Key Points

• 160 plasmids featuring an entire NPRS engineering platform, consisting of donor, acceptor and already cloned expression plasmids, allowing the production of new-to-nature non-ribosomal peptide libraries in E. coli (Fig. 2).

• 9 expression plasmids encoding 3 native NRPS clusters split on 3 plasmids each (Fig. 3, Tab. 1).

• 9 acceptor plasmids for insertion of any donor module containing negative selection mCherry cassettes for reliable Golden Gate cloning (Fig. 4, Tab. 2).

• 35 donor plasmids encoding standardized NRPS modules (Fig. 5, Tab. 3).

• 105 characterized expression plasmids allowing the expression of 42'875 hybrid NRPS by combinatorial transformation (Fig. 5, Tab. 3).

• Establishing a new Golden Gate standard for NRPS XUT^I module exchange, the NRPieceS standard.

• 2 expression plasmids with special A domains for production of peptides containing an azide-handle for chemical functionalisation (Fig. 8, Tab. 6).

• 3 compatible expression backbones  (Fig. 6, Tab 4) and 2 split intein pairs (Fig. 7, Tab 5) allowing the expression of a single fusion protein from 3 separate plasmids as well as a Golden Gate donor backbone, usable for any other project.

Design

Non-ribosomal peptide synthetases (NRPS) generate diverse bioactive compounds, including antibiotics, by assembling peptides in a modular fashion. To tap into this biosynthetic potential, E. coli was chosen for its genetic accessibility and close relation to NRPS-rich genera Photorhabdus and Xenorhabdus. Large NRPS gene clusters were split across three plasmids using the XUT^I standard, facilitating both cloning and modular swapping. Reassembly in E. coli relied on two split inteins (gp41-8 and NrdJ-1), reconstructing functional enzymes^[1]. We built a platform from three native clusters Chaiyaphumine^[2], Szentiamid^[3] and Xentrivalpeptide^[4]. We created tripartite Golden Gate acceptor plasmids for all three clusters and provided 35 NRPS XUT^I donor module, chosen through phylogenetic relation, to produce novel peptides. This modular and rational approach expands chemical diversity and accelerates new bioactive peptide discovery (Fig. 1).

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Build

NRPS native parts

All native cluster and exchange units were obtained from gDNA of the source strain via PCR while also removing BsaI restriction sites. Primers were designed to add 30 bp overhangs for Gibson assembly into the respective backbone. In a second round of Gibson cloning one module per native cluster was replaced with a mCherry cassette for negative selection in the subsequent Golden Gate cloning step.

Golden Gate Cloning

Our library is constructed as a Golden Gate system utilizing donor and acceptor vectors that can be combined interchangeably. Both the donor and acceptor vectors contain BsaI recognition sites with complementary overhangs, which result in the introduction of two amino acid "scar" residues, alanine and serine, flanking the inserted exchange unit. For characterization, we combined our 35 exchange units with the 3 Chaiyaphumine acceptor vectors to generate 105 Golden Gate plasmids. The library can be easily expanded by Golden Gate cloning the Szentiamid and Xentrivalpeptide acceptor vectors and creating 210 more expression vectors.

Parts collection

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Native Expression Plasmids

3 initiaton plasmids 3 elongation plasmids 3 termination plasmids

Golden Gate acceptor plasmids

3 initiaton plasmids 3 elongation plasmids 3 termination plasmids

Exchange units and Golden Gate expression plasmids

Alanine

Arginine

Arginine

Arginine

Asparagine

Asparagine

Aspartic acid

beta-Alanine

beta-Alanine

Diaminobutyric acid

Glutamine

Glutamic acid

Glutamic acid

Glycine

Histidine

Isoleucine

Isoleucine

Leucine

Lysine

Lysine

Lysine

Phenylalanine

Phenylalanine

Phenylalanine

Proline

Proline

Serine

Threonine

Threonine

Tryptophan

Tryptophan

Tyrosine

Tyrosine

Valine

Valine

Epimerization:

no

yes

yes

no

yes

no

no

yes

no

yes

no

yes

no

yes

yes

yes

no

yes

yes

yes

no

yes

no

no

yes

no

yes

yes

no

no

no

yes

no

yes

no

Source cluster:

