MptriA, an Acetyltransferase Gene Involved in Pigment Biosynthesis in

3 days ago - Monascus pigments (Mps) have been used as food colorants for several centuries in Asian countries. MptriA is a putative acetyltransferase...
0 downloads 8 Views 2MB Size
Subscriber access provided by UNIV OF DURHAM

Bioactive Constituents, Metabolites, and Functions

MptriA, an acetyltransferase gene involved in pigment biosynthesis in M. purpureus YY-1 Bin Liang, Xin-jun Du, Ping Li, Chanchan Sun, and Shuo Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00661 • Publication Date (Web): 10 Apr 2018 Downloaded from http://pubs.acs.org on April 11, 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 42

Journal of Agricultural and Food Chemistry

1

Title:

2

MptriA, an acetyltransferase gene involved in pigment biosynthesis in M.

3

purpureus YY-1

4 5

Authors:

6

Bin Lianga, Xinjun Dua, Ping Lia, Chanchan Suna,Shuo Wang a, *

7

Affiliation:

8

a

9

Technology), Ministry of Education, Tianjin 300457, China

Key Laboratory of Food Nutrition and Safety (Tianjin University of Science &

10 11

* Corresponding Author:

12

Shuo Wang

13

Key Laboratory of Food Nutrition and Safety (Tianjin University of Science &

14

Technology), Ministry of Education, Tianjin 300457, China

15

Tel: 86-22-60912484

16

Fax: 86-22-60912484

17

E-mail: [email protected]

1

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

18

ABSTRACT

19

Monascus pigments (Mps) have been used as food colorants for several centuries in

20

Asian countries. MptriA is a putative acetyltransferase gene involved in the MPs biosynthesis.

21

In order to analyze the function of MptriA, an MptriA disruption strain (∆MptriA) and a

22

complementation strain (∆MptriA::MptriA) were successfully obtained In addition to the loss

23

of color, the disruption of MptriA had little effect on the phenotypes during growth on four

24

different medium. The ∆MptriA strain showed decreased pigment and citrinin production

25

during the liquid-fermentation process. Transcriptional analysis showed that the expression of

26

several genes involved in the synthesis of pigments and citrinin was down-regulated in

27

∆MptriA. These results demonstrated that the role of MptriA was to transfer an acyl group to

28

the pyranoquinone structure of the polyketide chromophore during Monascus pigment

29

biosynthesis and to influence the citrinin biosynthesis pathway. This study contributes to the

30

exploration of pigment biosynthesis in M. purpureus.

31

KEYWORDS: M. purpureus; MptriA gene; Pigments; Disruption; Complementation

2

ACS Paragon Plus Environment

Page 2 of 42

Page 3 of 42

Journal of Agricultural and Food Chemistry

32

INTRODUCTION

33

As significant traditional edible fungi, Monascus species have been used in food,

34

medicine and industry for more than one thousand years, and more than one billion people

35

consume Monascus-fermented products as part of their daily diet1, 2, 3. One of the most famous

36

Monascus-fermented products, red fermented rice, has been used extensively as a natural food

37

colorant, folk medicine, fermentation starter in East and Southeast Asia4, 5, 6, 7. Meanwhile,

38

previous reports have shown that Monascus species can produce various natural and

39

functional secondary metabolites, such as Monascus pigments (Mps), monacolin K, and

40

γ-aminobutyric acid (GABA)8-10. Therefore, the utility of Monascus species has attracted the

41

attention of many research teams.

