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Recently, substantial levels of acidic d-amino acids, such as d-aspartate and d-glutamate, have been identified in many organisms, from bacteria to mammals, suggesting that acidic d-amino acids have multiple physiological significances. Although acidic d-amino acids found in animals primarily originate from foodstuffs and/or bacteria, the d-aspartate-synthesizing enzyme aspartate racemase is identified in various animals. In eukaryotic organisms, acidic d-amino acids are primarily degraded by the flavoenzyme d-aspartate oxidase (DDO). DDO is found in multiple eukaryotic organisms and may play important roles in acidic d-amino acid utilization, elimination, and intracellular level regulation. Moreover, owing to its perfect enantioselectivity and stereoselectivity, DDO may be a valuable tool in several biotechnological applications, including the identification and quantification of acidic d-amino acids. In this mini-review, previous DDO reports are summarized and the potential bioengineering and biotechnological applications of DDO are discussed.Key Points・Occurrence and distribution ofd-aspartate oxidase.・Fundamental properties ofd-aspartate oxidase of various eukaryotic organisms.・Biotechnological applications and potential engineering ofd-aspartate oxidase.

We employed genomic mutagenesis to the yeast Rhodotorula gracilis using Zeocin as a mutagen to develop enhanced carotenoid-producing mutant strains. The yeast mutant strains with enhanced carotenoid pigmentation were produced on agar plates containing Zeocin at a concentration of less than 1.5 µg/mL. The optimum concentration for producing enhanced carotenoid-pigmentation mutant strains was 0.25 µg/mL. The production of β-carotene, torulene, and torularhodin in an enhanced pigmentation strain were 1.3, 1.65, and 1.5 times higher than in the wild-type strain. These results suggested that genomic mutagenesis of the yeast using Zeocin could be applied for efficiently producing enhanced carotenoid-producing mutant strains.

Tris(2-chloroethyl) phosphate (TCEP) is a haloalkyl phosphate flame retardant and plasticizer that has been recognized as a global environmental contaminant. Sphingobium sp. strain TCM1 can utilize TCEP as a phosphorus source. To identify the phosphomonoesterase involved in TCEP utilization, we identified four putative alkaline phosphatase (APase) genes, named SbphoA , SbphoD1 , SbphoD2 , and SbphoX-II , in the genome sequence. Following expression of these genes in Escherichia coli , APase activity was confirmed for the SbphoA and SbphoX-II gene products but was not clearly observed for the SbphoD1 and SbphoD2 gene products, owing to their accumulation in inclusion bodies. The single deletion of either SbphoA or SbphoX-II retarded the growth and reduced the APase activity of strain TCM1 cells on medium containing TCEP as the sole phosphorus source; these changes were more marked in cells with the SbphoX-II gene deletion. In contrast, the deletion of either SbphoD1 or SbphoD2 had no effect on cell growth or APase activity. The double deletion of SbphoA and SbphoX-II resulted in the complete loss of cell growth on TCEP. These results show that SbPhoA and SbPhoX-II are involved in the utilization of TCEP as a phosphorus source and that SbPhoX-II is the major phosphomonoesterase involved in TCEP utilization.

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 .

Sphingobium sp. strain TCM1 can significantly degrade chlorinated organophosphorus flame retardants, such as tris(2-chloroethyl) phosphate. The PhoK of strain TCM1 (Sb-PhoK) is the main alkaline phosphatase (APase) that catalyzes the last step in the degradation pathway. Here, we purified and characterized Sb-PhoK produced in E. coli, and analyzed the regulation of Sb-phoK gene expression in strain TCM1. The recombinant Sb-PhoK was produced in the mature form, lacking a putative signal peptide, and formed a homodimer. Purified Sb-PhoK exhibited 384 U/mg of specific activity at 37 °C. The optimum temperature was 50 °C, and Sb-PhoK was completely inactivated when incubated at 60 °C for 10 min. The optimum pH was 10, with stability observed at pH 6.0–10.5. Sb-PhoK was suggested to contain two Ca2+ and one Zn2+ per subunit, but excess addition of Zn2+ into the reaction mixture markedly inhibited the enzyme activity. Sb-PhoK showed phosphatase activity against various phosphorylated compounds, except for bis(p-nitrophenyl) phosphate, indicating that it is a phosphomonoesterase with broad substrate specificity. The Km and kcat for p-nitrophenyl phosphate were 2.31 mM and 1270 s−1, respectively, under optimal conditions. The enzyme was strongly inhibited by vanadate, dithiothreitol, and SDS, but was highly resistant to urea and Triton X-100. Sb-phoK gene expression was regulated by the inorganic phosphate concentration in culture medium, and was induced at a low inorganic phosphate concentration. The deletion of Sb-phoB gene resulted in no induction of Sb-phoK gene even at a low inorganic phosphate concentration, confirming that Sb-PhoK is a member of Pho regulon.

d- Amino acid oxidase (DAAO) is a valuable flavoenzyme capable of being used in various practical applications, such as in determining d -amino acids and producing a material for semisynthetic cephalosporins, requiring higher thermal stability, higher catalytic activity, and broad substrate specificity. In this study, we isolated the thermophilic fungus Rasamsonia emersonii strain YA, which can grow on several d -amino acids as the sole nitrogen source, from a compost and characterized DAAO (ReDAAO) of the fungus. ReDAAO expressed in Escherichia coli exhibited significant oxidase activity against various neutral and basic d- amino acids, in particular hydrophobic d -amino acids. In addition, the enzyme also significantly acted on cephalosporin C, a starting material for semisynthetic antibiotics, and d- Glu, a general substrate for d- aspartate oxidase but not for DAAO, showing its unique and practically useful substrate specificity. The apparent k cat and K m values of the enzyme toward good substrates were comparable to those of higher catalytic fungal DAAOs, and the thermal stability ( T 50 value of ~60 °C) was comparable to that of a thermophilic bacterial DAAO and significantly higher than that of other eukaryotic DAAOs. These results highlight the great potential of ReDAAO for use in practical applications.

