Overexpression of Monacolin K Biosynthesis Genes in the Monascus

Subscriber access provided by TULANE UNIVERSITY

Biotechnology and Biological Transformations

Overexpression of Monacolin K Biosynthesis Genes in the Monascus purpureus Azaphilone Polyketide Pathway Chan Zhang, Jian Liang, Anan Zhang, Shuai Hao, Han Zhang, Qianqian Zhu, Baoguo Sun, and Chengtao Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05524 • Publication Date (Web): 08 Feb 2019 Downloaded from http://pubs.acs.org on February 8, 2019

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 26

Journal of Agricultural and Food Chemistry

1

Overexpression of Monacolin K Biosynthesis Genes in the Monascus purpureus

2

Azaphilone Polyketide Pathway

3

Chan Zhanga,b,∗ , Jian Lianga, Anan Zhanga, Shuai Haoa,b, Han Zhanga, Qianqian Zhua, Baoguo

4

Suna,b, and Chengtao Wanga,b,∗

5

a

6

& Business University (BTBU)，Beijing 100048,China.

7

b

8

Business University (BTBU), Beijing 100048, China.

9

∗ Corresponding author:

Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology

Beijing Engineering and Technology Research Center of Food Additives, Beijing Technology &

10

Chan Zhang & Chengtao Wang: Beijing Advanced Innovation Center for Food Nutrition and

11

Human Health, Beijing Engineering and Technology Research Center of Food Additives, Beijing

12

Technology & Business University, No. 11 Fucheng Road, Haidian District, Beijing, 100048,

13

China.

14

E-mail address: [email protected] (Chan Zhang)

15

[email protected] (Chengtao Wang).

16

ACS Paragon Plus Environment


17

ABSTRACT

18

Monascus purpureus is one of the important food and drug microbial resources

19

through the production of a variety of secondary metabolites, including monacolin K,

20

a well-recognized cholesterol-lowering agent. However, the high production costs and

21

naturally low contents of monacolin K have restricted its large-scale production. Thus,

22

in this study we sought to improve the production of monacolin K in M. purpureus

23

through overexpression of four genes (mokC, mokD, mokE, and mokI). Four

24

overexpression

25

conversion, which resulted in a 234.3%, 220.8%, 89.5%, and 10% increase in the yield

26

of monacolin K, respectively. The overexpression strains showed clear changes to the

27

mycelium surface with obvious folds and the spores with depressions, whereas the

28

pBC5 mycelium had a more full structure with a flatter surface. Further investigation

29

of these strains can provide the theoretical basis and technical support for the

30

development of functional Monascus varieties.

strains were successfully constructed by protoplast electric shock

31 32 33

KEYWORDS: Monacolin K; Gene overexpression; Monascus purpureus; mokC;

34

mokD; mokE; mokI

35


Page 2 of 26

Page 3 of 26


36

INTRODUCTION

37

Monascus spp. are one of the medicinal and edible filamentous fungi in East

38

Asian countries1, have long played a significant role in local life and culture, such as

39

medicine, wine, fermented bean curd and food coloring industries, and have received

40

attention worldwide owing to their diverse products, including abundant beneficial

41

metabolites2. Monascus species produce various secondary metabolites, including

42

monacolins, pigments, γ-aminobutyric acid, and citrinin3-5.

43

In 1979, Endo6 first isolated an active substance from the fermentation broth of

44

Monascus ruber, which strongly inhibited cholesterol synthesis and named it

45

monacolin K. Monacolin K can block the activity of 3-hydroxy-3-methyl glutaryl

46

coenzyme A (HMG-CoA) reductase as a competitive inhibitor7 in cholesterol

47

biosynthesis. Thus, monacolin K shows great potential for broad clinical application;

48

indeed, it is currently considered as one of the most effective drugs available for the

49

treatment of hyperlipidemia8. In addition, monacolin K has been reported to prevent

50

Alzheimer's disease, stroke, and also has ability to regulate kidney immunity9.

51

Manzoni10 first identified the biosynthetic pathway of lovastatin in Aspergillus

52

terreus in 2002. In brief, lovastatin biosynthesis is catalyzed by lovastatin nonaketide

53

synthase (LNKS) using acetyl-CoA and malonyl-CoA as substrates under the action

54

of the formation of polyketone compounds11. In 2008, Chen et al.12 screened a gene

55

cluster containing monacolin K synthase (mokA-mokI) in the Monascus pilosus

56

genome and through a series of experiments determined high similarity to the

57

lovastatin gene cluster in A. terreus (lovB-lovI).



58

With respect to industrial production, Monascus fermentation largely relies on

59

the Monascus strain used, and thus identifying or engineering Monascus with

60

excellent performance and high production is a critical factor for further industrial

61

development13 toward achieving economic benefits for the Monascus industry14. With

62

continuous progress in mold transformation methods, direct seeding can now be

63

achieved by the knockout and overexpression of specific genes in Monascus15.

