Creating a New Pathway in Corynebacterium glutamicum for the

Subscriber access provided by YORK UNIV

Biotechnology and Biological Transformations

Creating a New Pathway in Corynebacterium glutamicum for the Production of Taurine as a Food Additive Young-Chul Joo, Young Jin Ko, Seung Kyou You, Sang Kyu Shin, Jeong Eun Hyeon, Almisned Shuaa Musaad, and Sung Ok Han J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05093 • Publication Date (Web): 05 Dec 2018 Downloaded from http://pubs.acs.org on December 6, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 41

Journal of Agricultural and Food Chemistry

1

Creating a New Pathway in Corynebacterium glutamicum for the Production of Taurine

2

as a Food Additive

3 4

Young-Chul Joo,† Young Jin Ko,† Seung Kyou You,† Sang Kyu Shin,† Jeong Eun Hyeon,‡

5

Almisned Shuaa Musaad,† and Sung Ok Han*,†

6 7

†Department

8

‡Institute

9

of Korea

of Biotechnology, Korea University, Seoul 02841, Republic of Korea

of Life Science and Natural Resources, Korea University, Seoul 02841, Republic

10 11 12 13 14 15 16 17 18 19 20 21 22 23

1 Environment ACS Paragon Plus


24

ABSTRACT

25

Taurine is a biologically and physiologically valuable food additive. However,

26

commercial taurine production mainly relies on environmentally harmful chemical synthesis.

27

Herein, for the first time in bacteria, we attempted to produce taurine in metabolically

28

engineered Corynebacterium glutamicum. The taurine-producing strain was developed by

29

introducing cs, cdo1, and csad genes. Interestingly, while the control strain could not produce

30

taurine, the engineered strains successfully produced taurine via the newly introduced

31

metabolic pathway. Furthermore, we investigated the effect of a deletion strain of the

32

transcriptional repressor McbR gene on taurine production. As a result, sulfur accumulation

33

and L-cysteine biosynthesis were reinforced by the McbR deletion strain, which further

34

increased the taurine production by 2.3-fold. Taurine production of the final engineered strain

35

Tau11 was higher than in other previous reported strains. This study demonstrated a potential

36

approach for eco-friendly biosynthesis as an alternative to the chemical synthesis of a food

37

additive.

38 39

KEYWORDS: taurine production, new pathway, metabolic engineering, Corynebacterium

40

glutamicum, food additive

41 42 43 44 45 46


Page 2 of 41

Page 3 of 41

47


INTRODUCTION

48

Taurine (2-aminoethanesulfonic acid) is a very important sulfur-containing and

49

nonpeptidic amino acid, and it is of increasing interest because it is a biologically and

50

physiologically valuable nutrient used as an additive in food and drinks, animal feed, and

51

medicine.1-3 In many animals, including mammals and humans, taurine is involved in a

52

variety of crucial functions, such as antioxidation, bile salt formation, cell proliferation and

53

viability, detoxification, immunomodulation, membrane stabilization, neuromodulation,

54

neuroprotection, neurotransmission, oxidative stress inhibition, osmoregulation, the

55

regulation of calcium homeostasis and phosphorylation, and the stimulation of glycolysis and

56

glycogenesis.1, 2, 4-6 In particular, taurine intake is very important for people with taurine

57

deficiencies, such as preterm infants, children receiving total parenteral nutrition, and blind-

58

loop syndrome patients.5

59

In general, taurine is produced using three methods: extraction from food material,

60

enzymatic synthesis, and chemical synthesis.3 Taurine in food material has been reported to

61

exist mostly in poultry, fish, shellfish, shrimp, squid, algae, and other animals (including

62

mammals), but it does not exist in plants, such as vegetables, fruits, or grains (except for

63

cactus pears).7-10 Natural taurine is produced by extraction and purification from taurine-rich

64

food material.11 However, this natural taurine is unsuitable for medicinal use because it is

65

possible to contain animal-derived byproducts such as antibiotic residues, veterinary

66

medicinal products, hormones, other amino acids, and dipeptides.3,

67

enzymatic synthesis method consisting of two-step reactions for taurine production from

68

keratin raw material was previously developed. The first step is the preparation of keratin

69

hydrolysate by the extensive hydrolysis of the proteases isolated from the animal pancreas,


12-14

In addition, the


70

followed by the conversion of its hydrolysate to taurine using the enzyme mixture extracted

71

from the animal liver.3 Moreover, commercial taurine production mainly relies on chemical

72

synthesis.1, 3 A typical chemical synthesis method for taurine production is based on two-step

73

reactions. The first step is the synthesis of 2-aminoethylsulfuric acid from the

74

monoethanolamine and sulfuric acid by heat treatment. The second step is the production of

75

taurine from 2-aminoethylsulfuric acid with an acidic solution sodium sulfite by heat

76

treatment.1, 3, 11 However, chemical synthesis may carry various environmental and health

77

concerns due to chemical waste and organic solvents.15

78

The taurine biosynthetic pathway has been well studied in mammals. In this pathway,

79

taurine is synthesized by the L-cysteine sulfinic acid pathway from the L-cysteine substrate.4,

80

6, 8, 11

81

by L-cysteine dioxygenase (CDO).16 Subsequently, L-cysteine sulfinic acid is decarboxylated

82

to hypotaurine by L-cysteine sulfinic acid decarboxylase (CSAD),17 followed by spontaneous

83

oxidation to taurine (Figure 1A).6,

84

remains poorly studied.11 Several taurine biosynthetic pathways have been reported in chick

85

and fish which convert the 2-aminoacrylate-derived L-cysteine sulfonic acid (L-cysteic acid)

86

to taurine by CSAD or L-cysteine sulfonic acid decarboxylase (CAD) (Figure 1B).8, 11 The

87

two metabolic pathways of the mammals and some non-mammals were significantly

88

different in the starting metabolites such as L-methionine and L-serine, enzymes involved in

89

taurine biosynthesis, intermediates, and cofactors, etc (Figure 1AB).

In the L-cysteine sulfinic acid pathway, L-cysteine is oxidized to L-cysteine sulfinic acid

18

In non-mammals, the taurine biosynthetic pathway

90

In contrast to animals, including the mammals and some non-mammals, the

91

biosynthetic pathways of taurine in plants and bacteria are currently unknown,4, 8, 9 but its

92

biological functions are only well known. The growth stimulatory response and cell


Page 4 of 41

Page 5 of 41


93

membrane protective effects from exogenous taurine in the plant Triticum aestivum have

94

been reported.19 The TauABCD transport system in the bacterium Escherichia coli has been

95

reported to be used for taurine catabolism as a source of sulfur under conditions of sulfate or

96

L-cysteine

97

exogenous taurine has been reported in the yeast Saccharomyces cerevisiae.21 In addition,

98

the physiological roles of taurine remain unclear in plants and bacteria. However, a previous

99

study of fungal taurine biosynthesis uniquely suggested that the yeast Yarrowia lipolytica

100

could synthesize taurine from L-cysteine via L-cysteine sulfinic acid through a pathway

101

involving CDO and L-glutamate decarboxylase 1 (GAD1).22

starvation.20 Moreover, improved freezing and oxidative stress tolerance due to

102

To create a new metabolic pathway, the L-cysteine sulfonic acid-synthesizing enzyme

103

is needed to achieve the aims of this study, and its activity should not overlap with the L-

104

cysteine sulfinic acid pathway. Interestingly, L-cysteine sulfonic acid synthase (MA3297, CS,

105

L-cysteate

106

cysteine sulfonic acid and phosphate, and it has previously been reported to contribute to

107

coenzyme M biosynthesis in the euryarchaeon M. acetivorans.23 In addition, CS showed

108

specificity to sulfite, and did not catalyze the conversion of O-phospho-L-serine into L-

109

cysteine.23

synthase) catalyzes the conversion of O-phospho-L-serine and sulfite into L-

110

Corynebacterium glutamicum is an amino acid-producing bacterium generally

111

regarded as having a safe (GRAS) status.24 Therefore, it is a suitable strain for taurine

112

production with respect to food additives and medicine. Moreover, sufficient sulfur substrate

113

is needed to synthesize taurine, and the mechanism of sulfur assimilation has been well

114

studied in C. glutamicum.24 In particular, the transcriptional regulator McbR is involved in

115

the transcriptional repression of the CysN (sulfate adenylyltransferase subunit 1), CysD



116

(sulfate adenylyltransferase subunit 2), CysH (phosphoadenosine-phosphosulfate reductase),

117

CysI (sulfite reductase), CysX (sirohydrochlorin ferrochelatase), CysY (ferredoxin), and

118

CysK (O-acetyl-L-serine sulfhydrylase) genes involved in sulfur assimilation and L-cysteine

119

biosynthesis in C. glutamicum.25-29 However, the effect of McbR knockout on taurine

120

biosynthesis from

121

perspective.

