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Agmatine Production by Aspergillus oryzae is Elevated by Low pH During Solid-State 1
Cultivation 2
3
Naoki Akasaka,a Saori Kato,
b Saya Kato,
b Ryota Hidese,
b Yutaka Wagu,
c Hisao Sakoda,
a 4
Shinsuke Fujiwarab# 5
6
aInstitute of Applied Microbiology, Marukan Vinegar Co. Ltd., Kobe, Hyogo, Japan 7
bDepartment of Bioscience, Graduate School of Science and Technology, Kwansei Gakuin 8
University, Sanda, Hyogo, Japan 9
cBio’c Co. Ltd., Muro-cho, Toyohashi, Aichi, Japan 10
11
12
Running Head: Efficient Production of Agmatine by A. oryzae 13
14
#Address correspondence to: Shinsuke Fujiwara, [email protected] 15
16
KEYWORDS: Aspergillus oryzae, saccharification, rice syrup, polyamine, agmatine 17
AEM Accepted Manuscript Posted Online 25 May 2018Appl. Environ. Microbiol. doi:10.1128/AEM.00722-18Copyright © 2018 American Society for Microbiology. All Rights Reserved.
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18
Abbreviations used in this manuscript: NO, nitric oxide; MPF, multiple parallel 19
fermentation; LAB, lactic acid bacteria; ADC, arginine decarboxylase; GABA, -20
aminobutyric acid; SF, simple alcohol fermentation; HPLC, high-performance liquid 21
chromatography; GC, gas chromatography; GlcNAc, N-acetylglucosamine; PLP, pyridoxal 22
phosphate; ODC, ornithine decarboxylase; LC-MS/MS, liquid chromatography-tandem 23
mass spectrometry; LB, lysogeny broth medium; YPD, yeast-peptone-dextrose medium; 24
TCA, trichloroacetic acid; PBCV-1 DC, Paramecium bursaria chlorella virus-1 25
decarboxylase. 26
27
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ABSTRACT Sake (rice wine) produced by multiple parallel fermentation (MPF) involving 28
Aspergillus oryzae (strain RW) and Saccharomyces cerevisiae under solid-state cultivation 29
conditions contained 3.5 mM agmatine, while that produced from enzymatically 30
saccharified rice syrup by S. cerevisiae contained <0.01 mM agmatine. Agmatine was also 31
produced in ethanol-free rice syrup prepared with A. oryzae under solid-state cultivation 32
(3.1 mM) but not under submerged cultivation, demonstrating that A. oryzae in solid-state 33
culture produces agmatine. The effect of cultivation conditions on agmatine production was 34
examined. Agmatine production was boosted at 30°C and reached the highest level (6.3 35
mM) at pH 5.3. The addition of L-lactic, succinic, and citric acids reduced the initial culture 36
pH to 3.0, 3.5, and 3.2, respectively, resulting in further increase in agmatine accumulation 37
(8.2, 8.7, and 8.3 mM, respectively). Homogenate from a solid-state culture exhibited a 38
maximum L-arginine decarboxylase (ADC) activity (74 pmol min-1
g-1
) at pH 3.0 at 30°C; 39
that from a submerged culture exhibited an extremely low activity (<0.3 pmol min-1
g-1
) 40
under all conditions tested. These observations indicated that efficient agmatine production 41
in ethanol-free rice syrup is achieved by an unidentified low pH-dependent ADC induced 42
during solid-state cultivation of A. oryzae, even though A. oryzae lacks ADC orthologs and, 43
instead, possesses four ornithine decarboxylases (ODC1–4). Recombinant ODC1 and 44
ODC2 exhibited no ADC activity at acidic pH (pH 4.0>), suggesting that other 45
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decarboxylases or an unidentified ADC is involved in agmatine production. 46
IMPORTANCE It has been speculated that, in general, fungi do not synthesize agmatine 47
from L-arginine because they do not possess genes encoding for arginine decarboxylase. 48
Numerous preclinical studies have shown that agmatine exerts pleiotropic effects on 49
various molecular targets, leading to an improved quality of life. In the present study, we 50
first demonstrated that L-arginine was a feasible substrate for agmatine production by the 51
fungus Aspergillus oryzae RW. We observed that the productivity of agmatine by A. oryzae 52
RW was elevated at low pH only during solid-state cultivation. A. oryzae is utilized in the 53
production of various oriental fermented foods. The saccharification conditions optimized 54
in the current study could be employed not only in the production of an agmatine-55
containing ethanol-free rice syrup but also in the production of many types of fermented 56
foods, such as soy sauce (shoyu), rice vinegar, etc., as well as novel therapeutic agents and 57
nutraceuticals. 58
59
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INTRODUCTION 60
Polyamines, such as putrescine, spermidine, and spermine (1,2), have gained 61
attention as agents that prevent the deterioration of the quality of life associated with aging. 62
These biogenic amines can extend longevity and alleviate age-related pathologies, 63
including the decline of the locomotor activity, cognitive dysfunction, and chronic 64
inflammation, mainly by inducing autophagy in model organisms (3-8). Agmatine, a 65
decarboxylated derivative of L-arginine, is one of the natural polyamines and a promising 66
candidate substance for human health promotion (9,10). Numerous preclinical studies have 67
demonstrated that agmatine exerts pleiotropic modulatory effects on various molecular 68
targets, which suggests its possible application as a therapeutic agent and a nutraceutical 69
(9,10). For instance, agmatine can function as a scavenger of reactive oxygen species and 70
protect the mitochondria in the brain cells (11). An excess of nitric oxide (NO) caused by 71
an elevated expression of NO synthases in the hippocampus and frontal cortex of aged rats 72
is converted to the deleterious oxidant peroxinitrite, which induces inflammation and tissue 73
damage, resulting in cognitive deficits (12). Agmatine supplementation significantly 74
improves the age-related memory and learning impairments in rat by inhibiting the NO 75
synthase activities (13). A more detailed analysis revealed that long-term oral intake of 76
agmatine reverses hormonal perturbations, such as insulin resistance, enhances urea 77
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synthesis, and represses weight gain (obesity) induced by a high-fat diet. These effects are 78
linked to the agmatine-induced metabolic rate increase (an increase in the expression of 79
uncoupling proteins) and fatty acid oxidation via the activation of carnitine biosynthesis. 80
Carnitine is a compound that mediates the transfer of long-chain fatty acids to the 81
mitochondria for -oxidation (14). 82
It is well known that fermented foods contain high amounts of polyamines, which 83
are derived from food ingredients (e.g., soybean) and produced by microorganisms 84
involved in their fermentation (15,16). Okamoto et al. (15) and Galgano et al. (16) reported 85
that sake (rice wine), a Japanese traditional alcohol beverage, contains more agmatine than 86
other fermented foods [agmatine content (nmol/g): Japanese rice wine, 880; beer, 37; 87
cheese, not detected; and yogurt, not detected]; no agmatine is detected in rice, suggesting 88
that the amine is generated by microorganisms involved in fermentation (15,16). The 89
Japanese rice wine is produced in the course of a complex fermentation process, a so-called 90
multiple parallel fermentation (MPF), with a simultaneous saccharification of rice by the 91
filamentous fungus Aspergillus oryzae and ethanol fermentation by Saccharomyces 92
cerevisiae (17). Since A. oryzae produces and secretes vast amounts of hydrolyzing 93
enzymes that degrade solid raw materials composed of starches and proteins, the fungus is 94
also essential for the production of various oriental fermented foods, such as the Japanese 95
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soy sauce (shoyu), rice syrup (amazake), and rice vinegar, and the Chinese soybean paste 96
(Chang) and cereal wine (Huang-jiu) (18,19). During MPF, nitric acid-reducing bacteria 97
and lactic acid bacteria (LAB) spontaneously propagate, and produce nitrous acid and lactic 98
acid, respectively, which reduce the environmental pH and prevent contamination with 99
undesirable microbes (17). Although previous studies suggested that S. cerevisiae, nitric 100
acid-reducing bacteria, and/or LAB are involved in agmatine accumulation in the Japanese 101
rice wine (15,16), it remains unclear how the amine is produced. 102
In general, polyamine biosynthesis via arginine decarboxylation to generate 103
agmatine, a reaction catalyzed by arginine decarboxylase (ADC), is conserved in plants, 104
bacteria, and archaea (Fig. 1) (2,16). A. oryzae RIB40 and S. cerevisiae K7 are a laboratory 105
strain isolated in 1950 that exhibit unique characteristics (e.g., amylase and protease 106
production) typical of those of strains used for Japanese rice wine brewing (20), and an 107
industrial strain for Japanese rice wine production, respectively. Genome analyses predicted 108
that these two strains do not possess orthologous genes encoding ADC in their genomes 109
(21,22). This appears to agree with a previous finding that fungi synthesize putrescine only 110
from L-ornithine (Fig. 1) (23). A recent study revealed that Aspergillus niger possesses a 111
unique pathway for agmatine catabolism involving a novel ureohydrolase (4-112
guanidnobutyrase), with agmatine converted to succinate via -aminobutyric acid (GABA) 113
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by a series of catabolizing enzymes (Fig. 1) (24). According to a comparative genomics 114
analysis, A. oryzae RIB40 might harbor a corresponding agmatine catabolic pathway, 115
converting L-arginine to putrescine via L-ornithine as the major route for polyamine 116
biosynthesis (Fig. 1) (21). 117
In the current study, we focused on A. oryzae and S. cerevisiae that are widely 118
used for Japanese rice wine production, and investigated their capability to produce 119
agmatine. The strains were cultivated in a mixture of steamed rice and water. Based on the 120
generated data, A. oryzae was responsible for agmatine production; the fungus produced 121
agmatine during solid-state cultivation, but not under submerged cultivation; and the 122