42

Among secondary metabolites of Monascus spp., pigments used as food additives for

43

several centuries in Asian countries4, have been supposed as polyketides11, 12. Pigments

44

produced by Monascus species can be divided into three major groups: red pigments

45

(monascorubramine

46

rubropunctatin), and yellow pigments (monascin and ankaflavin)13. To date, at least 90 kinds

47

of Mps have been identified1, 14, and many showed multifarious biological activities such as

48

preventing hypertension15, and lowering cholesterol16, hypolipidemic effects17, and

49

anti-obesity18, 19, anti-tumor20, and anti-cancer activities21. Therefore, it is important to select a

50

high pigment-producing strain using molecular biological methods and optimize the

51

fermentation conditions to improve pigment production.

and

rubropunctamine),

orange

pigments

(monascorubrin

and

52

Analysis of the genomes of M. pilosus, M. purpureus, and M. ruber via bioinformatics

53

and RT-PCR showed that the Mps gene cluster contains a minimum of 16 genes, namely,

3

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

54

MpigA (nonreducing polyketide synthase, NR-PKS), MpigB (transcription factor), MpigC

55

(dehydrogenase), MpigD (3-O-transacetylase), MpigE (dehydrogenase), MpigF (monoamine

56

oxidase), MpigG (oxidoreductase), MpigH (dehydrogenase), MpigI (transcription factor),

57

MpigJ (fatty acid synthase, α subunit), MpigK (fatty acid synthase, β subunit), MpigL

58

(ankyrin), MpigM (P450-monooxygenase), MpigN/O (monooxygenase), MpigP (unknown

59

function), and MpigQ (transporter)3,

60

themselves to studying the Mps biosynthesis pathway, several steps and the identities of

61

related enzymes remain unclear or controversial1, 3. Mps biosynthesis is believed to follow a

62

polyketide pathway, in which the PKS genes have been shown to be extremely important to

63

the biosynthetic pathways of Mps, owning to targeted inactivation of MpPKS5 in M.

64

purpureus or pksPT in M. ruber gave rise to loss of pigment24, 25. In addition to PKS, several

65

genes involved in pigment biosynthesis have been investigated. Xie, et al.26 identified a

66

pigment biosynthesis regulatory gene (pigR) in M. ruber M7, which upregulated pigment

67

production. Targeted deletion of mrflbA, Mgb1 and Mgg1 resulted in phenotypic alterations

68

such as decreased vegetative growth and asexual sporulation and altered citrinin and pigment

69

production27, 28. Liu, et al.29 obtained an MpigE (as well as mppC in M. purpureus) gene

70

deletion strain (∆MpigE), which yielded only four kinds of yellow pigment and very few red

71

pigments but had no influence on citrinin. Liu, Zhou, Yi and Zhao23 reported that a mutant d

72

in which an approximately 30-kb region of the pigment gene cluster from M. ruber M7 was

73

deleted could induce the accumulation of high levels of M7PKS-1, which has been previously

74

shown to be an initial intermediate of Mps. Balakrishnan, et al.30 discovered a reductase gene,

75

mppE, that controls the biosynthesis of the yellow pigments, ankaflavin and monascin in the

22, 23

. Even though many researchers have devoted

4

ACS Paragon Plus Environment

Page 4 of 42

Page 5 of 42

Journal of Agricultural and Food Chemistry

76

azaphilone polyketide pathway. Although many scientists3, 25, 31, 32 have contributed to the

77

prediction of parts of the synthetic pathway of Mps, the identities of related genes involved in

78

pigment biosynthesis remain unclear or controversial, which limits the practical industrial

79

application of Monascus.

80

In our previous studies3, transcriptional differences in M. purpureus YY-1 grown in

81

different medium on the eighth day of growth indicated that MptriA is upregulated when M.

82

purpureus is grown in rice medium (high-yield pigment states). Therefore, we predicted that

83

the MptriA gene plays a very significant role in the production of pigments. Previous reports

84

have shown that MptriA homologs are found in two relevant azaphilone biosynthetic gene

85

clusters: azaD and cazE, which are involved in the biosynthesis of azanigerones and

86

chaetoviridin, respectively33, 34. In this study, we constructed a putative acetyltransferase

87

MptriA gene-deletion mutant of M. purpureus YY-1 and its revertant strain to investigate the

88

role of MptriA, and the results revealed that ∆MptriA caused little phenotypic alterations in

89

addition to colors and played a vital role in the production of some secondary metabolites,

90

such as pigments and citrinin. This work will guide further exploration of the function of

91

MptriA in the biosynthetic pathways of pigments in M. purpureus.