64

Through continuous exploration and efforts, gene overexpression and functional

65

validation have been successfully achieved for the Monascus protein-coding gene

66

mokH16, and the transcription factors LaeA and MpLaeA17,18. In 2013, Lee et al.17

67

overexpressed MpLaeA in M.pilosus, monacolin K and pigment production increased

68

significantly. In 2016, Liu et al.18 knocked out the MrLaeA gene in M. ruber, resulting

69

in a decrease in the secondary metabolite production, especially monascus and citrinin.

70

Therefore, it was speculated that the regulatory factors encoded by laeA, which also

71

have a regulatory effect on the synthesis of monacolin K in Monascus. This previous

72

work has provided a good foundation for the transformation of Monascus. Based on

73

this technology, in the present study, we aimed to optimize the production of

74

monacolin K in M. purpureus by modifying the targeted genes in the biosynthesis

75

cluster. In order to explore the regulation of gene clusters on monacolin K

76

biosynthesis in M. purpureus and to achieve the high yield of monacolin K, our study

77

intended to use the laboratory-preserved M. purpureus M1 (strain No. CGMCCC 3.0568)

78

as the starting strain, through the four genes (mokC, mokD, mokE, and mokI) were

79

overexpressed to construct monacolin K high-yield strains.


Page 4 of 26

Page 5 of 26


80

MATERIALS AND METHODS

81

Fungal Strain and Culture Conditions M. purpureus M1, a stable producer of

82

monacolin K, was obtained from the Chinese General Microbiological Culture

83

Collection Center (strain No. CGMCCC 3.0568). M. purpureus M1 was maintained on

84

potato dextrose agar for 5 days at 30°C and cultured with 50 mL seed medium

85

containing (per liter): 30 g glucose, 15 g soybean powder, 1 g MgSO4·7H2O, 2 g

86

KH2PO4, 70 g glycerol, 2 g NaNO3,and 10 g peptone at a neutral pH. Then the seed

87

solution was incubated on fermentation medium (20 g/L rice powder, 1 g/L

88

MgSO4·7H2O, 2 g/L ZnSO4·7H2O, 2.5 g/L KH2PO4, 90 g/L glycerol, 5 g/L NaNO3, and

89

10 g/L peptone) for 12 days at 25°C with 150 rpm.

90

Overexpression of mokC, mokD, mokE, and mokI Gene overexpression is a widely

91

used technique in the molecular biology of filamentous fungi. To engineer a

92

high-yield monacolin K Monascus strain, mokC, mokD, mokE, and mokI from M.

93

purpureus M1 were overexpressed in M1. Gene fragments of mokC (1575 bp), mokD

94

(792 bp), mokE (1083 bp), and mokI (1815 bp) were amplified by polymerase chain

95

reaction (PCR) respectively using specific primers (Table 1) by Invitrogen (Shanghai,

96

China), and cloned in the restriction sites of the pBC-hygro plasmid (Miaoling

97

Bioscience & Technology Ltd. Co., Wuhan, China). The resulting pBC-hygro

98

derivatives were designated pBC-hygro-mokC, pBC-hygro-mokD, pBC-hygro-mokE,

99

and pBC-hygro-mokI, respectively (Fig. 1). The ligation products were transformed

100

into Escherichia coli DH5α competent cells, and then the pBC-hygro plasmids were

101

cultured, selected, and verified. Recombinant plasmid size verification, using



102

pBC-hygro-mokC as an example, The ligated PCR product fragment of pBC-Hygro

103

plasmid and mokC gene was double digested with QuickCut Sma I and QuickCut Not

104

I, and the digested product was purified and ligated into E. coli competent DH5α at 34

105

µg/mL chloramphenicol. The positive transformants were screened on the plates, and

106

the plasmid was extracted. The recombinant plasmid was detected by electrophoresis

107

with the original plasmid. As shown in Fig.1 (b), the length of the recombinant

108

plasmid was about 8400 bp, which was consistent with the expected size.

109

Overexpression constructs were introduced into M. purpureus through

110

protoplast electric shock conversion19, 20 under the following conditions: voltage of 3

111

kV/cm, pulse time of 4 ms, capacitance of 25 µF, and resistance of 400 Ω (Fig. 2a).

112

The strains receiving the pBC-hygro plasmid were screened on hygromycin plates of

113

different concentrations to obtain hygromycin-resistant strains. To verify the genetic

114

stability of the different transformants, the transformants introduced with the

115

recombinant plasmids were passaged on the hygromycin selection (10 µg/mL

116

hygromycin) plate for five generations, and 10 single colonies were randomly selected

117

for further analysis (Fig. 2b).