L-cysteine

has not yet been studied from a metabolic engineering

122

For creating a new metabolic pathway for taurine production, we constructed C.

123

glutamicum recombinant strains, in which the mcbR gene was deleted and CS, CDO1, and/or

124

CSAD were overexpressed. In this study, we investigated the effects of single or synergistic

125

activities between the L-cysteine sulfinic acid pathway and/or the L-cysteine sulfonic acid

126

pathway for taurine production. Furthermore, we examined the effects of McbR knockout on

127

sulfur assimilation and L-cysteine biosynthesis. The control strain, Tau1 (empty pMT-tac

128

vector), could not produce taurine, but the engineered strains of Tau5 (L-cysteine sulfinic

129

acid pathway), Tau6 (L-cysteine sulfonic acid pathway), and Tau7 (combinatorial pathway)

130

successfully produced taurine. In the McbR deletion strain, Tau8, sulfur accumulation and L-

131

cysteine biosynthesis increased compared to the control strain, Tau1. Taurine production was

132

highest in the final engineered strain, Tau11 (CS, CDO1, and CSAD overexpression and

133

McbR knockout), during growth on glucose. These results demonstrated a potential approach

134

for the production of taurine as a food additive in a bacterium with a generally recognized as

135

safe (GRAS) status.

136 137

MATERIALS AND METHODS

138

Strains, Plasmids, Media, and Growth Conditions


Page 6 of 41

Page 7 of 41


139

The reagents used in this study were purchased from Sigma-Aldrich (St. Louis,

140

Missouri, USA). The pBluescriptR-CDO1 and pSPORT1-CSAD plasmids were obtained

141

from the Korea Human Gene Bank, Medical Genomics Research center, KRIBB, Korea.

142

Genomic DNA from Methanosarcina acetivorans ATCC 35395D-5 was purchased from the

143

American Type Culture Collection (ATCC, Manassas, Virginia, USA). E. coli DH5α was

144

used for all recombinant DNA manipulation. E. coli strains were grown for 24 h in a rotary

145

shaker at 37 °C and 200 rpm using Luria-Bertani medium containing 50 μg/mL ampicillin.

146

Transformation of plasmid into E. coli was performed by heat shock using the methods

147

described by Froger and Hall.30 C. glutamicum ATCC 13032 was used as the recombinant

148

host strain for taurine production. C. glutamicum strains were grown for 24 h in a rotary

149

shaker at 30 °C and 200 rpm using brain heart infusion medium containing 25 µg/mL

150

kanamycin for seed culture and preculture. Transformation of plasmid into C. glutamicum

151

was performed by electroporation using the methods described by van der Rest et al.31 All

152

bacterial strains and plasmids used in this study are listed in Table 1. For the main cultures,

153

the precultures were inoculated to an OD600 of 0.7 in 50 mL CGXII broth containing 40 g/L

154

glucose, 25 µg/mL kanamycin and 1 mM isopropyl thio-β-D-galactoside (IPTG) in 500-mL

155

baffled flasks. Taurine was produced for 10 h or 24 h in a rotary shaker at 30 °C and 200

156

rpm.

157 158

DNA Manipulations and Construction of Plasmids

159

Genomic DNA from M. acetivorans C2A ATCC 35395D-5 and C. glutamicum

160

ATCC 13032, and the pBluescriptR-CDO1 and pSPORT1-CSAD plasmids were used as

161

PCR templates for the CS (GenBank no. AAM06667.1), McbR (GenBank no. AUI02306.1),



162

CDO1 (GenBank no. AAH24241.1) and CSAD (GenBank no. NP_001346055.1) genes,

163

respectively. Bacterial genomic DNA was isolated using a Genomic DNA Purification Kit

164

(Promega, Madison, Wisconsin, USA). The cs, cdo1, csad genes, and the mcbR gene

165

fragments were amplified by PCR with Taq DNA polymerase (Solgent, Daejeon, Korea)

166

using the oligonucleotide primers provided in Table 2. The cs-csad sequence containing a

167

ribosomal binding site region and the ΔmcbR deletion were constructed using overlap

168

extension PCR32 with the overlapping oligonucleotide primers indicated in Table 2. PCR

169

products were purified using a PCR purification kit (GeneAll, Seoul, Korea). These purified

170

products were ligated into the respective restriction enzyme (Takara Bio, Otsu, Japan) sites

171

in the pMT-tac plasmid using T4 DNA ligase (Enzynomics, Daejeon, Korea) as indicated in

172

Table 2. Plasmids were extracted from E. coli or C. glutamicum transformants using the

173

plasmid purification kit (GeneAll, Seoul, Korea). DNA sequencing was performed at the

174

Cosmogenetech facility (Cosmogenetech Co., Ltd, Seoul, Korea).

175 176

Enzyme Assay Related to Taurine Biosynthesis

177

Cells from the Tau1, Tau2, Tau3, and Tau4 strains were grown in CGXII broth,

178

harvested after 24 h by centrifugation (16,600 x g at 4 °C for 15 min), and washed twice in

179

50 mM Tris-HCl (pH 8.5). Enzymes (CS, CDO1, or CSAD) were extracted according to the

180

methods described by Joo et al.24 The enzyme kinetic analysis of CS,23 CDO1,33 and CSAD4

181

was performed according to the previously reported methods. The values of kinetic

182

parameters was calculated from the non-linear regression using the Michaelis-Menten kinetic

183

model (Figure S1). The reactions were performed for 30 min in a rotary shaker at 30 °C and

184

200 rpm in 10 mL of 50 mM Tris-HCl (pH 8.5) containing 5 g/L dry cell weight (DCW) and


Page 8 of 41

Page 9 of 41


185

the specific substrates (10 mM O-phospho-L-serine, 10 mM L-cysteine, 10 mM L-cysteine

186

sulfonic acid, or 10 mM L-cysteine sulfinic acid). The specific activity was defined as L-

187

cysteine sulfonic acid, L-cysteine sulfinic acid, taurine, or taurine plus hypotaurine produced

188

in μmol per min per g DCW.

189 190

Analytical Methods

191

In this study, taurine production was compared in exponential phase. C. glutamicum

192

cell growth was estimated at OD600 using an UV–Vis spectrophotometer (Mecasys Co., Ltd,

193

Seoul, Korea). DCW was calculated on the basis of the correlation using the methods

194

described by Joo et al.24 Unless otherwise stated, the sampling for measurements of

195

intracellular amino acids was performed in triplicate from 1 mL of CGXII culture medium at

196

10 h or 4 h intervals from 0 h to 24 h using the methods described by Joo et al.24 All amino

197

acids were analyzed using a high-performance liquid chromatography (HPLC) system

198

(Binary HPLC pump model 1528, autosampler model 2707, dual λ absorbance detector

199

model 2487, Waters, Milford, MA, USA) using a Supelcosil LC-18-DB HPLC Column (5

200

μm particle size, L × I.D. 25 cm 4.6 mm, Sigma-Aldrich, St. Louis, Missouri, USA) and a

201

Waters AccQ-Tag Amino Acid Analysis System. Glucose concentrations measured using the

202

DNS assay method.34 The concentrations of sulfate, sulfite, and sulfide were determined

203

using the methods described by Joo et al. and Abdel-Latif.24, 35

204 205

RESULTS AND DISCUSSION

206

Construction of a Taurine Biosynthetic Pathway in C. glutamicum



207

As production host of taurine for food additive, we chose the GRAS bacterium C.

