Use of a head-to-tail peptide cyclase to prepare hybrid RiPPs

Cyclotides and lanthipeptides are cyclic peptide natural products with promising bioengineering potential. No peptides have been isolated that contain both structural motifs defining these two families, an N-to-C cyclised backbone and lanthionine linkages. We combined their biosynthetic machineries to produce hybrid structures that possess improved activity or stability, demonstrate how the AEP-1 plant cyclase can be utilised to complete the maturation of the sactipeptide subtilosin A, and present head-to-tail cyclisation of the glycocin sublancin. These studies show the plasticity of AEP-1 and its utilisation alongside other post-translational modifications.


Materials for cloning
Oligonucleotides were obtained from Integrated DNA Technologies.Cloning was performed using restriction enzymes, Q5 polymerase (for PCR), NEBridge® Golden Gate Assembly BsaI-HF (for Golden Gate assembly), and NEB Hifi DNA Assembly (for Gibson isothermal assembly) purchased from New England Biolabs (NEB).All polymerase chain reactions were carried out on a C1000 thermal cycler (Bio-Rad).DNA sequencing was performed by ACGT, Inc. Antibiotics for culturing cells were purchased from GoldBio.Media components were purchased from Bacto TM (Thermo Fisher).The two PCR products were gel-purified and re-assembled using Gibson assembly Cloning of pRSF : His-lctA_NGL : lctM Plasmid pRSF : His-lctA : lctM was used as template for PCR using primers: lctA_NGL_R gcggccgcttaAAGACCATTagagcagcaagtaaatacaaattgcc lctA_NGL_F cttgctgctctAATGGTCTTtaagcggccgcataatgctt The PCR product was gel-purified and assembled using Gibson assembly.Cloning of pETDuet : His-SboA : AlbA and variant constructs All pETDuet backbones used in this study had the BsaI cut site removed for Golden Gate cloning.The PCR product was gel-purified and re-assembled using Golden Gate assembly to yield pETDuet : His-SboA_NGL : AlbA.This plasmid was then amplified with primers:

Plasmid constructs
The two PCR products were gel-purified and re-assembled with Golden Gate assembly to yield corresponding variant constructs.

Strains and culturing
Unless stated otherwise, E. coli NEB Turbo and E. coli BL21(DE3)-T1 R were used as hosts for cloning and protein expression, respectively.This plasmid was used as template for QuikChange mutagenesis to mutate Cys247 residue of OaAEP1 to Ala, which was shown to improve the kinetic efficiency of this enzyme. 5The resulting plasmid was called pBHRSF184 : His6_SUMO_OAAEP (C247A).This plasmid was used to transform Shuffle T7 E. coli (NEB).Single colony was cultured overnight, followed by subculturing into 2 L of LB(+kan) (37 o C, 200 rpm).When OD600 reached 0.6-0.8, the culture was cooled in an ice-water bath for 5-10 min and induced with 0.25 mM IPTG, then returned to the shaker (18 o C, 200 rpm) for 18-20 h, resulting in ~4-5 g cell pellet/L culture.