d -Aspartate oxidase (DDO) is a valuable enzyme that can be utilized in the determination of acidic d -amino acids and the optical resolution of a racemic mixture of acidic amino acids, which require its higher stability, higher catalytic activity, and higher substrate-binding affinity. In the present study, we identified DDO gene (TdDDO) of a thermophilic fungus, Thermomyces dupontii , and characterized the recombinant enzyme expressed in Escherichia coli . In addition, we generated a variant that has a higher substrate-binding affinity. The recombinant TdDDO expressed in E. coli exhibited oxidase activity toward acidic d -amino acids and a neutral d -amino acid, d -Gln, with the highest activity toward d -Glu. The K m and k cat values for d -Glu were 2.16 mM and 217 s −1 , respectively. The enzyme had an optimum pH and temperature 8.0 and 60 °C, respectively, and was stable between pH 5.0 and 10.0, with a T 50 of ca. 51 °C, which was much higher than that in DDOs from other origins. Enzyme stability decreased following a decrease in protein concentration, and externally added FAD could not repress the destabilization. The mutation of Phe248, potentially located in the active site of TdDDO, to Tyr residue, conserved in DDOs and d -amino acid oxidases, markedly increased substrate-binding affinity. The results showed the great potential of TdDDO and the variant for practical applications.

Sphingobium sp. strain TCM1 can degrade tris(2-chloroethyl) phosphate (TCEP) to inorganic phosphate and 2-chloroethanol. A phosphotriesterase (PTE), phosphodiesterase (PDE) and phosphomonoesterase (PME) are believed to be involved in the degradation of TCEP. The PTE and PME that respectively catalyze the first and third steps of TCEP degradation in TCM1 have been identified. However, no information has been reported on a PDE catalyzing the second step. In this study, we identified, purified, and characterized a PDE capable of hydrolyzing haloalkyl phosphate diesters. The final preparation of the enzyme had a specific activity of 29 µmol min −1  mg −1 with bis( p -nitrophenyl) phosphate (B p NPP) as the substrate. It also possessed low PME activity with p -nitrophenyl phosphate ( p NPP) as substrate. The catalytic efficiency ( k cat / K m ) with B p NPP was significantly higher than that with p NPP, indicating that the enzyme prefers the organophosphorus diester to the monoester. The enzyme degraded bis(2,3-dibromopropyl) phosphate, bis(1,3-dichloro-2-propyl) phosphate and bis(2-chloroethyl) phosphate, suggesting that it is involved in the metabolism of haloalkyl organophosphorus triesters. The primary structure of the PDE from TCM1 is distinct from those of typical PDE family members and the enzyme belongs to the polymerase and histidinol phosphatase superfamily.

d-Aspartate oxidase (DDO) is a peroxisomal flavoenzyme that catalyzes the oxidative deamination of acidic d-amino acids. In the yeast Cryptococcus humicola strain UJ1, the enzyme ChDDO is essential for d-Asp utilization and is expressed only in the presence of d-Asp. Pyruvate carboxylase (Pyc) catalyzes the conversion of pyruvate to oxaloacetate and is involved in the import and activation of certain peroxisomal flavoenzymes in yeasts. In this study, we analyzed the role of Pyc in the expression of ChDDO gene in C. humicola strain UJ1. PYC gene disruption (∆Chpyc1) in strain UJ1 resulted in growth retardation on glucose and NH4Cl medium. The growth was restored by supplying oxaloacetate from l-Asp or α-ketoglutarate by a transaminase. On the other hand, the supply of oxaloacetate from d-Asp by ChDDO was not able to prevent growth retardation because of a significant decrease in ChDDO gene expression at the transcriptional level. The addition of pyruvate significantly decreased ChDDO gene transcription in the ∆Chpyc1 strain but increased the same in the wild-type strain, even though the intracellular pyruvate content was similar in both strains. These results suggest that ChDDO gene expression might be regulated by pyruvate metabolism, as well as by the presence of d-Asp.

d-aspartate oxidase (DDO) catalyzes the oxidative deamination of acidic d-amino acids, and its production is induced by d-Asp in several eukaryotes. The yeast Cryptococcus humicola strain UJ1 produces large amounts of DDO (ChDDO) only in the presence of d-Asp. In this study, we analyzed the relationship between d-Asp uptake by an amino acid permease (Aap) and the inducible expression of ChDDO. We identified two acidic Aap homologs, named “ChAap4 and ChAap5,” in the yeast genome sequence. ChAAP4 deletion resulted in partial growth defects on d-Asp as well as l-Asp, l-Glu, and l-Phe at pH 7, whereas ChAAP5 deletion caused partial growth defects on l-Phe and l-Lys, suggesting that ChAap4 might participate in d-Asp uptake as an acidic Aap. Interestingly, the growth of the Chaap4 strain on d- or l-Asp was completely abolished at pH 10, suggesting that ChAap4 is the only Aap responsible for d- and l-Asp uptake under high alkaline conditions. In addition, ChAAP4 deletion significantly decreased the induction of DDO activity and ChDDO transcription in the presence of d-Asp. This study revealed that d-Asp uptake by ChAap4 might be involved in the induction of ChDDO expression by d-Asp.