208

glutamicum strain that its genome is completely sequenced and the molecular biology and

209

metabolic engineering tools are well developed.36 This strain was also well studied for the

210

central carbon metabolism, physiology, main regulation, metabolic pathway in detail.36

211

Furthermore, this strain is traditionally employed for industrial scale production of L-amino

212

acids.36 In particular, L-cysteine, sulfite, and sulfide are certainly needed to synthesize taurine,

213

these biosynthetic pathways, mechanisms, and transcriptional regulations have been well

214

previously studied in C. glutamicum, a host strain in this study.24-29 Thus, C. glutamicum

215

strain is suitable a taurine-producing host with consideration of food additive, but no attempt

216

has been made to use it to produce taurine using a metabolic engineering approach. In order

217

to develop a taurine-producing strain by metabolic engineering in C. glutamicum which

218

taurine biosynthetic pathway is not preserved, the introduction of the following enzyme is

219

necessary: the widely known CDO and CSAD enzymes (the used to construct L-cysteine

220

sulfinic acid pathway), and the newly introduced CS enzyme (the used to construct L-cysteine

221

sulfonic acid pathway).

222

For that reason, in this study, metabolic engineering strategies for taurine production

223

in the GRAS bacterium C. glutamicum were designed to connect the O-phospho-L-serine and

224

L-cysteine

225

first strategy focused on the construction of the L-cysteine sulfonic acid pathway, consisting

226

of CS and CSAD from O-phospho-L-serine, and the L-cysteine sulfinic acid pathway,

227

consisting of CDO1 and CSAD from L-cysteine (Figure 1C). The next strategy focused on

228

whether the synergistic effect of the combined pathway (consisting of CS, CDO1, and CSAD)

229

could improve taurine production (Figure 1C). The final strategy focused on the effect of

routes of the taurine metabolic pathway (Figure 1C). For taurine biosynthesis, the


Page 10 of 41

Page 11 of 41


230

deleting the mcbR gene to improve taurine production, L-cysteine biosynthesis, and sulfur

231

assimilation (Figure 1C).

232

CS enzyme showed a substrate's affinity Km of 3.1 ± 0.2 mM O-phospho-L-serine, a

233

maximal velocity Vmax of 5.3 ± 0.3 μM s-1, a turnover number kcat of 16.6 ± 0.9 s-1 and a

234

catalytic efficiency kcat/Km of 5.3 ± 0.4 μM-1 s-1 (Table S1). In the case of CDO1 enzyme for

235

L-cysteine

236

revealed for Km, Vmax, kcat, and kcat/Km, respectively (Table S1). Furthermore, CSAD enzyme

237

for L-cysteine sulfinic acid or L-cysteine sulfonic acid as the substrates determined the Km of

238

1.8 ± 0.1 or 5.6 ± 0.3 mM, Vmax of 516.9 ± 0.9 or 8.3 ± 0.4 μM s-1, kcat of 3.9 ± 0.2 or 9.9 ±

239

0.5 s-1, and kcat/Km of 2.1 ± 0.2 or 1.8 ± 0.1 μM-1 s-1, respectively (Table S1). The results

240

presented in Table 3 show that the specific substrates (O-phospho-L-serine, L-cysteine, L-

241

cysteine sulfonic acid, or L-cysteine sulfinic acid) did not show specific activities of the CS,

242

CDO1, or CSAD enzymes in the control strain Tau1 (empty pMT-tac vector). The specific

243

activity of the engineered strain Tau2 (overexpressing CS) were approximately 13.6 ± 0.1

244

μmol/min/g DCW with the O-phospho-L-serine substrate (Table 3). These findings indicate

245

that the CS enzyme leads to the biosynthesis of L-cysteine sulfonic acid from O-phospho-L-

246

serine. Furthermore, the specific activity of the engineered strain Tau3 (overexpressing

247

CDO1) was approximately 18.4 ± 0.4 μmol/min/g DCW with the L-cysteine substrate (Table

248

3). These findings indicate that the CDO1 enzyme leads to the biosynthesis of L-cysteine

249

sulfinic acid from L-cysteine. In the engineered strain Tau4 (overexpressing CSAD), the

250

specific activity was approximately 7.9 ± 0.2 μmol/min/g DCW with the L-cysteine sulfonic

251

acid substrate and was approximately 11.2 ± 0.1 μmol/min/g DCW with the L-cysteine

252

sulfinic acid substrate (Table 3). These findings indicate that the CSAD enzyme converts not

as the substrate, values of 6.8 ± 0.4, 20.2 ± 1.0, 13.6 ± 0.7, and 2.0 ± 0.1 were



253

only L-cysteine sulfinic acid to hypotaurine but also L-cysteine sulfonic acid to taurine. The

254

presence of taurine (0.7 ± 0.0 mmol/L) in the engineered strain Tau4 suggests spontaneous

255

hypotaurine oxidation (Table 3). The hypotaurine oxidation to taurine still remains unclear.

256

Previous studies have suggested that hypotaurine was converted to taurine by hypothetical

257

NAD+-dependent hypotaurine dehydrogenase (oxidoreductase) or by the spontaneous

258

oxidation.6, 8, 18, 37, 38 The existence of hypotaurine-specific oxidoreductase was not known

259

until the present, hence it was regarded as spontaneous oxidation in this study. In the previous

260

study, the S. cerevisiae (pESC/CDO1–CSD) recombinant strain led to enhancement of the

261

accumulation level of hypotaurine rather than taurine by the reaction considered to be

262

spontaneous oxidation. In contrast, in the C. reinhardtii and Tetraselmis sp. strains, the

263

accumulation level of taurine was higher than hypotaurine. Therefore, the previous results

264

and the our results suggest that the conversion rates of taurine are probably due to their

265

differences as the activities of oxidoreductases or the levels of radical-scavenging reactions

266

for spontaneous oxidation in each microorganism species.39, 40 The engineered strains Tau2,

267

Tau3, and Tau4 were not reactive to other substrates beyond those mentioned.

268

In the mammals, taurine is synthesized by the L-cysteine sulfinic acid pathway

269

involving CDO1 and CSAD from L-cysteine as its substrate4, 6, 8, 11, 16, 17, whereas in the non-

270

mammals, taurine is synthesized by the L-cysteine sulfonic acid pathway via 2-aminoacrylate

271

or L-cysteine as its substrates.8, 11 However, taurine biosynthesis from O-phospho-L-serine as

272

a substrate has not yet been reported. According to our review of the literature, a taurine

273

pathway including 2-aminoacrylate, L-cysteine sulfinic acid, and L-cysteine sulfonic acid in

274

C. glutamicum has not yet been reported. The Kyoto Encyclopedia of Genes and Genomes

275

(KEGG) on-line pathway database (http://www.genome.jp/kegg/) has been widely used for


Page 12 of 41

Page 13 of 41


276

the analysis of metabolic pathways in previous studies.41-43 Our analysis based on this

277

database shows that a taurine pathway including 2-aminoacrylate, L-cysteine sulfinic acid, L-

278

cysteine sulfonic acid does not exist in C. glutamicum (data not shown).

279

We investigated the effects of single or synergistic activities between the L-cysteine

280

sulfinic acid and/or L-cysteine sulfonic acid pathways for taurine production. Our results

281

show that the control strain Tau1 (empty pMT-tac vector) could not produce taurine, but the

282

engineered strains Tau5 (L-cysteine sulfinic acid pathway), Tau6 (L-cysteine sulfonic acid

283

pathway), and Tau7 (combinatorial pathway) produced approximately 7.4 ± 0.3, 4.6 ± 0.1,

284

and 15.8 ± 0.7 mg/g DCW or 14.3 ± 0.8, 10.9 ± 0.5, and 28.8 ± 1.6 mg/g DCW of taurine

285

content at 10 h or 24 h, respectively (Figure 2). The taurine content in the engineered strain

286

Tau7 was approximately 2.0- and 2.7-fold higher than in the engineered strains of Tau5 and

287

Tau6 at 24 h, respectively (Figure 2). These results indicated that the engineered strains

288

successfully produced taurine via the newly introduced metabolic pathway (L-cysteine

289

sulfinic acid, L-cysteine sulfonic acid, or the combined pathway).

290 291

Effects of mcbR Deletion on Sulfur Assimilation and L-Cysteine Biosynthesis

292

To achieve the aims of this study, taurine biosynthesis requires not only L-cysteine

293

sulfinic acid and L-cysteine sulfonic acid as intermediates, but it also requires sufficient

294

supplies of sulfite and sulfide (Figure 1). However, the sulfur assimilatory reduction in C.

