Accepted Manuscript l-lactic acid production from starch by simultaneous saccharification and fermentation in a genetically engineered Aspergillus oryzae pure culture Satoshi Wakai, Toshihide Yoshie, Nanami Asai-Nakashima, Ryosuke Yamada, Chiaki Ogino, Hiroko Tsutsumi, Yoji Hata, Akihiko Kondo PII: DOI: Reference:

S0960-8524(14)01353-4 http://dx.doi.org/10.1016/j.biortech.2014.09.094 BITE 13984

To appear in:

Bioresource Technology

Received Date: Revised Date: Accepted Date:

18 July 2014 17 September 2014 18 September 2014

Please cite this article as: Wakai, S., Yoshie, T., Asai-Nakashima, N., Yamada, R., Ogino, C., Tsutsumi, H., Hata, Y., Kondo, A., l-lactic acid production from starch by simultaneous saccharification and fermentation in a genetically engineered Aspergillus oryzae pure culture, Bioresource Technology (2014), doi: http://dx.doi.org/10.1016/ j.biortech.2014.09.094

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

1

Title: L-lactic acid production from starch by simultaneous saccharification and

2

fermentation in a genetically engineered Aspergillus oryzae pure culture

3 4

Authors: Satoshi Wakaia,†, Toshihide Yoshieb,†, Nanami Asai-Nakashimaa, Ryosuke

5

Yamadaa, Chiaki Oginob, Hiroko Tsutsumic, Yoji Hatac, and Akihiko Kondob,*

6

†

These authors contributed equally to this work.

7 8

Affiliations:

9

a

Organization of Advanced Science and Technology, Kobe University, 1-1

10

Rokkodai-cho, Nada-ku, Kobe, Hyogo 657-8501, Japan

11

b

12

Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe, Hyogo 657-8501, Japan

13

c

14

Fushimi-ku, Kyoto, Kyoto 612-8385, Japan

Department of Chemical Science and Engineering, Graduate School of Engineering,

Research Institute, Gekkeikan Sake Co. Ltd., 101 Shimotoba-koyanagi-cho,

15 16

* Corresponding author: Akihiko Kondo

17

Department of Chemical Science and Engineering, Graduate School of Engineering,

18

Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe, Hyogo 657-8501, Japan

19

E-mail: [email protected]

20

Tel: +81-78-803-6196

21 22

Subject Classification: BIOPROCESSES (50.120 Submerged fermentation)

23 24

Keywords: Aspergillus oryzae, L-lactic acid, starch, lactate dehydrogenase

1

25

Abstract

26

Lactic acid is a commodity chemical that can be produced biologically. Lactic

27

acid-producing Aspergillus oryzae strains were constructed by genetic engineering. The

28

A. oryzae LDH strain with the bovine L-lactate dehydrogenase gene produced 38 g/L of

29

lactate from 100 g/L of glucose. Disruption of the wild-type lactate dehydrogenase gene

30

in A. oryzae LDH improved lactate production. The resulting strain A. oryzae LDH∆871

31

produced 49 g/L of lactate from 100 g/L of glucose. Because A. oryzae strains innately

32

secrete amylases, A. oryzae LDH∆871 produced approximately 30 g/L of lactate from

33

various starches, dextrin, or maltose (all at 100 g/L). To our knowledge, this is the first

34

report describing the simultaneous saccharification and fermentation of lactate from

35

starch using a pure culture of transgenic A. oryzae. Our results indicate that A. oryzae

36

could be a promising host for the bioproduction of useful compounds such as lactic

37

acid.

38 39 40

2

41 42

1. Introduction To create a sustainable society with an environmentally sound material cycle, it is

43

necessary to produce chemicals and fuels from renewable, inexpensive, and abundantly

44

available biomass resources such as starch and cellulose through fermentation processes

45

using microorganisms (van Zyl et al., 2012; Jang et al., 2012). Biomasses can be

46

classified as sugar-, starch-, and cellulose-based materials. Many sugar-based biomasses

47

are edible. Therefore, the utilization of such biomass as starting materials for

48

bioproduction would make a direct impact on food supplies. Although cellulosic

49

biomass has attracted attention because it is not a usable feedstock, cellulose is difficult

50

to break down. In contrast, starch is the most abundant hexose polymer in plants after

51

cellulose, and it can be hydrolyzed into simpler carbohydrates, including dextrin,

52

maltose, and glucose by treatment with acids and enzymes. However, efficient starch

53

hydrolysis requires the activity of α-1,4 and α-1,6-debranching hydrolases, debranching

54

enzymes, and transferases (van der Maarel et al., 2002).

55

Lactic acid is a commodity chemical that is widely used in the food, pharmaceutical,

56

textile, leather, and chemical industries (Abdel-Rahman et al., 2011). It can be produced

57

biologically, and it is a source of polylactate. Polylactate has been a focus of recent

58

studies for its use as a biodegradable alternative to petroleum-based plastic products

59

(Madhavan Nampoothiri et al., 2010). Therefore, there have been various reports on the

60

bioproduction of lactate by various microorganisms, including lactic acid bacteria

61

(Litchfield, 1996), Rhizopus species (Thitiprasert et al., 2011), recombinant yeasts

62

(Saitoh et al., 2005; Dequin and Barre, 1994; Ishida et al., 2006), and recombinant

63

Escherichia coli (Chang et al., 1999). Such bioproduction of lactate by microorganisms

64

requires biomass resources to avoid the consumption of petroleum resources. However,

3

65

these microorganisms do not have an inherent ability to degrade biomass. Therefore,

66

lactate bacteria and E. coli have been genetically engineered to decompose a specific

67

biomass (Narita et al., 2006; Okano et al., 2007; Okano et al., 2008), and co-cultivation

68

of biomass-degrading microorganisms has been studied (Kurosawa et al., 1998). The

69

filamentous fungus Aspergillus oryzae has a strong advantage over other organisms

70

because it produces various amylolytic enzymes such as Taka-amylase (alpha-amylase)

71

and glucoamylase (Sugimoto and Shoji, 2012).

