High-Throughput Engineering of Nonribosomal Extension Modules

Nonribosomal peptides constitute an important class of natural products that display a wide range of bioactivities. They are biosynthesized by large assembly lines called nonribosomal peptide synthetases (NRPSs). Engineering NRPS modules represents an attractive strategy for generating customized synthetases for the production of peptide variants with improved properties. Here, we explored the yeast display of NRPS elongation and termination modules as a high-throughput screening platform for assaying adenylation domain activity and altering substrate specificity. Depending on the module, display of A–T bidomains or C–A–T tridomains, which also include an upstream condensation domain, proved to be most effective. Reprograming a tyrocidine synthetase elongation module to accept 4-propargyloxy-phenylalanine, a noncanonical amino acid that is not activated by the native protein, illustrates the utility of this approach for altering NRPS specificity at internal sites.


Materials and methods
All chemicals were purchased from Sigma Aldrich and enzymes from NEB unless otherwise stated.Primers (Table S1) were synthesized by Microsynth AG (Switzerland).Propargyl glycine was obtained from Bachem, whereas 4-propargyloxy phenylalanine was synthesized as described by Kries et al. 1 Media and buffer components, kits, and enzymes were used as received from specified commercial suppliers.Expression media and buffers were prepared using purified H2O (Nanopure system, Barnstead).

Molecular cloning
The plasmids pSU18His-TycA and pTrc99a-TycB1, which respectively encode the Cterminally His6-tagged TycA protein and the first module of TycB, were obtained from Grünewald et al. 2 Detailed descriptions of the construction of plasmids pSU18His-TycA, pSU18His-TycApPhe, pTrc99a-TycB1SrfTEP26G, pTrc99a-TycB, pTrc99a-TycC, and pMG211-Sfp were reported previously. 1,3,4Plasmid pCT302 5 was used for all yeast experiments but a NheI restriction site was replaced with an NdeI restriction site; the XhoI restriction site was also moved upstream of the c-myc tag, and all other XhoI sites were removed. 6The cloning protocol for generating pCTRB-TycA A-T and pCTRB-W227S TycA A-T, which contains the W227S mutation, was previously described. 3Rs were conducted using Phusion HF Polymerase (NEB) and GC buffer according to the supplier's protocol (10 µL GC buffer 50-100 ng DNA template, 0.5 µM primer, 0.2 mM dNTPs and 0.5 µL Phusion Polymerase in 50 µL total volume).PCR products were purified by 1% agarose gel electrophoresis and isolated using the Zymoclean TM Gel DNA Recovery Kit (Zymo Research).The resulting PCR products were digested with the appropriate restriction enzymes in CutSmart Buffer at 37 °C for 1 h followed by a purification step using DNA Clean & Concentrator-5 Kit (Zymo Research).The restriction digest of the vector backbone (pSU18-His or pTr99a or pCTRB) vectors was conducted as described for the inserts but the incubation time was increased to 4 h.The vector backbone was also gel-purified and extracted with the Zymoclean TM Gel DNA Recovery Kit (Zymo Research).The ligation of digested PCR products and vector backbone (at a 6:1 ratio, 20-60 ng vector backbone) was performed at room temperature for 15 min using T4 DNA ligase.The ligation product was purified using the DNA Clean & Concentrator-5 Kit, and the plasmid was transformed into electrocompetent E. coli HM0079 cells.0.8 mL SOC medium (0.5% yeast extract, 2% tryptone, 10 mM NaCl, 2.5 mM MgCl2, 10 mM MgSO4, 20 mM glucose) was immediately added to the cells.The transformed cells were grown at 37 °C, 230 rpm for 1 h. 100 µL of the cell solution was plated on LB agar plates supplemented with the appropriate antibiotic (sodium ampicillin for pTrc99a and chloramphenicol for pSU18-His).5 mL LB pre-cultures containing sodium ampicillin (100 ug/mL) or chloramphenicol (30 µg/mL) were inoculated with a single E. coli colony and incubated at 37 °C, 230 rpm overnight.A 1.5 mL culture was centrifuged at 21,000 x g.Plasmids were extracted using the ZR Plasmid Miniprep Pure kit (Zymo Research) and sequenced at Microsynth AG (Balgach Switzerland).

