Unique Initiation and Termination Mechanisms Involved in the Biosynthesis of a Hybrid Polyketide-Nonribosomal Peptide Lyngbyapeptin B Produced by the Marine Cyanobacterium Moorena bouillonii

Lyngbyapeptin B is a hybrid polyketide-nonribosomal peptide isolated from particular marine cyanobacteria. In this report, we carried out genome sequence analysis of a producer cyanobacterium Moorena bouillonii to understand the biosynthetic mechanisms that generate the unique structural features of lyngbyapeptin B, including the (E)-3-methoxy-2-butenoyl starter unit and the C-terminal thiazole moiety. We identified a putative lyngbyapeptin B biosynthetic (lynB) gene cluster comprising nine open reading frames that include two polyketide synthases (PKSs: LynB1 and LynB2), four nonribosomal peptide synthetases (NRPSs: LynB3, LynB4, LynB5, and LynB6), a putative nonheme diiron oxygenase (LynB7), a type II thioesterase (LynB8), and a hypothetical protein (LynB9). In vitro enzymatic analysis of LynB2 with methyltransferase (MT) and acyl carrier protein (ACP) domains revealed that the LynB2 MT domain (LynB2-MT) catalyzes O-methylation of the acetoacetyl-LynB2 ACP domain (LynB2-ACP) to yield (E)-3-methoxy-2-butenoyl-LynB2-ACP. In addition, in vitro enzymatic analysis of LynB7 revealed that LynB7 catalyzes the oxidative decarboxylation of (4R)-2-methyl-2-thiazoline-4-carboxylic acid to yield 2-methylthiazole in the presence of Fe2+ and molecular oxygen. This result indicates that LynB7 is responsible for the last post-NRPS modification to give the C-terminal thiazole moiety in lyngbyapeptin B biosynthesis. Overall, we identified and characterized a new marine cyanobacterial hybrid PKS-NRPS biosynthetic gene cluster for lyngbyapeptin B production, revealing two unique enzymatic logics.


S4
was fused with N-terminus His6 and the size of LynB7-N-His (LynB7) is 378 aa and 44.3 kDa (without Nterminal Met).
HPLC analysis was performed with a HPLC system (Hitachi ELITE LaChrom L-2130 Pump, L-2455 Diode Array Detector, SSC-2300 Column Oven, and SSC-3215 Degasser) with a COMOSIL 5C4-AR300 (4.6 ø × 250 mm; Nacalai Tesque Inc., Japan); 10 µL of solution was injected into the HPLC system. The mobile phase was a two-step linear gradient of 0.1% TFA and acetonitrile with 0.1% (v/v) TFA (35-60% (v/v) acetonitrile for 10 min and 60-62.5% (v/v) acetonitrile for 20 min) with a flow-rate of 1.0 mL/min at 28 °C. For the next analytical cycle, the column was washed with 90% (v/v) acetonitrile for 10 min. Elution was monitored at 280 nm. To isolate the methylated product, a linear gradient of 0.1% TFA and acetonitrile with 0.1% (v/v) TFA (50-60% (v/v) acetonitrile for 10 min) was used. The fractionated solution containing the methylated product was evaporated to remove acetonitrile and then lyophilized to obtain the sample for LC-ESI-MS analysis.
LC-ESI-MS analysis was performed with an LC-MS system (Shimadzu LCMS-2020, LC-20AD pump, and SPD-M20A detector) with a COMOSIL Protein-R (2.0 ø × 150 mm; Nacalai Tesque Inc., Japan); 3 µL of solution was injected into the LC-MS system. The mobile phase was a two-step linear gradient of 0.1% formic acid and acetonitrile with 0.1% (v/v) formic acid (35-50% (v/v) acetonitrile for 10 min and 50-60% (v/v) acetonitrile for 30 min) with a flow-rate of 0.1 mL/min for acetoacetyl-ACP and 0.2 mL/min for methylated product at 40 °C. For the next analytical cycle, the column was washed with 90% (v/v) acetonitrile for 10 min.
Elution was monitored at 280 nm.
HPLC analysis was performed with a HPLC system (Hitachi Chromastar 5110 Pump, 5310 Column Oven, 5430 Diode Array Detector, and SSC-3215 Degasser) with a ODS 100Z (4.6 ø × 250 mm; TOSOH, Japan); 10 µL of the solution was injected into the HPLC system. The mobile phase was a linear gradient of water and CH3OH (40-70% (v/v) CH3OH for 20 min) with a flow-rate of 1.0 mL/min at 40 °C. Elution was monitored at 240 nm. For the next analytical cycle, the column was washed with 90% (v/v) CH3OH for 10 min. LC-APCI-MS analysis was performed with the above-mentioned LC-MS system with an ODS-100Z (2.0 ø × 150 mm); An aliquot (2 µL) of solution was injected into the system. The mobile phase was a linear gradient of water and CH3OH (40-70% (v/v) CH3OH for 20 min) with a flow-rate of 0.5 mL/min at 40 °C. Elution was monitored at 240 nm.