72

Aspergillus oryzae has been used in Japan for more than a 1,000 years for the

73

production of fermented foods such as sake, miso, and soy sauce, and it is ‘‘genetically

74

recognized as safe’’ by the US Food and Drug Administration (Barbesgard et al., 1992).

75

It is also capable of secreting large amounts of proteins into the culture medium, making

76

it an excellent host for industrial enzyme production. For these reasons, A. oryzae has

77

been used as an enzyme producer in industry (Fleissner and Dersch, 2010). In addition,

78

its potential use in chemical production has been investigated. Based on its genome

79

sequence (Machida et al., 2005), the wild-type A. oryzae strain RIB40 was genetically

80

engineered for bioproduction of fatty acids and malic acid (Tamano et al., 2013; Brown

81

et al., 2013).

82

In this paper, we report the bioproduction of lactate from sugars and starch by a

83

recombinant A. oryzae strain pure culture without addition of amylolytic enzymes. First,

84

a recombinant A. oryzae strain that expresses a bovine lactate dehydrogenase was

85

constructed. Then, we measured L-lactate production from feedstock by the recombinant

86

A. oryzae strain. We determined that A. oryzae is a promising host for bioproduction.

87 88

4

89

2. Material and Methods

90

2.1. Strains and media

91

The strains used in this study are summarized in Table 1. The A. oryzae strains

92

RIB40 and NSPlD1 were used as a wild-type strain and a DNA recombination host

93

strain, respectively. NSPlD1 was derived from A. oryzae RIB40 (Maruyama and

94

Kitamoto, 2008). The filamentous fungal strains of Rhizopus oryzae were used as a

95

source of the lactate dehydrogenase (LDH) gene.

96

The A. oryzae strains were cultivated on potato dextrose agar (PDA; Nissui

97

Pharmaceutical, Tokyo, Japan). A. oryzae auxotrophic strains were cultivated in

98

Czapek-Dox (CD) medium (2% [w/v] D-glucose, 0.3% [w/v] NaNO3, 0.2% [w/v] KCl,

99

0.1% [w/v] KH2PO4, 0.05% [w/v] MgSO4·7H2O, and 0.8 M NaCl, 1.5% [w/v] agar, pH

100

6.0) with necessary supplements (0.0015% [w/v] methionine, 20 mM uridine, 0.2%

101

[w/v] uracil). The R. oryzae strains were also cultivated on PDA and liquid medium

102

(GPY medium; 3% [w/v] D-glucose, 0.2% [w/v] KCl, 0.1% [w/v] KH2PO4, 0.05% [w/v]

103

MgSO4·7H2O, 1% [w/v] Bacto-peptone [Difco Laboratories, Detroit, MI, USA], and

104

0.5% [w/v] yeast extract, pH 6.0).

105

Escherichia coli NovaBlue cells (Novagen, Inc., Madison, WI, USA) were

106

used as the cloning host for DNA manipulations. E. coli transformants were cultivated

107

in Luria-Bertani medium (1% [w/v] tryptone, 0.5% [w/v] yeast extract [Nacalai Tesque,

108

Kyoto, Japan], and 0.5% [w/v] NaCl) containing 0.1 mg/mL ampicillin.

109 110

2.2. Construction of an A. oryzae strain that expresses a heterologous lactate

111

dehydrogenase gene

112

The LDH-A gene sequence (GenBank accession number D90141) encoding

5

113

bovine (Bos taurus) LDH was optimized based on the codon usage of A. oryzae and

114

synthesized by GENEWIZ (South Plainfield, NJ, USA). The resulting gene was named

115

as bLDH. The predicted amino acid sequence encoded by the bLDH gene was the same

116

as that in the database.

117

The plasmid vector for the expression of bLDH was constructed as follows:

118

gene fragment encoding bLDH with a hexahistidine tag was amplified by PCR using

119

primers ascI-L-LDH-F and notI-6His-L-LDH-R. The primers used in this study are

120

summarized in Table 2. KOD-plus-Neo DNA polymerase (TOYOBO, Osaka, Japan)

121

was used to amplify bLDH by PCR. The pIS1 vector was digested with AscI and NotI,

122

and the amplified fragment was cloned into pISI, which contains the sodM promoter

123

and glaB terminator from A. oryzae (Ishida et al., 2004), using the In-Fusion™ PCR

124

Cloning System (TaKaRa Bio Inc., Shiga, Japan). The resulting plasmid, pIS1-bLDH,

125

was introduced into E. coli NovaBlue cells. The nucleotide sequence inserted into the

126

plasmid was verified by DNA sequencing (ABI PRISM 3130xl genetic analyzer;

127

Applied Biosystems, Foster City, CA, USA).

128

The genes encoding LDHs (roLDHA and roLDHB) were amplified from the

129

chromosomal DNA of R. oryzae strains. Each PCR product was digested with AscI and

130

NotI and then ligated to the AscI/NotI-double digested plasmid vector pIS1. The

131

resulting plasmids, pIS1-roLDHA and pIS1-roLDHB, were introduced into E. coli

132

NovaBlue cells, and then the nucleotide sequence was verified by DNA sequencing as

133

described above.

134

A. oryzae was transformed according to a previously described method

135

(Kitamoto, 2002) with some minor modifications. A. oryzae LDH was generated by

136

introducing the expression plasmid into A. oryzae NSPlD1. The resulting transformants

6

137

were subcultured on CD medium supplemented with 0.0015% (w/v) L-methionine, 20

138

mM uridine, and 0.2% (w/v) uracil.