productivity was substantially enhanced in response to acidic stimuli. This suggested that 123
agmatine production by the fungus is specifically induced under solid-state cultivation 124
conditions and is associated with acid-resistance mechanisms in solid-state culture. 125
126
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RESULTS 127
Identification of the agmatine-producing microorganism. To identify the 128
microorganism(s) responsible for agmatine production, saccharification with A. oryzae 129
strain RW (used for the industrial production of Japanese rice wine), MPF with S. 130
cerevisiae and A. oryzae RW, and simple alcohol fermentation (SF) with S. cerevisiae were 131
performed, as described in the Materials and methods. As a reference, steamed rice was 132
degraded with starch-hydrolyzing enzymes (-amylase and glucoamylase) at 50°C for 7 d. 133
In the case of saccharification and MPF, the steamed rice absorbed the water in the cultures 134
within 1 h; liquefaction of the steamed rice was nearly completed within 48 h in these two 135
experimental set-ups. Hence, the culture conditions in saccharification and MPF cultures 136
gradually changed from solid- to liquid-state cultivation during the first 2 d. In the 137
enzymatic degradation experiment, liquefaction of the steamed rice was completed within 138
several hours. The final ethanol concentrations in the rice wines obtained by MPF and SF 139
were 12.6% and 9.4%, respectively, whereas that in the liquefied steamed rice (rice syrup) 140
fermented with A. oryzae RW was 0.2% (Fig. 2A). High-performance liquid 141
chromatography (HPLC) analysis revealed that the agmatine level in the rice syrup (3.1 142
mM) was similar to that in the rice wine obtained from MPF (3.5 mM) (Fig. 2A and B). On 143
the other hand, only a trace amount of agmatine (<0.01 mM) was detected in the rice wine 144
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obtained by SF (Fig. 2A and B). Further, a trace amount of spermidine was detected in the 145
rice wine obtained by MPF and the rice syrup fermented by the RW strain (Fig. 2B). No 146
spermine was detected in the rice syrups liquefied by A. oryzae RW (Fig. 2B), in agreement 147
with the notion that fungi, except for the Saccharomycotina subphylum, do not synthesize 148
spermine (23,25). The enzymatically liquefied rice syrup contained no detectable putrescine, 149
spermidine, spermine, agmatine, and ethanol (Fig. 2A and B). These observations suggested 150
that A. oryzae RW, but not S. cerevisiae, is responsible for agmatine production. To confirm 151
this, A. oryzae RW was re-purified by isolating a colony started from a single conidium. 152
The isolate was aseptically cultivated at 20°C in a mixture of steamed rice and water with 153
5.6 mM L-lactic acid, as described in the MATERIALS AND METHODS. HPLC analysis 154
revealed that agmatine levels in the rice syrup increased from 0.4 ± 0.03 mM (here and 155
elsewhere, the data are presented as the mean ± standard deviation) on day 0 to 1.9 ± 0.05 156
on day 1, reaching 3.6 ± 0.04 mM on day 7 (Fig. 2C), confirming agmatine production by 157
A. oryzae RW. 158
The effect of cultivation temperature on agmatine production by A. oryzae 159
RW. MPF is generally performed at low temperature (10–20°C), to prevent volatilization of 160
the flavor compounds that render the rice wine aroma pleasant (26). A. oryzae grows well at 161
ca. 30°C (27-29), while hyphal viability rapidly decreases after a few minutes at 50°C. It 162
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was anticipated that the agmatine production by A. oryzae RW would be enhanced by 163
fungal cultivation at an optimal growth temperature. Accordingly, to investigate the effect 164
of the growth temperature on agmatine yield, A. oryzae RW was cultivated at 20, 30, 40, 165
and 50°C for 7 d, and the agmatine levels in the rice syrups were periodically determined. 166
The amount of N-acetylglucosamine (GlcNAc) in the cultures was also monitored, as an 167
index of hyphal growth. Steamed rice was almost completely liquefied within 24 h at 30°C 168
and 40°C, while longer (48 h) or shorter (9 h) incubation was required for its liquefaction at 169
20°C or 50°C, respectively. When the strain RW was cultivated at 30°C, the GlcNAc 170
content increased from 154 ± 14 g/gculture
(day 0) to 244 ± 11 g/gculture
(day 1) within the 171
first 24 h of cultivation; it was maintained at ca. 250 g/gculture
from day 2 on (Fig. 3A). At 172
20°C, the amount of GlcNAc gradually increased from 165 ± 4 g/gculture
on day 1, to a 173
maximum of 259 ± 2 g/gculture
on day 7; no obvious increase of the GlcNAc levels was 174
observed at 40°C and 50°C (Fig. 3A), which was indicative of no hyphal growth above 175
40°C. Agmatine production by A. oryzae RW was greatly affected by the cultivation 176
temperature. The maximum agmatine production was observed at 30°C: when the RW 177
strain was cultivated at 30°C, agmatine levels in the culture increased from 0.4 ± 0.1 mM 178
(day 0) to 4.3 ± 0.3 mM (day 3), reaching 6.3 ± 0.4 mM after 7 d fermentation; this was 179
markedly higher than the final yield at 20°C (3.8 ± 0.5 mM) (Fig. 3B). Cultivation at 40°C 180
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also facilitated agmatine production, especially at the early stage of fermentation (days 0 to 181
3), although the final agmatine yield in the rice syrup was lower at 40°C (4.8 ± 0.6 mM on 182
day 7) than at 30°C (Fig. 3B). The temperature of 50°C severely impaired agmatine 183
production, and the agmatine level in the rice syrup did not exceed 2 mM throughout the 184
cultivation (1.4 ± 0.3 mM on day 7) (Fig. 3B). These results suggested that the optimal 185
temperature for agmatine production is around 30°C. In the current study, we examined the 186
agmatine productivity of A. oryzae RW at 30°C in the following experiments. 187
The effect of organic acids on agmatine production by A. oryzae RW. During 188
MPF, LAB (such as Lactobacillus sakei) proliferate and reduce the environmental pH by 189
producing lactic acid, preventing the contamination by undesirable microbes (17). The 190
addition of food-grade lactic acid instead of LAB to the cultures has been recently 191
implemented in Japanese rice wine production to shorten the fermentation period. 192
Furthermore, S. cerevisiae produces organic acids, such as malic, succinic, and citric acids 193
(30-32), in addition to carbon dioxide, a byproduct of ethanol fermentation. Therefore, A. 194
oryzae is exposed to an acidic environment throughout the process of Japanese rice wine 195
production, until the ethanol concentration reaches levels that are lethal to the fungus. Since 196
agmatine is a highly basic compound, we hypothesized that agmatine production by A. 197
oryzae is linked to the resistance and adaptation to acidic stresses. The effects of L-lactic, 198
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succinic, and citric acids on agmatine production by A. oryzae RW were evaluated by 199
cultivating the fungus in the presence of these organic acids at 30°C. The GlcNAc content 200
in the cultures increased from ca. 130 g/gculture
(day 0) to 230 g/gculture
(day 1), and the 201
liquefaction of the steamed rice was nearly completed within 24 h in all experimental set-202
ups. The effect of the initial pH on agmatine production was examined by the addition of 203
various concentrations of L-lactic acid to cultures. The addition of 5.6, 22.5, or 111.3 mM 204
L-lactic acid to cultures lowered the initial pH on day 0 to pH 5.3 ± 0.2, 4.1 ± 0.3, or 3.0 ± 205
0.1, respectively (Table S1). L-lactic acid facilitated agmatine production by the RW strain 206
in a dose-dependent manner. When the culture was acidified with 111.3 mM L-lactic acid, 207
the agmatine levels sharply increased, from 0.9 ± 0.3 mM (day 0) to 6.0 ± 1.0 mM (day 2) 208
during a 2 d fermentation, reaching a maximum of 8.2 ± 0.5 mM on day 7 (Fig. 4A). 209
Further, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis 210
confirmed that the rice syrup fermented in the presence of 111.3 mM L-lactic acid contained 211
8.1 mM agmatine; this was 1.3 times more than the final agmatine yield in the rice syrup 212
fermented in the presence of 5.6 mM L-lactic acid (6.3 ± 0.4 mM on day 7) (Fig. 4A). The 213
addition of 22.5 mM L-lactic acid enhanced agmatine production to a similar extent, 214
although the final agmatine yield (6.9 ± 0.5 mM) was slightly lower than that in the rice 215
syrup fermented in the presence of 111.3 mM L-lactic acid (Fig. 4A). The addition of more 216
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than 111.3 mM L-lactic acid to the cultures inhibited both saccharification and agmatine 217
production (data not shown). The presence of 111.3 mM sodium L-lactate (the initial pH on 218
day 0 was 6.3 ± 0.1) did not promote agmatine production: the final yield of agmatine was 219
markedly lower in the rice syrup fermented in the presence of sodium L-lactate (3.3 ± 0.3 220
mM on day 7) than in that supplemented with L-lactic acid (Fig. 4A). This suggested that 221
the lowering of the pH was pivotal for enhanced agmatine production by A. oryzae RW 222
(Table S1 and Fig. 4A). To confirm this, strain RW was cultivated in a mixture of steamed 223
rice and water acidified with other organic acids, namely, succinic acid (55.6 mM) and 224
citric acid (36.9 mM). The addition of succinic or citric acids lowered the initial culture pH 225
to 3.5 ± 0.2 and 3.2 ± 0.01, respectively. As expected, these two organic acids substantially 226
boosted agmatine production, similarly to 111.3 mM L-lactic acid [agmatine levels on day 7 227
(mM): succinic acid, 8.7 ± 0.1; citric acid, 8.3 ± 0.4] (Fig. 4B). It is noteworthy that 228
succinic acid facilitated agmatine generation to a greater extent than L-lactic and citric acids, 229
particularly at an early fermentation stage (days 0 to 2), although no remarkable difference 230
was noted in the final agmatine yields in the three experimental set-ups (Fig. 4B). We then 231
