibitory concentrations (MICs) of tyrocidine A and

analogues were determined for both Gram-positive and Gram-

negative bacterial strains. Minimal hemolytic concentrations (MHCs)

were determined using human erythrocytes. Unless otherwise

indicated, three biological replicas were performed. This value was

Figure 4. Click modiﬁcation of tyrocidine. The clickable handle in

tyrocidine 2 enabled one-step modiﬁcation with 6-TAMRA-azide

(

12) by Cu(I)-catalyzed alkyne−azide cycloaddition to give 13.

Tyrocidine 7 was modiﬁed analogously to give the ﬂuorescent

derivative 14.

imparting, for example, modest antibiotic activity against

Gram-negative Escherichia coli and reduced hemolytic activity

(

Table 1). Much more drastic eﬀects could conceivably be

achieved by harnessing the biosynthetically installed handles

for combinatorial optimization of the bioactive core.

Diverse strategies for altering the recognition properties of A

21−28

domains have been reported,

including a high-throughput

platform that we recently established to enable general A-

domain engineering. The Trp-to-Ser mutation provides a

minimally invasive yet versatile strategy for repurposing Phe-

speciﬁc A domains for a variety of functionalized building

blocks. Importantly, these noncanonical amino acids are

eﬃciently processed on the pathway level to produce

functionalized, bioactive NRPs. Tailoring disparate non-

ribosomal codes for functionalized analogues will allow the

site-selective incorporation of these bioorthogonal groups at

any peptide position to integrate synthetic biology and

bioorthogonal chemistry for the isolation, visualization, and

combinatorial optimization of NRP therapeutics.

Figure 3. Nonribosomal synthesis of functionalized tyrocidines. (A)

Tyrocidine analogues produced by the reprogrammed tyrocidine

synthetases. (B) HPLC chromatograms of in vitro reactions with

W227S TycA/TycB/TycC containing all wild-type amino acids and

supplemented with (i) additional L-Phe, (ii) O-propargyl-L-Tyr, (iii)

p-bromo-, (iv) p-iodo-, or (v) p-benzoyl-L-Phe. (C) HPLC chromato-

grams of in vitro reactions with TycA/W2742S TycB/TycC

containing all wild-type amino acids and supplemented with (i)

additional L-Phe, (ii) O-propargyl-L-Tyr, (iii) p-bromo-, (iv) p-iodo-,

or (v) p-benzoyl-L-Phe. * indicates the supplemented amino acid, #

the DKP side product, and ‡ a tyrocidine analogue containing two

noncanonical amino acids. Canonical amino acids elute together in

the injection peak, which is not shown.

ASSOCIATED CONTENT

■

sı Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/jacs.1c00925.

ﬂuorophore completely abolished bioactivity when introduced

at position 4, installation of the same modiﬁcation at position 1

led to more nuanced modulation of the therapeutic properties,

Complete experimental procedures and additional

kinetic and characterization data, including Tables S1−

S4 and Figures S1−S5 (PDF)

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Journal of the American Chemical Society

Tachibana, K.; Yokoyama, S. Regioselective Carbon-Carbon Bond

Communication

Formation in Proteins with Palladium Catalysis; New Protein

Chemistry by Organometallic Chemistry. ChemBioChem 2006, 7,

AUTHOR INFORMATION

Corresponding Author

Donald Hilvert − Laboratory of Organic Chemistry, ETH

Zurich, 8093 Zurich, Switzerland; orcid.org/0000-0002-

■

10) Wang, J.; Chen, Q.; Shan, Y.; Pan, X.; Zhang, J. Activity-Based

34−139.

941-621X ; Email: hilvert@org.chem.ethz.ch

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Target Identification of Bioactive Molecules. Trends Anal. Chem.

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David L. Niquille − Laboratory of Organic Chemistry, ETH

Zurich, 8093 Zurich, Switzerland; orcid.org/0000-0002-

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Ines B. Folger − Laboratory of Organic Chemistry, ETH

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(12) Gibson, D. G.; Young, L.; Chuang, R.-Y.; Venter, J. C.;

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Sophie Basler − Laboratory of Organic Chemistry, ETH

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594-2670

Complete contact information is available at:

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†

D.L.N. and I.B.F. contributed equally.

Notes

(15) Wenzel, M.; Rautenbach, M.; Vosloo, J. A.; Siersma, T.;

Aisenbrey, C. H. M.; Zaitseva, E.; Laubscher, W. E.; van Rensburg,

W.; Behrends, J. C.; Bechinger, B.; Hamoen, L. W. The Multifaceted

Antibacterial Mechanisms of the Pioneering Peptide Antibiotics

Tyrocidine and Gramicidin S. mBio 2018, 9, No. e00802-18.

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

This work was supported by ETH Zurich and the Swiss

National Research Foundation. The authors thank Prof. M. A.

Marahiel for supplying strain HM0079 and plasmid

pSU18_tycA, Prof. J. Piel for the A. baumannii and S. aureus

strains, Dr. M.-O. Ebert and the staﬀ of the NMR Service of

the Laboratory of Organic Chemistry at ETH Zurich for

support with the NMR analyses, H.-M. Fischer for assistance

with radiochemical experiments, L. Bertschi from the Mass

Spectrometry Service of the Laboratory of Organic Chemistry

at ETH Zurich for technical support, and D. A. Hansen, A.

Camus, H. Kries, and S. Studer for helpful discussions.

(16) Trauger, J. W.; Kohli, R. M.; Mootz, H. D.; Marahiel, M. A.;

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