A putative lichenysin A synthetase operon in Bacillus licheniformis: initial characterization

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Abstract

Certain Bacillus licheniformis strains isolated from oil wells have been shown to produce a very effective biosurfactant, lichenysin A, which is structurally similar to another less active lipopeptide, surfactin. Surfactin, like many small peptides in prokaryotes and lower eukaryotes, is synthesized non-ribosomally by multi-enzyme peptide synthetase complex. Analysis of several peptide synthetases of bacterial and fungal origin has revealed a high degree of sequence conservation. Two 35-mer oligonucleotides derived from highly conserved motifs (‘core I’ and ‘core II’) of surfactin synthetase were used to identify the cloned putative operon of lichenysin A synthetase lchA from B. licheniformis BNP29, a strain not amenable to genetic manipulation in a BAC system (F-plasmid-based bacterial artificial chromosome) based on Escherichia coli and its single-copy plasmid F-factor. A 32.4 kb fragment containing lichenysin A biosynthesis locus was sequenced and analysed. The structural architecture of putative lichenysin A synthetase protein containing seven amino acid (aa) activation-thiolation, two epimerization and one thioesterase domains is discussed in terms of its similarity to surfactin and other peptide synthetases. The 100 aa peptide chain situated between the highly conserved signature sequences FDXX and NXYGPTE(IV)X within amino acid binding domains of peptide synthetases is proposed to be a minimal block dictating the substrate specificity of the enzymes. A new operon-type structure has been localized directly upstream from the lichenysin A synthetase genes which, on the basis of sequence determination, potentially encode a four-member ABC-type transport system involved in product secretion.

Keywords

Lipopeptide
Lichenysin A
Surfactin
Peptide synthetase
PlchA promoter

Abbreviations

aa, amino acid(s)
Ap, ampicillin
B., Bacillus
bp, base pair(s)
Cm, chloramphenicol
E., Escherichia
kb, kilobase(s)
kDa, kilodalton(s)
lchA, putative operon of lichenysin A synthetase
nt, nucleotide(s)
ORF, open reading frame
P, promoter
PCR, polymerase chain reaction
R, resistance
RBS, ribosome-binding site(s)
SD, Shine-Dalgarno (sequence)
srfA, surfactin synthetase operon

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