295

glutamicum depends on many associated enzymes, thus the metabolic pathway engineering

296

is not simple. Previously, the involvement of the transcriptional regulator McbR has been

297

well studied in sulfur assimilation and L-cysteine biosynthesis in C. glutamicum.25-29 The

298

McbR transcriptional regulator is involved in the transcriptional repression of the cysN, cysD,



299

cysH, cysI, cysX, cysY, and cysK genes involved in sulfur assimilation and L-cysteine

300

biosynthesis in C. glutamicum.25-29 However, the effects of McbR knockout on sulfur

301

assimilation and taurine biosynthesis from L-cysteine have not yet been studied.

302

As a result, the engineered strain Tau8 (McbR knockout) decreased the

303

concentrations of O-phospho-L-serine and L-serine by approximately 1.2- or 1.2-fold more

304

and 2.0- or 2.2-fold more, respectively, compared with the engineered strain Tau1 (native

305

McbR) at 10 h or 24 h (Figure 3A, B). In contrast, the engineered strain Tau8 had an

306

approximately 3.5- or 3.0-fold higher L-cysteine concentration compared with the engineered

307

strain Tau1 at 10 h or 24 h (Figure 3C). As previously reported, the enhanced L-cysteine

308

biosynthesis is due to increased mRNA transcription and protein expression of the CysK

309

enzyme following deletion of the gene encoding the McbR transcriptional repressor in C.

310

glutamicum.27, 28, 44 These previous results also showed a significant decrease in L-serine and

311

an increase in L-cysteine in the C. glutamicum McbR deletion strain compared with wild-

312

type.28, 29 Furthermore, O-phospho-L-serine has been reported to be converted to L-serine by

313

O-phospho-L-serine phosphatase (SerB) in C. glutamicum.45 Therefore, in our results, the

314

decreases in O-phospho-L-serine and L-serine are probably due to their use as a precursor in

315

L-serine

316

taurine, a sustained and sufficient supply of intermediate precursors such as O-phospho-L-

317

serine and L-serine must be achieved. Herein, the inadequate O-phospho-L-serine and L-

318

serine levels were overcomed by the use of an L-serine-producing C. glutamicum strain

319

ATCC 21586 (Figure S2B). In particular, the engineered strain Tua12 (C. glutamicum ATCC

320

21586, overexpression of CS-CDO1-CSAD) showed approximately 1.7-, 2.2-, and 2.9-fold

321

increases in the intracellular concentrations of O-phospho-L-serine, L-serine, and L-cysteine

and L-cysteine biosynthesis, respectively. However, for the effective production of


Page 14 of 41

Page 15 of 41


322

than the engineered strain Tau7 (C. glutamicum ATCC 13032, overexpression of CS-CDO1-

323

CSAD) at 24 h (Figure S2). Furthermore, the intracellular concentrations of O-phospho-L-

324

serine, L-serine, L-cysteine, and taurine of the recombinant strain Tau7 were 4.6-, 4.9-, 4.2-,

325

and 5.2-fold higher than the extracellular concentrations, respectively (Figure S2A). As a

326

result, taurine production from the accumulation of abundant intermediate precursors in the

327

engineered strain Tau12 (376.4 ± 12.4 mg/L of intracellular taurine) significantly increased

328

compared to the engineered strain Tau7 (224.4 ± 13.2 mg/L of intracellular taurine) at 24 h

329

(Figure S2). By the detection of extracellular taurine, there is a possibility that a taurine

330

exporter exists in C. glutamicum cell membrane.

331

As depicted in Figure 3D, E, F, the engineered strain Tau8 showed approximately

332

1.4-, 2,1-, and 3.6-fold or 1.9-, 2.5-, and 4.2-fold increases in the molar concentrations of

333

intracellular sulfate, sulfite, and sulfide, respectively, than the engineered strain Tau1 at 10 h

334

or 24 h. The deletion of the mcbR gene has been reported to increase the mRNA transcription

335

and protein expression of the cysN, cysD, cysH, cysI, cysX, and cysY genes in C.

336

glutamicum.25, 28 Furthermore, it has been reported that C. glutamicum strains lacking the

337

cysN, cysD, cysH, cysI, cysX, or cysY genes were also unable to grow on inorganic sulfate

338

and sulfite sources because they could not activate sulfur assimilatory reduction.26 These

339

results indicated that sulfur assimilation and L-cysteine biosynthesis were strongly affected

340

by deletion of the mcbR gene.

341 342

Enhancing Taurine Biosynthesis by Deletion of the mcbR Gene

343

The taurine-producing strain was developed by introducing a newly constructed

344

metabolic pathway. However, to substantially increase taurine production, it was necessary



345

to increase the concentrations of both the L-cysteine intermediate precursor and the supply

346

of sulfite and sulfide through the combinatorial taurine pathway in a C. glutamicum mcbR

347

deletion strain.

348

After McbR deletion, the recombinant strain Tau8 (empty pMT-tac vector) not

349

produced taurine, but the engineered strains Tau9 (L-cysteine sulfinic acid pathway), Tau10

350

(L-cysteine sulfonic acid pathway), and Tau11 (combinatorial pathway) produced

351

approximately 12.9 ± 0.4, 15.0 ± 0.5, and 41.6 ± 1.2 mg/g DCW or 25.5 ± 1.5, 30.5 ± 1.9,

352

and 62.0 ± 2.4 mg/g DCW of taurine content at 10 h or 24 h, respectively (Figure 4). The

353

taurine content in the engineered strain Tau11 was approximately 3.2- and 2.4-fold or 3.2

354

and 2.0-fold higher than in the engineered strains Tau9 and Tau10 at 10 h or 24 h, respectively

355

(Figure 4). These results indicated that the McbR deletion strains more effectively produced

356

taurine through the L-cysteine sulfinic acid, L-cysteine sulfonic acid, or the combined

357

pathway via enhanced metabolism of sulfur and L-cysteine.

358

In the L-cysteine sulfonic acid pathway, the taurine content in the McbR deletion

359

strain, Tau9, was approximately 1.8-fold higher than in strain Tau5 at 24 h (Figure 2 and

360

Figure 4). Compared with the above results, the McbR deletion strain Tau8 had an O-

361

phospho-L-serine production that was approximately 83.8% of that of the engineered strain

362

Tau1 at 24 h (Figure 3A). This result suggests that despite the decrease of O-phospho-L-

363

serine, its level does not affect the metabolic flux toward taurine biosynthesis. Moreover, the

364

McbR deletion strain Tau8 increased the intracellular sulfite concentration approximately

365

2.5-fold compared with strain Tau1 at 24 h (Figure 3E). In general, sulfite is a highly reactive

366

and potentially toxic compound in many organisms, including bacteria; therefore, it is

367

converted to a sulfur-containing amino acid via mechanisms to oxidatively detoxify sulfite.46


Page 16 of 41

Page 17 of 41


368

This result suggests that with increased sulfur assimilation, sulfite is used for taurine

369

biosynthesis via L-cysteine sulfonic acid through the mechanism used to oxidatively detoxify

370

sulfite.

371

In the L-cysteine sulfinic acid pathway, the taurine content in the McbR deletion

372

strain, Tau10, was approximately 2.8-fold higher than in strain Tau6 at 24 h (Figure 2 and

373

Figure 4). The McbR deletion strain Tau8 showed approximately 3.0- and 4.2-fold higher L-

374

cysteine and intracellular sulfide concentrations than strain Tau1 at 24 h (Figure 3C, F),

375

respectively. Moreover, the taurine production in strain Tau6 was lower than in strain Tau5;

376

however, the taurine production in the McbR deletion strain Tau10 was significantly

377

increased compared to strains Tau6 and Tau9 (Figure 2 and Figure 4). Based on these results,

378

it appears that the McbR deletion strain expressing an L-cysteine sulfinic acid pathway had

379

significantly improved taurine production due to an increase in the metabolic flux toward

380

taurine due to the sufficient supply of L-cysteine and sulfide substrates.

