In situ detection of the intermediates in the biosynthesis of surfactin, a lipoheptapeptide from Bacillus subtilis OKB 105, by whole-cell cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in combination with mutant analysis

摘要

An innovative technique to investigate the intermediates involved in the biosynthesis of the lipoheptapeptide surfactin from Bacillus subtilis OKB105 combining whole-cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) with targeted generation of knock-out mutants was demonstrated. This method allows efficient, sensitive detection of biosynthetic intermediates in a minimum of time directly at the outer surface of microbial cells picked from agar plates or in surface extracts prepared thereof. Biosynthesis of surfactin is encoded by the srf-operon which is organized into four open reading frames which have been attributed to three multifunctional NRPS enzymes (SrfA-C) and a thioesterase/acyltransferase enzyme SrfD. For the wild-type strain OKB 105 only the end product surfactin was found mass spectrometrically. For the detection of lipopeptide intermediates three plasmid- and transposon-insertion mutants were generated interrupting the surfactin assembly line at defined positions. Strain LAB 327 was mutated in the spacer region between enzymes SrfA and B. Here only SrfA was active with the lipotripeptide beta-OH-acyl-L-Glu-L-Leu-D-Leu as the end product. Mutant OKB 120 bears a transposon mutation in SrfB between the first and second amino acid activating modules SrfB1 and SrfB2. It showed all intermediates from the lipodi- until to the lipotetrapeptide beta-OH-acyl-L-Glu-L-Leu-D-Leu-L-Val. In LAB 223 SrfC was knocked out by a transposon mutation. It produced the lipohexapeptide beta-OHacyl-L-Glu-L-Leu-D-Leu-L-Val-L-Asp-D-Leu. Our work highlights the applicability and the potential of whole-cell MALDI-TOFMS as an innovative efficient tool for the analysis of intermediate steps of biosynthetic pathways.