General peptide expression and purification
Buffer A 20 mM HEPES, 500 mM NaCl, 10% glycerol, pH 8.0 Buffer B 20 mM HEPES, 300 mM NaCl, 300 mM imidazole, 10% glycerol, pH 8.0 Ulp-1 20 mM HEPES, 150 mM NaCl, 0.5 mM DTT, 10% glycerol, pH 8.0 Cell pellet was harvested at 5000xg, 10 min and resuspended (5 mL/g pellet) in Buffer A + 10 mM imidazole.To ensure lysis, cells were passed three times through an Avestin Homogenizer C3 (10,000-15,000 psi) and clarified by centrifugation at 12,000xg for 30 min.The supernatant was decanted into Ni resin (2 mL resin / L culture), and slowly rotated in a cold room for 30-45 min.The resin was collected by centrifugation at 1,000xG for 5 min, then loaded to an empty gravity column (Bio-Rad).The column was washed with 4-5 CV of ATP wash buffer (Buffer A + 5 mM MgCl2, 5 mM ATP, 50 mM imidazole), followed by Wash buffer (Buffer A + 50 mM imidazole) until A280 was < 0.05.The ATP wash would release any bound chaperone protein (DnaK). 6Protein was eluted with 4 CV of Buffer B, concentrated using an Amicon 50K MWCO and the buffer was exchanged to Ulp-1 buffer using a PD-10 Column (Cytiva).The yield of protein was 5-6 mg/L culture.To the protein solution (~50 μM), His-tagged Ulp-1 protease was added to 0.2 μM final concentration and incubated at RT for 2 h to cleave off SUMO tag.After cleavage was completed, the reaction was concentrated using an Amicon 30K MWCO and the protein was further purified with Size-Exclusion Chromatography: Column Superdex 75 Increase 10/300GL SEC buffer 20 mM HEPES, 150 mM NaCl, pH 8.0 Flow rate 0.4 mL/min Zymo-OaAEP1 eluted after 10 mL.
To activate autolysis activity of OaAEP1, TCEP (0.5 mM final) and EDTA (1 mM final) was added to Zymo-OaAEP1 protein solution.Next, aliquots of 1 M aqueous NaOAc pH 3.6 was added to acidify the solution such that pH < 4.5 (this usually requires 1:10 to 1:20 volume), followed by incubation at RT overnight.After this period, there would be white flocculent which contained some precipitated proteins.The pH was brought to ~ 6-7 by addition of aliquots of 1 M HEPES pH 7.0 followed by centrifugation to remove the precipitate.
Finally, the supernatant, which contained core OaAEP1 and the cap domain, was concentrated using Amicon 10K MWCO and stored at -70 o C.

Purification of native subtilosin A
Purification of native subtilosin A was performed following a published procedure with slight modification. 7For 1-L culture scale, 25 g of Amberlite XAD16 resin (Fisher) was soaked in 10 vol of water for 30 min, followed by 10 vol of MeOH for 30 min to swell the resin.Then the resin was soaked in 10 vol of water and the water drained before use.
B. subtilis 168 was cultured overnight in LB media at 37 o C, then sub-cultured (1:100) into 1 L of LB media at 37 °C and 220 rpm until an OD600 of 0.8 was reached.Afterwards the shaking was reduced to 100 rpm and the cells were cultured for additional 24 h.Following centrifugation at 5000xg for 10 min, the culture supernatant was collected and acidified by addition of TFA to 0.5% final concentration.The supernatant was clarified again at 5000xg for 10 min, then decanted into 25 g of XAD16 resin prepared as described above.The suspension was shaken at 100 rpm and room temperature overnight.The next day, the resin was collected by applying vacuum through a 600-mL glass frit, then washed twice with 200 mL of DI water.The compound was eluted by addition of 100 mL of MeOH with intermittent swirling for 15-20 min.The elution step was repeated once.The eluent fractions were combined and evaporated with a rotavap until a brown residue remained.The residue was dissolved with 100 mL of n-butanol:water (1:1 vol).The upper organic phase was collected, evaporated with a rotovap to almost dryness (cloudy yellow suspension).The suspension was dissolved with 30-40 mL of methanol:water (1:1 vol), filtered and further purified with a RP-HPLC system.

