syn-Elimination of glutamylated threonine in lanthipeptide biosynthesis

Methyllanthionine (MeLan) containing macrocycles are key structural features of lanthipeptides. They are formed typically by anti-elimination of l-Thr residues followed by cyclization of l-Cys residues onto the (Z)-dehydrobutyrine (Dhb) intermediates. In this report we demonstrate that the biosynthesis of lanthipeptides containing the d-allo-l-MeLan macrocycle such as the morphogenetic lanthipeptide SapT proceeds through (E)-Dhb intermediates formed by net syn-elimination of l-Thr.


Small scale heterologous production and isolation of peptides
Heterologous production (general protocol) Chemically competent E. coli DH10β cells were co-transformed with pTRC33 GluRS_tRNA Glu , pCDFDuet-1 SptBa_SptC, and either pRSFDuet-1 His6-MBP-SptA(1-37)_SptBb or pRSFDuet-1 His6-MBP-SptA(1-35), and plated onto a Lysogen Broth (LB) agar plate containing 17 µg/mL kanamycin, 17 µg/mL spectinomycin, and 8 µg/mL chloramphenicol. The plate was left to incubate at 37 o C overnight and a single colony was picked the next day and grown overnight in 5 mL of Terrific Broth (TB) media containing 0.4% glycerol and the appropriate antibiotics. After overnight growth, 1 mL of the culture was used to inoculate 100 mL of fresh TB media containing 0.4% glycerol and appropriate antibiotics. The 100 mL culture was initially incubated at 37 o C while shaking at 160 rpm with monitoring OD600. Once OD600 reached 0.8, the temperature of the incubator was lowered to 18 o C and 50 µL of 1 M IPTG (isopropyl β-D-1-thiogalactopyranoside) was added. The culture was then grown for an additional 18 h at 18 ºC. The cells were then harvested at 5000 xg for 20 min at 4 o C. Cell pellet was stored at -80 ºC until further use.

Isolation and Analysis of Peptides
The cell pellet was resuspended in 5 mL of start buffer (20 mM Tris, 500 mM NaCl, pH 7.5) and lysed by sonication. Sample was then centrifuged at 25,000 xg for 45 min at 4 o C. The supernatant was isolated and loaded onto a pre-equilibrated Ni-NTA column containing 0.5 mL of HisPur Ni-NTA resin. The column was then washed with 5 mL of wash buffer (20 mM Tris, 500 mM NaCl, 30 mM imidazole, pH 7.5) and His6-MBP tagged peptide was eluted with 5 mL of elution buffer (20 mM Tris, 500 mM NaCl, 500 mM imidazole, pH 7.5). The elution fractions were concentrated using an Amicon Ultra-15 filter (3 kDa MW CO) by centrifugation at 4,500 xg. The buffer was then exchanged to storage buffer (20 mM Tris, 500 mM KCl, pH 7.5) by repeating the centrifugation step.
The His6-MBP tag was removed by treating the sample in storage buffer with His6-TEV protease (10:1 sample:protease) overnight. Samples were then desalted using C18 Ziptips (Agilent) and analyzed by matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry by cospotting the sample with super DHB onto a MALDI plate. MALDI-TOF MS data was acquired on a Bruker UltrafleXtreme MALDI TOF/TOF mass spectrometer. Large scale heterologous production and isolation of mSptA(1-37) Large scale heterologous production of MBP-mSptA(1-37) was performed on a 15 L scale by scaling up the small scale procedure described above using 15 individual 4 L flasks containing 1 L media for each flask.

Trypsin digestion of mSptA(1-37) and HPLC purification
mSptA(1-37) that was purified by HPLC was digested with trypsin (Promega, sequencing grade) using 1 mg of peptide and 20 µg of trypsin in 5 mL of buffer containing 2 mM Tris, 50 mM KCl, 1% glycerol, pH 7.5. The sample was then subjected to HPLC purification according to the conditions described above. The C-terminal fragment of mSptA(1-37) (ITDEDLPTASVYDhbQG) eluted at 60:40 A:B with a retention time of 21.2 -21.6 min. The desired processed peptide was separated from unprocessed peptide (ITDEDLPTASVYTQG) that eluted earlier with a retention time of 21.0 -21.2 min. We did not observe any other peaks with masses corresponding to dehydrated peptides, but cannot rule out that we removed very minor amounts of other such peptides.
The digestion and purification was repeated using an additional 1 mg of mSptA(1-37). Elution fractions containing the desired peptide were combined and lyophilized to yield 400 of µg mSptA(1-37)trypsin for NMR studies.

Trypsin digestion of mSptA(1-37) and analysis by LC-MS and LC-MS/MS
mSptA(1-37) (50 µg) was digested with 1 µg of trypsin (Promega, sequencing grade) in 50 mM Tris buffer, pH 7.5 at 37 o C for 3 h. After 3 h, the sample was desalted using C18 Ziptips and eluted into a 10 µL solution of 4:1 MeCN:H2O containing 0.1% formic acid. The digested sample was loaded onto an AdvanceBio Peptide Plus (2.7 µm particle size, 150 x 2.1 mm) column and subjected to LC-MS and LC-MS/MS according to a previously reported procedure for mSptA. 1 0.523 a These ions could suggest that Thr24 might be dehydrated but we do not favor this interpretation and instead suggest that these arrive from loss of H2O from b5 an b6 ions, which are observed. If these dehydrated ions came from a singly dehydrated precursor in which Thr24 were dehydrated, we would expect to see ions with non-dehydrated Thr35, which were not observed.

NMR experiments and analysis
Peptide (400 µg) was dissolved in 70% acetonitrile-d3/30% H2O and data were acquired on an Agilent VNMRS 750 MHz spectrometer with a 5-mm triple-resonance HCN probe at 25 o C. Chemical shifts were referenced to residual MeCN solvent peak at 1.97 ppm. Table S3 below contains all peak assignments for mSptA(1-37)trypsin.      Figure S5. 1 H-1 H NOESY spectrum of previously characterized prochlorosin 2.10 that contains two (Z)-Dhb residues. 3 NOEs are observed between the methyl protons of Dhb residues and amide protons of the same Dhb residue. These NOEs are absent for mSptA(1-37)trypsin (Figure 5 and S4).