Caerulomycin and collismycin antibiotics share a trans-acting flavoprotein-dependent assembly line for 2,2’-bipyridine formation

Linear nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) template the modular biosynthesis of numerous nonribosomal peptides, polyketides and their hybrids through assembly line chemistry. This chemistry can be complex and highly varied, and thus challenges our understanding in NRPS and PKS-programmed, diverse biosynthetic processes using amino acid and carboxylate building blocks. Here, we report that caerulomycin and collismycin peptide-polyketide hybrid antibiotics share an assembly line that involves unusual NRPS activity to engage a trans-acting flavoprotein in C-C bond formation and heterocyclization during 2,2’-bipyridine formation. Simultaneously, this assembly line provides dethiolated and thiolated 2,2’-bipyridine intermediates through differential treatment of the sulfhydryl group arising from l-cysteine incorporation. Subsequent l-leucine extension, which does not contribute any atoms to either caerulomycins or collismycins, plays a key role in sulfur fate determination by selectively advancing one of the two 2,2’-bipyridine intermediates down a path to the final products with or without sulfur decoration. These findings further the appreciation of assembly line chemistry and will facilitate the development of related molecules using synthetic biology approaches.

Q Exactive HF mass spectrometer (Thermo Fisher Scientific Inc., USA), and the data were analyzed using pymzML, pFind 3

and Thermo Xcalibur. High resolution ESI-MS (HR-ESI-MS) analysis for
small molecules was carried out on an Agilent 6230B Accurate Mass TOF LC/MS System (Agilent Technologies Inc., USA), and the data were analyzed using Agilent MassHunter Qualitative Analysis software. NMR data were recorded on an Agilent 500 MHz PremiumCompact+ NMR spectrometer (Agilent Technologies Inc., USA).
In general, the genes coding for the above proteins were amplified by PCR individually from the CAEproducing strain Actinoalloteichus cyanogriseus or the COL-producing strain Streptomyces roseosporus, both of which are listed in Supplementary Table 1, using the corresponding primers listed in Supplementary Table 3. The PCR products were first cloned into the vector pMD19-T for sequencing to confirm the fidelity and then into the expression vector pET-28a(+) (for N-terminal 6 × His-tagged proteins or N-terminal 6 × His and Sumo-tagged proteins), pSJ5 (for N-terminal 6 × His and Trx-tagged proteins), pQ8 (for N-terminal 6 × His and MBP-tagged proteins), or pET-37b(+) (for The culture of each E. coli transformant was incubated in Luria-Bertani (LB) medium containing 50 μg/mL kanamycin at 37°C and 220 rpm until the cell density reached 0.6-0. 8

Chemical synthesis of 4-hydroxy-2,2'-bipyridine-6-carboxyloyl-S-CoA (2,2'-bipyidinyl-S-CoA).
The precursor 4-hydroxy-2,2'-bipyridinyl-6-carboxylic acid hydrobromide (20) was synthesized according to the method described previously 4 .  were expressed in E.coli BAP1. All the mutant proteins were purified to homogeneity, and then concentrated according to the procedures for the native proteins described above. To examine O2 dependence under anaerobic conditions, gas exchange for O2 elimination was conducted in an anaerobic glovebox overnight before the incubation of the related reaction mixture at 30˚C for 1 h. All assays were performed at least in triplicate and each had at least two parallel samples.

In vitro assays of PCP S-aminoacylation on CaeA2 by nanoLC-MS/MS. The CaeA2 recombinant
protein that was purified from E.coli BAP1 was subjected to complete or partial protease hydrolysis with trypsin, Glu-C or chymotrypsin as well as a variety of their combinations to map at 30℃ for 20 min (leading to complete digestion) and then with 0.6 g of chymotrypsin (sequencing grade, Promega Corp., USA) at 30℃ for 10 min (leading to partial digestion that retains the C-terminal sequence F2042VSR). To obtain the engineered sequence SLGGDSIMGIQL2042VSR, the CaeA2 F2042L recombinant protein that was purified from E.coli BAP1 and its variants different in S-(amino)acylation underwent complete digestion with trypsin (4 g) and chymotrypsin (0.6 g) at 30℃ for 20 min.
To identify the target sequence that contains the Ppant-modified active-site L-serine residue, the raw MS data was processed using the pFind software by setting Ppant as a variable posttranslational modification. The selected sequence was then validated by HR-MS/MS analysis of the parent ion, its associated fragmented peptide ions and particularly the characteristic Ppant ejection ion 6 .
To prepare L-cysteinyl-S-CaeA2 F2042L (3), the reaction was conducted at 30˚C for 10 min in a 50 L