Identification and enzymatic properties of arginine decarboxylase from Aspergillus oryzae.

Aspergillus oryzae spores, when sprinkled onto steamed rice and allowed to propagate, are referred to as rice "koji." Agmatine, a natural polyamine derived from arginine through the action of arginine decarboxylase (ADC), is abundantly produced by solid state-cultivated rice koji of A. oryzae RIB40 under low pH conditions, despite the apparent absence of ADC orthologs in its genome. Mass spectrometry imaging revealed that agmatine was accumulated inside rice koji at low pH conditions, where arginine was distributed. ADC activity was predominantly observed in substrate mycelia and minimally in aerial mycelia. Natural ADC was isolated from solid state-cultivated A. oryzae rice koji containing substrate mycelia, using ammonium sulfate fractionation, ion exchange, and gel-filtration chromatography. The purified protein was subjected to sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE), and the detected peptide band was digested for identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The gene AO090102000327 of strain RIB40 was identified, previously annotated as phosphatidylserine decarboxylase (PSD), and encoded a 483-amino acid peptide. Recombinant protein encoded by AO090102000327 was expressed in Escherichia coli cells cultivated at 20°C, resulting in the detection of 49 kDa and 5 kDa peptides. The protein exhibited pyruvoyl-dependent decarboxylase activity, favoring arginine over ornithine and showing no activity with phosphatidylserine. The gene was designated Ao-adc1. Ao-ADC1 expression in rice koji at pH 4-6 was confirmed through western blotting using the anti-Ao-ADC1 serum. These findings indicate that Ao-adc1 encodes arginine decarboxylase involved in agmatine production.IMPORTANCEGene AO090102000327 in A. oryzae RIB40, previously annotated as a PSD, falls into a distinct clade when examining the phylogenetic distribution of PSDs. Contrary to the initial PSD annotation, our analysis indicates that the protein encoded by AO090102000327 is expressed in the substrate mycelia area of solid state-cultivated A. oryzae rice koji and functions as an arginine decarboxylase (ADC). The clade to which Ao-ADC1 belongs includes three other Ao-ADC1 paralogs (AO090103000445, AO090701000800, and AO090701000802) that presumably encode ADC rather than PSDs. Regarding PSD, AO090012000733 and AO090005001124 were speculated to be nonmitochondrial and mitochondrial PSDs in A. oryzae RIB40, respectively.

Leucine-Responsive Regulatory Protein in Acetic Acid Bacteria Is Stable and Functions at a Wide Range of Intracellular pH Levels.

Acetic acid bacteria grow while producing acetic acid, resulting in acidification of the culture. Limited reports elucidate the effect of changes in intracellular pH on transcriptional factors. In the present study, the intracellular pH of Komagataeibacter europaeus was monitored with a pH-sensitive green fluorescent protein, showing that the intracellular pH decreased from 6.3 to 4.7 accompanied by acetic acid production during cell growth. The leucine-responsive regulatory protein of K. europaeus (KeLrp) was used as a model to examine pH-dependent effects, and its properties were compared with those of the Escherichia coli ortholog (EcLrp) at different pH levels. The DNA-binding activities of EcLrp and KeLrp with the target DNA (Ec-ilvI and Ke-ilvI) were examined by gel mobility shift assays under various pH conditions. EcLrp showed the highest affinity with the target at pH 8.0 (Kd [dissociation constant], 0.7 µM), decreasing to a minimum of 3.4 µM at pH 4.0. Conversely, KeLrp did not show significant differences in binding affinity between pH 4 and 7 (Kd, 1.0 to 1.5 µM), and the highest affinity was at pH 5.0 (Kd, 1.0 µM). Circular dichroism spectroscopy revealed that the α-helical content of KeLrp was the highest at pH 5.0 (49%) and was almost unchanged while being maintained at >45% over a range of pH levels examined, while that of EcLrp decreased from its maximum (49% at pH 7.0) to its minimum (36% at pH 4.0). These data indicate that KeLrp is stable and functions over a wide range of intracellular pH levels. IMPORTANCE Lrp is a highly conserved transcriptional regulator found in bacteria and archaea and regulates transcriptions of various genes. The intracellular pH of acetic acid bacteria (AAB) changes accompanied by acetic acid production during cell growth. The Lrp of AAB K. europaeus (KeLrp) was structurally stable over a wide range of pH and maintained DNA-binding activity even at low pH compared with Lrp from E. coli living in a neutral environment. An in vitro experiment showed DNA-binding activity of KeLrp to the target varied with changes in pH. In AAB, change of the intracellular pH during a cell growth would be an important trigger in controlling the activity of Lrp in vivo.

The therapeutic and nutraceutical potential of agmatine, and its enhanced production using Aspergillus oryzae.