Design strategy for generating subtilosin A using OaAEP1
N-to-C cyclized natural product peptides have a specific site of enzymatic cyclization, but after cyclization that site becomes traceless.Hence, as has been pointed out previously, 8 alternative sites of cyclization are possible by designing the cyclization substrate such that upon cyclization an identical structure is formed (circular permutation of the cyclization site).In our case, subtilosin is naturally cyclized by amide bond formation between the amino group of Asn1 and the carboxylate of Gly35 (Fig. 2, main text).Since the OaAEP enzyme recognizes the NGL sequence and cleaves after Asn, we reasoned that the subtilosin ring could also be made by cyclization of the amino group of Lys2 and the carboxylate of an Asn placed at position 36 with the removal of Asn1 (Fig. 2).
Expression and purification of SboA(NGL) and its variants Expression was done using LB media (+amp/cam) supplemented with Trace Metal mix (Teknova T1001).E. coli BL21-DE3(T1 R ) was co-transformed with pETDuet : His-SboA_NGL : AlbA and pACYC-sufABCDSE, which contained genes for assembly of Fe-S clusters.Overnight culture from single colony was sub-cultured (1:200) into LB media, and cultured at 37 o C, 220 rpm for 2-3 h until OD600 ~ 0.5-0.7.The culture was incubated on ice for 5-10 min, induced with 0.25 mM IPTG and cultured at 18 o C, 80 rpm overnight (18-20 h).Cell pellet was harvested and purified according to the protocol in General peptide expression and purification.Eluted peptide was desalted with a C8 SPE column (ThermoFisher HyperSep, 500 mg resin bed) and lyophilized to dryness.Final yield of full-length peptide was ~3 mg/L culture.

Protease digest of SboA(NGL) and its variants
• Lys-N digestion: Full-length SboA peptide was dissolved in water to 100 μM.HEPES 1 M pH 7.0 was added to 100 mM final concentration, followed by Lys-N to 1 μM final concentration.The reaction was incubated at 37 o C for 3 h.• Arg-C digestion: Full-length peptide was dissolved in water to 100 μM and sonicated for 5 min to facilitate solubilization.Then 10X buffer (500 mM Tris pH 7.6, 10 mM CaCl2) was added to 1X, followed by 1 M DTT to 10 mM final concentration.Arg-C was added to 1-2 μM.The reaction was incubated at 37 o C for 2 h.• Lys-C digestion of native subtilosin A and cyclized products from OaAEP-1 cyclization of SboA(NGL)(-N1): Native subtilosin A and the two cyclized products obtained from OaAEP-1 cyclization of SboA(NGL)(-N1) (shown in Fig. S1a) were dissolved in 50 mM HEPES, pH 7.0 to 100 μM final concentration.Lys-C was then added to 2 μM final concentration and the reaction was incubated at 37 o C for 2 h.Presence of hydrolyzed product for each peptide was checked with MALDI-TOF MS.

OaAEP1 cyclization of SboA_NGL and its variants
Core peptide was dissolved in 20 mM sodium phosphate, pH 6.0 to 50-100 μM final concentration.
OaAEP1 was added to 1 μM and the reaction was incubated at 37 o C for 1 h.

2-PCA reaction with native subtilosin A and OaAEP-1-cyclized SboA(NGL)(-N1)
2-Pyridinecarboxaldehyde (2-PCA) is known to react exclusively with an exposed N-terminus of a peptide, and not with a lysine side chain, in part because the initial imine adduct can cyclize with the amide of the residue at position 2 of the peptide chain. 9Native subtilosin A and the cyclized isomers obtained from OaAEP-1 cyclization of SboA(NGL)(-N1) on HPLC (shown in Fig. S1a) were dissolved in 20 mM sodium phosphate (pH 7.5) to 20 μM final concentration, followed by 2-pyridinecarboxaldehyde (2-PCA) to 2 mM and the reaction was incubated at 37 o C for 90 min.
The reaction was acidified by the addition of 10% formic acid to 1% final concentration, and analyzed by MALDI-TOF MS.The reaction was stopped prior to completion because transamination products of the starting material, containing an N-terminal aldehyde (M-1 Da), were observed.Such products were not observed for a standard synthetic peptide and have not been reported in other studies using 2-PCA.Apparently, the cyclization step with the amide of the second residue that makes 2-PCA selective for the N-terminal amine is slow in the context of subtilosin compared to linear peptides, which is likely caused by the constrained structure of the mature product (Figure S1e).When the cyclization step is slow, isomerization of the initially formed imine between 2-PCA and the starting peptide and hydrolysis leads to N-terminal aldehyde formation.The ion chromatogram from 10.8 to 13.6 min, which spans the elution time of all relevant species, was extracted and deconvoluted.

