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.

Complete Genome Sequence of Latilactobacillus sp. Strain WDN19, a High-d-Aspartate-Producing Lactic Acid Bacterium Isolated from a Japanese Pickle.

We report here the complete genome sequence of Latilactobacillus sp. strain WDN19, isolated from a Japanese pickle. This strain can produce a large amount of d-aspartate in the culture broth. The genome consists of a circular chromosome (1,967,462 bp; GC content, 41.88%) and a circular plasmid (66,648 bp; GC content, 35.08%).

Development of an enzymatic screening method for d-aspartate-producing lactic acid bacteria.

d-Aspartate (d-Asp) is an important intermediate for synthetic penicillin and an endogenous amino acid that plays important roles in the endocrine and nervous systems in animals including humans. Lactic acid bacteria (LABs) have been used as probiotics in humans, and some LAB species produce d-Asp as a component of cell wall peptidoglycan. LAB strains with greater d-Asp production would therefore be valuable for industrial d-Asp production. In this study, we developed an enzymatic screening method for d-Asp-producing LABs and isolated a strain with high d-Asp production. The d-Asp concentration in the culture medium was colorimetrically estimated up to 4 mM using d-aspartate oxidase (ChDDO) from the yeast Cryptococcus humicola strain UJ1 coupled with horseradish peroxidase, although a more accurate determination required correction because of interference by the medium component Mn2+. We isolated 628 LAB strains from various foods and screened them for d-Asp production using the enzymatic d-Asp assay method. The screening identified 13 d-Asp-producing LAB strains, which were suggested to belong to the genera Latilactobacillus, Levilactobacillus, Lactococcus, and Enterococcus. d-Asp production ability was likely to widely differ among the strains in the same genera and species. One strain, named strain WDN19, produced much higher d-Asp levels (1.84 mM), and it was closely related to Latilactobacillus curvatus. These results indicated that the enzymatic screening method was useful for identifying and isolating d-Asp-producing LABs rapidly and easily, and it might provide novel findings regarding d-Asp production by LABs.