139 140 141

2.3. Construction of a lactate dehydrogenase-deletion strain of A. oryzae LDH The A. oryzae LDH gene was deleted by using pyrG as a marker, according to a

142

previously described method (Maruyama and Kitamoto, 2008) with some minor

143

modifications. The pyrG gene was amplified by PCR using the primer pair

144

pyrG-F/pyrG-R and A. oryzae RIB40 genomic DNA as a template. The 1.3-kb upstream,

145

0.3-kb upstream, and 1.3-kb downstream flanking regions of the AO090023000871

146

gene, which encodes lactate dehydrogenase, were amplified from A. oryzae RIB40

147

genomic DNA by PCR using the primer sets AO871-up1300F/AO871-up1300R,

148

AO871-up300F/AO871-up300R, and AO871-down1000F/AO871-down1000R,

149

respectively. The 1.3-kb upstream and pyrG fragments were fused, and the 0.3-kb

150

upstream and 1.3-kb downstream fragments were fused. Both these fragments were then

151

fused in tandem and cloned into the HindIII and XbaI sites of the pIS1 vector, which

152

contains genes for ampicillin resistance (Ampr) and the Coli origin (ori), without the

153

niaD marker, by using the In-Fusion™ PCR Cloning System. The resulting plasmid was

154

named pIS1-∆871 and was introduced into E. coli NovaBlue cells.

155

The DNA fragment used to generate the AO090023000871 deletion was

156

amplified by PCR using the same primers that were used for constructing pIS1-∆871.

157

The deletion fragment for the AO090023000871 gene was introduced into A. oryzae

158

LDH. The resulting transformant was plated on CD agar supplied with 0.0015% (w/v)

159

L-methionine. The

160

genomic DNA.

deletion of the AO090023000871 gene was confirmed by PCR using

7

161

The gene-complemented auxotrophic strains, NSPlD1-pyrG and LDHpyrG,

162

were generated by transformation with the pIS1-pyrG and pyrG gene fragment,

163

respectively. The resulting transformants were subcultured on CD agar supplemented

164

with 0.0015% (w/v) L-methionine.

165 166 167

2.4. Lactate fermentation A PY-based medium (0.2% [w/v] KCl, 0.1% [w/v] KH2PO4, 0.05% [w/v]

168

MgSO4·7H2O, 1% [w/v] Bacto-peptone [Difco Laboratories], and 0.5% [w/v] yeast

169

extract, pH 6.0) was used for lactate fermentation. Glucose (3–30% [w/v]), various

170

starches (soluble [Nacalai Tesque], potato [Nacalai Tesque], wheat [Nacalai Tesque],

171

and corn [Nacalai Tesque]), or maltose was added to the PY-based medium as a

172

substrate for lactate production. Calcium carbonate (CaCO3) was added to the medium

173

at a final concentration of 3% (w/v). The spores of transformants and the wild-type

174

strain were inoculated into the test medium (15 mL) at a density of 1 × 106 spores/mL in

175

a 50-mL test tube (ϕ30 mm). Fermentation was performed at 30°C on an orbital shaker

176

with a speed of 200 rpm. In the scaled-up fermentation, the spores of transformants

177

were inoculated into the test medium (100 mL) at a density of 1 × 106 spores/mL in a

178

500-mL shake flask.

179 180 181

2.5. Analytical methods The concentration of organic acids (lactic acid, malic acid, and succinic acid)

182

was measured by HPLC using an organic acid analysis system (solvent delivery system,

183

LC-20AB; column, Shim-pack SCR-102H; column temperature, 50°C; detector,

184

SPD-20A; Shimadzu Co., Kyoto, Japan). Five millimolar p-toluenesulfonic acid was

8

185

used as the mobile phase, and 20 mM bis-Tris containing 5 mM p-toluenesulfonic acid

186

and 10 µM EDTA were mixed just before detection to enhance sensitivity. The optical

187

purity (enantiomeric excess) of L-lactic acid was defined as follows: (L-lactic acid –

188

D-lactic

189

acids was measured using a D-/L-lactic acid kit (Megazyme, Wicklow, Ireland).

190

acid)/(L-lactic acid + D-lactic acid) × 100. The concentration of L- and D-lactic

The concentration of glucose was determined using the Wako Glucose CII-Test

191

kit (Wako Pure Chemical Industries, Osaka, Japan). The amount of total sugar,

192

corresponding to both starch and starch hydrolysis products, was colorimetrically

193

determined by the phenol-sulfuric acid reaction (Dubois et al. 1956). Alpha amylase

194

activity was determined using the alpha-amylase kit (Kikkoman Corp., Chiba, Japan).

195 196 197 198

2.6. Nucleotide sequence accession number The optimized bovine LDH-A gene sequence has been submitted to the DDBJ/EMBL/GenBank under accession number AB908309.

199 200

3. Results and Discussion

201 202 203

3.1. Construction of lactate-producing A. oryzae strains The wild-type A. oryzae strain RIB40 encodes an L-lactate dehydrogenase;

204

however, it does not accumulate detectable amounts of lactate. To improve the lactate

205

productivity of this microorganism, we first constructed A. oryzae strains that express

206

roLDHA or roLDHB from the lactate-producing filamentous fungus Rhizopus oryzae.

207

These transformants did not accumulate detectable amounts of lactate during a 14-day

208

fermentation. Because bLDH-expressing Saccharomyces cerevisiae produces large

9

209

amounts of L-lactic acid (Saitoh et al., 2005), we constructed a bLDH-expressing A.

210

oryzae strain (A. oryzae LDH). The A. oryzae LDH strain produced 38 g/L of lactate

211

from 100 g/L of glucose during a 14-day fermentation (Fig. 1a). An optical purity

212

analysis of the lactate produced showed that it was 99.9% L-lactic acid. The

213

concentration of the lactate produced was lower than that of the lactate produced by the

214

bLDH-expressing S. cerevisiae (Saitoh et al., 2005).

215

We expected that the wild-type lactate dehydrogenase (encoding on gene

216

AO090023000871) of A. oryzae LDH might hinder the accumulation of lactate due to

217

catalysis of the backward reaction from lactate to pyruvate. Therefore, the gene was

218

disrupted by homologous recombination with a pyrG gene fragment containing the

219

sequence flanking the targeted gene (Fig. 2a). Disruption of AO090023000871 in the

220

resulting transformant A. oryzae LDH∆871 was verified by colony-direct PCR analysis.

221

PCR using A. oryzae LDH∆871 spores and primers yielded a single 4.6-kb band (Fig.

