Chimeric Leader Peptides for the Generation of Non-Natural Hybrid RiPP Products

Combining biosynthetic enzymes from multiple pathways is an attractive approach for producing molecules with desired structural features; however, progress has been hampered by the incompatibility of enzymes from unrelated pathways and intolerance toward alternative substrates. Ribosomally synthesized and posttranslationally modified peptides (RiPPs) are a diverse natural product class that employs a biosynthetic logic that is highly amenable to engineering new compounds. RiPP biosynthetic proteins modify their substrates by binding to a motif typically located in the N-terminal leader region of the precursor peptide. Here, we exploit this feature by designing leader peptides that enable recognition and processing by multiple enzymes from unrelated RiPP pathways. Using this broadly applicable strategy, a thiazoline-forming cyclodehydratase was combined with enzymes from the sactipeptide and lanthipeptide families to create new-to-nature hybrid RiPPs. We also provide insight into design features that enable control over the hybrid biosynthesis to optimize enzyme compatibility and establish a general platform for engineering additional hybrid RiPPs.

. DNA spin columns were purchased from Epoch Life Sciences, and DNA sequencing was performed by the Roy J. Carver Biotechnology Center (UIUC). All polymerase chain reactions (PCRs) were conducted on an S1000 thermal cycler (Bio-Rad).
Cloning. Plasmids were constructed using REs and DNA ligase or by Gibson assembly (GA). For RE cloning, the target gene was amplified with Q5 polymerase, and after PCR cleanup using the QIAquick PCR Purification Kit Protocol, the amplicon was digested with restriction enzymes for ligation into a similarly digested and purified vector. For amplifications which gave more than one product, a gel extraction was performed. Ligations were performed with T4 DNA ligase at 23 °C for 1 h (100 ng vector and 3-fold excess insert). For GA, the standard protocol was followed with guidance from the NEBuilder Assembly tool (nebuilder.neb.com). Inserts were generated using Q5 polymerase and purified as above.
Vector backbones were linearized using PCR or restriction enzymes and similarly purified.
Genes for the cyclodehydratase (HcaF and HcaD), 1 lanthipeptide synthetase (ProcM and NisB/C), 2 dehydroalanine reductase (NpnJ), 3 and flavoenzyme decarboxylase (MibD) 4 were cloned previously from their native organisms. All new plasmids used in this study are listed in Table S1 and were constructed using primers listed in Table S2. The subtilosin radical SAM gene (AlbA) and its precursor peptide (SboA) were cloned from Bacillus subtilis strain 168 genomic DNA.
The hybrid peptides were built from HcaA, SboA, or ProcA precursor peptides through overlap extension PCR and initially cloned into a modified pET28 vector with an N-terminal maltose-binding protein (MBP) affinity tag (pET28-MBP). This intermediate was then used to clone MBP-tagged hybrid peptides into duet vectors or to express the peptide for in vitro activity assays. The NisA-derived hybrid peptide was synthesized as a gblock (IDT) for subsequent cloning and was cloned directly into a duet vector in frame with a His 6 tag. Duet vectors pACYCDuet, pCDFDuet, pETDuet, and pRSFDuet were used for coexpression in E. coli (see Table S1), as has been well established, 5 and genes were sequentially cloned into each multiple cloning site. To insert the cyclodehydratase HcaF and HcaD genes into a single multiple cloning site, the genes were taken together as a single amplicon from pETDuet-HcaF-HcaD.
Site-directed mutagenesis. Changes to the sequence of hybrid peptides were made with cloned PFU or KOD polymerase (a proofreading DNA polymerase isolated from Thermococcus kodakaraensis) and the site-directed mutagenesis (SDM) primers in Table S2 following the Agilent QuikChange protocol.
In vivo production of modified hybrid peptides. BL21(RIPL-DE3) chemically competent cells were cotransformed with pairs of duet vectors (Table S2) and selected with 100 µg/mL ampicillin for pETDuet, 50 µg/mL kanamycin for pRSFDuet or pET28, and 34 µg/mL chloramphenicol for pACYCDuet. Initial cultures were started from single colonies and grown in 10 mL of Luria−Bertani (LB) broth in 18 mm glass culture tubes at 37 °C supplemented with appropriate antibiotics for several hours, and then used to inoculate 0.5 or 1 L of the same media. Cells were grown to ~0.8 OD 600 , put on ice for 10 min, and induced with 0.6 mM IPTG (isopropyl β-D-1-thiogalactopyranoside) at 22 °C except for co-expressions containing AlbA, which were shaken at 100 rpm in non-baffled flasks to reduce aeration (see Figure S3). 6 After 18 h induction, cells were harvested at 4000 ´ g for 15 min at 4 °C, washed with TBS (Tris-buffered saline: 25 mM Tris pH 8.0, 150 mM NaCl), and centrifuged again at 4000 ´ g for 10 min at 4 °C. Cell pellets were stored for up to 1 week at -20 °C.