Agmatine, a natural polyamine produced from arginine by arginine decarboxylase, was first discovered in 1910, but its physiological significance was disregarded for a century. The recent rediscovery of agmatine as an endogenous ligand for α2-adrenergic and imidazoline receptors in the mammalian brain suggests that this amine may be a promising therapeutic agent for treating a broad spectrum of central nervous system-associated diseases. In the past two decades, numerous preclinical and several clinical studies have demonstrated its pleiotropic modulatory functions on various molecular targets related to neurotransmission, nitric oxide synthesis, glucose metabolism, polyamine metabolism, and carnitine biosynthesis, indicating potential for therapeutic applications and use as a nutraceutical to improve quality of life. An enzymatic activity of arginine decarboxylase which produces agmatine from arginine was low in mammals, suggesting that a large portion of the agmatine is supplemented from diets and gut microbiota. In the present review, we focus on and concisely summarize the beneficial effects of agmatine for treating depression, anxiety, neuropathic pain, cognitive decline and learning impairment, dependence on drugs, and metabolic diseases (diabetes and obesity), since these fields have been intensively investigated. We also briefly discuss agmatine content in foodstuffs, and a simple approach for enhancing agmatine production using the filamentous fungus Aspergillus oryzae, widely used for the production of various Asian fermented foods.

Agmatine Production by Aspergillus oryzae Is Elevated by Low pH during Solid-State Cultivation.

Sake (rice wine) produced by multiple parallel fermentation (MPF) involving Aspergillus oryzae (strain RW) and Saccharomyces cerevisiae under solid-state cultivation conditions contained 3.5 mM agmatine, while that produced from enzymatically saccharified rice syrup by S. cerevisiae contained <0.01 mM agmatine. Agmatine was also produced in ethanol-free rice syrup prepared with A. oryzae under solid-state cultivation (3.1 mM) but not under submerged cultivation, demonstrating that A. oryzae in solid-state culture produces agmatine. The effect of cultivation conditions on agmatine production was examined. Agmatine production was boosted at 30°C and reached the highest level (6.3 mM) at pH 5.3. The addition of l-lactic, succinic, and citric acids reduced the initial culture pHs to 3.0, 3.5, and 3.2, respectively, resulting in a further increase in agmatine accumulation (8.2, 8.7, and 8.3 mM, respectively). Homogenate from a solid-state culture exhibited a maximum l-arginine decarboxylase (ADC) activity (74 pmol · min-1 · µg-1) at pH 3.0 at 30°C; homogenate from a submerged culture exhibited an extremely low activity (<0.3 pmol · min-1 · µg-1) under all conditions tested. These observations indicated that efficient agmatine production in ethanol-free rice syrup is achieved by an unidentified low-pH-dependent ADC induced during solid-state cultivation of A. oryzae, even though A. oryzae lacks ADC orthologs and instead possesses four ornithine decarboxylases (ODC1 to ODC4). Recombinant ODC1 and ODC2 exhibited no ADC activity at acidic pH (pH < 4.0), suggesting that other decarboxylases or an unidentified ADC is involved in agmatine production.IMPORTANCE It has been speculated that, in general, fungi do not synthesize agmatine from l-arginine because they do not possess genes encoding arginine decarboxylase. Numerous preclinical studies have shown that agmatine exerts pleiotropic effects on various molecular targets, leading to an improved quality of life. In the present study, we first demonstrated that l-arginine was a feasible substrate for agmatine production by the fungus Aspergillus oryzae RW. We observed that the productivity of agmatine by A. oryzae RW was elevated at low pH only during solid-state cultivation. A. oryzae is utilized in the production of various Asian fermented foods. The saccharification conditions optimized in the current study could be employed not only in the production of an agmatine-containing ethanol-free rice syrup but also in the production of many types of fermented foods, such as soy sauce (shoyu), rice vinegar, etc., as well as for use as novel therapeutic agents and nutraceuticals.

Leucine responsive regulatory protein is involved in methionine metabolism and polyamine homeostasis in acetic acid bacterium Komagataeibacter europaeus.