222

2b), which corresponds to the expected size of a DNA fragment containing pyrG and the

223

AO090023000871 flanking region (Fig. 2a). In contrast, a similar PCR using A. oryzae

224

LDH spores amplified a 3.9-kb DNA fragment (Fig. 2b), which corresponds to the

225

length of the AO090023000871 gene alone (Fig. 2a). These results indicate that the

226

AO090023000871 region was successfully deleted and replaced with a pyrG fragment

227

in the A. oryzae LDH∆871 genome.

228

To examine the production of lactate by A. oryzae LDH∆871, batch

229

fermentation tests were conducted using glucose as a carbon source. After 7 d, A. oryzae

230

LDH∆871 produced 45 g/L of lactate from 100 g/L of glucose (Fig. 1a). This

231

concentration was higher than that of lactate produced by A. oryzae LDH, and the

232

production period was also shorter (Fig. 1a). The A. oryzae LDH strain has a growth

10

233

deficiency owing to the pyrG gene deletion, and has delayed glucose consumption in

234

the early growth phase (Fig. 1b). To examine the influence of the growth deficiency on

235

the production of lactate, A. oryzae LDHpyrG, which is complemented by the pyrG

236

gene, was constructed. A. oryzae LDHpyrG showed wild-type-like glucose consumption

237

and similar levels of lactate production as compared to A. oryzae LDH (Fig. 1a and b).

238

In contrast, the wild-type A. oryzae strain (RIB40), an auxotrophic strain (NSPlD1), and

239

a pyrG-complemented auxotrophic strain (NSPlD1-pyrG) did not produce detectable

240

amounts of lactate during a 14-day fermentation (Fig. 1a). These results indicate that A.

241

oryzae LDH∆871 expressed functional bLDH and that deletion of the native LDH

242

improved lactate production; however, the reasons for this are not known.

243 244 245

3.2. Lactate production from glucose by the transgenic A. oryzae LDH∆871 The effect of the initial glucose concentration on lactate fermentation was

246

examined with the aim of increasing the concentration of the lactate produced.

247

Fermentation of 150 g/L of glucose produced 70 g/L of lactate (Fig. 3a). This yield

248

(47%; as g-lactate/g-glucose) was similar to that obtained with 100 g/L of glucose

249

(48%); however, the time needed to achieve the maximum concentration of lactate using

250

150 g/L was longer than the time required using 100 g/L. The lactate concentration and

251

yield in a fermentation test with 30 g/L of glucose were lower than with 100 g/L of

252

glucose. In contrast, fermentation with 200 and 300 g/L of glucose produced a higher

253

concentration of lactate than with 100 g/L of glucose; however, the yield decreased

254

significantly, and the glucose was not completely consumed even after 14 days of

255

fermentation (Fig. 3b). In addition, the amounts of by-products such as malate and

256

succinate were positively related to glucose concentration (Fig. 3c and d).

11

257

The effect of the shape of the fermentation vessel on fermentation was also

258

studied. Fermentation in the shaker flask resulted in similar levels of lactate production

259

(49 g/L) and glucose consumption (within 10-day fermentation) (Fig. 3a and b). This

260

result indicates that the shape of the fermentation vessels does not influence lactate

261

production.

262

The A. oryzae LDH∆871 strain used approximately one-half of the total

263

glucose to produce lactate, and the remaining half of the glucose was used as a carbon

264

source for other metabolic processes. The intermediate metabolites in the tricarboxylic

265

acid (TCA) cycle, malate and succinate, were also detected (Fig. 3c and d). Because the

266

amounts of these compounds increased as the concentration of glucose increased, excess

267

amounts of glucose may induce pyruvate run-off into the TCA cycle. Therefore, the

268

maximal conversion rate of lactate from glucose by the transgenic A. oryzae LDH∆871

269

strain is approximately 50%. The transgenic A. oryzae strain can produce lactic acid,

270

while this titer and productivity (conversion rate and long fermentation time) were low

271

compared with previous reports by using other microorganisms (Castillo Martinez et al.,

272

2013). In the future, the effective production of lactic acid by the transgenic A. oryzae

273

may be necessary to improve by metabolic engineering and examining fermentation

274

style (jar fermentation and immobilized fermentation).

275 276 277

3.3. Simultaneous saccharification and fermentation of starches A. oryzae produces amylases that break down starch to glucose. To determine

278

whether A. oryzae LDH∆871 directly produces lactate from starch, fermentation tests

279

were conducted with starch rather than glucose as a carbon source. The A. oryzae

280

LDH∆871 strain produced 30 g/L of lactate from 100 g/L of soluble starch after 7 d of

12

281

fermentation (Fig. 4a). Similar amounts of lactate were also produced from other

282

insoluble starches (potato, corn, and wheat). During the fermentation, 2–5 g/L of

283

glucose, as a decomposition product of starch, was transiently detected on days 2–4;

284

however, the transiently accumulated glucose was rapidly broken down (Fig. 4b and c).

285

The optical purity of the L-lactate produced from various starches was >99.9%.

286

Fermentation tests were also conducted with maltose, which is an intermediate

287

compound of starch degradation. Fermentation of 100 g/L of maltose yielded 33 g/L of

288

lactate (Fig. 4a). Furthermore, the fermentation of maltose resulted in rapid

289

consumption of maltose and transient accumulation of glucose on day 2 (Fig. 4b and c).

290

The fermentation from a molecule with such lower molecular weight did not improve

291

lactate productivity.

292

To our knowledge, this is the first study to show that a pure A. oryzae culture

293

without addition of amylolytic enzyme can produce lactate from starch through

294

simultaneous saccharification and fermentation. Direct lactate production from starch

295

using a mixed culture system of Aspergillus awamori and Streptococcus lactis has been

296

previously reported (Kurosawa et al., 1988), and fermentation by this mixed culture

297

produced 25 g/L of lactate from 50 g/L of starch. Our pure culture system is simpler and

298

more useful than the abovementioned mixed culture fermentation system. However, the

299

concentration and productivity by the A. oryzae pure culture is low compared with those

300

by simultaneous saccharification and fermentation using other microorganisms (Yen et

301

al., 2010; Jin et al., 2003) and co-utilization of microorganisms and amylolytic enzymes

302

(Nguyen et al., 2013; Zhou et al., 2013).