118

Monacolin K Production The supernatants of fermentation medium were analyzed

119

by high-performance liquid chromatography (HPLC) fitted with a InertsilODS-3 C18

120

column at 25°C (5 µm, 150 × 4.6 mm) after filtration of the supernatant through a

121

0.45-µm filter. The HPLC parameters were as follows: the mobile phase, 0.1%

122

H3PO4/methanol (1:3, v/v); flow rate, 1 mL/min; and detection by ultraviolet

123

spectroscopy at a wavelength of 237 nm21-23. A standard monacolin K compound was


Page 6 of 26

Page 7 of 26


124

used to confirm the HPLC analysis.

125

Reverse Transcription-Quantitative PCR (RT-qPCR) Analysis of Monacolin K

126

Biosynthetic Gene Clusters The Monascus mycelia were obtained when the

127

monacolin K concentration peaked. Total mycelial RNA was extracted by RNAprep

128

Pure Plant Kit (Tiangen-bio, Beijing, China), and first-strand cDNA was synthesised

129

by FastQuant RT Kit (with gDNase; Tiangen-bio, Beijing, China), with the FQ-RT

130

Primer Mix. RT-qPCR was conducted to monitor gene expression levels using the

131

SYBR Green PCR master mix (Tiangen-bio, Beijing, China). Primers for mokA-mokI

132

(GenBank accession no. DQ176595.1) and GAPDH (GenBank accession no.

133

HQ123044.1) were designed by Beacon Designer 8 software to amplify a portion of

134

the nine genes (Table 1).

135

RT-qPCR was performed using a CFX96 Real-Time PCR Detection System

136

version (Bio-Rad, Hercules, CA, USA) as previously described24,25 with the following

137

amplification program: 95 °C for 15 min, followed by a three-step PCR (40 cycles of

138

denaturation at 95 °C for 10 s, annealing at 60 °C for 30 s, and extension at 72 °C for

139

30 s). Amplification was performed using Super Real Pre Mix Plus (SYBR Green) for

140

detection of the fluorophore SYBR green with fluorescein. Relative expression levels

141

were calculated by the 2-∆∆Ct method24. All values were normalised using the

142

housekeeping reference expression level of the GAPDH gene. RT-qPCR was carried

143

out in triplicate for each sample.

144

Scanning Electron Microscopy (SEM) of the Monascus Mycelium SEM was used

145

to observe the morphological differences in mycelia of the different strains. For SEM,



146

the mycelium cells of samples were fixed in 25% glutaraldehyde solution in

147

phosphate-buffered saline (PBS)

148

solution in PBS buffer, pH 7.2. The cells were dehydrated with different

149

concentrations of ethanol（30%，50%，70%，80%，90%，100%） in distilled water,

150

being left for 10min at each stage, and centrifuged at 12, 000 rpm for 5 min. The cells

151

were replaced with isoamyl acetate-ethanol solution(1:1, v:v), and the cells were

152

resuspended in each solvent for 10 min, centrifuged at 12, 000 rpm for 5 min. The

153

samples were added a certain volume of hexamethyl disilazane, and were dried to a

154

powder at 60˚C. After primary fixation, the mycelia were coated with gold palladium

155

for 2 min. Photomicrographs were then acquired using a VEGA 3LMU/LMH SEM

156

(TESCAN, Brno, Czech Republic).

157

RESULTS AND DISCUSSION

buffer (12h, 25°C), and rinsed with 0.1M H3PO4

158

Monacolin K, also known as lovastatin, is involved in cholesterol biosynthesis,

159

and can suppress the activity of HMG-CoA reductase as a competitive inhibitor15, 26.

160

The monacolin K biosynthetic gene clusters in Monascus have gained substantial

161

research attention, and the strategies for synthesizing the bioactivity of monacolin K

162

are achieved through regulation of gene clusters13. The monacolin K biosynthetic gene

163

cluster was identified according to similarities with lovastatin synthetic genes in

164

Aspergillus, and nine genes (mokA-mokI) were determined to be associated with

165

monacolin K synthesis27,28. The mokA-deficient mutant strain in M. pilosus

166

BCRC38072 cannot produce monacolin K, indicating that mokA encodes the

167

polyketide synthase responsible for monacolin K biosynthesis in this strain. In


Page 8 of 26

Page 9 of 26


168

addition, the mokB-deficient mutant of M. pilosus NBRC4480 cannot produce

169

monacolin K, but rather accumulates monacolin J, indicating that mokB is responsible

170

for synthesis of the diketide side chain of monacolin K. Overexpression of the mokH

171

gene in M. pilosus results in significantly higher monacolin K production than that

172

detected in wild-type strains, indicating that mokH positively regulates monacolin K

173

production.

