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

Abstract

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.

Keywords: Aspergillus oryzae; agmatine; polyamine; rice syrup; saccharification.

FIG 1

The putative pathways of polyamine biosynthesis and agmatine catabolism in A. oryzae, predicted from the genome sequence of A. oryzae RIB40. ADC, arginine decarboxylase; arginase, arginine ureohydrolase; agmatinase, agmatine ureohydrolase; ODC, ornithine decarboxylase; SPD synthase, spermidine synthase; AO, amine oxidase; GBald DH, 4-guanidinobutyraldehyde dehydrogenase; GBase, 4-guanidinobutyrase (4-guanidinobutyrate ureohydrolase); TA, γ-aminobutyrate transaminase; SSA DH, succinate-semialdehyde dehydrogenase; dcSAM, decarboxylated S-adenosylmethionine; MTA, methylthioadenosine; α-KG, α-ketoglutarate. The dotted arrow indicates the pathway predicted to be absent in A. oryzae (decarboxylation of l-arginine by ADC).

FIG 2

The identification of the microorganism involved in agmatine production. (A) The concentrations of agmatine and ethanol in rice wines and rice syrups. The steamed rice was fermented with S. cerevisiae and A. oryzae RW (MPF) or only with A. oryzae RW (saccharification [Sac.]) at 20°C. As references, the steamed rice was enzymatically degraded at 50°C with α-amylase and glucoamylase (En.), and S. cerevisiae was cultivated at 30°C in the rice syrup obtained after enzymatic degradation of steamed rice (SF). All fermentations and the enzymatic degradation were conducted for 7 days, and the levels of agmatine and ethanol in the resultant rice wines and rice syrups were quantified by HPLC and GC, respectively (n = 1). Black bars, agmatine concentration (in millimolar); white bars, ethanol (% [vol/vol]). ND, not detected. (B) HPLC profiles of rice wines made via MPF and SF, and rice syrups obtained by saccharification with A. oryzae RW and enzymatic degradation of steamed rice. The chromatograms are consistent with the data shown in panel A. The samples were diluted 40 times with distilled water prior to HPLC analyses. (a to e) 10 μM standards (a), MPF (b), SF (c), saccharification (d), enzymatic degradation (e). P1, putrescine (4.8 min); P2, spermidine (9.2 min); P3, agmatine (14.4 min); P4, spermine (18.6 min); IS, internal standard (caldopentamine, 32.9 min). (C) Agmatine level in a rice syrup obtained by the aseptic cultivation of A. oryzae RW (in millimolar). A. oryzae RW was aseptically cultivated at 20°C, as described in Materials and Methods. The levels of agmatine accumulated in the rice syrup were periodically determined by HPLC. The experiments were performed in triplicate, and the error bars represent standard deviations.

FIG 3

The effect of cultivation temperature on agmatine production by A. oryzae RW. The steamed rice was fermented with A. oryzae RW at various temperatures, and the concentration of agmatine in the rice syrup was periodically monitored. The amount of GlcNAc in the cultures was also determined, to evaluate hyphal growth. (A) GlcNAc (micrograms per gram of culture). (B) Agmatine levels in the rice syrup (in millimolar). The experiments were performed in triplicate, and the error bars represent standard deviations. Circles, 20°C; squares, 30°C; triangles, 40°C; diamonds, 50°C.

FIG 4

The effect of organic acids on agmatine production by A. oryzae RW and RIB40. The steamed rice was fermented by A. oryzae RW or RIB40 at 30°C in the presence of organic acids, and the concentration of agmatine in the rice syrup was periodically determined. (A) The effect of l-lactic acid and sodium l-lactate on the agmatine production by A. oryzae RW. Circles, 5.6 mM l-lactic acid; squares, 22.5 mM l-lactic acid; triangles, 111.3 mM l-lactic acid; diamonds, 111.3 mM sodium l-lactate. (B) The effect of succinic and citric acids on agmatine production by A. oryzae RW. Triangles, 111.3 mM l-lactic acid; crosses, 55.6 mM succinic acid; bars, 36.9 mM citric acid. (A and B) The experiments were performed in triplicate, and the error bars represent standard deviations. (C) Agmatine levels in the rice syrup fermented with A. oryzae RIB40 (mM, n = 1). Open circles, with 111.3 mM l-lactic acid; closed circles, without l-lactic acid.

FIG 5

The effect of culture conditions on agmatine production by A. oryzae RW. A. oryzae RW was cultivated in a liquefied rice medium, composed of mashed steamed rice and water acidified with 111.3 mM l-lactic acid (submerged culture). The fungus was also grown under solid-state cultivation (saccharification of steamed rice using the solid starter culture) in the presence of 111.3 mM l-lactic acid. Agmatine levels in the culture supernatants were periodically determined by HPLC. GlcNAc content in cultures was determined to estimate hyphal growth. (A) GlcNAc (in micrograms per gram of culture). (B) Agmatine levels in the culture supernatants (in millimolar). (A and B) Open circles, submerged culture; closed circles, solid-state culture. The experiments were performed in triplicate, and the error bars represent standard deviations.

FIG 6

The effect of additional l-arginine on agmatine production by A. oryzae RW. The solid starter culture of A. oryzae RW was incubated with l-arginine in the presence or absence of l-lactic acid at 30°C. The amount of agmatine accumulated in the supernatant was periodically measured by HPLC. The experiments were performed in triplicate, and the error bars represent standard deviations. Crosses, no l-arginine and l-lactic acid; diamonds, 10 mM l-arginine; circles, 10 mM l-arginine and 5.6 mM l-lactic acid; squares, 10 mM l-arginine and 22.5 mM l-lactic acid; triangles, 10 mM l-arginine and 111.3 mM l-lactic acid.

FIG 7

The agmatine-yielding activity of the homogenate of A. oryzae RW cells. The homogenates of the solid starter culture and hyphae obtained from a submerged culture were incubated with l-arginine in the presence of PLP at selected pH values (3.0, 4.0, 5.0, or 6.0) and temperatures (20, 30, 40, 50, or 60°C) for 60 min. The pH and temperature dependence activity assays were performed at 30°C and at pH 3.0, respectively. As a reference, powdered steamed rice, disrupted with liquid nitrogen, was evaluated in the in vitro assays. The activity in the cell homogenates was defined in terms of pmol of agmatine per min per μg of GlcNAc. In the reference experiment, the activity was normalized per weight (in milligrams) of the powdered steamed rice (pmol agmatine · min⁻¹ · [mg powdered steamed rice]⁻¹). (A) pH dependency of the activity. (B) Temperature dependency of the activity. (A and B) Squares, submerged culture; closed circles, solid starter culture; triangles, powdered steamed rice. The assays were performed in triplicate, and the error bars represent standard deviations.

FIG 8

Decarboxylase activity of the A. oryzae RW ODCs with l-ornithine and l-arginine. The ODCs of A. oryzae RW were expressed in E. coli, and the recombinant proteins obtained in soluble form (ODC1 and ODC2) were analyzed by enzyme assays to determine ODC or ADC activity by monitoring putrescine or agmatine levels, respectively, in reaction mixtures. (A) SDS-PAGE with Coomassie brilliant blue staining of the purified recombinant proteins. The purified ODC1 and ODC2 proteins are indicated in their respective lanes. (B) pH dependency of the ODC activity (nmol putrescine · min⁻¹ · mg⁻¹). (C) pH dependency of the ADC activity (nmol agmatine · min⁻¹ · mg⁻¹). (B and C) Circles, ODC1; squares, ODC2. The assays were performed in triplicate, and the error bars represent standard deviations.