HPLC purification of SboA(NGL) and its variants
When enzymatic reaction was completed, reaction was diluted (1:1 volume) with MeOH, centrifuged to remove insoluble material and injected onto a RP-HPLC system using following conditions:

LC-MS/MS analysis of SboA variants
The sample was acidified by addition of 10% TFA to 1% final concentration, centrifuged to remove insoluble debris and injected to a LC-MS system.General qTOF condition was used.The buffer of the eluant fraction from Ni purification was exchanged to LahT150 buffer (20 mM HEPES pH 8.0, 0.5 mM TCEP) using an Amicon 3K MWCO, making sure the imidazole concentration was < 50 mM.LahT150 10 was added to 5 μM final concentration and the reaction was incubated at RT overnight.To quench the reaction, 10% TFA stock was added to 0.5% final concentration.The reaction was filtered and injected onto a RP-HPLC system using the following condition: The core peptide generally elutes around 20-25 min.

HPLC purification of ProcA2.8(15RGD)_NGL and ProcA2.8(16RGD)_NGL
When the enzymatic reaction was completed, the reaction was diluted (1:1 volume) with MeOH, centrifuged to remove insoluble material and injected onto a RP-HPLC system using the following conditions:

8(16RGD)_NGL binding to integrin
Binding affinity of ProcA2.8 variants for αβV3 integrin was determined via a fluorescence polarization competition assay as previously reported. 2In brief, peptide was titrated against fixed concentration of αβV3 integrin (R&D Systems) and fluorescein-c(RGDyK) (Anaspec).At equilibirum, fluorescence polarization was measured and the Ki was then determined from the IC50 value and KD value of probe and target protein.The experimental details are as followed.The obtained data was converted to anisotropy value and plotted against peptide concentration and fitted to a dose response function using Origin Pro software: Where: y = anisotropy value, A1 = minimum anisotropy, A2 = maximum anisotropy, p = Hill's coefficient (fixed at -1), x = competitor concentration.The obtained IC50 was used to calculate inhibition constant Ki using the Cheng-Prussoff equation: Where: S is probe concentration, and KD is the binding constant of probe (13 nM).

Expression and purification of LctA_NGL
Expression was done using TB media (+kan).E. coli BL21-DE3(T1 R ) was transformed with pRSF : His-lctA_NGL : lctM.Cells were cultured, harvested and purified according to the protocol in  N1).The presence of a 2-PCA adduct with peak 1 but not subtilosin A standard indicates a free N-terminal α-amine group in the former, consistent with an iso-peptide cyclized product.(e) NMR structure of subtilosin (PDB 1pxq) 13 where Asn1, Lys2 (N-terminal residue in SboA(NGL)(-N1)), and Gly35 (last residue) are shown in stick format.Figure S2.MS/MS analysis of OaAEP1-hydrolyzed product of SboA(NGL)(-N1).Residues in green were matched as dehydrated amino acids, which result from collision-induced breakage and tautomerization of the sactionine linkage. 14Assignments of b/y ions are shown in Table S1.S2 and  S3.Abbreviations: Cam = carbamidomethyl.Figure S4.Bioactivity of native sublancin ( ) and head-to-tail cyclized sublancin (peptide from panel d, Fig. S3) ( ).Bacteria were treated with peptide at the indicated concentrations, grown in LB media at 30 o C and the OD600 was measured after 6 h. 15Indicator strain: B. subtilis 168 ∆SPβ.lctA_NGL core peak 2 ( ), and its head-to-tail cyclic product ( ).Bacteria were treated with peptide at the indicated concentrations, grown in GM17 media at 30 o C and the OD600 was measured after 18 h.Indicator strain: L. lactis cremoris.

