Metagenomes of complex microbial consortia derived from different soils as sources for novel genes conferring formation of carbonyls from short-chain polyols on Escherichia coli.
Metagenomic DNA libraries from three different soil samples (meadow, sugar beet field, cropland) were constructed. The three unamplified libraries comprised approximately 1267000 independent clones and harbored approximately 4.05 Gbp of environmental DNA. Approximately 300000 recombinant Escherichia coli strains of each library per test substrate were screened for the production of carbonyls from short-chain (C2 to C4) polyols such as 1,2-ethanediol, 2,3-butanediol, and a mixture of glycerol and 1,2-propanediol on indicator agar. Twenty-four positive E. COLI clones were obtained during the initial screen. Fifteen of them contained recombinant plasmids, designated pAK201-215, which conferred a stable carbonyl-forming phenotype on E. coli Sequencing revealed that the inserts of pAK201-215 encoded 26 complete and 14 incomplete predicted protein-encoding genes. Most of these genes were similar to genes with unknown functions from other microorganisms or unrelated to any other known gene. The further analysis was focused on the 7 plasmids (pAK204, pAK206, pAK208, and pAK210-213) recovered from the positive clones, which exhibited an NAD(H)-dependent alcohol oxidoreductase activity with polyols or the correlating carbonyls as substrates in crude extracts. Three genes (ORF6, ORF24, and ORF25) conferring this activity were identified during subcloning of the inserts of pAK204, pAK211, and pAK212. The sequences of the three deduced gene products revealed no significant similarities to known alcohol oxidoreductases, but contained putative glycine-rich regions, which are characteristic for binding of nicotinamide cofactors.
Knietsch
Anja
A
Abteilung Allgemeine Mikrobiologie, Göttingen, Germany.
Waschkowitz
Tanja
T
Bowien
Susanne
S
Henne
Anke
A
Daniel
Rolf
R
D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology / Q000235:genetics* / Q000302:isolation & purification; D003001:Cloning, Molecular; D003470:Culture Media; D004269:DNA, Bacterial / Q000235:genetics; D017753:Ecosystem; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015698:Genomic Library; D008969:Molecular Sequence Data; D011108:Polymers / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D012988:Soil Microbiology
D003470:Culture Media; D004269:DNA, Bacterial; D011108:Polymers; C024617:polyol; D000429:Alcohol Oxidoreductases