92

MATERIALS AND METHODS

93

Fungal strains, culture medium, and growth conditions. M. purpureus YY-1 obtained

94

from Gutian Shenghua Monascus Ltd. of Ningde City (Fujian Province, China) was used for

95

the generation of the ∆MptriA strain3. The ∆MptriA strain was used to generate the

96

∆MptriA::MptriA strain. For phenotypic characterization, four kinds of medium were used,

97

namely, potato dextrose agar medium (PDA), malt extract agar medium (MA), Czapek yeast

5

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 6 of 42

98

extract agar medium (CYA), and glycerol nitrate agar medium (25%) (G25N)35. For

99

sporulation, M. purpureus YY-1 was grown on COM medium (30 g of glucose, 3 g of peptone,

100

0.5 g of KH2PO4, 0.5 g of MgSO4 per liter, pH 5.5-6.0). For total DNA extraction and to

101

screen the constructed strains, MA medium supplemented with the appropriate antibiotic was

102

used. All strains were maintained on MA slants at 28°C. Minimal medium (MM), induction

103

medium

104

tumefaciens-mediated transformation (ATMT)36. Luria-Bertani medium (LB), supplemented

105

with antibiotic when necessary, was used to cultivate Escherichia coli for propagating

106

plasmids. M. purpureus YY-1 and its derivatives and A. tumefaciens were grown at 28°C.

107

Fungal spores and A. tumefaciens were co-cultured at 24°C for 3 days. E. coli DH5α was

108

grown at 37°C for routine cloning. All strains and plasmids used in this study are listed in

109

Table 1.

(IM)

and

co-cultivation

medium

(CM)

were

used

for

Agrobacterium

110

DNA extraction. Fungal genomic DNA was isolated from mycelia grown on cellophane

111

membranes covering MA plates using the cetyltrimethylammonium bromide (CTAB) method

112

described by Shao, et al.37.

113

Cloning and analysis of the MptriA gene. A pair of primers, MptriA-F/MptriA-R

114

(Table 2), was designed to amplify the MptriA gene. PCR was carried out to amplify the

115

MptriA gene from the genome of M. purpureus YY-13, and the protocol was as follows: initial

116

denaturation at 94°C for 2 min; 30 amplification cycles of 98°C for 10 s, 55°C for 30 s, and

117

68°C for 2.5 min; and a final extension step at 72°C for 10 min; a TProfessional thermal

118

cycler (Biometra, Germany) was used for the PCR. The amino acid sequence encoded by

119

MptriA

was

predicted

using

SoftBerry's

6

ACS Paragon Plus Environment

FGENESH

program

Page 7 of 42

Journal of Agricultural and Food Chemistry

120

(http://linux1.softberry.com/berry.phtml), and the MptriA functional regions were analyzed

121

using the Pfam 30.0 program (http://pfam.xfam.org/). The homology of the deduced amino

122

acid sequence was analyzed using the BLASTP program on the NCBI web site

123

(http://blast.ncbi.nlm.nih.gov/Blast.cgi).

124

∆MptriA strain construction. To construct the MptriA disruption mutant, the 5′ and 3′

125

flanking regions (2355 bp and 2297 bp, respectively) of the MptriA gene were amplified with

126

the primer pairs triA5-F/triA5-R and triA3-F/triA3-R using KOD-FX DNA polymerase

127

(Toyobo, Japan) (Table 2). The PCR products of the 5’ and 3’ flanking regions were purified

128

and cloned into the pEASY-Blunt vector (Transgen, China) to generate pEBTL and pEBTR,

129

respectively. Successful cloning of the inserts into the resulting plasmids was verified by

130

sequencing. Then, pEBTL was digested with KpnI and ApaI and ligated into the

131

corresponding sites of pAg1-H3 (a vector containing the hygromycin phosphotransferase

132

gene hph) to generate pAgHL. Then, both pEBTR and pAgHL were digested with AscI and

133

SbfI and ligated with T4 DNA ligase to generate the plasmid pAgHLR. A 2-kb SbfI-digested

134

DNA fragment containing the neomycin phosphotransferase resistance gene (neo) from the

135

plasmid pAgHN was inserted into the corresponding sites of pAgHLR, yielding pAgHNLR.