303 304

Furthermore, the concentration of lactate produced from starch using our system was lower than that of lactate produced from glucose. Starch and maltose are

13

305

broken down into glucose and then metabolized to pyruvate through glycolysis. In

306

wild-type cells, pyruvate is then metabolized through the TCA cycle; however, in the

307

recombinant strain, it is converted into lactate. Pyruvate represents a key branch point

308

for lactate production and the TCA cycle. It has been reported that the concentration of

309

glucose affects the expression of enzymes in the glycolysis pathway and the TCA cycle

310

(Maeda et al., 2004). Therefore, metabolic engineering would be useful to improve

311

lactate production by the A. oryzae strain.

312

Although for the simultaneous saccharification and fermentation by A. oryzae,

313

the concentration of lactate produced from starch was less than that produced from

314

glucose, it is useful because A. oryzae can utilize not only soluble starch but also

315

insoluble starch without pretreatment such as gelatinization. The structure of starch

316

from different biomass types varies. For some structures, starches are difficult to

317

efficiently degrade using enzymatic reactions in industrial applications. Because A.

318

oryzae produces different types of amylases, it may be useful for decomposing such

319

versatile starch-based biomass.

320 321 322

3.4. Improvement of lactate production by addition of glucose Aspergillus oryzae shows a high capacity for starch decomposition because the

323

expression of some amylases is induced by starch and its hydrolysates. However, the

324

overexpression of a protein requires energy. Therefore, the low yield of lactate

325

production in transgenic A. oryzae cells degrading a starch substrate may reflect such

326

energy expenditure. Because the induction of amylase in A. oryzae is regulated by

327

carbon catabolite repression by glucose (Ichinose et al., 2014), we tested the production

328

of lactate from a mixture of glucose and starch to improve the yield of lactate by

14

329

repressing the induced expression of amylase. With a mixture of glucose (50 g/L) and

330

starch (50 g/L), 45 g/L of lactate were produced after 7 d (Fig. 5a). The concentration of

331

lactate produced from a mixture of starch and glucose was higher than that produced

332

from 100 g/L of soluble starch and was similar to that produced from 100 g/L of glucose.

333

After 4 d of fermentation, the consumption rate of total sugar in the fermentation of

334

starch alone was slower than that achieved in the fermentation of the mixed substrate

335

(Fig. 5b). The glucose-consumption rate in the fermentation test using the mixed

336

substrate was slower than that achieved in the fermentation of glucose alone (Fig. 5c).

337

The supernatant after the 1-day cultivation with SPY medium showed 8.2

338

U/mL alpha-amylase activity and a distinct 48-kDa protein band in the SDS-PAGE

339

analysis (Fig. 5d). This activity level reached 12.7 U/mL after 2 d of cultivation. In

340

contrast, the alpha-amylase activity in the cultivation with glucose-supplied SPY

341

medium was only 1.8 and 7.1 U/mL after 1-day and 2-day cultivations, respectively, and

342

the 48-kDa protein band in the supernatant after 1 d was weaker than after 2 d and

343

during cultivation with SPY medium (Fig. 5d). In contrast, the cultivation with GPY

344

medium did not show detectable activity or a visible protein band after 1 d of cultivation

345

(Fig. 5d). Only 2.2 U/mL of alpha amylase activity was observed after 7 d of cultivation.

346

These results indicate that the addition of glucose to SPY medium induced carbon

347

catabolite repression and then reduced the expression of alpha-amylase during the early

348

growth phase. This repression would alter energy conversion (consumption) and carbon

349

flux in the cells, because the lactate production was slightly improved. Therefore,

350

efficient production of lactate in transgenic A. oryzae may be necessary to improve the

351

regulation of amylolytic enzyme expression and metabolic engineering, including

352

repressing run-off into the TCA cycle.

15

353 354

4. Conclusion

355

Recently, the demand for lactate has been increasing considerably because of

356

its great potential for use in the production of biodegradable plastics. However, the high

357

costs of bioproduction of lactate from petroleum and bio-based materials have limited

358

the large-scale application of polylactate. In this work, a genetically engineered A.

359

oryzae strain produced lactate from starch by simultaneous saccharification and

360

fermentation. The process used a pure culture of A. oryzae and was simpler than

361

conventional methods with mixed cultures or co-utilization of enzymes. Therefore, our

362

results strongly indicate that A. oryzae is a promising host microorganism for the

363

bioproduction of compounds.

364 365

Acknowledgements

366

We are grateful to Professor Kitamoto (Tokyo University) for providing A.

367

oryzae NSPlD1. We would like to thank Professor Tsuneo Yamane (Chubu University)

368

for his valuable suggestions. This work was supported by the Special Coordination

369

Funds for Promoting Science and Technology, the Creation of Innovation Centers for

370

Advanced Interdisciplinary Research Areas (Innovative Bioproduction Kobe) from

371

Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.

372 373

References

374 375 376

1.

Abdel-Rahman, M.A., Tashiro, Y., Sonomoto, K., 2011. Lactic acid production from lignocellulose-derived sugars using lactic acid bacteria: overview and limits. J.

16

377 378

Biotechnol. 156, 286–301. doi: 10.1016/j.jbiotec.2011.06.017. 2.

379 380

Barbesgard, P., Heldt-Hansen, H.P., Didenrichsen, B., 1992. On the safety of Aspergillus oryzae; a review. Appl. Microbiol. Biotechnol. 36, 569–572.

3.

Brown, S.H., Bashkirova, L., Berka, R., Chandler, T., Doty, T., McCall, K.,

381

McCulloch, M., McFarland, S., Thompson, S., Yaver, D., Berry, A., 2013.

382

Metabolic engineering of Aspergillus oryzae NRRL 3488 for increased production

383

of L-malic acid. Appl. Microbiol. Biotechnol. 97, 8903–8912. doi:

384

10.1007/s00253-013-5132-2.