cultivated A. oryzae RW at 30°C under solid-state condition in the presence of 5.6 or 111.3 232
mM L-lactic acid to periodically monitor the pH values and organic acids, as well as 233
agmatine, in the cultures. The final agmatine yields on day 7 in the cultures supplemented 234
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with 5.6 or 111.3 mM L-lactic acid were 5.9 or 9.2 mM, respectively, which was consistent 235
with the data indicated in Fig. 4A. When the culture was acidified with 5.6 mM L-lactic 236
acid, the culture pH increased from pH 5.0 to pH 5.4 on day 1, and then gradually 237
decreased to reach pH 4.8 on day 7 (Fig. S1A). In the case of cultivation with 111.3 mM L-238
lactic acid, the similar pH increase was observed during the first 24 h of cultivation (day 0, 239
pH 3.1; and day 1, pH 3.5), whereas the pH values were maintained at ca. pH 3.6 from day 240
2 on (Fig. S1A). The concentration of L-lactic acid in the culture supplemented with 5.6 241
mM L-lactic acid nearly unchanged throughout the cultivation (ca. 5 mM) (Fig. S1B). In 242
contrast, the concentration of L-lactic acid in the culture with 111.3 mM L-lactic acid 243
dropped from 110.4 mM on day 0 to 86.3 mM on day 1, and was then maintained at around 244
80 mM until the end of cultivation (Fig. S1B). HPLC analysis also revealed that ca. 1–4 245
mM succinic, citric, and malic acids accumulated in the both cultures after 7 d fermentation 246
(Fig. S1C–D). 247
We also evaluated agmatine production by A. oryzae RIB40 (20,21). A similar 248
low-pH dependency of agmatine production was observed with that strain as with strain 249
RW [agmatine levels on day 7 (mM): without L-lactic acid, 2.0; with 111.3 mM L-lactic 250
acid, 4.0] (Fig. 4C), even though the highest achieved agmatine levels were lower than 251
those achieved with strain RW. 252
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The effect of culture conditions on agmatine production by A. oryzae. In A. 253
oryzae, thousands of genes are differentially transcribed in solid-state and submerged 254
culture conditions (33,34), and particular proteins (e.g., glucoamylase) are specifically 255
expressed in the solid-state culture (35). To investigate whether A. oryzae RW produced 256
agmatine under submerged culture conditions, the fungus was cultivated in a liquefied rice 257
medium composed of mashed steamed rice and water supplemented with 111.3 mM L-lactic 258
acid. As a reference, solid-state fermentation (saccharification of steamed rice) with the 259
solid starter culture was also carried out, in the presence of 111.3 mM L-lactic acid. Under 260
the submerged conditions, GlcNAc content gradually increased from 1.6 ± 0.02 g/gculture
261
(day 0) to 134 ± 8 g/gculture
(day 5) (Fig. 5A) with a concomitant decrease of the medium 262
viscosity. This suggested degradation of starch by hydrolyzing enzymes secreted by the 263
growing fungal cells. In the case of saccharification, GlcNAc levels increased from 60 ± 264
0.3 g/gculture
(day 0) to 169 ± 5 g/gculture
(day 3), reaching 185 ± 29 g/gculture
on day 5 265
(Fig. 5A). Agmatine was produced specifically under solid-state cultivation conditions: 266
agmatine levels in the rice syrup obtained by saccharification reached 9.9 ± 0.1 mM after a 267
5 d fermentation. However, the amine levels in the supernatant of the submerged culture 268
were negligible throughout the cultivation (<0.02 mM; Fig. 5B), even though both media 269
were acidified with L-lactic acid. Further, when A. oryzae RW was cultivated in a yeast-270
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peptone-dextrose (YPD) liquid medium containing 111.3 mM L-lactic acid, or in the 271
liquefied rice medium lacking L-lactic acid, no agmatine accumulated in the culture 272
supernatants (data not shown). Together with the observation that A. oryzae RW and RIB40 273
produced agmatine, to some extent, in the absence of acidic stimuli (saccharification by the 274
RW strain in the presence of sodium L-lactate, Fig. 4A; and by the RIB40 strain in the 275
absence of L-lactic acid, Fig. 4C), these findings suggested that solid-state cultivation is 276
required for agmatine production by A. oryzae. 277
The effect of additional L-arginine on agmatine production by A. oryzae RW. 278
The supernatants from solid-state cultures on day 0 already contained 0.5–1.0 mM 279
agmatine (Figs. 2–5), which had accumulated during pre-incubation of the solid starter 280
culture in water with organic acids (see MATERIALS AND METHODS). This suggested 281
that the strain RW could promptly produce large amounts of agmatine in response to the 282
acidic stimuli if sufficient amounts of substrates were supplied. To verify this, the solid 283
starter culture of A. oryzae RW was incubated with L-arginine, a possible substrate for 284
agmatine synthesis (Fig. 1), in the presence or absence of various concentrations of L-lactic 285
acid, at 30°C. The addition of L-arginine alone to the suspension resulted in the 286
accumulation of 1.1 ± 0.02 mM agmatine during the incubation period (Fig. 6). By contrast, 287
no increase in agmatine was observed and its concentration remained below 0.3 mM in the 288
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reference experiment (no L-arginine and L-lactic acid) (Fig. 6). These results supported the 289
speculation that L-arginine is a substrate for agmatine synthesis in A. oryzae RW. Next, the 290
solid starter culture was incubated with L-lactic acid and L-arginine; L-lactic acid enhanced 291
agmatine generation in a concentration-dependent manner [agmatine levels after 120 min 292
(mM): 5.6 mM L-lactic acid, 2.0 ± 0.05; 22.5 mM L-lactic acid, 2.8 ± 0.04; and 111.3 mM 293
L-lactic acid, 3.7 ± 0.2) (Fig. 6). The pH values of reaction mixtures, supplemented with no 294
L-arginine and L-lactic acid (reference experiment), L-arginine alone, L-arginine and 5.6 295
mM L-lactic acid, L-arginine and 22.5 mM L-lactic acid, or L-arginine and 111.3 mM L-296
lactic acid, were maintained at ca. pH 6.0, 9.0, 8.5, 6.0, or 3.0, respectively, throughout the 297
incubation. These results indicated the low pH-dependent agmatine production from L-298
arginine by A. oryzae RW. 299
The effect of pH and temperature on the agmatine-yielding activity of A. 300
oryzae RW cell homogenate. We next analyzed the pH and temperature dependencies of L-301
arginine decarboxylase activity as the agmatine-yielding activity by an in vitro assay 302
involving cell homogenates of A. oryzae RW. Homogenates of the solid starter culture and 303
of hyphal aggregates from a submerged culture in YPD liquid medium were incubated with 304
L-arginine in the presence of pyridoxal phosphate (PLP) at various pH values and 305
temperatures, and the resultant agmatine content in the reaction mixtures was determined. 306
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When the assays were performed at 30°C, the solid-culture homogenate exhibited a 307
maximum activity [74 ± 14 (pmolagmatine
) min-1
(gGlcNAc
)-1
] at pH 3.0 (Fig. 7A). The 308
activity decreased to approximately one-tenth of the maximum at pH 4.0 [8.1 ± 2.5 309
(pmolagmatine
) min-1
(gGlcNAc
)-1
]. No agmatine-yielding activity was detected above pH 5.0 310
(Fig. 7A). The observed low pH optimum for the activity suggested that the enzyme 311
responsible for agmatine production was an extracellular enzyme. To assess this possibility, 312
extracellular fraction was extracted from the solid starter culture of A. oryzae RW as 313
described in the MATERIALS AND METHODS. To confirm a successful obtaining of the 314
extracellular fraction, starch hydrolyzing activity, which is typical indicator of extracellular 315
enzymes such as -amylase and glucoamylase (18), was measured in advance. The 316
increasing of reducing sugar in the supernatant of the reaction mixture was confirmed. In 317
contrast, the fraction showed no agmatine-yielding activity when incubated with L-arginine 318
and PLP at pH 3.0 at 30°C for 60 min (data not shown), indicating that the enzyme for 319
agmatine synthesis was not, at least, an extracellular enzyme. According to the temperature-320
dependence activity assays, the maximum activity of the solid starter culture homogenate 321
was observed at ca. 30–40°C [activity, in (pmolagmatine
) min-1
(gGlcNAc
)-1
: 30°C, 74 ± 14; 322
40°C, 69 ± 2], and an appreciable activity was also detected at 50°C and 60°C [ca. 40 323
(pmolagmatine
) min-1
(gGlcNAc
)-1
] (Fig. 7B). These observations were consistent with the data 324
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shown in Figs. 3 and 4. By contrast, the homogenate of hyphae from a submerged culture 325
exhibited an extremely low agmatine-yielding activity under all conditions tested [<0.3 326
(pmolagmatine
) min-1
(gGlcNAc
)-1
]; this activity was markedly lower than the activity of the 327
solid starter culture homogenate (Fig. 7), which was in agreement with the data presented in 328
Fig. 5B. No agmatine accumulation was observed during the incubation of powdered 329
steamed rice in the presence of L-arginine and PLP (Fig. 7). 330
L-ornithine and L-arginine decarboxylase activities of ornithine 331
decarboxylases (ODCs) of A. oryzae RW. Whole-genome sequencing analysis predicted 332
that A. oryzae RIB40 harbors no ADC; instead, the strain was anticipated to carry four 333
genes encoding for ornithine decarboxylases (ODCs) in its genome [AO090023000771 334
(XP_001821277), AO090026000097 (XP_001821595), AO090038000189 335
(XP_023093031), and AO090026000380 (XP_001821844)] (21). A unique PLP-dependent 336
decarboxylase of Paramecium bursaria chlorella virus-1 [PBCV-1 DC (NP_0485549)] 337
shares high similarities on the amino acid sequence and protein structure levels with 338
eukaryotic ODCs, including Trypanosoma brucei ODC [tbODC (1QU4)] (36). However, 339
PBCV-1 DC functions as an ADC since the enzyme prefers L-arginine to L-ornithine as a 340
substrate (37). One of the determinants of the altered substrate specificity is a structural 341
