Engineered biosynthesis of peptide antibiotics

Stachelhaus, Torsten; Schneider, Axel; Marahiel, Mohamed A.

doi:10.1016/0006-2952(96)00111-6

Cited by 62 publications

(61 citation statements)

References 55 publications

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“…These comparisons may provide good techniques for elucidating the boundary of the functional domain that directs engineered peptide synthesis (22,31,33,34).…”

Section: Discussionmentioning

confidence: 99%

Cloning, Sequencing, and Characterization of the Iturin A Operon

2001

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Bacillus subtilis RB14 is a producer of the antifungal lipopeptide iturin A. Using a transposon, we identified and cloned the iturin A synthetase operon of RB14, and the sequence of this operon was also determined. The iturin A operon spans a region that is more than 38 kb long and is composed of four open reading frames, ituD, ituA, ituB, and ituC. The ituD gene encodes a putative malonyl coenzyme A transacylase, whose disruption results in a specific deficiency in iturin A production. The second gene, ituA, encodes a 449-kDa protein that has three functional modules homologous to fatty acid synthetase, amino acid transferase, and peptide synthetase. The third gene, ituB, and the fourth gene, ituC, encode 609-and 297-kDa peptide synthetases that harbor four and two amino acid modules, respectively. Mycosubtilin, which is produced by B. subtilis ATCC 6633, has almost the same structure as iturin A, but the amino acids at positions 6 and 7 in the mycosubtilin sequence are D-Ser3L-Asn, while in iturin A these amino acids are inverted (i.e., D-Asn3L-Ser). Comparison of the amino acid sequences encoded by the iturin A operon and the mycosubtilin operon revealed that ituD, ituA, and ituB have high levels of homology to the counterpart genes fenF (79%), mycA (79%), and mycB (79%), respectively. Although the overall level of homology of the amino acid sequences encoded by ituC and mycC, the counterpart of ituC, is relatively low (64%), which indicates that there is a difference in the amino acid sequences of the two lipopeptides, the levels of homology between the putative serine adenylation domains and between the asparagine adenylation domains in the two synthetases are high (79 and 80%, respectively), implying that there is an intragenic domain change in the synthetases. The fact that the flanking sequence of the iturin A synthetase coding region was highly homologous to the flanking sequence that of xynD of B. subtilis 168 and the fact that the promoter of the iturin A operon which we identified was also conserved in an upstream sequence of xynD imply that horizontal transfer of this operon occurred. When the promoter was replaced by the repU promoter of the plasmid pUB110 replication protein, production of iturin A increased threefold.Many Bacillus subtilis strains produce a small peptide(s) with a long fatty moiety, the so-called lipopeptide antibiotics. The peptide portions of these compounds contain ␣-amino acids with a D configuration and are produced nonribosomally with templates of the multifunctional peptide synthetases. As in the synthesis of the peptide antibiotic gramicidin S, the peptide chain grows in a defined sequence by moving on the template of the multifunctional peptide synthetase (18,35,44). On the basis of the structural relationships, the lipopeptides that have been identified in B. subtilis are generally classified into three groups: the surfactin group (27), the plipastatin-fengycin group (16,41,42), and the iturin group (17). The members of the surfactin and plipastatin-fengycin groups are ...

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“…These comparisons may provide good techniques for elucidating the boundary of the functional domain that directs engineered peptide synthesis (22,31,33,34).…”

Section: Discussionmentioning

confidence: 99%

Cloning, Sequencing, and Characterization of the Iturin A Operon

2001

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“…Hopefully, the approach described here, which was developed to enable a broader investigation of the type II PKSs, will complement other, newly emerging approaches to antibiotic discovery (28). These include several genome projects involving known pathogens (20) to provide additional targets for conventional screening programs and de novo drug design, new strategies in combinatorial biology (12,31,32,45,65,66) to yield novel scaffolding for decorative (bio-)chemistry, and attempts to access the immense marine biodiversity (30).…”

Section: Discussionmentioning

confidence: 99%

A study of iterative type II polyketide synthases, using bacterial genes cloned from soil DNA: a means to access and use genes from uncultured microorganisms