385

4.

Castillo Martinez, F.A., Balciunas, E.M., Salgado, J.M., Dominguez Gonzalez, J.M.,

386

Converti, A., Oliveira, R.P.D.S. 2013. Lactic acid properties, applications and

387

production: A review. Trends Food Sci. Technol. 30, 70–83.

388

5.

Chang, D.E., Jung, H.C., Rhee, J.S., Pan, J.G., 1999. Homofermentative production

389

of D- or L-lactate in metabolically engineered Escherichia coli RR1. Appl. Environ.

390

Microbiol. 65, 1384–1389.

391

6.

Dequin, S., Barre, P., 1994. Mixed lactic acid-alcoholic fermentation by

392

Saccharomyces cerevisiae expressing the Lactobacillus casei L(+)-LDH.

393

Biotechnology (N Y) 12, 173–177.

394

7.

Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F., 1956.

395

Colorimetric method for determination of sugars and related substances. Anal.

396

Chem. 28, 350–356.

397

8.

Fleissner, A., Dersch, P., 2010. Expression and export: recombinant protein

398

production systems for Aspergillus. Appl. Microbiol. Biotechnol. 87, 1255–1270.

399

doi: 10.1007/s00253-010-2672-6.

400

9.

Ichinose, S., Tanaka, M., Shintani, T., Gomi, K., 2014. Improved α-amylase

17

401

production by Aspergillus oryzae after a double deletion of genes involved in

402

carbon catabolite repression. Appl. Microbiol. Biotechnol. 98, 335–43. doi:

403

10.1007/s00253-013-5353-4.

404

10. Ishida, H., Hata, Y., Kawato, A., Abe, Y., Kashiwagi, Y., 2004. Isolation of a novel

405

promoter for efficient protein production in Aspergillus oryzae. Biosci. Biotechnol.

406

Biochem. 68, 1849–1857.

407

11. Ishida, N., Saitoh, S., Onishi, T., Tokuhiro, K., Nagamori, E., Kitamoto, K.,

408

Takahash,i H., 2006. The effect of pyruvate decarboxylase gene knockout in

409

Saccharomyces cerevisiae on L-lactic acid production. Biosci. Biotechnol. Biochem.

410

70, 1148–1153.

411

12. Jang, Y.S., Kim, B., Shin, J.H., Choi, Y.J., Choi, S., Song, C.W., Lee, J., Park, H.G.,

412

Lee, S.Y., 2012. Bio-based production of C2-C6 platform chemicals. Biotechnol.

413

Bioeng. 109, 2437–2459. doi: 10.1002/bit.24599.

414

13. Jin, B., Huang, L.P., Lant, P., 2003. Rhizopus arrhizus – a producer for

415

simultaneous saccharification and fermentation of starch waste materials to

416

L(+)-lactic

417 418

acid. Biotechnol. Lett. 25, 1983–1987.

14. Kitamoto, K., 2002. Molecular biology of the Koji molds. Adv. Appl. Microbiol. 51, 129–153.

419

15. Kurosawa, H., Ishikawa, H., Tanaka, H., 1998. L-Lactic acid production from starch

420

by coimmobilized mixed culture system of Aspergillus awamori and Streptococcus

421

lactis. Biotechnol. Bioeng. 31, 183–187.

422 423 424

16. Litchfield, J.H., 1996. Microbiological production of lactic acid. Adv. Appl. Microbiol. 42, 45–95. 17. Madhavan Nampoothiri, K., Nair, N.R., John, R.P., 2010. An overview of the recent

18

425

developments in polylactide (PLA) research. Bioresour. Technol. 101, 8493–8501.

426

doi: 10.1016/j.biortech.2010.05.092.

427

18. Maeda, H., Sano, M., Maruyama, Y., Tanno, T., Akao, T., Totsuka, Y., Endo, M.,

428

Sakurada, R., Yamagata, Y., Machida, M., Akita, O., Hasegawa, F., Abe, K., Gomi,

429

K., Nakajima, T., Iguchi, Y., 2004. Transcriptional analysis of genes for energy

430

catabolism and hydrolytic enzymes in the filamentous fungus Aspergillus oryzae

431

using cDNA microarrays and expressed sequence tags. Appl. Microbiol. Biotechnol.

432

65, 74–83.

433

19. Machida, M., Asai, K., Sano, M., Tanaka, T., Kumagai, T., Terai, G., Kusumoto, K.,

434

Arima, T., Akita, O., Kashiwagi, Y., Abe, K., Gomi, K., Horiuchi, H., Kitamoto, K.,

435

Kobayashi, T., Takeuchi, M., Denning, D.W., Galagan, J.E., Nierman, W.C., Yu, J.,

436

Archer, D.B., Bennett, J.W., Bhatnagar, D., Cleveland, T.E., Fedorova, N.D., Gotoh,

437

O., Horikawa, H., Hosoyama, A., Ichinomiya, M., Igarashi, R., Iwashita, K.,

438

Juvvadi, P.R., Kato, M., Kato, Y., Kin, T., Kokubun, A., Maeda, H., Maeyama, N.,

439

Maruyama, J., Nagasaki, H., Nakajima, T., Oda, K., Okada, K., Paulsen, I.,

440

Sakamoto, K., Sawano, T., Takahashi, M., Takase, K., Terabayashi, Y., Wortman,

441

J.R., Yamada, O., Yamagata, Y., Anazawa, H., Hata, Y., Koide, Y., Komori, T.,

442

Koyama, Y., Minetoki, T., Suharnan, S., Tanaka, A., Isono, K., Kuhara, S.,

443

Ogasawara, N., Kikuchi, H., 2005. Genome sequencing and analysis of Aspergillus

444

oryzae. Nature 438, 1157–1161.

445

20. Maruyama, J., Kitamoto, K., 2008. Multiple gene disruptions by marker recycling

446

with highly efficient gene-targeting background (∆ligD) in Aspergillus oryzae.

447

Biotechnol. Lett. 30, 1811–1817. doi: 10.1007/s10529-008-9763-9.

