Mechanism of high D-aspartate production in the lactic acid bacterium Latilactobacillus sp. strain WDN19.

D-Aspartate (D-Asp) is a useful compound for a semisynthetic antibiotic and has potentially beneficial effects on humans. Several lactic acid bacteria (LAB) species produce D-Asp as a component of cell wall peptidoglycan. We previously isolated a LAB strain (named strain WDN19) that can extracellularly produce a large amount of D-Asp. Here, we show the factors that contribute to high D-Asp production ability. Strain WDN19 was most closely related to Latilactobacillus curvatus. The D-Asp production ability of strain WDN19 in a rich medium was 13.7-fold higher than that of L. curvatus DSM 20019. A major part of D-Asp was synthesized from L-Asp contained in the medium by aspartate racemase (RacD). During their cultivation, the RacD activity in strain WDN19 was higher than in strain DSM 20019, especially much higher in the early exponential growth phase because of the higher racD transcription and the higher activity of RacD itself of strain WDN19. In a synthetic medium, the extracellular production of D,L-Asp was observed in strain WDN19 but not in strain DSM 20019. The addition of L-asparagine (L-Asn) to the medium increased and gave D,L-Asp production in strains WDN19 and DSM 20019, respectively, suggesting L-Asp synthesis by L-asparaginase (AsnA). The L-Asn uptake ability of the strains was similar, but the AsnA activity in the middle exponential and early stationary growth phases and intracellular D,L-Asp was much higher in strain WDN19. In their genome sequences, only an aspartate aminotransferase gene was found among L-Asp-metabolizing enzymes, except for RacD, but was disrupted in strain WDN19 by transposon insertion. These observations indicated that the high D-Asp production ability of strain WDN19 was mainly based on high RacD and AnsA activities and L-Asp supply. KEY POINTS: • Strain WDN19 was suggested to be a strain of Latilactobacillus curvatus. • Extracellular high d-Asp production ability was not a common feature of L. curvatus. • High d-Asp production was due to high RacD and AnsA activities and l-Asp supply.