381

In the combined pathway, the taurine content in the McbR deletion strain Tau11 was

382

approximately 2.1-fold higher than in the native McbR strain Tau7 at 24 h (Figure 2 and

383

Figure 4). Furthermore, the taurine content of combined pathway (McbR deletion strain

384

Tau11, 62.0 ± 2.4 mg/g DCW) was higher than the sum of the individual L-cysteine sulfonic

385

acid (McbR deletion strain Tau9, 25.5 ± 1.5 mg/g DCW) and L-cysteine sulfinic acid (McbR

386

deletion strain Tau10, 30.5 ± 1.9 mg/g DCW) pathways at 24 h (Figure 4). This result was

387

also true for the recombinant strain Tau7 (native McbR, combinatorial pathway). These

388

results, presented in Figure 2 and 4, show that the recombinant strains Tau7 and Tau1, which

389

express the combined pathway, exhibit synergistic taurine production compared with the

390

other strains. A similar study has used such synergistic effects between the threonine pathway



391

and the citramalate pathway in E. coli to improve 1-propanol production and to increase

392

metabolic flux.47 We suggest that the recombinant strains Tau7 and Tau11 had significantly

393

improved taurine production due to the overexpression of enzymes from the L-cysteine

394

sulfinic acid pathway and the L-cysteine sulfonic acid pathway that then increased the

395

metabolic flux toward taurine rather than the accumulation of sulfur. In previous studies, only

396

the effects of McbR on the transcription of sulfur metabolism genes and fundamental amino

397

acid biosynthesis in C. glutamicum were described.25-29 However, this work provides an

398

important metabolic engineering approach for taurine production enhanced by the effects of

399

McbR knockout on sulfur and L-cysteine metabolism.

400 401

Taurine Production in Shake Flasks

402

The final engineered strain, Tau11, reached stationary phase with a cell biomass of

403

approximately 8.3 ± 0.3 g/L at 24 h (Figure 5). The glucose was completely consumed to

404

support cell growth and taurine production by 24 h (Figure 5). After cultivation for 24 h, the

405

final engineered strain, Tau11, produced approximately 517.0 ± 12.7 mg/L of taurine, with a

406

volumetric productivity of approximately 21.5 ± 0.5 mg/L/h, and a specific productivity of

407

approximately 2.6 ± 0.0 mg/g DCW/h (Figure 5).

408

The microbial taurine production levels are summarized in Table 4. A S. cerevisiae

409

(pESC/CDO1–CSD) recombinant strain produces approximately 10.7 ± 1.9 mg/g DCW of

410

taurine content under H2O2-treated conditions, which was the previous highest production

411

reported (Table 4).9 Taurine production was identified in wild-type strains of Y. lipolytica

412

(under high L-methionine and L-cystine conditions) and Synechococcus sp. (in the presence

413

of L-cysteine sulfinic acid) but was not detected in cultures of Synechococcus sp. in the


Page 18 of 41

Page 19 of 41


L-cysteine

sulfinic acid substrate (Table 4).4,

22

414

absence of the

Wild-type strains of

415

Chlamydomonas reinhardtii (in the presence of 6 mM L-serine), Ostreococcus tauri (in the

416

presence of 6 mM sodium sulfate), and Tetraselmis sp. (in the presence of 1.6% (w/v) sea

417

salt) produced approximately 0.2 ± 0.0, 1.5 ± 0.0, and 1.6 ± 0.3 mg/g DCW of taurine content,

418

respectively (Table 4).8 However, in absence of serine, sodium sulfate, and sea salt, taurine

419

production was very low.8 In this study, in the final engineered strain, Tau11, the maximum

420

taurine content reached was approximately 62.0 ± 1.5 mg/g DCW at 24 h, which is the highest

421

reported taurine content in microorganisms (Table 4). However, we have not achieved the

422

high level of taurine production compared to other amino acids such as L-glutamate, L-lysine,

423

and L-hydroxyproline of the previous studies.48-50 This may be due to the degradation of

424

taurine, an inadequate intermediate level, the ineffective inducer of host strain, or the non-

425

optimal codon usage of heterologous enzyme in C. glutamicum. Therefore, the solutions are

426

required such as the effective overexpression of synthesis-related genes of taurine and

427

intermediate by IPTG-replacement inducer, the removal of taurine degradation-related genes,

428

the codon optimization of heterologous enzymes, the introduction of hypotaurine

429

dehydrogenase and taurine exporter. In future work, our aim will be to reach the mass

430

production of taurine by the fed-batch fermentation of C. glutamicum strain that have been

431

resolved these problems by metabolic engineering.

432

Herein, for the first time in bacteria, we successfully produced taurine via a newly

433

introduced metabolic pathway in a metabolically engineered C. glutamicum strain. The

434

present work has also provided a solid base for the future industrial production of taurine in

435

microorganism. This study demonstrated a potential approach for eco-friendly taurine

436

production as an alternative to its chemical synthesis for use as a food additive.



437 438

ASSOCIATED CONTENT

439

Supporting Information

440

The Supporting Information is available free of charge via the Internet at http://pubs.acs.org.

441

The non-linear regression of CS, CDO1, and CSAD, the extracellular and intracellular

442

concentrations of amino acids, kinetic parameters data (PDF).

443 444

AUTHOR INFORMATION

445

Corresponding Author

446

*Telephone:

+82-2-3290-3151. Fax: +82-2-3290-3151. E-mail: [email protected]

447 448

ORCID

449

Sung Ok Han: 0000-0002-2400-2882

450 451

Funding

452

This research was supported by the National Research Foundation of Korea (NRF) grant

453

funded by the Korea government (MSIP) (No. 2018R1A2B2003704).

454 455

Notes

456

The authors do not have any conflicts of interest.

457


Page 20 of 41

Page 21 of 41


458

REFERENCES

459

(1) Bondareva, O. M.; Lopatik, D. V.; Kuvaeva, Z. I.; Vinokurova, L. G.; Markovich, M. M.;

460 461 462 463 464

Prokopovich, I. P. Synthesis of taurine. Pharm. Chem. J. 2008, 42, 142–144. (2) Bouckenooghe, T.; Remacle, C.; Reusens, B. Is taurine a functional nutrient? Curr. Opin. Clin. Nutr. Metab. Care 2006, 9, 728–733. (3) Bulychev, E. Y.; Rubanyak, N. Y. Commercial synthesis of 2-aminoethanesulfonic acid (taurine). Pharm. Chem. J. 2013, 46, 740–741.

465

(4) Agnello, G.; Chang, L. L.; Lamb, C. M.; Georgiou, G.; Stone, E. M. Discovery of a

466

substrate selectivity motif in amino acid decarboxylases unveils a taurine biosynthesis

467

pathway in prokaryotes. ACS Chem. Biol. 2013, 8, 2264–2271.

468

(5) Birdsall, T. C. Therapeutic applications of taurine. Altern. Med. Rev. 1998, 3, 128–136.

469

(6) Huxtable, R. J. Physiological actions of taurine. Physiol. Rev. 1992, 72, 101–163.

470

(7) Laidlaw, S. A.; Grosvenor, M.; Kopple, J. D. The taurine content of common foodstuffs.

471

JPEN, J. Parenter. Enteral Nutr. 1990, 14, 183–188.

472

(8) Tevatia, R.; Allen, J.; Rudrappa, D.; White, D.; Clemente, T. E.; Cerutti, H.; Demirel, Y.;

473

Blum, P. The taurine biosynthetic pathway of microalgae. Algal Res. 2015, 9, 21–26.

474

(9) Honjoh, K.; Matsuura, K.; Machida, T.; Nishi, K.; Nakao, M.; Yano, T.; Miyamoto, T.;

475

Iio, M. Enhancement of menadione stress tolerance in yeast by accumulation of

476

hypotaurine and taurine: co-expression of cDNA clones, from Cyprinus carpio, for

477

cysteine dioxygenase and cysteine sulfinate decarboxylase in Saccharomyces cerevisiae.

478

Amino Acids 2010, 38, 1173–1183.

479 480

(10) Stintzing, F. C.; Schieber, A.; Carle, R. Phytochemical and nutritional significance of cactus pear. Eur. Food Res. Technol. 2001, 212 396–407.



481 482

(11) Salze, G. P.; Davis, D. A. Taurine: a critical nutrient for future fish feeds. Aquaculture 2015, 437, 215–229.

483

(12) Verdon, E.; Hurtaud‐Pessel, D.; Thota, J. R. A role for high‐resolution mass

484

spectrometry in the high‐throughput analysis and identification of veterinary medicinal

485

product residues and of their metabolites in foods of animal origin, High‐Throughput

486

Analysis for Food Safety, 2014.