174

The present study was designed to verify the effects of mokC, mokD, mokE, mokI

175

in monacolin K biosynthesis in M. purpureus M1. These genes were overexpressed in

176

M. purpureus M1 to construct high monacolin K yielding strains, which were used to

177

explore the regulation effect of these four genes on monacolin K metabolism in M.

178

purpureus. After introduction of the pBC-hygro plasmid to Monascus competent cells

179

through protoplast click conversion, nine strains of Monascus (designated pBC1,

180

pBC2, pBC3, pBC4, pBC5, pBC6, pBC7, pBC8, and pBC10) were obtained after five

181

generations of inheritance and selection. Based on the gene transcription levels,

182

metabolism, and verification results, we confirmed that the mokC, mokD, mokE and

183

mokI gene overexpression strains were successfully constructed (Fig. 1).

184

The RNA of Monascus pBC5 and M. purpureus M1 was extracted and

185

reverse-transcribed into cDNA, using the hygroma-hygrogel-R and hygro-R primers.

186

The pBC5 clearly harbor hygromycin gene (1000 bp) suggesting that the pBC hygro

187

plasmid was successfully transformed into the wild-type M. purpureus (Fig. 3),

188

whereas the M. purpureus M1 strain could not amplify the hygromycin gene fragment.

189

This result verified that the transformant of the pBC-hygro plasmid was successfully



Page 10 of 26

190

constructed and determined the suitability of the pBC5 strain as a control strain in the

191

subsequent analyses.

192

Eight hygromycin-resistant strains were obtained by passaging of the

193

mokC-overexpressing strain (Cn strain, where n indicates the strain number 1-8) for

194

five generations, and were cultured at the same time as the control strain pBC5. The

195

content of monacolin K in the fermentation broth was determined by HPLC. The yield

196

of monacolin K in the control strain pBC5 was 72.5 mg/L (Fig. 4a), whereas that in the

197

mokC overexpression strain C8 was 242.4 mg/L (Fig. 4b), representing a 234.3%

198

increase. Therefore, strain C8 was used as the candidate mokC-overexpressing strain.

199

Eight

200

mokD-overexpressing strain (Dn) for five generations. The yield of monacolin K in

201

strain D8 was 232.6 mg/L (Fig. 4c), representing a 220.8% increase compared with the

202

production

203

mokD-overexpressing strain. Eight hygromycin-resistant strains were obtained by

204

passage of the mokE-overexpressing strain (En) for five generations. The monacolin

205

K yield of strain E3 was 137.4 mg/L (Fig. 4d), representing a 89.5% increase compared

206

with

207

mokE-overexpressing strain. Five hygromycin-resistant strains were obtained by

208

passage of the mokI-overexpressing strain (In) for five generations. The monacolin K

209

yield of strain I1 was 79.7 mg/L (Fig. 4e), representing a 10% increase compared to

210

that

211

mokI-overexpressing strain.

hygromycin-resistant

that

of

level

of

pBC5.

of

pBC5.

strains

pBC5.

Therefore,

Therefore,

Therefore,

were

strain

strain

obtained

D8 was

E3 was

I1 strain

was


by

used

used

used

passage

as

as

as

the

the

the

of

the

candidate

candidate

candidate

Page 11 of 26


212

The PCR results demonstrated that strains pBC5, C8, D8, E3, and I1 of M.

213

purpureus could significantly amplify the hygromycin gene fragment of about 1000 bp,

214

thus validating the successful construction of these transformants of pBC-hygro

215

plasmids (Fig. 3). The expression levels of monacolin K synthesis-related genes in the

216

overexpression strains were assessed on day 8 when stable overexpression was

217

achieved.

218

Overexpression of mokC in C8 upregulated the expression of the other genes to

219

different degrees. The expression level of the mokC gene was 63.2-fold higher than

220

that in the control pBC5. The levels of mokA, mokH, and mokI were increased by

221

0.1–0.6-fold, whereas the expression level of mokF was decreased by 0.9-fold. In D8,

222

the expression level of the mokC gene was 40.6-fold higher than that in the control

223

pBC5. The expression levels of mokD, mokH, and mokI genes were increased by

224

3.3-fold, 3.7-fold, and 1.9-fold, with smaller increase for mokA, mokE, and mokG

225

between 0.1–0.8-fold. By contrast, the expression levels of mokB and mokF were

226

decreased 0.1-fold and 0.7-fold, respectively (Fig. 5b). In E3, the mokC expression

227

level showed the greatest enhancement, with a 40.6-fold increase compared to that of

228

the control. The expression levels of mokE, mokH, and mokI genes were increased by

229

3.3-fold, 3.7-fold, and 1.9-fold, although the increase for mokA, mokE, and mokG was

230

small, between 0.1–0.8-fold. By contrast, the expression levels of mokB and mokF

231

were decreased by 0.1-fold and 0.7-fold, respectively (Fig. 5c). In I1, the expression

232

level of the mokI gene was 4.1-fold higher than that in the control pBC5. the

233

expression levels of mokA, mokD, mokC, mokE, mokB, mokH, and mokG were all



234

significantly higher than that in the control pBC5, with increases of 4.1-, 3.5-, 3.2-, 3.0-,

235

2.7-, 2.0-, 1.8-, and 1.6-fold, respectively (Fig. 5d).