rearrangement at the active site accompanied by a substitution of a key active-site residue 342
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(Asp to Glu) at position 296 in PBCV-1 DC (position 332 in tbODC) (38). These findings 343
led us to hypothesize that the ODCs of A. oryzae might catalyze the decarboxylation of L-344
arginine. 345
In the current study, genes of A. oryzae RW, homologous to AO090023000771, 346
AO090026000097, AO090038000189, and AO090026000380 of A. oryzae RIB40, were 347
designated as odc1, odc2, odc3, and odc4, respectively. The loci of the odc genes in A. 348
oryzae RW were PCR-amplified, and their nucleotide sequences were determined. The 349
exon-intron structures of these genes were predicted based on those of the homologous 350
genes in the strain RIB40; the predicted molecular masses of ODC1, ODC2, ODC3, and 351
ODC4 were 50, 44, 50, and 47 kDa, respectively. A comparative analysis revealed that the 352
coding regions of odc1, odc3, and odc4, respectively, harbor 2, 5, and 4 nucleotide 353
substitutions compared with the corresponding genes in A. oryzae RIB40, some of which 354
resulted in amino acid replacements [ODC1, Ala369Thr (G1105A); ODC3, Phe77Val 355
(A230T), Arg174Trp (C520T), Val339Met (G1015A), and Asp425Gly (A1274G); ODC4, 356
Thr359Ile (C1076T)]. The nucleotide sequence of the odc2 coding regions was identical to 357
that of exons of AO090026000097. The ODCs of A. oryzae RW shared 42−47% or 36−40% 358
amino acid sequence identity with tbODC (36) or PBCV-1 DC (37,38), respectively. 359
Homology searches (BLASTP) also revealed that almost all the active site residues, such as 360
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Ala67 and Lys69 in tbODC (36-38), were conserved in the four orthologs of A. oryzae RW 361
(and RIB40), except for the replacement of Asp332 (in tbODC) with Glu at positions 314, 362
341, and 332 in ODC2, ODC3, and ODC4, respectively. These amino acid substitutions 363
corresponded with Glu296 in PBCV-1 DC. 364
The four odc genes in A. oryzae RW were separately cloned into an expression 365
plasmid pET28a, and the recombinant proteins were produced in Escherichia coli. ODC1 366
and ODC2 were produced in a soluble form, whereas the other two proteins were produced 367
as insoluble aggregates. ODC1 and ODC2 were purified (Fig. 8A), and their decarboxylase 368
activities toward L-ornithine or L-arginine were determined at 30°C in enzyme assays, by 369
monitoring putrescine or agmatine levels, respectively, in reaction mixtures. These 370
recombinant proteins showed the highest ODC activities at pH 6.0 among the conditions 371
tested. Further, the ODC activity of ODC1 [549 ± 98 (nmolputrescine
) min-1
mg-1
] was ca. 2.5 372
times higher than that of ODC2 [211 ± 31 (nmolputrescine
) min-1
mg-1
] (Fig. 8B). At pH 5.0, 373
the activities of ODC1 [329 ± 29 (nmolputrescine
) min-1
mg-1
] and ODC2 [15 ± 2 374
(nmolputrescine
) min-1
mg-1
] decreased to 60% and 7% of their maximum, and no ODC 375
activity was detected when ODC1 and ODC2 were incubated at pH 3.0 and below pH 4.0, 376
respectively (Fig. 8B). ODC1 exhibited no ADC activity under all the conditions tested 377
[<0.03 (nmolagmatine
) min-1
mg-1
] (Fig. 8C). ODC2, on the other hand, exhibited a slight 378
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decarboxylase activity for L-arginine at pH 6.0 [2.8 ± 0.2 (nmolagmatine
) min-1
mg-1
] (Fig. 379
8C), although this activity was ca. 80 times lower than that of the maximal ODC activity of 380
ODC2 at pH 6.0 (Fig. 8B). 381
382
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DISCUSSION 383
In the current study, we showed that A. oryzae RW produced agmatine during the 384
early fermentation stage (days 0 to 2) (Figs. 3−5) in parallel with the transition of culture 385
conditions from solid-state to submerged cultivation, caused by the liquefaction of steamed 386
rice, and that the agmatine production by A. oryzae RW was substantially enhanced in 387
response to acidic stimuli, i.e., organic acids (Fig. 4). By contrast, A. oryzae RW produced 388
no agmatine in submerged culture even when the media were acidified with L-lactic acid 389
(Fig. 5). Furthermore, homogenate of a solid starter culture prepared in the absence of 390
acidic stress (see MATERIALS AND METHODS) exhibited agmatine-yielding activity, 391
while that of the hyphal aggregates from a submerged culture did not (Fig. 7). This was 392
consistent with the observation that A. oryzae RW and RIB40 produced an appreciable 393
amount of agmatine in the absence of organic acids (Fig. 4). These observations suggested 394
that the solid-state culture is required for the agmatine production by A. oryzae, and that the 395
enzymes responsible for agmatine synthesis are induced specifically during solid-state 396
cultivation but not during submerged cultivation. At the same time, the enhanced agmatine 397
production caused by organic acids may be associated with the mechanism of resistance to 398
acidic environments, resembling the bacterial acid-resistance system involving amino acid 399
decarboxylation (39). During E. coli exposure to an acidic environment, a series of PLP-400
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dependent decarboxylases (e.g., GadA/B and AdiA) decarboxylate glutamic acid and 401
arginine to form GABA and agmatine, respectively, with a concomitant consumption of an 402
intracellular proton (39-41). This contributes to the homeostatic maintenance of the 403
cytoplasmic pH (39-41). As for the fungal growth environment, many aspects of the solid-404
state culture are different from those of the submerged culture, such as water activity, media 405
homogeneity, and the availability of nutrients and gasses (42). Numerous genes are 406
differentially expressed in solid-state and submerged cultures of A. oryzae (34), allowing 407
the fungus to adapt to the environmental differences. The exact physiological significance 408
of the agmatine production specific for solid-state culture remains elusive. A. oryzae might 409
have acquired the ability to produce agmatine to adapt to solid-state cultivation conditions, 410
and that ability might have been potentiated to resist acidic stresses in solid-state culture 411
over the long history of rice wine production. 412
When A. oryzae RW was cultivated under solid-state condition in the presence of 413
5.6 or 111.3 mM L-lactic acid, pH of the both cultures increased during the first 24 h of 414
cultivation (Fig. S1). The data shown in Figs. 6 and 7 suggested that L-arginine was a 415
substrate for agmatine. The pH increase at the beginning of cultivation might be due to the 416
rapid accumulation of agmatine via L-arginine decarboxylation, accompanied with the 417
consumption of protons in the environments. In the case of the cultivation with 111.3 mM 418
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L-lactic acid, the levels of L-lactic acid sharply dropped within the first 24 h of fermentation 419
(Fig. S1), suggesting the assimilation of the acid by the growing fungal cells. This also 420
might be a cause of the observed pH increase at the early fermentation stage. We also found 421
the accumulation of small amounts of succinic, citric, and malic acids, which are known to 422
be produced by fungi belonging to the genus Aspergillus, including A. oryzae (43). The pH 423
values of the culture with 5.6 mM L-lactic acid gradually decreased from day 2 on, while 424
those of the culture with 111.3 mM L-lactic acid nearly unchanged after reaching pH 3.5 on 425
day 1 (Fig. S1). This might be explained by the difference in the buffering capacities of the 426
both cultures exerted by the additional L-lactic acid: the culture with 111.3 mM L-lactic acid 427
would have a higher buffering capacity than that with 5.6 mM L-lactic acid since the former 428
contained a greater amount of residual L-lactic acid (ca. 80 mM) than the later (ca. 5 mM) 429
(Fig. S1), and the accumulation of other organic acids (i.e., succinic, citric, and malic acids) 430
would manifest as the pH decrease in the culture supplemented with 5.6 mM L-lactic acid. 431
However, the pH decrease observed in the culture with 5.6 mM L-lactic acid at the middle 432
to late fermentation stage (day 2 to 7) would have little effect on agmatine production 433
because the culture condition was shifted from solid-state to submerged cultivation within 434
the first 24 h of cultivation, in which A. oryzae RW produces no agmatine (Figs. 5 and 7). 435
Together with the data shown in Figs. 4–7, it is suggested that acidic stimuli during the 436
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solid-state cultivation (lowering of the initial pH in saccharification) is essential to enhance 437
agmatine production by the fungus. 438
In saccharification experiments, the highest agmatine yield was achieved at 30°C, 439
which was accompanied by a concomitant increase in GlcNAc levels (Fig. 3). Cultivation 440
at 40°C also enhanced agmatine production to a similar extent while no cell growth was 441
observed (Fig. 3), indicating that the hyphal growth (an increase in the number of viable 442
cell), as well as the cultivation temperature and acidic stimuli, are important for 443
maximizing the agmatine production by A. oryzae RW. On the other hand, in vitro assays 444
with a homogenate of the solid starter culture revealed that the optimal temperature for the 445
activity of enzymes responsible for agmatine synthesis was ca. 30–40°C (Fig. 7). These 446
results suggest that the optimal temperature for agmatine production, where the maximal 447
enzymatic activity and the sufficient cell growth are achieved, lies between 30°C and 40°C. 448
The agmatine production by A. oryzae RW might be further facilitated by cultivating the 449
fungus at the exact optimal temperature, which remains to be determined. 450
It should be noted that, in general, fungi do not possess ADC (23). Consistent with 451
this, the analysis of the A. oryzae RIB40 genome predicted that the fungus does not harbor 452