136

The plasmid pAgHNLR was transformed into A. tumefaciens AGL-1 via ATMT as described

137

previously38 with the exception that cellophane was used instead of nitrocellulose membrane

138

during the co-culture phase. The A. tumefaciens AGL-1 clones containing pAgHNLR were

139

incubated for transformation with M. purpureus YY-1 to yield the fungal transformants. All

140

the fungal transformants were selected on MA plates supplemented with 200 µg/mL

141

hygromycin B and 500 µg/mL cefotaxime. Hygromycin-resistant and neomycin-sensitive

7

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

142

strains were selected, and ∆MptriA was confirmed by PCR analysis using the internal primers

143

YtriA1-F/YtriA1-R, external the outer primers YtriA2-F/YtriA2-R and cross-validation

144

primers YtriA3-F/YtriA3-R, and YtriA3-F/YtriA3-R (Table 2).

145

Complementation of ∆MptriA with MptriA of M. purpureus YY-1. To further verify

146

whether all the differences exhibited by the ∆MptriA strain were caused by the disruption of

147

MptriA, this gene was complemented. For complementation, the entire MptriA gene along

148

with a 760-bp upstream region containing the putative promoter region of the gene and a

149

599-bp downstream region was amplified from wild-type M. purpureus YY-1 with the

150

primers pair triA-F/triA-R and inserted into pEASY-Blunt to generate pEBtriA (Table 2).

151

Successful cloning of the insert into the resulting plasmid was verified by sequencing. Then,

152

the plasmids pEBtriA and pAgHN were digested with SacI and KpnI, and the 2727-bp DNA

153

fragment containing the intact MptriA was inserted into the corresponding sites of pAgHN to

154

generate pAgHNtriA. Finally, the plasmid pAgHNtriA was transformed into A. tumefaciens

155

AGL-1; and then the A. tumefaciens AGL-1 clones containing pAgHNtriA were incubated for

156

transformation with the ∆MptriA strain by ATMT, as described previously, to yield the

157

MptriA-complementation strain (∆MptriA::MptriA) by ATMT as described previously38.

158

Transformants were selected on MA plates supplemented with 20 µg/mL neomycin and 500

159

µg/mL cefotaxime at 28ºC. Neomycin-resistant strains were selected. The complementation

160

was confirmed by PCR amplification with the primer pairs YtriA1-F/YtriA1-R and

161

Neo-F/Neo-R (Table 2).

162

Southern hybridization analysis. To further verify the homologous recombination

163

events, Southern hybridization analysis was conducted. For Southern blot assays, the DIG

8

ACS Paragon Plus Environment

Page 8 of 42

Page 9 of 42

Journal of Agricultural and Food Chemistry

164

High Prime DNA Labeling and Detection Starter Kit II (Roche, Germany) was used

165

according to the manufacturer’s protocol. The DNA (20 µg) of the M. purpureus YY-1,

166

putative ∆MptriA and ∆MptriA::MptriA strains were digested with XhoI. Probe 1 and probe 2

167

were amplified via PCR with the primer pairs ProtriA-F/ProtriA-R and Prohph-F/Prohph-R

168

(Table 2), respectively. Probe 1 and probe 2 were used to verify the MptriA disruptant, and the

169

∆MptriA::MptriA strain was confirmed with probe 1.