K G C A T C S I G A A C L V D G P I P D F E I A G A T G L F G L W G N
Solvent A: Water + 0.1% TFA B: MeCN + 0.1% TFA Column MacNagel® 5 µm C18 100 Å, LC Column 250 x 10 mm Gradient 4 mL/min 0-5 min: 10-40% B 5-25 min: 40-60% B 25-30 min: 60-80% B The collected fractions were checked with MALDI-TOF MS.Under this condition, subtilosin A elutes around 27 min.The final yield was ~ 0.6 mg per L culture.

Figure
Figure S1.(a) Extracted ion chromatograms of native subtilosin A standard and the products of the OaAEP1 reaction with SboA(NGL)(-N1).(b) MS-MS analysis of native subtilosin A and the two products produced by OaAEP1.Neither product nor the authentic standard resulted in any fragment ions consistent with head-to-tail cyclization.As shown in Fig. S2, the linear analog readily fragments.(c) MALDI-TOF MS analysis of Lys-C digestions of native subtilosin A standard and the products of the OaAEP1 reaction with SboA(NGL)(-N1).Only native subtilosin A and the OaAEP1 peak 2 product were hydrolyzed (M+18), consistent with cleavage of an amide bond inside the macrocycle.Peak 1 peptide was not hydrolyzed by endoproteinase LysC, consistent with the side chain of Lys1 being involved in amide bond formation by OaAEP1.(d) HR-ESI analysis of 2-PCA reaction with native subtilosin A and cyclization isomers from OaAEP-1 cyclization of SboA(NGL)(-N1).The presence of a 2-PCA adduct with peak 1 but not subtilosin A standard indicates a free N-terminal α-amine group in the former, consistent with an iso-peptide cyclized product.(e) NMR structure of subtilosin (PDB 1pxq)13 where Asn1, Lys2 (N-terminal residue in SboA(NGL)(-N1)), and Gly35 (last residue) are shown in stick format.

Figure
Figure S3.MALDI-TOF MS analysis of (a) His-SUMO-tev-SunAcore_NGL co-expressed in E. coli with the S-glycosyl transferase SunS, 12 and treated with TEV protease in vitro to remove the SUMO tag; (b) TCEP-reduced, and iodoacetamide (IAA) alkylated peptide from panel a; (c) OaAEP1 treated peptide from panel a resulting in loss of the C-terminal Gly-Leu motif and an additional water from N-to-C cyclization; and (d) TCEP-reduced and IAA-alkylated product of the cyclic peptide resulting from (c).The square brackets connecting Cys residues in the chemical structures represent disulfide bonds.(e) ESI-MS/MS analysis of peptide in panel b; (f) ESI MS/MS analysis of the peptide in panel d.For the cyclized sample, the sugar moiety was labile under our condition for collision induced dissociation.Assignments of b/y ions are shown in TablesS2 and S3.Abbreviations: Cam = carbamidomethyl.

Figure
Figure S5.ESI-MS/MS analysis of (a) c15RGD and (b) c16RGD.Since the peptides were headto-tail cyclized, the MS/MS spectra did not show characteristic b/y ions under similar CID condition that readily fragmented the non-cyclized variants (see Fig.S6).The -44 Da species are likely decarboxylated products of the parent ions.

Figure
Figure S8.(a,c) HPLC-purified LctA after co-expression with LctM and cleavage by trypsin.(b,d) OaAEP1 treatment of the core peptides.(e) Bioactivity of native lacticin 481 ( ), non-cyclized B. subtilis 168 was cultured in 5 mL of LB media, followed by gDNA extraction with PureLink™ Genomic DNA Mini Kit (ThermoFisher).The gDNA was used as template for PCR reactions with primers: For MS/MS analysis, the qTOF was run in targeted mode, fragmenting [M+3H] 3+ precursor ions using collision voltage of 20-25 V.

Table S1 .
Assigned b/y ions for Figure S2.