Xenorhabdus khoisanae DSM 25463, Xenoamicin synthetase XUT2

Xenorhabdus mauleonii DSM 17908, Fitayylide derivat synthetase XUT6

Xenorhabdus cabanillasii JM26, Bicornitin derivat synthetase XUT6

Xenorhabdus cabanillasii JM26, Bicornitin derivat synthetase XUT3

Xenorhabdus cabanillasii JM26, Fabclavine synthetase XUT4

Xenorhabdus innexi DSM 16336, Fabclavine synthetase XUT3

Xenorhabdus khoisanae DSM 25463, unknown synthetase XUT6

Xenorhabdus nematophila ATCC 19061, Xenematide synthetase XUT4

Xenorhabdus innexi DSM 16336, Fitayylide synthetase XUT3

Photorhabdus kayaii DSM 15194, unknown synthetase XUT3

Xenorhabdus romanii DSM 17910, Intrazentin derivat synthetase XUT4

Photorhabdus temperata subs. thracensis DSM 15199, unknown synthetase XUT5

Xenorhabdus beddingii DSM 4764, unknown synthetase XUT2

Xenorhabdus szentirmaii DSM 16338, unknown synthetase XUT8

Xenorhabdus miraniensis DSM 17902, unknown synthetase XUT13

Xenorhabdus KK7.4, Xenobactin synthetase XUT5

Xenorhabdus KK7.4, Fiayylide derivat synthetase XUT6

Xenorhabdus cabanillasii JM26, Taxlllaide derivat synthetase XUT6

Photorhabdus luminescense IT4.1, unknown synthetase XUT3

Xenorhabdus hominickii DSM 17903, PAX synthetase XUT6

Xenorhabdus mauleonii DSM 17908, PAX synthetase XUT3

Xenorhabdus mauleonii DSM 17908, Protegomycin derivat synthetase XUT3

Xenorhabdus PB61.4, Chaiyaphumine synthetase XUT2

Photorhabdus luminescens subs. laumondii TT01 DSM_15139, GxpS synthetase XUT3

Xenorhabdus PB61.4, Chaiyaphumine synthetase XUT4

Xenorhabdus nematophila ATCC 19061, Xenoamicin synthetase XUT11

Xenorhabdus KK7.4, Fitayylide derivat synthetase XUT2

Photorhabdus temperata K122, unknown synthetase XUT4

Xenorhabdus KK7.4, Xenobactin synthetase XUT3

Xenorhabdus hominickii DSM 17903, Xenematide/​Taxlllaide synthetase XUT5

Xenorhabdus KJ12.1, Xenoprotide synthetase XUT3

Xenorhabdus innexi DSM 16336, Fitayylide synthetase XUT5

Xenorhabdus stockiae DSM 17904, Xenoprotide derivat synthetase XUT3

Xenorhabdus hominickii DSM 17903, unknown synthetase XUT3

Xenorhabdus KK7.4, Xentivialpeptide synthetase XUT6

Part name: BBa_25GRKS3J BBa_25JQ5Y0E BBa_2546K4QE BBa_25I5QU6P BBa_253F5MN6 BBa_25W6NAAK BBa_25CZ42LP BBa_25ODH20W BBa_25C8C5FV BBa_255Z007P BBa_25DUZZVM BBa_25L885HE BBa_25498JTE BBa_25631CKS BBa_25A33AG4 BBa_25RUHHBG BBa_25SPBXEH BBa_25L2LT9Z BBa_25QM73MD BBa_257L92YL BBa_25E3QF9W BBa_25B6CBPG BBa_25JVF1RU BBa_25P50E3H BBa_256QSWQN BBa_2544KP0W BBa_25NMTD8Y BBa_25E7OHKD BBa_255BCE4G BBa_25SPZFCP BBa_25055HQG BBa_25APRBKS BBa_25KE336Y BBa_25F2MQ35 BBa_25X9PUZ6

A

R

R

R

N

N

D

ßA

ßA

Dab

Q

E

E

G

H

I

I

L

K

K

K

F

F

F_L

P

P

S

T

T

W

W

Y

Y

V

V

2

3

4

Chaiyaphumine
Xenorhabdus sp.
PB61.4

BBa_25O5HCVX BBa_25V5G86W BBa_253DFKWX BBa_25696F6U BBa_251I5HRE BBa_25FFJF3V BBa_251SWGLU BBa_25JQ6UGB BBa_25GX3ZJ4 BBa_2585O7JE BBa_257NQ6XB BBa_25HJOUSX BBa_25M17972 BBa_25CZFJ68 BBa_25MBCAYC BBa_25ZGNUI4 BBa_252KOVW6 BBa_25XRQSSG BBa_25HPRG5E BBa_251OOHAS BBa_259MYIUM BBa_25HSSATK BBa_257TGFX2 BBa_253M7EBF BBa_252ZQMN6 BBa_25YLDO0C BBa_25HDI0R9 BBa_25B9NJO8 BBa_25RNFPM8 BBa_25LMDCTV BBa_25J3CYXN BBa_25ZGVXKU BBa_25TRMRJH BBa_25UH5WSK BBa_25U8NYFH BBa_25P3Q1BQ BBa_25NILA3H BBa_25A661VA BBa_25V2SUON BBa_25NILA3H BBa_25N1JNJU BBa_25L7FO2G BBa_25NILA3H BBa_25DVND4I BBa_25S3RX4R BBa_25D8391D BBa_25HXHLBU BBa_25LZPWWI BBa_25NBWMXC BBa_25G6XSNC BBa_25ATPF1V BBa_255IBIXB BBa_251C3KOQ BBa_25M92T4P BBa_258D3BQX BBa_256CPU7S BBa_2590HRJX BBa_25BPT7FT BBa_25JCZSGT BBa_250BLCIQ BBa_25IY3OJV BBa_25N5CBXT BBa_25TJHBMA BBa_25JQL2V1 BBa_25TBBHMJ BBa_25N8PT1Z BBa_2590ZIEO BBa_258GSVNQ BBa_2508MJ0Y BBa_25WCBOUE BBa_25U0C9DU BBa_25TFXXFO BBa_25TIP463 BBa_25P6EHSR BBa_25Y2DS8P BBa_25T4ZUXN BBa_250NPZCE BBa_25GPAMZF BBa_253IIP3G BBa_25T38H3M BBa_25AXTKU8 BBa_25RVBZA5 BBa_25DR5PII BBa_25V5EA9I BBa_257K6LLL BBa_25AUL0PL BBa_25SC77SH BBa_25XEC79C BBa_25VFHJ3A BBa_25CSBUH7 BBa_25MDJISM BBa_25RYS8II BBa_25HBVKHA BBa_25CCQHIT BBa_25IE7WWB BBa_25ADPKBO BBa_25C47GU4 BBa_25Q44PSG BBa_25FDWYMQ BBa_255GP9OC BBa_259OT3UF BBa_2571R42T BBa_25STMUQJ BBa_25MF1AUK BBa_2525JBI3

