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Evolution of cyclizing 5-aminolevulinate synthases in the biosynthesis of actinomycete secondary metabolites: outcomes for genetic screening techniques.

Petříčková K, Chroňáková A, Zelenka T, Chrudimský T, Pospíšil S, Petříček M, Krištůfek V - Front Microbiol (2015)

Bottom Line: Supporting information was acquired through analysis of actinomycete genomic sequence data available in GenBank and further genetic or metabolic characterization of selected strains.Comparison of 16S rRNA taxonomic identification and BOX-PCR profiles provided evidence for numerous horizontal gene transfers of biosynthetic genes or gene clusters within actinomycete populations and even from non-actinomycete organisms.Our results underline the importance of environmental and evolutionary data in the design of efficient techniques for identification of novel producers.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbiology, Czech Academy of Sciences, v. v. i. Prague, Czech Republic.

ABSTRACT
A combined approach, comprising PCR screening and genome mining, was used to unravel the diversity and phylogeny of genes encoding 5-aminolevulinic acid synthases (ALASs, hemA gene products) in streptomycetes-related strains. In actinomycetes, these genes were believed to be directly connected with the production of secondary metabolites carrying the C5N unit, 2-amino-3-hydroxycyclopent-2-enone, with biological activities making them attractive for future use in medicine and agriculture. Unlike "classical" primary metabolism ALAS, the C5N unit-forming cyclizing ALAS (cALAS) catalyses intramolecular cyclization of nascent 5-aminolevulinate. Specific amino acid sequence changes can be traced by comparison of "classical" ALASs against cALASs. PCR screening revealed 226 hemA gene-carrying strains from 1,500 tested, with 87% putatively encoding cALAS. Phylogenetic analysis of the hemA homologs revealed strain clustering according to putative type of metabolic product, which could be used to select producers of specific C5N compound classes. Supporting information was acquired through analysis of actinomycete genomic sequence data available in GenBank and further genetic or metabolic characterization of selected strains. Comparison of 16S rRNA taxonomic identification and BOX-PCR profiles provided evidence for numerous horizontal gene transfers of biosynthetic genes or gene clusters within actinomycete populations and even from non-actinomycete organisms. Our results underline the importance of environmental and evolutionary data in the design of efficient techniques for identification of novel producers.

No MeSH data available.


Related in: MedlinePlus

Amino acid sequence alignment of antibiotic-related cyclizing 5-aminolevulinate synthases (ALASs; black) with representatives of standard proteobacterial and human ALAS (gray). Original organisms abbreviated as follows S. – Streptomyces, Sac. – Saccharothrix, K. – Kitasatospora, R. – Rhodobacter, H. – Homo. Amino acid residues directly involved in binding of substrates and the PLP cofactor indicated in bold with arrows (Astner et al., 2005). Residues different from classical ALAS and strictly conserved in cALAS boxed gray and outlined in black when residues are directly connected to the enzyme activity. Conservative amino acid stretches used for the screening PCR method design are indicated.
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Figure 1: Amino acid sequence alignment of antibiotic-related cyclizing 5-aminolevulinate synthases (ALASs; black) with representatives of standard proteobacterial and human ALAS (gray). Original organisms abbreviated as follows S. – Streptomyces, Sac. – Saccharothrix, K. – Kitasatospora, R. – Rhodobacter, H. – Homo. Amino acid residues directly involved in binding of substrates and the PLP cofactor indicated in bold with arrows (Astner et al., 2005). Residues different from classical ALAS and strictly conserved in cALAS boxed gray and outlined in black when residues are directly connected to the enzyme activity. Conservative amino acid stretches used for the screening PCR method design are indicated.

Mentions: Available actinomycete genomes were scanned for putative ALAS-coding regions using a standard BlastP algorithm with well characterized cALAS as queries (cut off set to 40% identity and 60% similarity). The protein sequences retrieved were inspected for conservation of ALAS-typical regions (Figure 1, Supplementary Table S3).