487

(13) Schonherr, J. Analysis of products of animal origin in feeds by determination of

488

carnosine and related dipeptides by high-performance liquid chromatography. J. Agric.

489

Food Chem. 2002, 50, 1945–1950.

490

(14) EFSA Report for 2014 on the results from the monitoring of veterinary medicinal

491

product residues and other substances in live animals and animal products. EFSA

492

supporting publication 2016, 2016:EN-923.

493 494 495 496

(15) Li, C. J.; Trost, B. M. Green chemistry for chemical synthesis. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 13197–13202. (16) Joseph, C. A.; Maroney, M. J. Cysteine dioxygenase: structure and mechanism. Chem. Commun. (Cambridge, U. K.) 2007, 3338–3349.

497

(17) Tang, X. W.; Hsu, C. C.; Sun, Y.; Wu, E.; Yang, C. Y.; Wu, J. Y. Multiplicity of brain

498

cysteine sulfinic acid decarboxylase – purification, characterization and subunit structure.

499

J. Biomed. Sci. 1996, 3, 442–453.

500 501

(18) Morris, J. H.; Rogers, Q. R. The metabolic basis for the taurine requirement of cats. Adv. Exp. Med. Biol. 1992, 315, 33–44.


Page 22 of 41

Page 23 of 41


502

(19) Hao, L. H.; He, P. Q.; Liu, C. Y.; Chen, K. S.; Li, G. Y. Physiological effects of taurine

503

on the growth of wheat (Triticum aestivum L.) seedlings. J. Plant Physiol. Mol. Biol.

504

2004, 30, 595–598.

505 506

(20) Eichhorn, E.; van der Ploeg, J. R.; Leisinger, T. Deletion analysis of the Escherichia coli taurine and alkanesulfonate transport systems. J. Bacteriol. 2000, 182, 2687–2695.

507

(21) Honjoh, K.; Machida, T.; Nishi, K.; Matsuura, K.; Soli, K. W.; Sakai, T.; Ishikawa, H.;

508

Matsumoto, K.; Miyamoto, T.; Iio, M. Improvement of freezing and oxidative stress

509

tolerance in Saccharomyces cerevisiae by taurine. Food Sci. Technol. Res. 2007, 13, 145–

510

154.

511

(22) Hébert, A.; Forquin-Gomez, M. P.; Roux, A.; Aubert, J.; Christophe, J.; Heilier, J. F.;

512

Landaud, S.; Bonnarme, P.; Beckerich, J. M. New insights into sulfur metabolism in

513

yeasts as revealed by studies of Yarrowia lipolytica. Appl. Environ. Microbiol. 2013, 79,

514

1200–1211.

515

(23) Graham, D. E.; Taylor, S. M.; Wolf, R. Z.; Namboori, S. C. Convergent evolution of

516

coenzyme M biosynthesis in the Methanosarcinales: cysteate synthase evolved from an

517

ancestral threonine synthase. Biochem. J. 2009, 424, 467–478.

518

(24) Joo, Y. C.; Hyeon, J. E.; Han, S. O. Metabolic design of Corynebacterium glutamicum

519

for production of L-cysteine with consideration of sulfur-supplemented animal feed. J.

520

Agric. Food Chem. 2017, 65, 4698–4707.

521

(25) Rückert, C.; Milse, J.; Albersmeier, A.; Koch, D. J.; Pühler, A.; Kalinowski, J. The dual

522

transcriptional regulator CysR in Corynebacterium glutamicum ATCC 13032 controls a

523

subset of genes of the McbR regulon in response to the availability of sulphide acceptor

524

molecules. BMC Genomics 2008, 9, 483.



525

(26) Rückert, C.; Koch, D. J.; Rey, D. A.; Albersmeier, A.; Mormann, S.; Pühler, A.;

526

Kalinowski, J. Functional genomics and expression analysis of the Corynebacterium

527

glutamicum fpr2-cysIXHDNYZ gene cluster involved in assimilatory sulphate reduction.

528

BMC Genomics 2005, 6, 121.

529

(27) Rey, D. A.; Nentwich, S. S.; Koch, D. J.; Ruckert, C.; Puhler, A.; Tauch, A.; Kalinowski,

530

J. The McbR repressor modulated by the effector substance S-adenosylhomocysteine

531

controls directly the transcription of a regulon involved in sulphur metabolism of

532

Corynebacterium glutamicum ATCC 13032. Mol. Microbiol. 2005, 56, 871–887.

533

(28) Kromer, J. O.; Bolten, C. J.; Heinzle, E.; Schroder, H.; Wittmann, C. Physiological

534

response of Corynebacterium glutamicum to oxidative stress induced by deletion of the

535

transcriptional repressor McbR. Microbiology 2008, 154, 3917–3930.

536

(29) Kromer, J. O.; Heinzle, E.; Schroder, H.; Wittmann, C. Accumulation of

537

homolanthionine and activation of a novel pathway for isoleucine biosynthesis in

538

Corynebacterium glutamicum McbR deletion strains. J. Bacteriol. 2006, 188, 609–618.

539 540

(30) Froger, A.; Hall, J. E. Transformation of plasmid DNA into E. coli using the heat shock method. J. Visualized Exp. 2007, 253.

541

(31) van der Rest, M. E.; Lange, C.; Molenaar, D. A heat shock following electroporation

542

induces highly efficient transformation of Corynebacterium glutamicum with xenogeneic

543

plasmid DNA. Appl. Microbiol. Biotechnol. 1999, 52, 541–545.

544 545

(32) Bryksin, A.; Matsumura, I. Overlap extension PCR cloning. Methods Mol. Biol. 2013, 1073, 31–42.

546

(33) Arjune, S.; Schwarz, G.; Belaidi, A. A. Involvement of the Cys-Tyr cofactor on iron

547

binding in the active site of human cysteine dioxygenase. Amino Acids 2015, 47, 55–63.


Page 24 of 41

Page 25 of 41


548

(34) Hyeon, J. E.; Jeon, W. J.; Whang, S. Y.; Han, S. O. Production of minicellulosomes for

549

the enhanced hydrolysis of cellulosic substrates by recombinant Corynebacterium

550

glutamicum. Enzyme Microb. Technol. 2011, 48, 371–377.

551 552

(35) Abdel-Latif, M. S. New spectrophotometric method for sulfite determination. Anal. Lett. 1994, 27, 2601–2614.

553

(36) Wieschalka, S.; Blombach, B.; Bott, M.; Eikmanns, B. J. Bio-based production of

554

organic acids with Corynebacterium glutamicum. Microb. Biotechnol. 2013, 6, 87–102.

555

(37) Jacobsen, J. G.; Smith, L. H. Biochemistry and physiology of taurine and taurine

556

derivatives. Physiol. Rev. 1968, 48, 424–511.

557

(38) Grove, R. Q.; Karpowicz, S. J. Reaction of hypotaurine or taurine with superoxide

558

produces the organic peroxysulfonic acid peroxytaurine. Free Radical Biol. Med. 2017,

559

108, 575–584.

560

(39) Sugio, T.; White, K. J.; Shute, E.; Choate, D.; Blake, R. C. Existence of a hydrogen

561

sulfide:ferric ion oxidoreductase in iron-oxidizing bacteria. Appl. Environ. Microbiol.

562

1992, 58, 431–433.

563 564

(40) Stecchini, M. L.; Del Torre, M.; Munari, M. Determination of peroxy radical-scavenging of lactic acid bacteria. Int. J. Food Microbiol. 2001, 64, 183–188.

565

(41) Karpe, A. V.; Beale, D. J.; Godhani, N. B.; Morrison, P. D.; Harding, I. H.; Palombo, E.

566

A. Untargeted metabolic profiling of winery-derived biomass waste degradation by

567

Penicillium chrysogenum. J. Agric. Food Chem. 2015, 63, 10696–10704.

568

(42) Liu, J. Y.; Chang, M. C.; Meng, J. L.; Feng, C. P.; Zhao, H.; Zhang, M. L. Comparative

569

proteome reveals metabolic changes during the fruiting process in Flammulina velutipes.

570

J. Agric. Food Chem. 2017, 65, 5091–5100.



571

(43) Huang, Z.; Zhang, S.; Xu, Y.; Li, L.; Li, Y. Metabolic effects of the pksCT gene on

572

Monascus aurantiacus Li As3.4384 using gas chromatography–time-of-flight mass

573

spectrometry-based metabolomics. J. Agric. Food Chem. 2016, 64, 1565–1574.