Novel
derivatives

Backbones

Split inteins

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Azide incorporation plasmids

Test

High-throughput

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To characterize the part and minimize other effects on production, the Golden Gated expression plasmids were each individually combined with the native Chaiyaphumine. This approach enables comparable results for interpreting the functioning of non-ribosomal peptide synthetases (NRPS). For efficient characterization of the exchange units, we implemented a high-throughput testing system capable of expressing over 100 hybrid NRPS simultaneously. Instead of cultivating in 10 ml flask cultures, we opted to grow the combinations in 24-well plates. They were cultured over 3 days at 25°C and 200 rpm. The peptides were extracted from the XPP3 media using XAD16-N beads, followed by elution from the beads with a methanol:acetonitrile (MeOH:ACN) mixture. These extracts were subsequently analyzed by LC-MS. The results can be found in the 'Characterizing our Parts' section in our results page and on the corresponding parts pages in the registry.

Upscaling

With only small quantities of our peptides available, applications were initially restricted to basic activity screening. As part of our goal to establish a comprehensive discovery platform, we sought to address the subsequent steps. To measure peptide production not only qualitatively but also quantitatively, reference compounds are required, which we obtained through purification from larger culture volumes for the native Chaiyaphumine (Initation BBa_25KICM3F, Elongation BBa_257KLRC3 and Termination BBa_25G3Z1O2) and the derivative Chaiyaleucine (Initation BBa_25KICM3F, Elongation BBa_257KLRC3, Termination BBa_25M92T4P).

Learn / Outlook

Correlation of production

Characterization of our exchange units with Chaiyaphumine revealed several key factors affecting the functionality of hybrid NRPS assemblies. The position of the exchange unit within the assembly line probably influences peptide production, with modules at the XUT4 position yielding functional peptides in 86% of cases, compared to 54% at XUT3 and 40% at XUT2, likely due to fewer disruptive interactions with condensation (C) domains at later positions. The type of condensation domain also might impact success rates: LCL domains supported 73% functional constructs, E-DCL domains performed similarly, while CE domains showed only 24% success. However, all CE domain clusters had low phylogenetic relatedness to Chaiyaphumine, confounding direct attribution. The Phylogenetic relation between donor and acceptor modules, assessed through combined sequence similarity of thioesterase (TE) and upstream thiolation (T) domains, also correlates strongly with production success. Donor modules sharing over 55% T-TE similarity with the Chaiyaphumine cluster yielded peptides in 39 out of 51 tested constructs, whereas those below 26% similarity succeeded in only 20 out of 54. This highlights that phylogenetic relation shows a strong correlation towards NRPS compatibility. Nonetheless, limitations arise when closely related clusters lack the desired amino acid or even a TE domain, indicating that additional predictive metrics are required for more comprehensive optimization.

Application

Our platform is designed to construct a versatile peptide library, enabling teams to screen for bioactivity and accelerate antibiotic discovery by harnessing the vast, largely unexplored chemical diversity of non-ribosomal peptides(NRPs). By making this modular system accessible, we hope that any lab can derivatize and engineer peptides for diverse applications beyond antibiotics, including anticancer agents like bleomycin^[5], immunosuppressants such as cyclosporin^[5], biopesticides (zwittermicin A^[6]), and biosurfactants (surfactin^[7]). The platform also supports hit-to-lead optimization, as illustrated by “Chaiyavaline,” where future studies could systematically substitute amino acids to pinpoint residues essential for bioactivity. While functionalizing peptides with azide groups enables click chemistry for attaching moieties like siderophores to improve uptake and pharmacological properties. Importantly, the modular NRPS parts can also be repurposed for creative new functions, as demonstrated by the 2014 RiboSURF iGEM team, who used A domains to load nonproteinogenic amino acids onto tRNAs. We invite all iGEM teams to explore new applications for our toolkit and expand the possibilities of synthetic biology together.