ADC orthologs, while possessing four ODC orthologs (21). Further, an extensive study of 453
A. niger evidenced the lack of ADC activity in the fungal mycelia (24). In the current study, 454
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however, we showed that A. oryzae RW produced agmatine (Figs. 2−5), with L-arginine as 455
a possible substrate for agmatine biosynthesis (Figs. 6 and 7). This suggested that A. oryzae 456
harbors ADC. PBCV-1 DC functions as an ADC despite a high amino acid sequence 457
identity and structural similarity shared with eukaryotic ODCs (37,38). According to Shah 458
et al., the shift in the substrate specificity of PBCV-1 DC was caused by a structural 459
rearrangement in the active site: a key active-site residue (Asp332 in tbODC), which forms 460
a hydrogen bond with the -amino group of putrescine, is replaced with Glu296 in PBCV-1 461
DC, interacting with the guanidino group of agmatine, and the helix containing 462
Asp332/Glu296 is shorter in PBCV-1 DC than in tbODC (38). These structural changes 463
enlarged the active-site pocket to accommodate the larger substrate (L-arginine), while 464
retaining the interactions between Glu296 and other active-site residues essential for 465
enzyme function (38). We found the same amino acid substitution in A. oryzae RW (and 466
RIB40) proteins ODC2, ODC3, and ODC4. The Asp-to-Glu substitution might be one of 467
the reasons why ODC2 exhibited a slight but detectable ADC activity (Fig. 8C). However, 468
ODC2 might not be involved in agmatine biosynthesis in A. oryzae RW because the 469
recombinant protein did not exhibit ADC activity under acidic pH conditions (below pH 4.0, 470
Fig. 8C), which was inconsistent with the results of in vitro assays with the homogenate of 471
a solid starter culture (Fig. 7). Further, the ADC activity of ODC2 was markedly lower than 472
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its decarboxylase activity with L-ornithine (Fig. 8). Thus, ODC2, as well as ODC1, would 473
function as ODCs in A. oryzae RW. Other functionally not annotated decarboxylases, such 474
as ODC3 and ODC4, which both harbor a substitution corresponding to Glu296 in PBCV-1 475
DC, might be responsible for the decarboxylation of L-arginine to form agmatine. Further 476
investigation is necessary to clarify the underlying mechanism of agmatine production in A. 477
oryzae. 478
Increasing evidence supports the beneficial effect of agmatine on the quality of life 479
(9,10). Various types of agmatine supplements are currently commercially available (44). 480
The Japanese rice wine contains a high amount of agmatine (15,16). However, continuous 481
intake of the rice wine can be associated with a health impairment risk because of its high 482
ethanol content (ca. 20%) (45). Based on their extensive use in fermented food production, 483
A. oryzae and its products have acquired a “Generally Recognized as Safe” status from the 484
US Food and Drug Administration and the World Health Organization (18). In the current 485
study, we demonstrated that A. oryzae RW led to the accumulation of substantial amounts 486
of agmatine (ca. 9 mM) in rice syrup (Figs. 4 and 5), without ethanol production (Fig. 2A). 487
It is hence expected that natural rice syrup fermented using A. oryzae RW would promote 488
or improve human health. The reported findings may hence be employed in the production 489
of not just a variety of fermented foods containing an increased amount of agmatine, but 490
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also in the production of safe and novel therapeutic agents and nutraceuticals. 491
492
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MATERIALS AND METHODS 493
Microbial strains. Conidia of the A. oryzae strain used for the industrial 494
production of the Japanese rice wine (Kikai-yo) were purchased from Bio’c Co. Ltd. 495
(Toyohashi, Aichi, Japan). In the current study, the strain was tentatively designated as RW 496
(rice wine). A. oryzae RIB40 (20,21) was obtained from the National Research Institute of 497
Brewing (Higashihiroshima, Hiroshima, Japan). Dried cells of S. cerevisiae used for 498
Japanese rice wine production (Iida Kanso Kobo) were purchased from Iida Brewing Co. 499
Ltd. (Yao, Osaka, Japan). E. coli DH5 (TaKaRa Bio, Ohtsu, Shiga, Japan), which was 500
used to construct expression plasmids for A. oryzae RW ODCs, was routinely cultivated at 501
37°C in the lysogeny broth (LB) medium containing 20 g/ml kanamycin. For the ODC 502
production, E. coli BL21 CodonPlus(DE3)-RIL (Agilent, Santa Clara, CA, USA) cells 503
harboring the appropriate expression plasmid were cultivated at 37°C in LB medium 504
containing 20 g/ml kanamycin and 20 g/ml chloramphenicol. 505
Preparation of koji (solid starter culture of A. oryzae). To prepare the solid 506
starter culture comprising rice and fungal hyphae (18), A. oryzae RW and RIB40 were 507
subjected to solid-state fermentation on steamed rice, as previously described (46), with 508
modifications. Specifically, 300 g of rice grains, which were polished to 90% of total 509
weight, was soaked in tap water for 3 h and steamed for 50 min. After cooling to 30°C, 230 510
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mg (6.9 × 107) of the A. oryzae RW or RIB40 conidia was inoculated to the steamed rice, 511
and the preparation was mixed well. The conidia-containing rice was overlaid with a gauze 512
(15 23 cm) moistened with water, wrapped with a cotton cloth (35 35 cm) in a plastic 513
container (16 23 5 cm), and incubated at 30°C for 45 h. At 20 and 29 h, the rice grains 514
with A. oryzae propagating on their surfaces were mixed upside-down to improve the 515
aeration and to lower the temperature of the culture (18). The resultant solid starter culture 516
was mixed after 45 h as described above, cooled at room temperature for 2 h, and used as 517
an A. oryzae cell inoculum in the subsequent fermentation experiments. 518
MPF, SF, and saccharification. Unless otherwise indicated, tap water sterilized 519
by autoclaving (121°C, 15 min) containing 5.6 mM L-lactic acid was used in all 520
fermentation experiments. For MPF, 20 mg of dried cells of S. cerevisiae was inoculated to 521
5 ml of the YPD liquid medium (47) containing 5.6 mM L-lactic acid; the culture was 522
statically incubated at 30°C for 16 h. Next, 1 ml of the culture was transferred to 100 ml of 523
the YPD liquid medium containing 5.6 mM L-lactic acid, and further cultivated at 30°C for 524
24 h. The cells were harvested by centrifugation (4°C, 8000 g, 5 min), resuspended in 4 525
ml of sterilized tap water, and kept at 20°C until use. For the experiment, 50 g of the solid 526
starter culture of A. oryzae RW was first suspended in 160 ml of sterilized tap water 527
containing L-lactic acid, and incubated at 20°C for 1.5 h to equilibrate the temperature of 528
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the suspension; 100 g of steamed rice and 1 ml of the yeast cell suspension were then added 529
to the mixture, for a total volume of 300 ml, and incubated at 20°C for 7 d. 530
For saccharification by A. oryzae RW or RIB40, the cultures were prepared as 531
described above but without the S. cerevisiae cells. To evaluate the effect of cultivation 532
temperature on the agmatine production by A. oryzae RW, the solid starter culture of A. 533
oryzae RW was first pre-incubated at 20, 30, 40, or 50°C for 1.5 h in the presence of L-534
lactic acid; steamed rice was then added and the cells were cultivated at each temperature 535
for 7 d. To analyze the impact of acidic stresses on the agmatine production, the solid 536
starter culture of A. oryzae RW was pre-incubated in 160 ml of sterilized tap water 537
containing 22.5 or 111.3 mM L-lactic acid, 55.6 mM succinic acid, or 36.9 mM citric acid, 538
at 30°C for 1.5 h. The steamed rice was then added to the suspensions, and the suspensions 539
were incubated at 30°C for 7 d. The initial concentrations of succinic and citric acids were 540
determined to adjust the molar concentration of protons dissociating from these organic 541
acids to that from 111.3 mM L-lactic acid. The effect of the lactate anion on agmatine 542
production was also investigated using water supplemented with 111.3 mM sodium L-543
lactate. As a reference, 150 g of the steamed rice was suspended in 160 ml of sterilized tap 544
water containing 5.6 mM L-lactic acid and degraded by 0.54 g of -amylase (Nagase 545
ChemteX, Osaka, Japan) and 0.36 g of glucoamylase (Nagase ChemteX) at 50°C for 7 d. 546
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For SF, 150 g of the steamed rice was suspended in 160 ml of sterilized water 547
supplemented with 5.6 mM L-lactic acid, and liquefied with -amylase (Nagase ChemteX) 548
and glucoamylase (Nagase ChemteX) at 50°C for 2 h. Then, 1 ml of the yeast cell 549
suspension was inoculated into the resultant liquefied steamed rice (i.e., the rice syrup) and 550
incubated at 30°C for 7 d. All cultures were mixed well with a sterilized spatula every day, 551
and the culture supernatants were periodically collected for subsequent HPLC, LC-MS/MS, 552
and gas chromatography (GC) analyses (see below). 553
Aseptic cultivation of A. oryzae RW. The RW strain was first re-purified by 554
isolating a colony that started from a single conidium, as follows: 10 mg of A. oryzae RW 555
conidia was suspended in 1 ml of saline [0.85% (wt/vol) NaCl] supplemented with 0.5% 556
(vol/vol) polyoxyethylene (10) octylphenyl ether (Wako Pure Chemical, Osaka, Japan) and 557
diluted in saline. Then, 100 l of a 10-fold dilution series (100 to 10
-7) was spread on 558
dextrose-yeast-peptone agar plates, composed of 3% (wt/vol) soluble starch, 1% (wt/vol) 559
peptone (BD Biosciences, San Jose, CA, USA), 0.5% (wt/vol) yeast extract (Wako Pure 560
Chemical), 0.2% (wt/vol) KCl, 0.1% (wt/vol) KH2PO4, 0.05% (wt/vol) MgSO4∙7H2O, and 561
2% (wt/vol) agar; the conidia were incubated at 30°C for 24 h. One of the colonies formed 562
on the agar plate was transferred to 4 g of steamed rice [prepared by autoclaving (121°C, 15 563