236

Therefore, the RT-qPCR analysis showed that maximal monacolin K

237

biosynthesis was reached on day 8 based previous study28, at which point most of the

238

related genes showed higher transcription in the overexpression strains.

239

The effect of monacolin K biosynthesis gene overexpression on fungal

240

morphology was assessed with SEM (5000× and 10, 000×). The mycelia of the

241

overexpression strains C8, D8, E3, I1 and the control strain pBC5 were observed for

242

structural differences. It could be seen from Fig. 6e and 6j that the pBC5 mycelium

243

was dense and mostly combined with a network connection, and it had spores on the

244

top or side of the mycelium, which was consistent with the Ji et al.29 description.

245

Under the same magnification, overexpression strains showed clear changes to

246

the mycelium surface with obvious folds and the mycelium length shorter, whereas

247

the pBC5 mycelium had a more full structure with a flatter surface (Fig. 6B). The

248

spores of the overexpression strains showed different degrees of depression, while the

249

pBC5 strain had smooth surface and no depression. Wang et al.30 have studied the

250

overexpression of the mokE gene, it was found that mokE gene had a certain influence

251

on the morphology of mycelium and spores in Monascus. It was speculated that the

252

overexpression of mokE gene promoted the length of mycelium to be shortened, and

253

the network structure between hyphae to be reduced, thereby promoting the

254

production of monacolin K. This was basically consistent with the results of the

255

experiment. It suggested that overexpression of four genes had a certain effect on the


Page 12 of 26

Page 13 of 26


256

morphological structure of Monascus31-33, which promoted the synthesis of monacolin

257

K. And Lv et al.34 have studied that hyphal diameter was highly correlated with the

258

biosynthesis of the Monascus yellow pigments. Many factors contributed to the

259

development of morphological form in submerged fermentation, and many cases

260

indicated that the fungal morphology could influence the productivity of fungal

261

fermentations to some extent35.

262

In summary, we successfully constructed mokC, mokD, mokE, and mokI

263

overexpression Monascus strains, which all showed substantially increased yields of

264

monacolin K, demonstrating a significant impact of these genes on monacolin K

265

anabolism and good candidates for producing high-yielding strains. Moreover, we

266

detected a folding phenomenon of parts of the mycelium surface in the overexpression

267

strains, indicating that these genes could also influence the mycelia morphology.

268

Together, these findings lay the foundation for further in-depth analysis of the

269

function of this gene cluster to uncover the complex network regulation mechanism of

270

the monacolin K synthesis pathway. Further exploration of these strains and

271

underlying mechanisms will help to achieve the industrial-scale production of

272

monacolin K toward its widespread clinical application to best exploit its

273

health-promoting benefits.

274

ABBREVIATIONS

275

HMG-CoA, 3-hydroxy-3-methyl glutaryl coenzyme A; LNKS, Lovastatin Nonaketide

276

Synthase; PCR, Polymerase Chain Reaction; HPLC, High-Performance Liquid

277

Chromatography;

RT-qPCR,

Reverse

Transcription-Quantitative


PCR;

SEM,


Page 14 of 26

278

Scanning Electron Microscopy; PBS, Phosphate-Buffered Saline.

279

ACKNOWLEDGEMENTS

280

This work was supported by Beijing Natural Science Foundation (Grant No.

281

KZ201810011015), Beijing Nova Program (Grant No. Z181100006218021), Support

282

Project of High-level Teachers in Beijing Municipal Universities in the Period of 13th

283

Five--year Plan (Grant No. CIT&TCD201804023), National Natural Science

284

Foundation of China (Grant No. 31301411, 31571801, and 31401669), National Key

285

Research

286

2016YFD0400502-02),The construct of innovation service ability--Science and

287

technology

288

2016-014213-000034), Beijing Municipal Science and Technology Project (Grant No.

289

Z171100002217019), and Beijing Excellent Talents Training Project (Grant No.

290

2016000020124G025).

291

CONFLICTS OF INTEREST

292

The authors declare that they have no conflicts of interest.

293

SUPPORTING INFORMATION

294

Supplementary data related to this article can be found at http://pubs.acs.org.

295

Supplementary Table 1. Primer sequences for gene overexpression.

and

Development

achievement

Program

(Grant

transformation--Upgrade

No.

project

2016YFD0400802,

(Grant

No.