The leucine responsive regulatory protein (Lrp) is a global transcription factor that regulates the expression of genes involved in amino acid metabolism. To identify metabolic pathways and related genes under the control of Lrp in the acetic acid bacterium Komagataeibacter europaeus, the Kelrp null mutant (KGMA7110), which requires supplementation of all 20 amino acids for normal growth, was cultivated in minimal media containing or lacking particular amino acids. The results confirmed that KGMA7110 was auxotrophic for methionine and its catabolites S-adenosylmethionine (SAM) and spermidine (SPD). Quantitative reverse-transcription PCR analysis revealed lower metK (SAM synthetase) and mdtI (SPD efflux pump) expression in KGMA7110 than in wild-type KGMA0119. By contrast, these genes were significantly up-regulated in the Kelrp mutant lacking the putative C-terminal ligand-sensing domain (KGMA7203), indicating abnormal regulation of target genes by the KeLrp variant in KGMA7203. KGMA7110 (0.69±0.27 µM) and KGMA7203 (4.90±0.61 µM) excreted lower and higher quantities of SPD, respectively, than KGMA0119 (2.28±0.26 µM). This was attributed to imbalanced carbon flow caused by Kelrp disruption that respectively attenuated and stimulated metK and mdtI expression. These findings indicate that KeLrp plays a key role in SAM biosynthesis and intracellular polyamine homeostasis in K. europaeus.

Polyamines in brown rice vinegar function as potent attractants for the spotted wing drosophila.

Vinegar produced by acetic acid bacteria is used as an attractant for fruit flies. Apple cider vinegar (ACV) and brown rice vinegar (BRV) are used as lures to detect Drosophila suzukii (also known as the spotted wing drosophila [SWD], a newly emerging invasive pest of soft-skinned fruits) and to capture Drosophila melanogaster, respectively. In the present study, we evaluated the attractiveness of BRV and ACV to SWD in laboratory trapping experiments using an upturned microcentrifuge tube with a pipette tip as a trap. We transferred SWD (approximately 20, 7-10 days old) to a glass vial containing a trap baited with BRV or ACV and counted the captured flies. BRV attracted more flies (52.88 ± 9.75%) than ACV (35.78 ± 7.47%) in 6 h. Based on high-performance liquid chromatography, we found that BRV contained greater amounts of putrescine (12.36 ± 0.44 µM) and spermidine (35.08 ± 4.34 µM) than ACV (putrescine, 0.31 ± 0.067 µM; spermidine, not detected). The attractiveness of ACV supplemented with putrescine (12 µM) and spermidine (35 µM) (68.56 ± 4.69%) was significantly higher than that of ACV, indicating that the enhanced attractiveness of BRV to SWD was accomplished by the additive effects of polyamines and other known attractive volatiles, such as acetic acid and acetoin. BRV is expected to be a powerful tool for the efficient management of SWD.

Change in the plasmid copy number in acetic acid bacteria in response to growth phase and acetic acid concentration.

Plasmids pGE1 (2.5 kb), pGE2 (7.2 kb), and pGE3 (5.5 kb) were isolated from Gluconacetobacter europaeus KGMA0119, and sequence analyses revealed they harbored 3, 8, and 4 genes, respectively. Plasmid copy numbers (PCNs) were determined by real-time quantitative PCR at different stages of bacterial growth. When KGMA0119 was cultured in medium containing 0.4% ethanol and 0.5% acetic acid, PCN of pGE1 increased from 7 copies/genome in the logarithmic phase to a maximum of 12 copies/genome at the beginning of the stationary phase, before decreasing to 4 copies/genome in the late stationary phase. PCNs for pGE2 and pGE3 were maintained at 1-3 copies/genome during all phases of growth. Under a higher concentration of ethanol (3.2%) the PCN for pGE1 was slightly lower in all the growth stages, and those of pGE2 and pGE3 were unchanged. In the presence of 1.0% acetic acid, PCNs were higher for pGE1 (10 copies/genome) and pGE3 (6 copies/genome) during the logarithmic phase. Numbers for pGE2 did not change, indicating that pGE1 and pGE3 increase their PCNs in response to acetic acid. Plasmids pBE2 and pBE3 were constructed by ligating linearized pGE2 and pGE3 into pBR322. Both plasmids were replicable in Escherichia coli, Acetobacter pasteurianus and G. europaeus, highlighting their suitability as vectors for acetic acid bacteria.

Effective trapping of fruit flies with cultures of metabolically modified acetic acid bacteria.