min) 3 g of rice grains moistened with 1.5 ml of tap water] in a test tube, and further 564
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incubated at 30°C for 7 d. Next, 5 ml of saline was added to the test tube, and the newly 565
formed conidia were suspended by gentle tapping. The conidial suspension was mixed with 566
an equal volume of 50% (wt/vol) glycerol, and stored at -80°C until use. An aliquot, 567
containing 2.3 106 conidia, was inoculated to 10 g of autoclaved rice (prepared as 568
described above) in a glass dish, and incubated at 30°C for 40 h to obtain the solid starter 569
culture. The resultant solid culture was suspended in 30 ml of sterilized tap water 570
containing 5.6 mM L-lactic acid, followed by a pre-incubation at 30°C for 1.5 h. In the 571
meantime, 15 g of rice grains, moistened with 7.5 ml of tap water, was autoclaved (121°C, 572
15 min), and the autoclaved rice was added to the suspension. The mixture was incubated at 573
20°C for 7 d, and culture supernatants were periodically collected for the subsequent HPLC 574
analysis. All procedures were carried out aseptically. 575
The effect of culture conditions on agmatine production by A. oryzae RW. To 576
examine the effect of culture conditions on agmatine production, the RW strain was 577
cultivated in a liquid medium consisting of mashed steamed rice and water, as follows: 15 g 578
of rice grains soaked in 75 ml of tap water were autoclaved (121°C, 15 min), and mashed 579
with a sterilized pestle. The mashed rice was mixed with separately autoclaved tap water 580
(75 ml) supplemented with 111.3 mM L-lactic acid, and the resultant preparation (160 ml) 581
was used as a liquefied rice medium. Next, 100 mg (3.0 107) of A. oryzae RW conidia 582
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was inoculated to this medium, and cultivated at 30°C with reciprocal shaking at 150 rpm 583
for 5 d. As a reference, solid-state cultivation (saccharification of steamed rice using the 584
solid starter culture of A. oryzae RW) was performed at 30°C, similarly to aseptic 585
cultivation of the fungus, with 111.3 mM L-lactic acid in the culture. Agmatine levels in 586
culture supernatants were periodically monitored by HPLC. 587
Determination of the hyphal growth of A. oryzae. The growth of A. oryzae 588
mycelia in cultures was evaluated by determining the amount of GlcNAc, which is the 589
building block of the major fungal cell wall constituent, chitin (43). Briefly, 2 g of the solid 590
starter culture were dried at 100°C for 1 h, and completely ground using a mortar. The 591
resultant powder was suspended in 10 ml of 50 mM phosphate buffer (pH 7.0), vigorously 592
vortex-mixed for 10 s, and recovered by centrifugation (10,000 g, 10 min). These 593
washing steps were repeated three times, and the resultant pellet was resuspended in 10 ml 594
of the phosphate buffer. Next, 10 mg of chitinase (Yatalase; TaKaRa Bio) was added to the 595
suspension, and incubated at 37°C for 1 h, with reciprocal shaking at 200 rpm. The 596
supernatant was collected as the GlcNAc fraction by centrifugation (10,000 g, 10 min). In 597
the case of saccharification or submerged cultivation of A. oryzae RW, 5 g of culture was 598
directly suspended in the phosphate buffer without drying. The amount of GlcNAc was 599
determined using a colorimetric method, as previously described (48). Then, 500 l of the 600
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GlcNAc fraction was mixed with 100 l of 0.8 M borate buffer (pH 9.1, adjusted with 601
KOH) and heated in boiling water for 3 min. After cooling in tap water, 3 ml of a coloring 602
solution composed of 10 mg/ml p-dimethylaminobenzaldehyde and 125 mM hydrochloric 603
acid in glacial acetic acid was added to the mixture, and further incubated at 37°C for 20 604
min, giving rise to purple coloration. The absorbance of the reaction mixtures at 585 nm 605
was measured, and the amount of GlcNAc in cultures (gGlcNAc
/gculture
) was estimated based 606
on a standard curve generated using 0.05, 0.1, 0.15, and 0.2 mol of GlcNAc (Wako Pure 607
Chemical). A solid starter culture containing 400–500 g/g GlcNAc was routinely used in 608
MPF and saccharification. 609
The effect of additional L-arginine on agmatine production by A. oryzae RW. 610
First, 50 g of the solid starter culture of A. oryzae RW was suspended in 100 ml of sterilized 611
tap water. 160 ml of separately sterilized water containing 5.6, 22.5, or 111.3 mM L-lactic 612
acid was then added to the suspension; the total volume of the suspension was adjusted to 613
300 ml. The suspension was incubated at 30°C for 1.5 h to equilibrate the temperature of 614
the suspension prior to assaying. Next, 500 mM L-arginine was added to the suspensions to 615
the final concentration of 10 mM, and further incubated at 30°C. The amount of agmatine 616
accumulated in the supernatants was monitored every 30 min for 120 min using HPLC. 617
The effect of pH and temperature on the agmatine-yielding activity of a 618
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homogenate of A. oryzae RW cells. The solid starter culture of A. oryzae RW was prepared 619
as described above. For a submerged culture, 200 mg (6.0 107) of conidia was inoculated 620
into 300 ml of the YPD liquid medium and cultivated at 30°C with reciprocal shaking at 621
150 rpm for 24 h. The aggregates of hyphae germinated from the conidia were harvested by 622
filtration through a filter paper (ADVANTEC Toyo Kaisha, Ltd., Tokyo, Japan) and washed 623
twice with saline. The solid starter culture and the hyphal aggregates from a submerged 624
culture were rapidly frozen in liquid nitrogen and ground into a fine powder using a pre-625
chilled mortar and a pestle. Next, 0.1 g of the powder was suspended in 1 ml of 50 mM 626
phosphate buffer (pH 7.0) containing a protease inhibitor cocktail (cOmplete Mini; Roche 627
Diagnostics, Mannheim, Germany), and the suspension was used as a cell homogenate in 628
the in vitro assay. The GlcNAc content of the suspension was determined as described 629
above. The effect of pH and temperature on the agmatine-yielding activity of the 630
homogenate was investigated as follows: after vigorous vortex-mixing, 100 l of the 631
suspension was added to 400 l of 25 mM citrate buffer (pH 3.0, 4.0, 5.0, or 6.0) 632
supplemented with 1 mM L-arginine and 0.1 mM PLP, and incubated at selected 633
temperatures (20, 30, 40, 50, or 60°C) for 60 min. The pH- and temperature-dependence 634
activity assays were performed at 30°C and pH 3.0, respectively. The reaction was stopped 635
by the addition of 50 l of 10% (wt/vol) trichloroacetic acid (TCA) (Wako Pure Chemical), 636
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which was followed by vigorous vortex-mixing and cooling on ice. The supernatants of the 637
reaction mixtures were recovered by centrifugation and subjected to HPLC analysis to 638
determine agmatine content. As a reference, the steamed rice was frozen in liquid nitrogen 639
and ground into a fine powder using a pre-chilled mortar and pestle, similarly to the 640
disruption of A. oryzae RW cells as described above. 0.1 g of the resultant powder was 641
suspended in 1 ml of 50 mM phosphate buffer (pH 7.0). Next, 100 l of the suspension was 642
mixed with 400 l of 25 mM citrate buffer (pH 3.0, 4.0, 5.0, or 6.0) containing 1 mM L-643
arginine and 0.1 mM PLP, and incubated at selected temperatures (20, 30, 40, 50, or 60°C) 644
for 60 min to confirm that no agmatine was generated. The agmatine-yielding activity of 645
the cell homogenates was defined as pmol of agmatine per min per g of GlcNAc 646
[(pmolagmatine
) min-1
(gGlcNAc
)-1
]. In the reference experiments, the activity was defined as 647
pmol of agmatine per min per mg of powdered steamed rice [(pmolagmatine
) min-1
(mgpowdered
648
steamed rice)
-1]. 649
Fractionation of extracellular enzymes. Extracellular fraction was carefully 650
obtained from the solid starter culture of A. oryzae RW according to the previously 651
described method (49) with slight modifications. 2 g of the solid starter culture of A. oryzae 652
RW was suspended in 10 ml of 50 mM sodium citrate buffer (pH 5.5) supplemented with 653
90 mM NaCl, 1 mM 2-mercaptoethanol, 2 mM dithiothreitol, and 1% (vol/vol) Protease 654
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Inhibitor Mixture for Fungal and Yeast Extracts (Wako Pure Chemical), and extracellular 655
proteins were extracted at 4°C for 1 h. The suspension was centrifuged, and the supernatant 656
was filtered through 0.45 m pore-size cellulose acetate filter (AS ONE, Osaka Japan). The 657
resultant filtrate was collected as a fraction with extracellular enzymes. To confirm that the 658
extracellular fraction was successfully obtained, its starch hydrolyzing activity was 659
measured in advance by incubating 5 ml of the fraction with 5 g of steamed rice suspended 660
in 5 ml of 50 mM sodium citrate buffer (pH 5.5) at 37°C for 1 h. The increasing of reducing 661
sugar in the supernatant of the reaction mixture was confirmed by titration (Fehling-662
Lehmann-Schoorl method) (50). 663
Expression and purification of ODCs of A. oryzae RW. The odc genes (odc1–4) 664
of A. oryzae RW, together with their 5ʹ-upstream and 3ʹ-downstream flanking regions (ca. 665
200 bp each), were PCR-amplified from the genomic DNA using the corresponding primer 666
pairs (odc1, odc1-up and odc1-down; odc2, odc2-up and odc2-down; odc3, odc3-up and 667
odc3-down; and odc4, odc4-up and odc4-down) (Table S2), which were designed based on 668
the genome sequence of strain RIB40 (21), and their nucleotide sequences were 669
determined. The full-length odc1, odc2, odc3, and odc4 genes (including introns) were 670
amplified from A. oryzae RW genomic DNA using the specific primer pairs odc1-Fw and 671
odc1-Rv; odc2-Fw and odc2-Rv; odc3-Fw and odc3-Rv; and odc4-Fw and odc4-Rv, 672
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respectively (Table S2). These primers contained a 15 b 5ʹ adaptor sequence homologous to 673
