Identification of a species-specific aminotransferase in Pediococcus acidilactici capable of forming α-aminobutyrate

Abstract

During cheese ripening, the bacterial strain Pediococcus acidilactici FAM18098 produces the non-proteinogenic amino acid, α-aminobutyrate (AABA). The metabolic processes that lead to the biosynthesis of this compound are unknown. In this study, 10 P. acidilactici, including FAM18098 and nine Pediococcus pentosaceus strains, were screened for their ability to produce AABA. All P. acidilactici strains produced AABA, whereas the P. pentosaceus strains did not. The genomes of the pediococcal strains were sequenced and searched for genes encoding aminotransferases to test the hypothesis that AABA could result from the transamination of α-ketobutyrate. A GenBank and KEGG database search revealed the presence of a species-specific aminotransferase in P. acidilactici. The gene was cloned and its gene product was produced as a His-tagged fusion protein in Escherichia coli to determine the substrate specificity of this enzyme. The purified recombinant protein showed aminotransferase activity at pH 5.5. It catalyzed the transfer of the amino group from leucine, methionine, AABA, alanine, cysteine, and phenylalanine to the amino group acceptor α-ketoglutarate. Αlpha-ketobutyrate could replace α-ketoglutarate as an amino group acceptor. In this case, AABA was produced at significantly higher levels than glutamate. The results of this study show that P. acidilactici possesses a novel aminotransferase that might play a role in cheese biochemistry and has the potential to be used in biotechnological processes for the production of AABA.

Mass spectrometry imaging–based assays for aminotransferase activity reveal a broad substrate spectrum for a previously uncharacterized enzyme

“…3 , S3 and S4 ). AABA has a similar structure to that as Ala, and other ATs that were able to use Leu, Ala, Met, and Phe are also able to use AABA as an amino donor ( 41 ). This finding also demonstrates that the easily scalable nature of the oxime MS assay enables detection of novel AT activity and also provides rich experimental data to facilitate structure–function analyses of the AT enzyme family.…”

The aminotransferase Aat initiates 3-phenyllactic acid biosynthesis in Pediococcus acidilactici

“…Both compounds, PLA and HPLA, exhibit antifungal activity (Lavermicocca et al, 2000). Additionally, it possesses the genes of the arginine deiminase pathway, which converts arginine to ornithine (Eugster et al, 2019;Wenger et al, 2020). This metabolic pathway releases carbon dioxide which could influence the eye formation in cheese.…”

“…This metabolic pathway releases carbon dioxide which could influence the eye formation in cheese. Finally, P. acidilactici can degrade serine and threonine and, in turn, form alanine and α-aminobutyric acid in medium and cheese (Eugster et al, 2019;Wenger et al, 2020). We assume that serine and threonine are converted to alanine and α-aminobutyric acid, respectively.…”

“…When we searched the genome sequence of P. acidilactici strain FAM 18098 for genes encoding aminotransferases, an aminotransferase class I/II-fold pyridoxal phosphate-dependent enzyme (GenBank Protein WP_159211138) was identified that may be involved in the formation of α-aminobutyric acid and alanine (Wenger et al, 2020). In order to analyze the enzymatic activity of this aminotransferase, the gene was cloned and overexpressed in Escherichia coli.…”

Roadmap for the integration of environmental microbiomes in risk assessments under EFSA's remit

“…Concurrently to the pursuit of these objectives, the Agroscope strain collections was to be characterised using HTS to identify their potential and enable their targeted use in agriculture and food sectors. The project was able to analyse bacterial species involved in cheese ripening (Moser et al, 2018;Wenger et al, 2020), study fungal biological control agents to control pest arthropods (Mayerhofer et al, 2019;Fernández-Bravo et al, 2021), and detect and characterise insect (Pedrazzini et al, 2021) and nematode (Koutsovoulos et al, 2020) The present document has been produced and adopted by the bodies identified above as author(s). In accordance with Article 36 of Regulation (EC) No 178/2002, this task has been carried out exclusively by the author(s) in the context of a grant agreement between the European Food Safety Authority and the author(s).…”