Acetoin in vinegar is an attractant to fruit flies when combined with acetic acid. To make vinegar more effective in attracting fruit flies with increased acetoin production, Komagataeibacter europaeus KGMA0119 was modified by specific gene disruption of the acetohydroxyacid isomeroreductase gene (ilvC). A previously constructed mutant lacking the putative ligand-sensing region in the leucine-responsive regulatory protein (KeLrp, encoded by Kelrp) was also used. The ilvC and Kelrp disruptants (KGMA5511 and KGMA7203, respectively) produced greater amounts of acetoin (KGMA5511, 0.11%; KGMA7203, 0.13%) than the wild-type strain KGMA0119 (0.069%). KGMA7203 produced a trace amount of isobutyric acid (0.007%), but the other strains did not. These strains produced approximately equal amounts of acetic acid (0.7%). The efficiency of fruit fly attraction was investigated with cultured Drosophila melanogaster. D. melanogaster flies (approximately 1,500) were released inside a cage (2.5 m by 2.5 m by 1.5 m) and were trapped with a device containing vinegar and a sticky sheet. The flies trapped on the sticky sheet were counted. The cell-free supernatant from KGMA7203 culture captured significantly more flies (19.36 to 36.96% of released flies) than did KGMA0119 (3.25 to 11.40%) and KGMA5511 (6.87 to 21.50%) cultures. Contrastingly, a 0.7% acetic acid solution containing acetoin (0.13%) and isobutyric acid (0.007%), which mimicked the KGMA7203 supernatant, captured significantly fewer flies (0.88 to 4.57%). Furthermore, the KGMA0119 supernatant with additional acetoin (0.13%) and isobutyric acid (0.007%) captured slightly more flies than the original KGMA0119 supernatant but fewer than the KGMA7203 supernatant, suggesting that the synergistic effects of acetic acid, acetoin, isobutyric acid, and unidentified metabolites achieved the efficient fly trapping of the KGMA7203 supernatant.

Enhanced production of branched-chain amino acids by Gluconacetobacter europaeus with a specific regional deletion in a leucine responsive regulator.

Vinegar with increased amounts of branched-chain amino acids (BCAAs; valine, leucine and isoleucine) is favorable for human health as BCAAs decrease diet-induced obesity and hyperglycemia. To construct Gluconacetobacter europaeus which produces BCAAs, leucine responsive regulator (GeLrp) is focused and two Gelrp mutants were constructed. Wild-type KGMA0119 didn't produce significant amount of valine (0.13 mM) and leucine (0 mM) and strain KGMA7110 which lacks complete Gelrp accumulated valine (0.48 mM) and leucine (0.11 mM) but showed impaired growth, and it was fully restored in the presence of essential amino acids. Strain KGMA7203 was then constructed with a nonsense mutation at codon Trp132 in the Gelrp, which leads a specific deletion at an estimated ligand-sensing region in the C-terminal domain. KGMA7203 produced greater quantities of valine (0.80 mM) and leucine (0.26 mM) and showed the same growth characteristics as KGMA0119. mRNA levels of BCAAs biosynthesis genes (ilvI and ilvC) and probable BCAAs efflux pump (leuE) were determined by quantitative reverse-transcription PCR. Expression rates of ilvI and ilvC in the two Gelrp disruptants were greater than those in KGMA0119. leuE was highly expressed in KGMA7110 only, suggesting that the accumulation in KGMA7110 culture was caused by increased expression of the biosynthesis genes and abnormal enhanced export of amino acids resulting in impaired cell growth. In contrast, KGMA7203 would achieve the high level production through enhanced expression of the biosynthesis genes without enhancing that for the efflux pump. KGMA7203 was considered advantageous for production of vinegar with higher amounts of valine and leucine.

An efficient method using Gluconacetobacter europaeus to reduce an unfavorable flavor compound, acetoin, in rice vinegar production.

Gluconacetobacter europaeus, one of the microorganisms most commonly used for vinegar production, produces the unfavorable flavor compound acetoin. Since acetoin reduction is important for rice vinegar production, a genetic approach was attempted to reduce acetoin produced by G. europaeus KGMA0119 using specific gene knockout without introducing exogenous antibiotic resistance genes. A uracil-auxotrophic mutant with deletion of the orotate phosphoribosyltransferase gene (pyrE) was first isolated by positive selection using 5-fluoroorotic acid. The pyrE disruptant designated KGMA0704 (ΔpyrE) showed 5-fluoroorotic acid resistance. KGMA0704 and the pyrE gene were used for further gene disruption experiments as a host cell and a selectable marker, respectively. Targeted disruption of aldC or als, which encodes α-acetolactate decarboxylase or α-acetolactate synthase, was attempted in KGMA0704. The disruption of these genes was expected to result in a decrease in acetoin levels. A disruption vector harboring the pyrE marker within the targeted gene was constructed for double-crossover recombination. The cells of KGMA0704 were transformed with the exogenous DNA using electroporation, and genotypic analyses of the transformants revealed the unique occurrence of targeted aldC or als gene disruption. The aldC disruptant KGMA4004 and the als disruptant KGMA5315 were cultivated, and the amount of acetoin was monitored. The acetoin level in KGMA4004 culture was significantly reduced to 0.009% (wt/vol) compared with KGMA0119 (0.042% [wt/vol]), whereas that of KGMA5315 was not affected (0.037% [wt/vol]). This indicates that aldC disruption is critical for acetoin reduction. G. europaeus KGMA4004 has clear application potential in the production of rice vinegar with less unfavorable flavor.