the plasmid vector pET28a (Merck, Darmstadt, Germany) (Table S2). The obtained DNA 674
fragments were separately ligated with the linearized plasmid vector using the seamless 675
ligation cloning extract method (51). The linearized plasmid vector was obtained from 676
pET28a via inverse PCR amplification using the primer pair pET28a-Fw and pET28a-Rv 677
(Table S2). The predicted introns of odc1, odc2, odc3, and odc4 harbored by intermediary 678
plasmids were removed by PCR-based site-directed mutagenesis (quick-change PCR) using 679
the appropriate primer pairs (Table S2), yielding plasmids pODC1, pODC2, pODC3, and 680
pODC4, respectively. These plasmids were separately introduced into E. coli BL21 681
CodonPlus(DE3)-RIL cells, and the resultant transformants were grown in the LB medium 682
containing 20 g/ml kanamycin and 20 g/ml chloramphenicol at 37°C. The expression of 683
the ODCs with N-terminal His tags was induced by the addition of 1 mM isopropyl -D-684
thiogalactopyranoside. After further incubation at 37°C for 4 h, the cells were harvested by 685
centrifugation, resuspended in buffer A [20 mM sodium phosphate (pH 7.5), 50 mM NaCl, 686
0.2 mM phenylmethylsulfonyl fluoride, 1 mM 2-mercaptoethanol, and 0.1 mM EDTA], and 687
disrupted by sonication. After removing the cell debris by centrifugation, each supernatant 688
was applied to a column packed with 3 ml of Ni-nitrilotriacetic acid agarose (Qiagen, 689
Hilden, Germany) and eluted with buffer B (buffer A supplemented with 100 mM 690
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imidazole). Each purified protein was dialyzed against buffer C [buffer A supplemented 691
with 10% (wt/vol) glycerol]. The concentrations of purified proteins were determined using 692
the Bradford dye-binding assay with BSA as a standard (52). 693
Determination of decarboxylase activities of A. oryzae RW ODCs with L-694
ornithine and L-arginine. To monitor the ODC and ADC activities of the recombinant 695
proteins, the products of enzymatic reactions were analyzed by HPLC. The reaction 696
mixtures (500 l) contained 0.1 mM substrate (L-ornithine or L-arginine) and 0.1 mM PLP 697
in 25 mM citrate buffer (pH 3.0, 4.0, 5.0, or 6.0). Each purified protein (5 g) was added to 698
the mixture and then incubated at 30°C for 60 min. The reaction was stopped by the 699
addition of 50 l of 10% (wt/vol) TCA, and the reaction mixture was filtered through a 700
0.45 m pore-size filter (Millex LH filter; Millipore, Bedford, MA, USA). The putrescine 701
or agmatine content in the filtrate was determined by HPLC to assess the ODC or ADC 702
activity, respectively. 703
Quantification of the polyamines, organic acids, and ethanol by HPLC and 704
GC. The polyamines and organic acids, or ethanol in the culture supernatants were 705
quantified using HPLC or GC, respectively, as previously described (47, 53). To extract the 706
polyamines, 1/10 volume of 10% (wt/vol) TCA was added to the samples and thoroughly 707
vortex-mixed. The mixtures were centrifuged, and the resultant supernatants were collected 708
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as the polyamine fractions. Caldopentamine or spermine was added to the fractions as an 709
internal standard at the final concentration of 10 M, and filtered through a 0.45 m pore-710
size filter (Millex LH filter; Millipore) prior to injection. 711
Quantification of agmatine by LC-MS/MS. Agmatine in the rice syrup, 712
fermented with A. oryzae RW, was analyzed by LC-MS/MS at a facility at Shimadzu 713
Techno-Research (Kyoto, Japan). Agmatine-d8, an octa-deuterated stable isotope of 714
agmatine, was purchased from Toronto Research Chemicals (Toronto, ON, Canada) and 715
used as an internal standard. The mobile phase was composed of 0.05% (vol/vol) formic 716
acid containing 10 mM nonafluorovaleric acid (A) and methanol (B). Steamed rice was 717
saccharified at 30°C using the solid starter culture of A. oryzae RW in the presence of 111.3 718
mM L-lactic acid for 7 d as described above. The supernatant of the resultant rice syrup was 719
diluted 100 times with water, and the diluent was further diluted 100 times with the mobile 720
phase (A:B, 65:35). A stock solution of 1000 M agmatine (agmatine sulfate; Tokyo 721
Chemical Industry, Tokyo, Japan) was diluted with the mobile phase (A:B, 65:35) to obtain 722
standard solutions of 0.01, 0.05, 0.1, 0.5, 1, 5, and 10 M, for the generation of a 723
calibration curve. Agmatine-d8 was added to the diluted rice syrup and the standards, at the 724
final concentration of 10 ng/ml, and an aliquot (1 l) of each sample was injected onto the 725
LC-MS/MS instrument. The calibration curve was generated by plotting the peak-area ratio 726
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of agmatine and agmatine-d8 (y) vs. the nominal agmatine concentration of the standards (x), 727
and was fit using the least-squares linear regression analysis with a weighting factor of 1/x2. 728
An LC system (Nexera X2; Shimadzu, Kyoto, Japan) coupled to an electrospray 729
ionization triple-quadrupole MS/MS (LCMS-8060; Shimadzu), operated in the positive 730
mode, was used for agmatine detection. Chromatographic separations were carried out at 731
40°C using an Inertsil ODS-3 column (2.1 mm inner diameter 50 mm, 3 m particle size; 732
GL Science, Tokyo, Japan). The flow rate of the mobile phase was set at 0.2 ml/min. For 733
the gradient elution program, methanol concentration was 35% for 4 min; then linearly 734
increased from 35% to 90% over 1 min; and was held for 3 min. Methanol concentration 735
was then reduced to 35% at 8.1 min and held for 2 min for re-equilibration (the total run 736
time was 10 min). The detection was carried out in the multiple-reaction monitoring mode 737
by monitoring the m/z transitions from 131 to 72 for agmatine, and from 139 to 80 for 738
agmatine-d8. 739
Nucleotide sequence accession numbers. The nucleotide sequences of odc1, 740
odc2, odc3, and odc4 of A. oryzae RW were deposited in the DDBJ, EMBL, and GenBank 741
databases under the accession numbers LC368598, LC368599, LC368600, and LC368601, 742
respectively. 743
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ACKNOWLEDGMENTS 744
Part of this work was funded by the Core to Core Program, which was supported 745
by the Japan Society for the Promotion of Science (JSPS) and the National Research 746
Council of Thailand (NRCT). 747
748
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DNA cloning kit. Biochem Biophys Rep 4:148-151. 903
52. Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram 904
quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 905
72:248-254. 906
53. Morimoto N, Fukuda W, Nakajima N, Masuda T, Terui Y, Kanai T, Oshima T, Imanaka 907
T, Fujiwara S. 2010. Dual biosynthesis pathway for longer-chain polyamines in the 908
hyperthermophilic archaeon Thermococcus kodakarensis. J Bacteriol 192:4991-5001. 909
910
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55
FIGURE LEGENDS 911
FIG 1 The putative pathways of polyamine biosynthesis and agmatine catabolism in A. 912
oryzae, predicted from the genome sequence of A. oryzae RIB40. ADC, arginine 913
decarboxylase; arginase, arginine ureohydrolase; agmatinase, agmatine ureohydrolase; 914
ODC, ornithine decarboxylase; SPD synthase, spermidine synthase; AO, amine oxidase; 915
GBald DH, 4-guanidinobutyraldehyde dehydrogenase; GBase, 4-guanidinobutyrase (4-916
guanidinobutyrate ureohydrolase); TA, -aminobutyrate transaminase; SSA DH, succinate-917
semialdehyde dehydrogenase; dcSAM, decarboxylated S-adenosylmethionine; MTA, 918
methylthioadenosine; -KG, -ketoglutarate. The dotted arrow indicates the pathway 919
predicted to be absent in A. oryzae (decarboxylation of L-arginine by ADC). 920
921
FIG 2 The identification of the microorganism involved in agmatine production. (A) The 922
concentrations of agmatine and ethanol in rice wines and rice syrups. The steamed rice was 923
fermented with S. cerevisiae and A. oryzae RW (MPF) or only with A. oryzae RW 924
(saccharification, Sac.) at 20°C. As references, the steamed rice was enzymatically 925
degraded at 50°C with -amylase and glucoamylase (En.), and S. cerevisiae was cultivated 926
at 30°C in the rice syrup obtained after enzymatic degradation of steamed rice (SF). All 927
fermentations and the enzymatic degradation were conducted for 7 d, and the levels of 928
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agmatine and ethanol in the resultant rice wines and rice syrups were quantified by HPLC 929
and GC, respectively (n = 1). Bars: black, agmatine (mM); white, ethanol (%, vol/vol). ND, 930
not detected. (B) The HPLC profiles of rice wines made via MPF and SF, and rice syrups 931
obtained by saccharification with A. oryzae RW and enzymatic degradation of steamed rice. 932
The chromatograms are consistent with the data shown in Fig. 2A. The samples were 933
diluted 40 times with distilled water prior to HPLC analyses. Panels: a, 10 M standards; b, 934
MPF; c, SF; d, saccharification; e, enzymatic degradation. Peaks: P1, putrescine (4.8 min); 935
P2, spermidine (9.2 min); P3, agmatine (14.4 min); P4, spermine (18.6 min); IS, internal 936
standard (caldopentamine, 32.9 min). (C) Agmatine level in a rice syrup obtained by the 937
aseptic cultivation of A. oryzae RW (mM). A. oryzae RW was aseptically cultivated at 20°C 938
as described in the MATERIALS AND METHODS. The levels of agmatine accumulated in 939
the rice syrup were periodically determined by HPLC. The experiments were performed in 940
triplicate, and the error bars represent standard deviations. 941
942
FIG 3 The effect of cultivation temperature on agmatine production by A. oryzae RW. The 943
steamed rice was fermented with A. oryzae RW at various temperatures, and the 944
concentration of agmatine in the rice syrup was periodically monitored. The amount of 945
GlcNAc in the cultures was also determined, to evaluate hyphal growth. (A) GlcNAc 946
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(g/gculture