574

(44) Rey, D. A.; Puhler, A.; Kalinowski, J. The putative transcriptional repressor McbR,

575

member of the TetR-family, is involved in the regulation of the metabolic network

576

directing the synthesis of sulfur containing amino acids in Corynebacterium glutamicum.

577

J. Biotechnol. 2003, 103, 51–65.

578

(45) Peters-Wendisch, P.; Stolz, M.; Etterich, H.; Kennerknecht, N.; Sahm, H.; Eggeling, L.

579

Metabolic engineering of Corynebacterium glutamicum for L-serine production. Appl.

580

Environ. Microbiol. 2005, 71, 7139–7144.

581 582 583 584

(46) Kappler, U.; Dahl, C. Enzymology and molecular biology of prokaryotic sulfite oxidation. FEMS Microbiol. Lett. 2001, 203, 1–9. (47) Shen, C. R.; Liao, J. C. Synergy as design principle for metabolic engineering of 1propanol production in Escherichia coli. Metab. Eng. 2013, 17, 12–22.

585

(48) Kim, J.; Fukuda, H.; Hirasawa, T.; Nagahisa, K.; Nagai, K.; Wachi, M.; Shimizu, H.

586

Requirement of de novo synthesis of the OdhI protein in penicillin-induced glutamate

587

production by Corynebacterium glutamicum. Appl. Microbiol. Biotechnol. 2010, 86,

588

911–920.

589

(49) Falcioni, F.; Buhler, B.; Schmid, A. Efficient hydroxyproline production from glucose

590

in minimal media by Corynebacterium glutamicum. Biotechnol. Bioeng. 2015, 112, 322–

591

330.


Page 26 of 41

Page 27 of 41


592

(50) Xu, J. Z.; Wu, Z. H.; Gao, S. J.; Zhang, W. Rational modification of tricarboxylic acid

593

cycle for improving L-lysine production in Corynebacterium glutamicum. Microb. Cell

594

Fact. 2018, 17, 105.

595 596



597

Page 28 of 41

FIGURE CAPTIONS

598 599

Figure 1. Metabolic engineering strategy for taurine production in C. glutamicum. (A)

600

Taurine pathway in mammals, (B) taurine pathway in non-mammals, and (C) a newly

601

constructed taurine pathway in GRAS bacterium C. glutamicum. The dashed arrow from

602

glucose indicates the involvement of multiple enzymatic steps in glycolysis and serine

603

metabolism. The large black arrow indicates the overexpression of CS, CDO1, and CSAD

604

enzymes leading to taurine biosynthesis. The L-cysteine sulfinic acid pathway is generally a

605

taurine pathway of mammals consisting of the CDO1 and CSAD enzymes. As a first attempt

606

at metabolic engineering, we used the L-cysteine sulfonic acid pathway, which is a new

607

pathway consisting of the CS and CSAD enzymes. The gray arrow indicates sulfur

608

assimilation. The gray bar indicates transcriptional repression. The X-mark indicates the

609

deletion of the mcbR gene. SerB, O-phospho-L-serine phosphatase; CysE,

610

acetyltransferase; CysK, O-acetyl-L-serine sulfhydrylase; CS,

611

synthase; CDO1, L-cysteine dioxygenase; CSAD, L-cysteine sulfinic acid decarboxylase;

612

McbR, transcriptional regulator (L-methionine and L-cysteine biosynthetic repressor); CysN,

613

sulfate adenylyltransferase subunit 1; CysD, sulfate adenylyltransferase subunit 2; CysH,

614

phosphoadenosine-phosphosulfate

615

sirohydrochlorin ferrochelatase; CysY, ferredoxin; DNA, deoxyribonucleic acid; ATP,

616

adenosine triphosphate; NADPH, nicotinamide adenine dinucleotide phosphate; PAP, 3'-

617

phosphoadenosine-5'-phosphophate; PAPS, 3'-phosphoadenosine-5'-phosphosulfate; MAT,

618

L-methionine

619

adenosylhomocysteinase; CBS,

reductase;

adenosyltransferase;

DNMT,

L-cystathionine

CysI,

DNA

L-cysteine

sulfite


sulfonic acid

reductase;

methyltransferase;

beta-synthase; CTH,

L-serine

CysX,

AHCY,

L-cystathionine

Page 29 of 41


620

gamma-lyase; SDH,

L-serine

dehydratase; PAPS-AS, 3′-phosphoadenylyl sulfate:2′-

621

aminoacrylate C-sulfotransferase; CAD, L-cysteine sulfonic acid decarboxylase.

622 623

Figure 2. Effects of overexpressing CS-CSAD (strain Tau5), CDO1-CSAD (strain Tau6),

624

and CS-CDO1-CSAD (strain Tau7) on taurine production. The white bar indicates taurine

625

content for 10 h and the black bar indicates taurine content for 24 h in the recombinant strains.

626

The engineered strains were incubated in 500 mL baffled flasks in 50 mL CGXII medium

627

containing 40 g/L glucose and 25 μg/mL kanamycin for 10 and 24 h at 30 °C at 200 rpm.

628

The data represent the averages of triplicates, and the error bars represent the standard

629

deviations.

630 631

Figure 3. Comparison of sulfur and L-cysteine metabolism in the engineered strains of Tau1

632

(native McbR) and Tau8 (McbR knockout). (A) O-Phospho-L-serine concentration, (B) L-

633

serine concentration, (C) L-cysteine concentration, (D) intracellular sulfate concentration, (E)

634

intracellular sulfite concentration, and (F) intracellular sulfide concentration. The white bar

635

indicates substance concentration for 10 h and the black bar indicates substance concentration

636

for 24 h in the recombinant strains. The engineered strains were incubated in 500 mL baffled

637

flasks in 50 mL CGXII medium containing 40 g/L glucose and 25 μg/mL kanamycin for 10

638

and 24 h at 30 °C at 200 rpm. The data represent the averages of triplicates, and the error bars

639

represent the standard deviations.

640 641

Figure 4. Effects of deletion of the mcbR gene with overexpression of CS-CSAD, CDO1-

642

CSAD, and CS-CDO1-CSAD on taurine production. The white bar indicates taurine content



643

for 10 h and the black bar indicates taurine content for 24 h in the recombinant strains. The

644

engineered strains were incubated in 500 mL baffled flasks in 50 mL CGXII medium

645

containing 40 g/L glucose and 25 μg/mL kanamycin for 10 and 24 h at 30 °C at 200 rpm.

646

The data represent the averages of triplicates, and the error bars represent the standard

647

deviations.

648 649

Figure 5. Taurine production, cell biomass, and glucose consumption in the final engineered

650

strain, Tau11. The circle symbol indicates taurine production. The square symbol indicates

651

cell biomass. The diamond symbol indicates glucose consumption. The engineered strain was

652

incubated in 500 mL baffled flasks in 50 mL CGXII medium containing 40 g/L glucose and

653

25 μg/mL kanamycin for 24 h at 30 °C at 200 rpm. The data represent the averages of

654

triplicates, and the error bars represent the standard deviations.