et al. 1997

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To examine as randomly as possible the role of the ␤-ketoacyl and acyl carrier protein (ACP) components of bacterial type II polyketide synthases (PKSs), homologs of the chain-length-factor (CLF) genes were cloned from the environmental community of microorganisms. With PCR primers derived from conserved regions of known ketosynthase (KS ␣ ) and ACP genes specifying the formation of 16-to 24-carbon polyketides, two CLF (KS ␤ ) genes were cloned from unclassified streptomycetes isolated from the soil, and two were cloned from soil DNA without the prior isolation of the parent microorganism. The sequence and deduced product of each gene were distinct from those of known KS ␤ genes and, by phylogenetic analysis, belonged to antibiotic-producing PKS gene clusters. Hybrid PKS gene cassettes were constructed with each novel KS ␤ gene substituted for the actI-ORF2 or tcmL KS ␤ subunit genes, along with the respective actI-ORF1 or tcmK KS ␣ , tcmM ACP, and tcmN cyclase genes, and were found to produce an octaketide or decaketide product characteristic of the ones known to be made by the heterologous KS ␣ gene partner. Since substantially less than 1% of the microorganisms present in soil are thought to be cultivatable by standard methods, this work demonstrates a potential way to gain access to a more extensive range of microbial molecular diversity and to biosynthetic pathways whose products can be tested for biological applications.The polyketide metabolites are a large group of structurally complex and diverse natural products, many of which are clinically valuable antibiotics or chemotherapeutic agents or have other useful pharmacological activities (48). Polyketides are synthesized by polyketide synthases (PKSs) which, like the related fatty acid synthases, are multifunctional enzyme assemblies that catalyze repeated decarboxylative condensations between enzyme-bound acylthioesters (52). The structural diversity of polyketides is a result of the different numbers and types of acyl units involved, coupled with various possibilities for enzyme-mediated reduction, dehydration, cyclization, and aromatization of the initial ␤-ketoacyl condensation products (52). Manipulation of the sequence or specificity of the PKS enzymes can lead to the production of novel secondary metabolites (28,29,33). The key to further exploitation of this approach to molecular diversity lies in an increased understanding of the genetics and biochemistry of the various PKS systems.Recent progress in the cloning and sequencing of microbial PKS genes has led to the identification of at least two architecturally different types of PKSs (28, 33). Modular type I PKSs, represented by erythromycin A (11, 17), avermectin (41), rapamycin (58), and soraphen (57), are discrete multifunctional enzymes sporting numerous unique active site domains. The domains are grouped into distinct modules that in turn control the sequential addition of acylthioester units to the growing polyketide chain and the subsequent modification of the respective condensation prod...

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“…These findings strongly support the functional integrities of single domains within multifunctional peptide synthetases. Directly downstream of the 3 end of the tycC gene, and probably transcribed in the tyrocidine operon, two tandem ABC transporters, which may be involved in conferring resistance against tyrocidine, and a putative thioesterase were found.The biosynthetic system required for the production of the cyclic decapeptide tyrocidine is one of the prototypes for a protein template-driven pathway to synthesize biologically active peptides by the nonribosomal mechanism (20,21,30,44,46). Large enzymes, called peptide synthetases, activate the amino acid constituents as aminoacyl adenylate at the expense of ATP and thioesterify them on the thiol moiety of an enzyme-attached cofactor, 4Ј-phosphopantetheine.…”

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confidence: 99%

The tyrocidine biosynthesis operon of Bacillus brevis: complete nucleotide sequence and biochemical characterization of functional internal adenylation domains

1997

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The cyclic decapeptide antibiotic tyrocidine is produced by Bacillus brevis ATCC 8185 on an enzyme complex comprising three peptide synthetases, TycA, TycB, and TycC (tyrocidine synthetases 1, 2, and 3), via the nonribosomal pathway. However, previous molecular characterization of the tyrocidine synthetase-encoding operon was restricted to tycA, the gene that encodes the first one-module-bearing peptide synthetase. Here, we report the cloning and sequencing of the entire tyrocidine biosynthesis operon (39.5 kb) containing the tycA, tycB, and tycC genes. As deduced from the sequence data, TycB (404,562 Da) consists of three modules, including an epimerization domain, whereas TycC (723,577 Da) is composed of six modules and harbors a putative thioesterase domain at its C-terminal end. Each module incorporates one amino acid into the peptide product and can be further subdivided into domains responsible for substrate adenylation, thiolation, condensation, and epimerization (optional). We defined, cloned, and expressed in Escherichia coli five internal adenylation domains of TycB and TycC. Soluble His 6 -tagged proteins, ranging from 536 to 559 amino acids, were affinity purified and found to be active by amino acid-dependent ATP-PP i exchange assay. The detected amino acid specificities of the investigated domains manifested the colinear arrangement of the peptide product with the respective module in the corresponding peptide synthetases and explain the production of the four known naturally occurring tyrocidine variants. The K m values of the investigated adenylation domains for their amino acid substrates were found to be comparable to those published for undissected wild-type enzymes. These findings strongly support the functional integrities of single domains within multifunctional peptide synthetases. Directly downstream of the 3 end of the tycC gene, and probably transcribed in the tyrocidine operon, two tandem ABC transporters, which may be involved in conferring resistance against tyrocidine, and a putative thioesterase were found.The biosynthetic system required for the production of the cyclic decapeptide tyrocidine is one of the prototypes for a protein template-driven pathway to synthesize biologically active peptides by the nonribosomal mechanism (20,21,30,44,46). Large enzymes, called peptide synthetases, activate the amino acid constituents as aminoacyl adenylate at the expense of ATP and thioesterify them on the thiol moiety of an enzyme-attached cofactor, 4Ј-phosphopantetheine. Subsequently, activated amino acids are condensed stepwise in an aminoto carboxy-terminal direction. Previous investigations at the protein chemical level and, in particular, comparisons of several genes encoding peptide synthetases have revealed the modular architecture of this class of enzymes (4,23,28,42,44,53). One module is defined to harbor all catalytic activities to incorporate a single amino acid residue into the peptide product. The modules coincide in number with the number of incorporated amino acids and are arr...

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Engineered biosynthesis of peptide antibiotics

Cited by 62 publications

References 55 publications

Cloning, Sequencing, and Characterization of the Iturin A Operon

Cloning, Sequencing, and Characterization of the Iturin A Operon

A study of iterative type II polyketide synthases, using bacterial genes cloned from soil DNA: a means to access and use genes from uncultured microorganisms

The tyrocidine biosynthesis operon of Bacillus brevis: complete nucleotide sequence and biochemical characterization of functional internal adenylation domains

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