448

21. Narita, J., Okano, K., Kitao, T., Ishida, S., Sewaki, T., Sung, M.H., Fukuda, H.,

19

449

Kondo, A., 2006. Display of α-amylase on the surface of Lactobacillus casei cells

450

by use of the PgsA anchor protein, and production of lactic acid from starch. Appl.

451

Environ. Microbiol. 72, 269–275.

452

22. Nguyen, C.M., Choi, G.J., Choi, Y.H., Jang, K.S., Kim, J.C., 2013. D-and L-lactic

453

acid production from fresh sweet potato through simultaneous saccharification and

454

fermentation. Biochem. Eng. J. 81, 40–46.

455

23. Okano, K., Kimura, S., Narita, J., Fukuda, H., Kondo, A., 2007. Improvement in

456

lactic acid production from starch using α-amylase-secreting Lactococcus lactis

457

cells adapted to maltose or starch. Appl. Microbiol. Biotechnol. 75, 1007–1013.

458

24. Okano, K., Zhang, Q., Shinkawa, S., Yoshida, S., Tanaka, T., Fukuda, H., Kondo, A.,

459

2008. Efficient production of optically pure D-lactic acid from raw corn starch by

460

using a genetically modified L-lactate dehydrogenase gene-deficient and

461

alpha-amylase-secreting Lactobacillus plantarum strain. Appl. Environ. Microbiol.

462

75, 462–467. doi: 10.1128/AEM.01514-08.

463

25. Saitoh, S., Ishida, N., Onishi, T., Tokuhiro, K., Nagamori, E., Kitamoto, K.,

464

Takahashi, H., 2005. Genetically engineered wine yeast produces a high

465

concentration of L-lactic acid of extremely high optical purity. Appl. Environ.

466

Microbiol. 71, 2789–2792.

467

26. Sugimoto, T., Shoji, H., 2012 Indigestible dextrin is an excellent inducer for

468

α-amylase, α-glucosidase and glucoamylase production in a submerged culture of

469

Aspergillus oryzae. Biotechnol. Lett. 34, 347–351. doi:

470

10.1007/s10529-011-0777-3.

471 472

27. Tamano, K., Bruno, K.S., Karagiosis, S.A., Culley, D.E., Deng, S., Collett, J.R., Umemura, M., Koike, H., Baker, S.E., Machida, M., 2013. Increased production of

20

473

fatty acids and triglycerides in Aspergillus oryzae by enhancing expressions of fatty

474

acid synthesis-related genes. Appl. Microbiol. Biotechnol. 97, 269–281. doi:

475

10.1007/s00253-012-4193-y.

476

28. Thitiprasert, S., Sooksai, S., Thongchul, N., 2011. In vivo regulation of alcohol

477

dehydrogenase and lactate dehydrogenase in Rhizopus oryzae to improve L-lactic

478

acid fermentation. Appl. Biochem. Biotechnol. 164, 1305–1322. doi:

479

10.1007/s12010-011-9214-2.

480

29. van Zyl, W.H., Bloom, M., Viktor, M.J., 2012. Engineering yeasts for raw starch

481

conversion. Appl. Microbiol. Biotechnol. 95(6), 1377–1388. doi:

482

10.1007/s00253-012-4248-0.

483

30. van der Maarel, M.J., van der Veen, B., Uitdehaag, J.C., Leemhuis, H., Dijkhuizen,

484

L., 2002. Properties and applications of starch-converting enzymes of the

485

alpha-amylase family. J. Biotechnol. 94, 137–155.

486

31. Yen, H.W., Kang, J.L., 2010. Lactic acid production directly from starch in a

487

starch-controlled fed-batch operation using Lactobacillus amylophilus. Bioprocess

488

Biosyst. Eng. 33, 1017–1023. doi: 10.1007/s00449-010-0426-6.

489

32. Zhou, X., Ye, L., Wu, J.C., 2013. Efficient production of L-lactic acid by newly

490

isolated thermophilic Bacillus coagulans WCP10-4 with high glucose tolerance.

491

Appl. Microbiol. Biotechnol. 97, 4309–4314. doi: 10.1007/s00253-013-4710-7.

492 493 494

21

495

Figure captions

496 497

Fig. 1 Lactate production from glucose by A. oryzae strains

498

Fermentation of glucose (100 g/L) by using the following strains: transgenic A. oryzae

499

LDH∆871 (filled squares), A. oryzae LDHpyrG (filled triangles), A. oryzae LDH (filled

500

circles), wild-type A. oryzae RIB40 (open squares), auxotrophic A. oryzae NSPlD1

501

(open circles), and pyrG-complemented auxotrophic A. oryzae NSPlD1-pyrG (open

502

triangles). The concentrations of lactate (a) and glucose (b) are plotted as the mean

503

values obtained in at least three independent measurements. Error bars represent the

504

standard deviation.

505 506

Fig. 2 Construction and verification of the transgenic strains A. oryzae LDH and A.

507

oryzae LDH∆871

508

Expression of bLDH in A. oryzae LDH cells was detected immunologically (a): M, size

509

marker; 1, cell extract from wild-type A. oryzae RIB40; 2, cell extract from A. oryzae

510

LDH. AO090023000871 deletion strategy using the pyrG gene (b). Verification of

511

AO090023000871 deletion by PCR amplification (c): M, marker; W, PCR product from

512

A. oryzae LDH; ∆, PCR product from A. oryzae LDH∆871.

513 514

Fig. 3 Lactate fermentation of glucose by A. oryzae LDH∆871

515

Fermentation experiments using test tubes were conducted with various glucose

516

concentrations using the transgenic A. oryzae strain LDH∆871. Open circles, 30 g/L;

517

closed circles, 100 g/L; open triangles, 150 g/L; closed triangles, 200 g/L; and open

518

squares, 300 g/L. A fermentation experiment using a shaker flask was conducted with

22

519

100 g/L glucose (filled squares). The concentrations of lactate (a), glucose (b), malate

520

(c), and succinate (d) are plotted as the mean values obtained from at least three

521

independent measurements. Error bars represent the standard deviation.