Protein expression and purification. BL21(DE3-RIPL) cells were transformed with plasmids containing the tagged proteins and selected using appropriate antibiotics. Starter cultures were grown in 10 mL LB with antibiotics and used to inoculate 1 L of similarly prepared media. After reaching an OD 600 of ~0.8, cells were induced overnight with IPTG. Next, cells were harvested by centrifugation at 4000 ´ g for 15 min at 4 °C, washed with TBS, and then subjected to centrifugation at 4000 ´ g for 10 min at 4 °C. Cell pellets were stored for up to 1 week at -20 °C. Proteins used for in vitro assays were purified using affinity chromatography via the MBP or His 6 tag.
MBP-tagged proteins were purified from E. coli cells using amylose resin (NEB Purification of modified hybrid peptides. All hybrid peptides except Hyp1.1 were MBP-tagged and purified as described above. Typically, >20 mg of MBP-tagged peptide (corresponding to >1 mg of core peptide after affinity tag removal) was obtained from 1 L of culture. Hyp1.1 was expressed as a His 6 -tagged peptide and purified by the following procedure. The harvested cells were resuspended in 25 mL of LanA In vitro modification assays. In vitro assays were performed with purified proteins and peptides as This solution (4 mL) was then added to the hydrolysate, which was again heated to 110 °C for 1 h. After cooling, the solvent was removed, and the residue was taken up in 3 mL of CH 2 Cl 2 . The suspension was then cooled to 0 °C and pentafluoropropionic acid (1 mL) was added dropwise. Next, the sample was heated to 110 °C for 30 min and allowed to cool. The solvent was again removed before suspending the derivatized product in MeOH (200 µL) prior to GCMS analysis.
The derivatized samples were analyzed by GC-MS (Agilent HP 6890N) using an Agilent CP-Chirasil-L-Val column (25 m ´ 0.25 mm ´ 0.12 µm), with co-injection of previously prepared DL-MeLan and LL-MeLan standards for stereochemical verification. 11 Samples were applied to the column via split injection and run according to the following temperature method: 150 °C injection for 3 min, then raised to 200 °C by 3 °C /min and held (21.6 min total runtime). The MS was operated using selected ion monitoring at 379 Da for the detection of MeLan isomers. Table S1. New plasmids generated for this study and their use in combinations. Hybrid peptides (Hyp) are listed in Figure 2, 3, 4, 5 and S19. HcaF-HcaD comprise a two-component, thiazoline-forming cyclodehydratase, NisB and NisC form a class I lanthipeptide synthetase, while ProcM is a class II lanthipeptide synthetase. Constructs for the tailoring enzymes MibD and NpnJ A are reported elsewhere. 3,8 The combinations of plasmids used for co-expression and the figure where the results are presented are also listed. Abbreviations: No., number; MCS, multiple cloning site; MBP, maltose-binding protein.         dehydratases, 13 the related thiopeptide dehydratases, which also contain an RRE, are leader-independent (red "x" in figure). 14 It is notable that RiPP pathways employ multiple enzymes that are dependent on certain recognition motifs and that these motifs tend to occupy distinct parts of the peptide (see panel b and c). [14][15][16] Leader proteases could also be considered leader-dependent and similarly tend to bind a part of the leader not used by other enzymes. 15,16 Nature might have evolved these physically separate binding sites to improve biosynthetic efficiency. Regardless, it appears that the recognition sequences are "plug and play" and simply need to be inserted into an existing peptide for a new enzyme to act on it.                        Table S1). The peptide was purified, digested with AspN, and analyzed by MALDI-TOF-MS. (d) The mass spectrum shows a several ions, but the most intense peak (labeled Hyp3.4a) corresponded to the desired hybrid product. (e) Deduced structure of the hybrid Hyp3.4a product after AspN digestion.