Profiling of d-alanine production by the microbial isolates of rat gut microbiota

Cindy J. Lee, Tian A. Qiu, Zhilai Hong, Zhenkun Zhang, Yuhao Min, Linzixuan Zhang, Lei Dai, Huimin Zhao, Tong Si, Jonathan V. Sweedler

Research output: Contribution to journalArticlepeer-review


d-alanine (d-Ala) and several other d-amino acids (d-AAs) act as hormones and neuromodulators in nervous and endocrine systems. Unlike the endogenously synthesized d-serine in animals, d-Ala may be from exogenous sources, e.g., diet and intestinal microorganisms. However, it is unclear if the capability to produce d-Ala and other d-AAs varies among different microbial strains in the gut. We isolated individual microorganisms of rat gut microbiota and profiled their d-AA production in vitro, focusing on d-Ala. Serial dilutions of intestinal contents from adult male rats were plated on agar to obtain clonal cultures. Using MALDI-TOF MS for rapid strain typing, we identified 38 unique isolates, grouped into 11 species based on 16S rRNA gene sequences. We then used two-tier screening to profile bacterial d-AA production, combining a d-amino acid oxidase-based enzymatic assay for rapid assessment of non-acidic d-AA amount and chiral LC–MS/MS to quantify individual d-AAs, revealing 19 out of the 38 isolated strains as d-AA producers. LC–MS/MS analysis of the eight top d-AA producers showed high levels of d-Ala in all strains tested, with substantial inter- and intra-species variations. Though results from the enzymatic assay and LC–MS/MS analysis aligned well, LC–MS/MS further revealed the existence of d-glutamate and d-aspartate, which are poor substrates for this enzymatic assay. We observed large inter- and intra-species variation of d-AA production profiles from rat gut microbiome species, demonstrating the importance of chemical profiling of gut microbiota in addition to sequencing, furthering the idea that microbial metabolites modulate host physiology.

Original languageEnglish (US)
Article numbere22446
JournalFASEB Journal
Issue number8
StatePublished - Aug 2022


  • amino acid
  • gastrointestinal microbiome
  • host microbial interactions
  • mass spectrometry
  • rats

ASJC Scopus subject areas

  • Genetics
  • Molecular Biology
  • Biochemistry
  • Biotechnology


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