655


Page 30 of 41

Page 31 of 41


Figure 1 A

B



C


Page 32 of 41

Page 33 of 41


Figure 2

Taurine content (mg/g DCW)

35 30 25 20 15 10 5 0

Tau1

Tau5

Tau6

Tau7



Page 34 of 41

Figure 3 B

250

200

200

200

150

100

L-Cysteine (mg/L)

250

150

100

50

50

0

Tau8

D

150

100

50

0

Tau1

0

Tau1

Tau8

E

Tau1

4

3

3

3

1

Sulfide (mM)

4

2

2

1

0

Tau8

2

1

0

Tau1

Tau8

F

4

Sulfite (mM)

Sulfate (mM)

C

250

L-Serine (mg/L)

O-Phospho-L-serine (mg/L)

A

0

Tau1

Tau8


Tau1

Tau8

Page 35 of 41


Figure 4

Taurine content (mg/g DCW)

70 60 50 40 30 20 10 0

Tau8

Tau9

Tau10 Tau11



Page 36 of 41

50

8

40

6

30

4

20

2

10

0

0

4

8

12

16

20

Time (hours)


0 24

800

600

400

200

0

Taurine (mg/L)

10

Glucose (g/L)

DCW (g/L)

Figure 5

Page 37 of 41


Table 1. Bacterial Strains and Plasmids Used in This Study strain or plasmid strains E. coli DH5α

description

source, reference, or target

F− ϕ80dlacΔ(lacZ)M15 Δ(lacZYA-argF)U169 endA1 recA1 hsdR17(rK−, mK+) deoR thi-1 phoA supE44 λ− gyrA96 relA1 E. coli DH5α, pMT-tac E. coli DH5α, pMT-tac::cs E. coli DH5α, pMT-tac::cdo1 E. coli DH5α, pMT-tac::csad E. coli DH5α, pMT-tac::cs-csad E. coli DH5α, pMT-tac::cdo1-csad E. coli DH5α, pMT-tac::cs-csad-cdo1 E. coli DH5α, pK18mobsacB E. coli DH5α, pK18mobsacB::ΔmcbR

Invitrogena

C. glutamicum ATCC 13032 ATCC 21586 Tau1 Tau2 Tau3 Tau4 Tau5 Tau6 Tau7 Tau8 Tau9 Tau10 Tau11 Tau12

wild-type strain, auxotrophic for biotin wild-type strain, auxotrophic for biotin, L-serine-producing strain C. glutamicum ATCC 13032, pMT-tac C. glutamicum ATCC 13032, pMT-tac::cs C. glutamicum ATCC 13032, pMT-tac:: cdo1 C. glutamicum ATCC 13032, pMT-tac::csad C. glutamicum ATCC 13032, pMT-tac::cs-csad C. glutamicum ATCC 13032, pMT-tac::cdo1-csad C. glutamicum ATCC 13032, pMT-tac::cs-csad-cdo1 C. glutamicum ATCC 13032, ΔmcbR, pMT-tac C. glutamicum ATCC 13032, ΔmcbR, pMT-tac::cs-csad C. glutamicum ATCC 13032, ΔmcbR, pMT-tac::cdo1-csad C. glutamicum ATCC 13032, ΔmcbR, pMT-tac::cs-csad-cdo1 C. glutamicum ATCC 21586, pMT-tac::cs-csad-cdo1

ATCCb ATCC 24 this study this study this study this study this study this study this study this study this study this study this study

plasmids pBluescriptR-CDO1 pSPORT1-CSAD pMT-tac pK18mobsacB pK18mobsacB::ΔmcbR pMT-tac::cs pMT-tac::cdo1 pMT-tac::csad pMT-tac::cs-csad pMT-tac::cdo1-csad pMT-tac::cs-csad-cdo1

pBluescriptR, carrying cdo1 gene pSPORT1, carrying csad gene lacI-P-tac-MCS-rrnBT1T2, KmR, and AmpR Integration vector, sacB, lacZα, MCS, and KmR pK18mobsacB, carrying mcbR fragment gene pMT-tac, carrying cs gene pMT-tac, carrying cdo1 gene pMT-tac, carrying csad gene pMT-tac, carrying cs and csad genes pMT-tac, carrying cdo1 and csad genes pMT-tac, carrying cs, csad, and cdo1genes

KHGBc KHGB 24 ATCC this study this study this study this study this study this study this study

EC1 EC2 EC3 EC4 EC5 EC6 EC7 EC8 EC9

aInvitrogen

24 this study this study this study this study this study this study this study this study

Corporation, Carlsbad, California, USA. bATCC, American Type Culture Collection, Manassas, Virginia, USA. cKHGB, Korea Human Gene Bank, Daejeon, Korea.



Table 2. List of Oligonucleotide Primers Used in This Study primer

sequencea

CS F′ (ClaI)

TAGCGCATCGATATGATAGCAATGGGAAGATTC

CS R′ (KpnI)

ATTAATGGTACCTCAGAGCTTCAGTTCCTC

CDO1 F′ (NotI)

GGGGCGGCCGCAAGGAGATATAGATGGAACAGACCGAAGTGCTGAAG

CDO1 R′ (NotI)

CCCGCGGCCGCTTAGTTGTTCTCCAGCGAGCCCG

CSAD F′ (ClaI)

GCGCATCGATATGGCTGACTCAAAACCACTCAGG

CSAD R′ (NotI)

ATTAGCGGCCGCATTTTGAGAACCCAGGAG

CS-CSAD F′ (ClaI)

CATATCGATATGATAGCAATGGGAAGATTCATATTAAAATG

CS-CSAD MR′

GTGGTTTTGAGTCAGCCATCTATATCTCCTTTCAGAGCTTCAGTTCCTCAAG

CS-CSAD MF′

GAGGAACTGAAGCTCTGAAAGGAGATATAGATGGCTGACTCAAAACCAC

CS-CSAD R′ (NotI)

CAGGCGGCCGCTCACAGGTCCTGGCCCAG

ΔMcbR F′ (XbaI)

GGGTCTAGAGAAAGTACGATGAAGCGTCC

ΔMcbR MR′

TTGGTCCACAACATCAGTCC

ΔMcbR MF′

GATGTTGTGGACCAATTCACGGAGGATACGATCAAT

ΔMcbR R′

TCATGGCCGAGACTCCTA

ΔMcbR Seq1-1

CTTTTTACCATTGCGAGAAGATTCCGC

ΔMcbR Seq1-2

CCCCGGTTTCCTGATTTTGTGC

ΔMcbR Seq2-1

TTCACGCAACAAACGTGGAACTC

ΔMcbR Seq2-2

TGACAGCACCAATCAGACATGTGATG

aRestriction

enzyme sites are italicized and bold, the nucleotide long 5′ extensions are underlined, and the ribosomal binding site (RBS) are blocked.


Page 38 of 41

Page 39 of 41


Table 3. Specific Activity in the CS-, CDO1-, or CSAD-Overexpressing C. glutamicum Strains using Specific Substrates strain Tau1

overexpression DCW (g/L) none 5

Tau1

none

5

Tau1

none

5

Tau1

none

5

Tau2

CS

5

Tau3

CDO1

5

Tau4

CSAD

5

Tau4

CSAD

5

spontaneous oxidationd

substratea (mmol/L) O-phospho-L-serine (10) L-cysteine (10) L-cysteine sulfonic acid (10) L-cysteine sulfinic acid (10) O-phospho-L-serine (10) L-cysteine (10) L-cysteine sulfonic acid (10) L-cysteine sulfinic acid (10) L-cysteine sulfinic acidderived hypotaurine

product (mmol/L) NDc

specific activity (μmol/min/g DCWb)

ND ND ND L-cysteine

sulfonic acid (2.0 ± 0.0) L-cysteine sulfinic acid (2.8 ± 0.1) taurine (1.2 ± 0.0) hypotaurine (1.0 ± 0.0) taurine (0.7 ± 0.0)

aSpecific

13.6 ± 0.1 18.4 ± 0.4 7.9 ± 0.2 11.2 ± 0.1e

activity was performed in the presence of substrate. bDCW was estimated on the basis of the correlation OD600 1 = 0.28 g DCW/L. cND, not detected. dSpontaneous oxidation was analyzed the reaction sample from L-cysteine sulfinic acid in the engineered strain Tau4. eThis specific activity of L-cysteine sulfinic acid in the engineered strain Tau4 was determined the product amount of hypotaurine with the oxidized form taurine from hypotaurine. All data represent means from three independent experiments performed in triplicate and ± indicate standard deviation from the mean.



Page 40 of 41

Table 4. Microbial Taurine Production strain

type

Yarrowia lipolytica

wild-type

taurine content (mg/g DCW)a +b

Synechococcus sp.

wild-type

+

4

Chlamydomonas reinhardtii

wild-type

0.2 ± 0.0

8

Ostreococcus tauri

wild-type

1.5 ± 0.0

8

Tetraselmis sp.

wild-type

1.6 ± 0.3

8

Saccharomyces cerevisiae

pESC/CDO1–CSD

10.7 ± 1.9

9

Corynebacterium glutamicum strain Tau11

ΔmcbR, pMT-tac::cs-csad-cdo1

62.0 ± 1.5

this study

aTaurine

reference or target 22

contents were unified based on the reported results. bThis is indicated the identification of the presence of taurine.


Page 41 of 41


TABLE OF CONTENTS


Creating a New Pathway in Corynebacterium glutamicum for the

Recommend Documents