Novel Metabolic Pathway for N -Methylpyrrolidone Degradation in Alicycliphilus sp. Strain BQ1

Abstract

The molecular mechanisms underlying the biodegradation of -methylpyrrolidone (NMP), a widely used industrial solvent that produces skin irritation in humans and is teratogenic in rats, are unknown. sp. strain BQ1 degrades NMP. By studying a transposon-tagged mutant unable to degrade NMP, we identified a six-gene cluster () that is transcribed as a polycistronic mRNA and encodes enzymes involved in NMP biodegradation. and the transposon-affected gene encode an -methylhydantoin amidohydrolase that transforms NMP to γ--methylaminobutyric acid; this is metabolized by an amino acid oxidase (NMPC), either by demethylation to produce γ-aminobutyric acid (GABA) or by deamination to produce succinate semialdehyde (SSA). If GABA is produced, the activity of a GABA aminotransferase (GABA-AT), not encoded in the gene cluster, is needed to generate SSA. SSA is transformed by a succinate semialdehyde dehydrogenase (SSDH) (NMPF) to succinate, which enters the Krebs cycle. The abilities to consume NMP and to utilize it for growth were complemented in the transposon-tagged mutant by use of the genes. Similarly, MG1655, which has two SSDHs but is unable to grow in NMP, acquired these abilities after functional complementation with these genes. In wild-type (wt) BQ1 cells growing in NMP, GABA was not detected, but SSA was present at double the amount found in cells growing in Luria-Bertani medium (LB), suggesting that GABA is not an intermediate in this pathway. Moreover, GABA-AT deletion mutants complemented with genes retained the ability to grow in NMP, supporting the possibility that γ--methylaminobutyric acid is deaminated to SSA instead of being demethylated to GABA.-Methylpyrrolidone is a cyclic amide reported to be biodegradable. However, the metabolic pathway and enzymatic activities for degrading NMP are unknown. By developing molecular biology techniques for sp. strain BQ1, an environmental bacterium able to grow in NMP, we identified a six-gene cluster encoding enzymatic activities involved in NMP degradation. These findings set the basis for the study of new enzymatic activities and for the development of biotechnological processes with potential applications in bioremediation.

Degradation of Recalcitrant Polyurethane and Xenobiotic Additives by a Selected Landfill Microbial Community and Its Biodegradative Potential Revealed by Proximity Ligation-Based Metagenomic Analysis

“…Exploring the BP8 metagenome, genes encoding enzymes presumably involved in the degradation of the PolyLack R additives were identified in several of the deconvoluted genomes. Genes for NMP degradation, similar to the ones reported for A. denitrificans BQ1 (Solís-González et al, 2018) were identified in the Paracoccus sp. BP8 genome.…”

Alicycliphilus : current knowledge and potential for bioremediation of xenobiotics

“…(, ) and Solís‐González et al . (). Anaerobic degradation of cyclohexanol in A. denitrificans K601 begins with its oxidation to cycohexanone catalysed by (1) cyclohexanol dehydrogenase, followed by the formation of 2‐cyclohexenone, performed by (2) cyclohexanone dehydrogenase.…”

“…In strain B1, but not in strain BQ1, a transposase‐encoding gene was identified next to the nmpA gene homologue, suggesting a horizontal transfer mechanism for the acquisition of this cluster in B1 strain (Solís‐González et al . ).…”

“…) were transferred for complementing a NMP − mutant in the strain BQ1 (Solís‐González et al . ). Unfortunately, the number of exconjugants obtained is very low, despite trying different conditions to improve the methodology, suggesting a natural recalcitrance in Alicycliphilus for acquiring exogenous DNA, and making this process complex and inefficient.…”

“…BQ1 (Solís‐González et al . ) and A. denitrificans strains BC and K601 (Loza‐Tavera Lab, unpublished) was successful by bi‐ or tri‐parental mating using different mobilizable plasmids and E. coli S17‐1, as donor strain. With this approach, a mini Tn5 transposon mutant library using the pUT/mini‐Tn5 Km R suicide plasmid was constructed in Alicycliphilus sp.…”