Modification of biologically active peptides: production of a novel lipohexapeptide after engineering of Bacillus subtilis surfactin synthetase.
The Bacillus subtilis strain ATCC 21332 produces the lipoheptapeptide surfactin, a highly potent biosurfactant synthesized by a large multimodular peptide synthetase. We report the genetic engineering of the surfactin biosynthesis resulting in the production of a novel lipohexapeptide with altered antimicrobial activities. A combination of in vitro and in vivo recombination approaches was used to construct a modified peptide synthetase by eliminating a large internal region of the enzyme containing a complete amino acid incorporating module. The remaining modules adjacent to the deletion were recombined at different highly conserved sequence motifs characteristic of amino acid incorporating modules of peptide synthetases. The primary goal of this work was to identify permissive fusion sites suitable for the engineering of peptide synthetase genes by genetic recombination. Analysis of the rearranged enzymes after purification from B. subtilis and from the heterologous host Escherichia coli revealed that the selection of the recombination site is of crucial importance for a successful engineering. Only the recombination at a specific HHII x DGVS sequence motif resulted in an active peptide synthetase. The expected lipohexapeptide was produced in vivo and first evidence of a reduced toxicity against erythrocytes and an enhanced lysis of Bacillus licheniformis cells was shown.
Symmank
Hanka
H
Institut für Kristallographie, Takustrasse 6 , Freie Universität Berlin, D-14195 Berlin, Germany.
Franke
Peter
P
Saenger
Wolfram
W
Bernhard
Frank
F
D020816:Amino Acid Motifs; D001412:Bacillus subtilis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000096:biosynthesis; D016296:Mutagenesis; D009154:Mutation; D010453:Peptide Synthases / Q000096:biosynthesis* / Q000737:chemistry; D010455:Peptides / Q000378:metabolism*; D015202:Protein Engineering; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D010455:Peptides; D010453:Peptide Synthases; C069542:surfactin synthetase