a b c on January 17, 2021 by guest...2018/05/21 · The final e thanol concentration s in the rice...

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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

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Running Head: Efficient Production of Agmatine by A. oryzae 13

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#Address correspondence to: Shinsuke Fujiwara, [email protected] 15

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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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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

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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

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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

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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 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 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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44

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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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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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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58

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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59

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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60

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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