Evolution of cyclizing 5-aminolevulinate synthases in the biosynthesis of actinomycete secondary metabolites: outcomes for genetic screening techniques.

Petříčková K, Chroňáková A, Zelenka T, Chrudimský T, Pospíšil S, Petříček M, Krištůfek V - Front Microbiol (2015)

Amino acid sequence alignment of antibiotic-related cyclizing 5-aminolevulinate synthases (ALASs; black) with representatives of standard proteobacterial and human ALAS (gray). Original organisms abbreviated as follows S. – Streptomyces, Sac. – Saccharothrix, K. – Kitasatospora, R. – Rhodobacter, H. – Homo. Amino acid residues directly involved in binding of substrates and the PLP cofactor indicated in bold with arrows (Astner et al., 2005). Residues different from classical ALAS and strictly conserved in cALAS boxed gray and outlined in black when residues are directly connected to the enzyme activity. Conservative amino acid stretches used for the screening PCR method design are indicated.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4525017&req=5

Figure 1: Amino acid sequence alignment of antibiotic-related cyclizing 5-aminolevulinate synthases (ALASs; black) with representatives of standard proteobacterial and human ALAS (gray). Original organisms abbreviated as follows S. – Streptomyces, Sac. – Saccharothrix, K. – Kitasatospora, R. – Rhodobacter, H. – Homo. Amino acid residues directly involved in binding of substrates and the PLP cofactor indicated in bold with arrows (Astner et al., 2005). Residues different from classical ALAS and strictly conserved in cALAS boxed gray and outlined in black when residues are directly connected to the enzyme activity. Conservative amino acid stretches used for the screening PCR method design are indicated.
Mentions: Available actinomycete genomes were scanned for putative ALAS-coding regions using a standard BlastP algorithm with well characterized cALAS as queries (cut off set to 40% identity and 60% similarity). The protein sequences retrieved were inspected for conservation of ALAS-typical regions (Figure 1, Supplementary Table S3).

Bottom Line: Supporting information was acquired through analysis of actinomycete genomic sequence data available in GenBank and further genetic or metabolic characterization of selected strains.Comparison of 16S rRNA taxonomic identification and BOX-PCR profiles provided evidence for numerous horizontal gene transfers of biosynthetic genes or gene clusters within actinomycete populations and even from non-actinomycete organisms.Our results underline the importance of environmental and evolutionary data in the design of efficient techniques for identification of novel producers.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbiology, Czech Academy of Sciences, v. v. i. Prague, Czech Republic.

ABSTRACT
A combined approach, comprising PCR screening and genome mining, was used to unravel the diversity and phylogeny of genes encoding 5-aminolevulinic acid synthases (ALASs, hemA gene products) in streptomycetes-related strains. In actinomycetes, these genes were believed to be directly connected with the production of secondary metabolites carrying the C5N unit, 2-amino-3-hydroxycyclopent-2-enone, with biological activities making them attractive for future use in medicine and agriculture. Unlike "classical" primary metabolism ALAS, the C5N unit-forming cyclizing ALAS (cALAS) catalyses intramolecular cyclization of nascent 5-aminolevulinate. Specific amino acid sequence changes can be traced by comparison of "classical" ALASs against cALASs. PCR screening revealed 226 hemA gene-carrying strains from 1,500 tested, with 87% putatively encoding cALAS. Phylogenetic analysis of the hemA homologs revealed strain clustering according to putative type of metabolic product, which could be used to select producers of specific C5N compound classes. Supporting information was acquired through analysis of actinomycete genomic sequence data available in GenBank and further genetic or metabolic characterization of selected strains. Comparison of 16S rRNA taxonomic identification and BOX-PCR profiles provided evidence for numerous horizontal gene transfers of biosynthetic genes or gene clusters within actinomycete populations and even from non-actinomycete organisms. Our results underline the importance of environmental and evolutionary data in the design of efficient techniques for identification of novel producers.

No MeSH data available.


Related in: MedlinePlus