Identification of the Last Unknown Genes in the Fermentation Pathway of Lysine

Abstract

Although the proteins of the lysine fermentation pathway were biochemically characterized more than thirty years ago, the genes encoding the proteins that catalyze three steps of this pathway are still unknown. We combined gene context, similarity of enzymatic mechanisms, and molecular weight comparisons with known proteins to select candidate genes for these three orphan proteins. We used a wastewater metagenomic collection of sequences to find and characterize the missing genes of the lysine fermentation pathway. After recombinant protein production and purification following cloning in Escherichia coli, we demonstrated that these genes (named kdd, kce, and kal) encode a L-erythro-3,5-diaminohexanoate dehydrogenase, a 3-keto-5-aminohexanoate cleavage enzyme, and a 3-aminobutyryl-CoA ammonia lyase, respectively. Because all of the genes of the pathway are now identified, we used this breakthrough to detect lysine-fermenting bacteria in sequenced genomes. We identified twelve bacteria that possess these genes and thus are expected to ferment lysine, and their gene organization is discussed.

Metabolic diversity among main microorganisms inside an arsenic-rich ecosystem revealed by meta- and proteo-genomics

“…Second, and unlike other Carnoulès bins, their genome carries drm and punA genes, suggesting that they can use purine as a carbon source (Schuch et al, 1999). Third, genes involved in amino acid fermentation were also identified in both bins, for example those of the lysine pathway, which converts lysine into butyrate, acetate and ammonia (Kreimeyer et al, 2007). Fourth, the CARN1 and CARN4 bins of 'Candidatus Fodinabacter communificans' carry the arginase-encoding gene rocF involved in urea biosynthesis, while a complete urease-encoding ure operon was present in Thiomonas sp.…”

New Insights into the Alternative d-Glucarate Degradation Pathway

“…The corresponding vectors were transformed into BL21 DE3 cells and the proteins produced and purified using immobilized metal affinity chromatography, anion exchange, and gel filtration. Sorbitol and betaine were added to the culture medium to improve the yield of soluble and properly folded recombinant protein (18). Under these conditions, at least 3 mg of pure and soluble protein were obtained per liter of culture.…”

Assignment of function to a domain of unknown function: DUF1537 is a new kinase family in catabolic pathways for acid sugars

“…Many of the widespread DUFs in bacteria are biologically essential, and the importance of prioritizing these DUFs has been recognized in recent years (10). Before this study, only one DUF family, the DUF849 family of 922 proteins, had been evaluated in large scale by a single report (11), which heavily relied on previously published enzymatic activity and catalytic mechanism/ liganded structure of one of its members (35,36). Recognizing the difficulties associated with DUF functional assignment, we attempt to apply an integrated "genomic enzymology" strategy that had only been used for functional characterization of members of families with known functions (37,38).…”