TycB variants
pCTRB-TycB2 A-T: To generate the plasmid for yeast surface display of the A and T domains of the second module of TycB, two PCR fragments were generated using the primer pairs H32/P43, P4n/P6n and the template pTrc99a-TycB, respectively (Table S1).
TycB2 gene library: To create space to accommodate pPhe, residues L1704, M1768, and M1803 in the active site of the TycB2 A domain were randomized using NNK codons.Initially, five fragments were generated using pTRc99a-TycB as a template and the following primer pairs: H32/P43, P4n/P45, P44/P49, P48/P51 and P50/P6n.In a second step, the first three fragments were assembled using primers H32 and P49 and the last two fragments using primers P48 and P6n.In a third step, the two assembled products were connected by overlap PCR using H32 and P6n (Table S1).The PCR product was then transformed into freshly prepared electrocompetent yeast cells together with the pCTRB vector backbone as described below.
pCTRB-M1768A/M1803I-TycB2 A-T: this plasmid was isolated from yeast after sorting the TycB2 library (see Sequencing plasmids from homologous recombination or library clones) pTrc99a-M1768A/M1803I-TycB: To introduce the mutations that confer pPhe specificity into the full-length TycB gene, two fragments were produced by PCR using the primer pairs G112/G79 and G80/G114 and pCTRB-M1768A/M1803I-TycB2 AT and pTrc99a-TycB as templates, respectively (Table 5).The two fragments were subsequently assembled by PCR.The purified and assembled PCR product was digested with XhoI and SacI and incubated for 1 h at 37 °C.The digested insert was purified using the Clean and Concentrator-5 kit (Zymo Research).The purified insert and the pTrc99a vector backbone were ligated using T4 DNA ligase in a ratio of 1:6 at 16 °C for 16 h.The ligated plasmid was purified using the Clean and Concentrator-5 kit (Zymo Research) and transformed by electroporation into electrocompetent HM0079 cells.The cells were resuspended in 800 µL SOC medium which contains 0.5% yeast extract, 2% tryptone, 10 mM NaCl, 2.5 mM MgCl2, 10 mM MgSO4, 20 mM glucose.The suspension was grown for 1 h at 37 °C and plated onto LB-agar plates containing 100 µg/mL ampicillin.The plates were incubated at 37 °C for 16 h.3 mL LB Miller medium were inoculated with a single colony and incubated at 37 °C, 230 rpm for 16 h.The plasmid was isolated using the ZymoPURE TM Plasmid Miniprep Kit and sequenced by Microsynth AG (Switzerland).A 20% (w/v) glycerol stock of the culture containing the plasmid was prepared and stored at -80 °C.

TycC variants
pCTRB-TycC3 A-T: To generate the plasmid for yeast surface display of the A and T domains of the third module of TycC, two PCR fragments were generated using the primer pairs H32/P43 and P16n/P18n and the templates pCTRB-TycA A-T and pTrc99a-TycC, respectively (Table S1).

Production of tyrocidine synthetase proteins in E. coli
To produce tyrocidine synthetase, the procedure reported by Niquille and Folger et al. 4 was adopted with minor adaptations.The constituent proteins of the synthetase were produced with an N-terminal (TycA) or C-terminal (TycB variants and TycC) His6-tag in E. coli strain HM0079. 2 The cell cultures were supplemented with chloramphenicol (30 μg/mL, PanReac AppliChem) for TycA production or with ampicillin (100 μg/mL, PanReac AppliChem) for TycB and TycC production.Briefly, 5 mL LB Miller broth containing the appropriate antibiotic were inoculated with a single HM0079 colony transformed with the desired plasmid (pSU18His-TycA, pTrc99a-TycB, pTrc99-M1786A/M1803I-TycB or pTrc99a-TycC) and incubated overnight at 37 °C, 230 rpm.The culture was prepared in a 2 L baffled flask, containing 800 mL modified Studier medium. 10The modified Studier medium is based on LB Miller broth to which MgSO4 (2 mM), glycerol (Acros, 1% (m/v)), Na2HPO4 (25 mM), KH2PO4 (FisherBio, 25 mM), NH4Cl (50 mM), and Na2SO4 (5 mM) were added.The medium was additionally supplemented with the appropriate antibiotic, inoculated at a 1:500 ratio with the pre-culture and incubated at 37 °C, 190 rpm for 4-6 h until an OD600 of around 2 was reached.At this point, protein expression was induced by adding isopropyl β-d-1thiogalactopyranoside (PanReac AppliChem, 250 µM), and the culture was incubated at 20 °C, 190 rpm for 20 h.The cells were centrifuged at 5,000 x g, 4 h for 20 min and the pellet was stored at -20 °C.