PXM

296 297

REFFERENCES

298

(1) Patel, S., Functional food red yeast rice (RYR) for metabolic syndrome amelioration: a review

299

on pros and cons. World Journal of Microbiology & Biotechnology 2016,32, 87.


Page 15 of 26


300

(2) Cheng, M. J.; Wu, M. D.; Chen, I. S.; Chen, C. Y.; Lo, W. L.; Yuan, G. F., Secondary metabolites

301

from the red mould rice of Monascus purpureus BCRC 38113. Natural Product Research 2010,24,

302

1719-1725.

303

(3) Ansari, M. P.; Puri, A.; Ali, M.; Panda, B. P., Five new secondary metabolites from Monascus

304

purpureus-fermented Hordeum vulgare and Sorghum bicolor. Natural Product Research 2013,27,

305

1848-1855.

306

(4) Chen, H. H.; Chen, Y. Y.; Yeh, J.; Jiang, C. M.; Wu, M. C., Immune-stimulated antitumor effect of

307

different molecular weight polysaccharides from on human leukemic U937 cells. CyTA - Journal of

308

Food 2014,12, 134-140.

309

(5) Dikshit, R.; Tallapragada, P., Statistical optimization of pigment production by Monascus

310

sanguineus under stress condition. Preparative Biochemistry 2014,44, 68-79.

311

(6) Su, Y. C.; Wang, J. J.; Lin, T. T.; Pan, T. M., Production of the secondary metabolites

312

gamma-aminobutyric acid and monacolin K by Monascus. Journal of Industrial Microbiology &

313

Biotechnology 2003,30, 41-46.

314

(7) Suzuki, N.; Imai, A., HMG-CoA reductase inhibitor lovastatin causes reversible cytoskeleton

315

perturbation by RhoA signalling suppression in peritoneal cell line Met5A. Journal of Obstetrics &

316

Gynaecology 2010,30, 404-407.

317

(8) Lee, C. L.; Wen, J. Y.; Hsu, Y. W.; Pan, T. M., The blood lipid regulation of Monascus-produced

318

monascin and ankaflavin via the suppression of low-density lipoprotein cholesterol assembly and

319

stimulation of apolipoprotein A1 expression in the liver. Journal of Microbiology Immunology &

320

Infection 2018, 51,27-37.

321

(9) Nezami, N.; Safa, J.; Salari, B.; Ghorashi, S.; Khosraviani, K.; Davarifarid, S.; Hashemiaghdam, Y.;



322

Nargabad, O. N.; Tabrizi, J. S., Effect of lovastatin therapy and withdrawal on serum uric acid level in

323

people with type 2 diabetic nephropathy. Nucleosides & Nucleotides 2012,31, 353-363.

324

(10) Manzoni, M.; Rollini, M., Biosynthesis and biotechnological production of statins by filamentous

325

fungi and application of these cholesterol-lowering drugs. Applied Microbiology & Biotechnology

326

2002,58, 555-564.

327

(11) Sakai, K.; Kinoshita, H.; Nihira, T., Identification of mokB involved in monacolin K biosynthesis

328

in Monascus pilosus. Biotechnology Letters 2009,31, 1911-1916.

329

(12) Chen, Y. P.; Tseng, C. P.; Liaw, L. L.; Wang, C. L.; Chen, I. C.; Wu, W. J.; Wu, M. D.; Yuan, G. F.,

330

Cloning and characterization of monacolin K biosynthetic gene cluster from Monascus pilosus. J Agric

331

Food Chem 2008,56, 5639-5646.

332

(13) Lee, C. L.; Hung, H. K.; Wang, J. J.; Pan, T. M., Improving the ratio of monacolin K to citrinin

333

production of Monascus purpureus NTU 568 under dioscorea medium through the mediation of pH

334

value and ethanol addition. J Agric Food Chem 2007,55, 6493-6502.

335

(14) Kang, B.; Zhang, X.; Wu, Z.; Qi, H.; Wang, Z., Effect of pH and nonionic surfactant on profile of

336

intracellular and extracellular Monascus pigments. Process Biochemistry 2013,48, 759-767.

337

(15) Yu, L. J.; Zhang, H. X.; Xie, Y. H.; Ma, S. M.; Liu, H.; Luo, Y. B., Optimization of Fermentation

338

Conditions for Higher Monacolin K Production by Monascus purpureus. Advanced Materials Research

339

2013,781-784, 1397-1402.

340

(16) Chen, Y. P.; Yuan, G. F.; Hsieh, S. Y.; Lin, Y. S.; Wang, W. Y.; Liaw, L. L.; Tseng, C. P.,

341

Identification of the mokH Gene Encoding Transcription Factor for the Upregulation of Monacolin K

342

Biosynthesis in Monascus pilosus. Journal of Agricultural & Food Chemistry 2013,58, 287-293.