522 523

Fig. 4 Lactate production from various starches and maltose by A. oryzae LDH∆871

524

Fermentation tests with soluble (open circles), potato (closed circles), wheat (closed

525

squares), corn (closed triangles) starch, and maltose (open squares) were conducted

526

using A. oryzae LDH∆871. The concentrations of lactate (a), total sugar (b), and glucose

527

(c) were plotted as the mean values obtained from at least three independent

528

measurements. Error bars represent the standard deviation.

529 530

Fig. 5 Lactate fermentation of a mixture of glucose and starch

531

Fermentation of 100 g/L glucose (open circles), 100 g/L soluble starch (open triangles),

532

and a mixture of 50 g/L glucose and 50 g/L soluble starch (closed circles) was

533

conducted using A. oryzae LDH∆871. The concentrations of lactate (a), total sugar (b),

534

and glucose (c) are plotted as the mean values obtained from at least three independent

535

measurements. Error bars represent the standard deviation. Alpha-amylase production

536

during lactate fermentation was analyzed by a SDS-PAGE (d): M, marker; and 0-4,

537

supernatant of each culture.

538

23

539

Tables

540 541

Table 1. Microorganisms and plasmids used in this study Strain and plasmid

E. coli strain NovaBlue

A. oryzae strains RIB40

Relevant features

Reference

endA1 hsdR17(r K12 -m K12 +) supE44 thi-I gyrA96 relA1 lac recA1/F’ [proAB+ lacIq Z∆M15::Tn10(Tetr)]

Novagen

Wild type

Machida (2005) Maruyama and Kitamoto (2008) This study

NSPlD1

niaD− sC− adeA− ∆argB::adeA− ∆ligD::argB ∆pyrG::adeA

NSPlD1-pyrG

niaD−::pIS1-pyrG[niaD pyrG] sC− adeA− ∆argB::adeA− ∆ligD::argB ∆pyrG::adeA niaD−::pIS1-LDH[niaD P-sodM::L-LDH::T-glaB] sC− adeA− ∆argB::adeA− ∆ligD::argB ∆pyrG::adeA niaD−::pIS1-bLDH[niaD P-sodM::L-LDH::T-glaB] sC− adeA− ∆argB::adeA− ∆ligD::argB ∆pyrG::adeA ∆Ao090023000871::pyrG niaD−::pIS1-LDH[niaD P-sodM::L-LDH::T-glaB] sC− adeA− ∆argB::adeA− ∆ligD::argB ∆pyrG::adeA pyrG

LDH

LDH∆871

LDHpyrG

R. oryzae strains NBRC 4705 Plasmids pIS1-bLDH pIS1-roLDHA pIS1-roLDHB pIS1-∆871 pIS1-pyrG

This study

This study

Wild type

NBRC

Vector for expression of bLDH [P-sodM::bLDH::T-glaB]; niaD marker Vector for expression of roLDHA [P-sodM::bLDH::T-glaB]; niaD marker Vector for expression of roLDHB [P-sodM::bLDH::T-glaB]; niaD marker Template for AO090023000871 gene disruption fragment [niaD pyrG] Vector for complement of pyrG gene without flanking region of AO090023000871 gene [niaD pyrG]

This study

542 24

This study This study This study This study

543

Table 2. Primers used in this study Primer

Sequence (5ʹ to 3ʹ) a,b,c

AscI-L-LDH-F

544

CACCCAAAGTCGAGGcgcgcc aaaaatgcagaacctcctgaaggaggagca tgtgc NotI-6His-L-LDH-R GTCGAGCATATGCGCggccgct caatggtgatgatgatgatggaactgcagc tccttttggatgccccacaggg AscI-ldhA-F aaaaaaggcgcgccatggtattacactcaaa ggt NotI-6His-ldhA-R aaagcggccgctcaatggtgatgatgatgat gacacctacttttacaaaa AscI-ldhB-F aaaaaaggcgcgccatggtactacattcaaa ggt NotI-6His-ldhB-R aaagcggccgctcaatggtgatgatgatgat gtgataaatattcaactcc AO871-up1300F TCTCCATACTAGAGCggcctgta gcgtagg AO871-up1300R AGTCCTCTCGGGCCAtgttcag gaggcttcaattgatctgtctgt pyrG-F TGGCCCGAGAGGACTattccga gggtgtgc pyrG-R GGTAATGTGCCCCAGgcttgtca gatatgt AO871-up300F CTGGGGCACATTACCatgctcaa tgacatcatcgccgtcctcctat AO871-up300R TGTTCAGGAGGCTTCaattgatc tgtctgt AO871-down1000F GAAGCCTCCTGAACActttgac attttcatgtgtgttgcgggatt AO871-down1000R ACGTACGATGTCCCTagtctagg actatatattcgaactataccg a Underlined letters, restriction enzyme sites

545

b

Bold letters, nucleotide sequence of the 6His tag.

546

c

Capital letters, nucleotide sequences for in-fusion reaction.

547 548 549

25

Experiments Construction of bLDH expression vector Construction of bLDH expression vector Construction of roLDHA expression vector Construction of roLDHA expression vector Construction of roLDHB expression vector Construction of roLDHB expression vector Deletion of AO090023000871 gene Deletion of AO090023000871 gene Deletion of AO090023000871 gene Deletion of AO090023000871 gene Deletion of AO090023000871 gene Deletion of AO090023000871 gene Deletion of AO090023000871 gene Deletion of AO090023000871 gene

550 551 552

Fig. 1

553 554

26

555 556 557 558

Fig. 2

559 560

27

561 562 563

Fig. 3

564 565

28

566 567 568

Fig. 4

569 570

29

571 572 573

Fig. 5

574 575

30

576

Highlights

577

・ Lactic acid-producing A. oryzae was constructed by genetic engineering.

578

・ A bLDH-expressing A. oryzae strain showed highest production among those

579

examined.

580

・ Pure culture of transgenic A. oryzae produced lactate from starch.

581

・ A. oryzae could be a promising host for bioproduction of useful compounds.

582 583

31