Protein purification
The heterologously expressed tyrocidine synthetase proteins were purified as reported by Niquille and Folger et al. 4 Briefly, to lyse the HM0079 cells containing the protein of interest, the thawed pellet was resuspended in 30 mL Tris-HCl (50 mM) buffer pH 8.0, supplemented with NaCl (500 mM), and glycerol (10% (w/v).Polymyxin (Apollo, 1 mg/mL), RNase A (AppliChem, 10 µg/mL), DNase (PanReac AppliChem10 µg/mL), and lysozyme (PanReac, 1 mg/ml) were added to increase lysis efficiency.The solution was incubated at 4 °C for 20 min before sonication at 60% amplitude in a Q700 sonicator (Qsonica) equipped with a 1/4 microtip (sonication time: 4 cycles of 30 s each with a 30 s break in between).The solution was centrifuged at 15,000 x g for 20 min at 4 °C and the supernatant containing the soluble protein was applied to pre-equilibrated Ni-NTA columns containing 5 mL Ni-beads (Quiagen).

Figure S2 .
Figure S2.Cleavage sites for excising NRPS elongation modules for functional display on yeast.(a) Sequence alignment of the linker regions connecting the C and A domains of all TycB and TycC modules with structurally characterized SrfA-C, which served as a reference.The N-terminal excision site selected for the A-T constructs is indicated by red triangles.Dark red indicates high conservation, whereas dark blue indicates high variability.(b) The C-terminal excision site for the A-T and C-A-T constructs (magenta triangles) and the N-terminal excision site for C-A-T constructs (yellow triangles).(c) Crystal structure of SrfA-C (PDB: 2VSQ, residues 398-510), 11 showing the N-terminal excision site for the A-T constructs (red).The C domain is colored cyan, the A domain orange/yellow, and linker regions dark blue.(d) Crystal structure of the TycC5-TycC6 T-C bidomain (PDB: 2JGP) 12 showing the N-terminal excision site for C-A-T constructs (yellow) and C-terminal (magenta) excision site for A-T and C-A-T constructs.The T and C domains are colored red and cyan, respectively.

Figure S3 .
Figure S3.Tyrocidine synthetase domains/modules chosen for yeast display, showing their native context within the megaenzyme.The segment below the synthetase indicates the N-and C-terminal amino acids of the display constructs with triangles, colored as in Figure S2, indicating the cleavage sites chosen.(a) The previously described TycA A-T bidomain. 3Its A domain (purple) contained the W227S mutation that confers pPhe specificity.The corresponding (b) A-T and (c) C-A-T constructs from TycB3 are shown.The A domain of TycB3 (magenta) contained the W2742S mutation to accommodate pPhe.(d) The analogous A-T construct from TycB2 was engineered in high-throughput to switch its substrate specificity from L-Phe to pPhe.(e) The A-T construct from TycC3 (yellow) and the C-A-T construct from TycC6 (orange) are illustrated.

Figure S5 :
Figure S5: The activity of TycB2 variants 1 (a), 2 (b), 3 (c) and 4 (d) displayed on the surface of yeast.Assays were performed with 0.1 mM ATP and 0.5 mM pPhe in the presence of 0.5 mM Phe as a competitive inhibitor.The fraction of cells in the pink quadrant correlates with the ability of the displayed A domain to activate pPhe and transfer it to the ppan cofactor of the T domain tethered to the cell surface.

Figure S7 :
Figure S7: MS/MS characterization of the products of the in vitro biosynthetic reaction catalyzed by TycA, M1768A/M1803I TycB and TycC in the presence of pPhe.(a) The selected mass of the major product that elutes at a retention time of 6.88-6.98(see Fig. 5 in the main text) is consistent with the propargyloxy-tyrocidine derivative 1 (calculated [M+H] + 1324.6725Da; found 1324.6730Da).(b)The side product that elutes at a retention time of 6.96-7.08min is wildtype tyrocidine A (calculated [M+H] + 1270.6619;found 1270.6634Da).(c) MS2 spectrum of the major product confirms its assignment to structure 1, with the noncanonical pPhe incorporated site-specifically as the third residue in the natural product analogue.The observed fragmentation masses (b-ions) are shown in blue below the single letter code of the sequence, the sites of fragmentation are marked with dashed lines, and the parent ion is highlighted with a red star.O stands for Orn and f for D-Phe; all other capital single letters refer to L-amino acids.
-W2742S-TycB3-A-T-c-myc (protein sequence displayed on yeast, bold amino acid marks start of NRPS protein and highlighted in blue is W2742S mutation) -W2742S-TycB3-C-A-T-c-myc (protein sequence displayed on yeast, bold amino acid marks start of NRPS protein and highlighted in blue is W2742S mutation)