343

(17) Lee,S. S.; Lee, J. H.; Lee, I., Strain improvement by overexpression of the laeA gene in Monascus


Page 16 of 26

Page 17 of 26


344

pilosus for the production of monascus-fermented rice. Journal of Microbiology & Biotechnology

345

2013,23, 959-965.

346

(18) Liu,Q.; Cai, L.; Shao, Y.; Zhou, Y.; Li, M.; Wang, X.; Chen, F., Inactivation of the global regulator

347

LaeA in Monascus ruber results in a species-dependent response in sporulation and secondary

348

metabolism. Fungal Biology 2016,120, 297-305.

349

(19) Xu, Z. W.; Zhang, Y.; Wang, Z. X.; Cao, Y.; Cui, L.; Li, S.; Liu, Q. Y., A Highly Efficient Protocol

350

for Transformation of Saccharomyces cerevisiae and Pichia pastoris by Electroporation. Acta

351

Scientiarum Naturalium Universitatis Sunyatseni 2010,49(3), 98-101.

352

(20) Sun, Q.; Wang, H.; Zhang, H.; Luo, H.; Shi, P.; Bai, Y.; Lu, F.; Yao, B.; Huang, H., Heterologous

353

production of an acidic thermostable lipase with broad-range pH activity from thermophilic fungus

354

Neosartorya fischeri P1. Journal of Bioscience & Bioengineering 2016,122, 539-544.

355

(21) Hong, S. Y.; Oh, J. H.; Lee, I., Simultaneous enrichment of deglycosylated ginsenosides and

356

monacolin K in red ginseng by fermentation with Monascus pilosus. Journal of the Agricultural

357

Chemical Society of Japan 2011,75, 1490-1495.

358

(22) Chan, Z.; Jian, L.; Le, Y.; Chai, S.; Zhang, C.; Sun, B.; Wang, C., Glutamic acid promotes

359

monacolin K production and monacolin K biosynthetic gene cluster expression in Monascus. Amb

360

Express 2017,7, 22.

361

(23) Perini, M.; Carbone, G.; Camin, F., Stable Isotope Ratio Analysis for authentication of Red Yeast

362

Rice. Talanta 2017,174, 228-233.

363

(24) Verderio, P.; Pizzamiglio, S.; Gallo, F.; Ramsden, S. C., FCI: an R-based algorithm for evaluating

364

uncertainty of absolute real-time PCR quantification. Bmc Bioinformatics 2008,9, 13.

365

(25) Schultzthater, E.; Frey, D. M.; Margelli, D.; Raafat, N.; Federmengus, C.; Spagnoli, G. C.; Zajac,



366

P., Whole blood assessment of antigen specific cellular immune response by real time quantitative PCR:

367

a versatile monitoring and discovery tool. Journal of Translational Medicine 2008,6, 58.

368

(26) Zhang, Z.; Ali, Z.; Khan, S. I.; Khan, I. A., Cytotoxic monacolins from red yeast rice, a Chinese

369

medicine and food. Food Chemistry 2016,202, 262-268.

370

(27) Sakai, K.; Kinoshita, H.; Shimizu, T.; Nihira, T., Construction of a Citrinin Gene Cluster

371

Expression System in Heterologous Aspergillus oryzae. Journal of Bioscience & Bioengineering

372

2008,106, 466-472.

373

(28) Zhang, C.; Liang, J.; Yang, L.; Sun, B.; Wang, C., De Novo RNA Sequencing and Transcriptome

374

Analysis of Monascus purpureus and Analysis of Key Genes Involved in Monacolin K Biosynthesis.

375

Plos One 2017,12, e0170149.

376

(29) Ji, Y. K.; Kim, H. J.; Oh, J. H.; Lee, I., Characteristics of Monascus sp. isolated from Monascus

377

fermentation products. Food Science & Biotechnology 2010,19, 1151-1157.

378

(30) Lin, L.; Wang, C.; Zhenjing, L. I.; Chen, M.; Shufen, W. U.; Ren, Z., Effect of mok E

379

Overexpression on Monacolin K Production and Morphology of Mycelia and Spores in Monascus.

380

Food Science 2018, 39, 45-49.

381

(31) Liu, Q.; Xie, N.; He, Y.; Wang, L.; Shao, Y.; Zhao, H.; Chen, F., MpigE, a gene involved in

382

pigment biosynthesis in Monascus ruber M7. Applied Microbiology & Biotechnology 2014,98, 285-296.