). (B) Agmatine levels in the rice syrup (mM). The experiments were performed 947
in triplicate, and the error bars represent standard deviations. Circles, 20°C; squares, 30°C; 948
triangles, 40°C; and diamonds, 50°C. 949
950
FIG 4 The effect of organic acids on agmatine production by A. oryzae RW and RIB40. 951
The steamed rice was fermented by A. oryzae RW or RIB40 at 30°C in the presence of 952
organic acids, and the concentration of agmatine in the rice syrup was periodically 953
determined. (A) The effect of L-lactic acid and sodium L-lactate on the agmatine production 954
by A. oryzae RW. Circles, 5.6 mM L-lactic acid; squares, 22.5 mM L-lactic acid; triangles, 955
111.3 mM L-lactic acid; and diamonds, 111.3 mM sodium L-lactate. (B) The effect of 956
succinic and citric acids on agmatine production by A. oryzae RW. Triangles, 111.3 mM L-957
lactic acid; crosses, 55.6 mM succinic acid; and bars, 36.9 mM citric acid. (A and B) The 958
experiments were performed in triplicate, and the error bars represent standard deviations. 959
(C) Agmatine levels in the rice syrup fermented with A. oryzae RIB40 (mM, n = 1). Open 960
circles, with 111.3 mM L-lactic acid; and closed circles, without L-lactic acid. 961
962
FIG 5 The effect of culture conditions on agmatine production by A. oryzae RW. A. oryzae 963
RW was cultivated in a liquefied rice medium, composed of mashed steamed rice and water 964
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acidified with 111.3 mM L-lactic acid (submerged culture). The fungus was also grown 965
under solid-state cultivation (saccharification of steamed rice using the solid starter culture) 966
in the presence of 111.3 mM L-lactic acid. Agmatine levels in the culture supernatants were 967
periodically determined by HPLC. GlcNAc content in cultures was determined to estimate 968
hyphal growth. (A) GlcNAc (g/gculture
). (B) Agmatine levels in the culture supernatants 969
(mM). (A and B) Open circles, submerged culture; closed circles, solid-state culture. The 970
experiments were performed in triplicate, and the error bars represent standard deviations. 971
972
FIG 6 The effect of additional L-arginine on agmatine production by A. oryzae RW. The 973
solid starter culture of A. oryzae RW was incubated with L-arginine in the presence or 974
absence of L-lactic acid at 30°C. The amount of agmatine accumulated in the supernatant 975
was periodically measured by HPLC. The experiments were performed in triplicate, and the 976
error bars represent standard deviations. Crosses, no L-arginine and L-lactic acid; diamonds, 977
10 mM L-arginine; circles, 10 mM L-arginine and 5.6 mM L-lactic acid; squares, 10 mM L-978
arginine and 22.5 mM L-lactic acid; and triangles, 10 mM L-arginine and 111.3 mM L-lactic 979
acid. 980
981
FIG 7 The agmatine-yielding activity of the homogenate of A. oryzae RW cells. The 982
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homogenates of the solid starter culture and hyphae obtained from a submerged culture 983
were incubated with L-arginine in the presence of PLP at selected pH values (3.0, 4.0, 5.0, 984
or 6.0) and temperatures (20, 30, 40, 50, or 60°C) for 60 min. The pH- and temperature-985
dependence activity assays were performed at 30°C and at pH 3.0, respectively. As a 986
reference, powdered steamed rice, disrupted with liquid nitrogen, was evaluated in the in 987
vitro assays. The activity in the cell homogenates was defined in terms of pmol of agmatine 988
per min per g of GlcNAc [(pmolagmatine
) min-1
(gGlcNAc
)-1
]. In the reference experiment, 989
the activity was normalized per weight (mg) of the powdered steamed rice [(pmolagmatine
) 990
min-1
(mgpowdered steamed rice
)-1
]. (A) pH dependency of the activity. (B) Temperature 991
dependency of the activity. (A and B) Squares, submerged culture; closed circles, solid 992
starter culture; and triangles, powdered steamed rice. The assays were performed in 993
triplicate, and the error bars represent standard deviations. 994
995
FIG 8 Decarboxylase activity of the A. oryzae RW ODCs with L-ornithine and L-arginine. 996
The ODCs of A. oryzae RW were expressed in E. coli, and the recombinant proteins 997
obtained in soluble form (ODC1 and ODC2) were analyzed by enzyme assays to determine 998
ODC or ADC activity, by monitoring putrescine or agmatine levels, respectively, in 999
reaction mixtures. (A) SDS-PAGE with Coomassie brilliant blue staining of the purified 1000
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recombinant proteins. The purified ODC1 and ODC2 proteins are indicated in their 1001
respective lanes. (B) pH dependency of the ODC activity [(nmolputrescine
) min-1
mg-1
]. (C) 1002
pH dependency of the ADC activity [(nmolagmatine
) min-1
mg-1
]. (B and C) Circles, ODC1; 1003
squares, ODC2. The assays were performed in triplicate, and the error bars represent 1004
standard deviations. 1005
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GBaldDH
HNH2N
COOH
NH
NH2 H2N
COOH
NH2
H2NNH2
HNH2N
NH
NH2
HN
NH
NH2OHCHN
NH
NH2HOOC NH2HOOC
H2N NH
NH2
ADCCO2
L-arginine
AOH2O, O2
NH3, H2O2
Agmatine
4-guanidinobutyraldehyde
L-ornithine
Agmatinase
Arginase
Urea
Urea
ODCCO2
Putrescine
NAD(P)+
H2ONAD(P)H
H+
4-guanidinobutyric acid
dcSAM MTA
Spermidine
γ-aminobutyricacid (GABA)
GBase
Urea
Succinatesemialdehyde
Succinic acid
TAα-KG
Glu
SSADH
NAD(P)+, H2O
NAD(P)H, H+
CHOHOOC
COOHHOOC
SPD synthase
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
ureohydrol ase; agmatinase, agmatine ureohydrolase; ODC, ornithine decarboxylase; SPD synthase,
spermidine synthase; AO, amine oxidase; GBald DH, 4-guanidinobutyraldehyde dehydrogenase; GBase, 4-
guanidinobutyrase (4-guanidinobutyrate ureohydrol ase); TA, γ-aminobutyrate transaminase; SSA DH,
succinate-semialdehyde dehydrogenase; dcSAM, decarboxylat ed S-adenosylmethionine; MTA,
methylthioadenosine; α-KG, α-ketoglutarat e. The dotted arrow indicates the pathway predicted to be absent
in A. oryzae (decarboxylation of L-arginine by ADC).
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0
5
10
15
0
1
2
3
4
Agm
atin
e (m
M)
A B
Eth
anol
(%, vol/vol)
MPF SF Sac. Retention time (min)
Inte
nsi
ty (
mV
)
0
100
200
0
200
400
0
200
400
0
200
400
0
200
400
0
P1 P2P3 P4 IS
b
c
10 20 30 40
e
d
a
En.
ND
ND
Time (day)
C
Agm
atin
e (m
M)
0
1
2
3
4
0 1 2 3 4 5 6 7
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 ri ce (SF). All
fermentations and the enzymatic degradation were conducted for 7 d, and the levels of agmatine and ethanol
in the resultant rice wines and rice syrups were quantifi ed by HPLC and GC, respectively (n = 1). Bars: black,
agmatine (mM); white, ethanol (%, vol/vol). ND, not detected. (B) The 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 Fig. 2A. The samples were diluted
40 times with distilled water prior to HPLC analyses. Panels: a, 10 µM standards; b, MPF; c, SF; d,
saccharifi cation; e, enzymatic degradation. Peaks: 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 (mM). A. oryzae RW was
aseptically cultivated at 20°C as described in the 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.
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0
100
200
300
400
0 1 2 3 4 5 6 7
A
Agm
atin
e (m
M)
Time (day)
B
Time (day)
Glc
NA
c (µ
g/g
culture
)
0
2
4
6
8
0 1 2 3 4 5 6 7
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 (µg/gculture). (B) Agmatine levels in the rice syrup (mM). The experiments were
performed in triplicate, and the error bars represent standard deviations. Circles, 20°C; squares, 30°C;
triangles, 40°C; and diamonds, 50°C.
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0
2
4
6
8
10
0 1 2 3 4 5 6 7
0
2
4
6
8
10
0 1 2 3 4 5 6 7
Ag
mat
ine
(mM
)A
Time (day)
B
Time (day)
Ag
mat
ine
(mM
)
0
2
4
6
8
10
0 1 2 3 4 5 6 7A
gm
atin
e (m
M)
C
Time (day)
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-l actic 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; and 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; and 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; and closed circles, without L-lactic acid.
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0
100
200
300
0 1 2 3 4 5
0
5
10
15
0 1 2 3 4 5
Agm
atin
e (m
M)
Time (day)Time (day)
Glc
NA
c (µ
g/g
culture
)
A B
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 ri ce 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 (µg/gculture). (B) Agmatine levels in the culture supernatants (mM).
(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.
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0
1
2
3
4
0 30 60 90 120
Ag
mat
ine
(mM
)
Time (min)
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; and triangles, 10 mM L-arginine and 111.3 mM L-lactic acid.
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0
20
40
60
80
100
10 20 30 40 50 60 70
0
20
40
60
80
100
2 3 4 5 6 7
Agm
atin
e-yie
ldin
g ac
tivity
[(p
mola
gm
atine)
min
-1(µ
gG
lcN
Ac)-1
or
(pm
ola
gm
atine)
min
-1(m
gp
ow
dere
dst
eam
ed r
ice)-1
]
pH Temperature (°C)
A B
30°C pH 3.0
Agm
atin
e-yie
ldin
g ac
tivity
[(p
mola
gm
atine)
min
-1(µ
gG
lcN
Ac)-1
or
(pm
ola
gm
atine)
min
-1(m
gp
ow
dere
dst
eam
ed r
ice)-1
]
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 [(pmolagmatine) min-1 (µgGlcNAc)-1]. In the reference experiment, the activity was normalized per
weight (mg) of the powdered steamed rice [(pmolagmatine) min-1 (mgpowdered 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; and triangles, powdered steamed rice. The assays were performed in triplicate,
and the error bars represent standard deviations.
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0
1
2
3
4
5
2 3 4 5 6 7
0
200
400
600
800
2 3 4 5 6 7
A
9766
45
30
20
kDa
Mar
ker
OD
C1
OD
C2
CB
AD
C a
ctiv
ity
(nm
ola
gm
ati
ne)
min
-1m
g-1
]
OD
C a
ctiv
ity
[(nm
olp
utr
esc
ine)
min
-1m
g-1
]
pH pH
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 [(nmolputrescine) min-1 mg-1]. (C) pH dependency of the ADC
activity [(nmolagmatine) min-1 mg-1]. (B and C) Circles, ODC1; squares, ODC2. The assays were performed in
triplicate, and the error bars represent standard deviations.
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