383

(32) Zhuang, Z.; Lohmar, J. M.; Satterlee, T.; Cary, J. W.; Calvo, A. M., The Master Transcription

384

Factor mtfA Governs Aflatoxin Production, Morphological Development and Pathogenicity in the

385

Fungus Aspergillus flavus. Toxins 2016,8, 29.

386

(33) Leiter, É.; Park, H. S.; Kwon, N. J.; Han, K. H.; Emri, T.; Oláh, V.; Mészáros, I.; Dienes, B.;

387

Vincze, J.; Csernoch, L., Characterization of the aodA, dnmA, mnSOD and pimA genes in Aspergillus


Page 18 of 26

Page 19 of 26


388

nidulans. Sci Rep 2016,6, 20523.

389

(34) Lv, J.; Zhang, B. B.; Liu, X. D.; Chan, Z.; Lei, C.; Xu, G. R.; Cheung, P. C. K., Enhanced

390

production of natural yellow pigments from Monascus purpureus by liquid culture: The relationship

391

between fermentation conditions and mycelial morphology. Journal of Bioscience & Bioengineering

392

2017,124,452-458.

393

(35) Papagianni, M., Fungal morphology and metabolite production in submerged mycelial processes.

394

Biotechnology Advances 2004,22, 189-259.

395 396



Page 20 of 26

397

Figure captions

398 399

Fig. 1 Construction of pBC-hygro-mokC (a) and agarose gel electrophoresis of

400

pBC-hygro-mokC(b), pBC-hygro-mokD(c), pBC-hygro-mokE(d), pBC-hygro-mokI(e)

401

recombinant plasmid size verification.

402

(b-e) Lane 1 indicates the DNA super helix marker; Lane 2 indicates a pBC-hygro-

403

mokC (b), pBC-hygro-mokD (c), pBC-hygro-mokE (d), pBC-hygro-mokI (e)

404

recombinant plasmid. Lane 3 indicates pBC-hygro original plasmid.

405

Fig.2 To screen the tolerated concentration of hyacinth B and electric shock

406

conditions in M. purpureus M1.(a) To screen the electric shock conditions, (b) To

407

screen the tolerated concentration of hyacinth B.

408

Fig. 3 PCR of the hygromycin B gene in M. purpureus pBC5 and overexpression

409

strains. M: DL2000 DNA Marker; Lane 1: pBC5; Lane 2: C8; Lane 3: D8; Lane 4: E3;

410

Lane 5: I1.

411

Fig. 4 Monacolin K contents of

412

genes.

413

(a) Monacolin K content of pBC-Hygro conversion in M. purpureus.(b) Monacolin K

414

content

415

mokD-overexpression strains. (d) Monacolin K content of mokE-overexpression

416

strains. (e) Monacolin K content of mokI-overexpression strains.

417

Fig. 5 Expression of genes related to monacolin K biosynthesis (mokA-mokI) of M.

418

purpureus pBC5 (control), and overexpression strains (a) C8, (b) D8, (c) E3, and (d)

of

mokC-overexpression

M. purpureus strains with overexpression four

strains.

(c)

Monacolin


K

content

of

Page 21 of 26


419

I1.

420

Fig. 6 The colony and SEM images of M. purpureus overexpression strains C8, D8,

421

E3, and I1, and pBC5 (control). (A) The colony morphology of five strains, (B) SEM

422

images of five strains at different magnifications: 5000× (a–e) and 10, 000× (f–j). (a-e):

423

C8, D8, E3, I1 and pBC5; (f-j):C8, D8, E3 I1 and pBC5. The red arrow represents the

424

spores depression.



Page 22 of 26

Table 1. Primer sequences for gene overexpression Primer

Sequence

Tm （℃）

mokC-F mokC-R mokD-F mokD-R mokE-F mokE-R mokI-F mokI-R hygro-F hygro-R

AAGGAAAAAAGCGGCCGCATGACAGTTCCGACAGATACG (NotI) TCCCCCGGGTCAGAGATCTTCGTCCCGAC (SmaI) GCTCTAGAATGCGTATCCAACGCACCC (XbaI) CCCAAGCTTTCACCCAATGACTCTAGCCC (HindIII) GCTCTAGAATGACCATCACCTTCACCCTAC (XbaI) CCCAAGCTTTTACCCCAACTTCACCACAAC (HindIII) GCTCTAGAATGGCTTCCCACCAGTCTG (XbaI) CCCAAGCTTCTAGACTCGTTCATCGCGG (HindIII) ATGCCTGAACTCACCGCGACG GATAAGGAAACGGGAGCCTGC

64

Fig.1


65.6 60.4 64.3 55

Page 23 of 26


Fig.2

Fig.3

Fig. 4



Fig.5


Page 24 of 26

Page 25 of 26


Fig.6



TOC Graphic


Page 26 of 26

Overexpression of Monacolin K Biosynthesis Genes in the Monascus

Recommend Documents