170

RNA isolation and complementary DNA preparation. Total RNA of M. purpureus

171

YY-1, ∆MptriA, and ∆MptriA::MptriA was isolated from mycelia after 48 h of cultivation

172

using the RNeasy® Plant Mini Kit (QIAGEN, Germany) according to the manufacturer’s

173

protocol. RNA concentration was determined by measuring the absorbance at 260 and 280 nm

174

(A260/A280), and RNA integrity was verified by visualization on 1% agarose gels. RNA

175

samples were stored at −80°C. For reverse transcription, total RNA (390 ng) was added to a

176

20 µL mixture derived from the PrimeScript™ RT Reagent Kit (Takara, Japan), and the

177

reaction conditions followed the manufacturer’s protocol.

178

Quantitative real-time PCR analysis. The changes in mRNA levels obtained by

179

RNA-seq were further validated by quantitative real-time PCR (qRT-PCR) of the MptriA gene.

180

Each reaction (20 µL) contained 10 µL of SYBR Premix Ex Taq II, 0.8 µL of 10 µM forward

181

primer, 0.8 µL of 10 µM reverse primer, 0.4 µL of ROX Reference Dye II (Takara, Japan), 2

182

µL of template cDNA, and 6 µL of ddH2O. All real-time PCRs were performed using the

183

Mastercycler ep realplex system (Eppendorf, Germany) with the following steps (two-step

184

PCR amplification, standard procedure): 30 s at 95°C, 40 cycles of 5 s at 95°C, and 34 s at

185

60°C. Sample melting curves were assessed to evaluate the specificity of the amplification.

9

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

186

GAPDH was used as the reference gene29. The primers used in this part are listed in Table 2.

187

Samples were analyzed in triplicate, and the experiments were repeated at least three times.

188

MP and Citrinin analysis. Three kinds of COM medium (100 mL) with appropriate

189

antibiotics were fermented by the wild-type strain M. purpureus YY-1, ∆MptriA or

190

∆MptriA::MptriA. After agitation with 170 rpm for 40 h at 30°C, 20 mL of the fermentation

191

mixtures of the above three strains were inoculated into 200 mL of RM medium (20 g of rice

192

powder, 20 g of glucose, 20 g of peptone, 2 g of NaNO3, 1.5 g of KH2PO4, 1 g of MgSO4 per

193

liter) with the appropriate antibiotics. The fermentation was carried out at 28ºC, and samples

194

were taken every other day from the 2nd day to the 18th day of fermentation to measure MP

195

and citrinin production. In addition, samples were harvested from 2 d to 8 d for RNA isolation

196

and transcriptional analysis by real-time PCR.

197

MP production was detected by following the method described by the Chinese

198

standards (GB 1816.15-2015) with slight modification. The supernatant was removed by

199

centrifugation from the fermentation broth, and the fungal mycelia were washed twice with

200

sterile water and then dried at 60°C in a hot air oven until a constant weight was attained.

201

Dried mycelia (0.02 g) were treated with 10 mL of 70% ethanol and heated at 60°C in water

202

for 1 h. After filtration, the absorbance values of the red, orange, and yellow pigments were

203

determined at the specific wavelengths 505 nm, 465 nm and 410 nm, respectively, using a 50

204

Bio UV-Visible spectrophotometer (Cary Varian, USA); 70% ethanol was used as a negative

205

control. Pigment yield was expressed as absorbance at the λmax of the pigment per g of dry M.

206

purpureus YY-1 mycelia39.

207

To detect citrinin production, HPLC was performed following the method described by

10

ACS Paragon Plus Environment

Page 10 of 42

Page 11 of 42

Journal of Agricultural and Food Chemistry

208

the Chinese standards (GB/T 5009.222-2008) with slight modification. After filtration and

209

drying as described above, 0.01 g of dried mycelia was extracted with 1 mL of

210

acetate/formic acid (TEF, 7:3:1 by volume) buffer. The prepared samples were filtered

211

through a 0.2-µm filter and analyzed by HPLC using a reverse-phase C18 column (5 µm, 4.6

212

mm×250 mm). The HPLC parameters were as follows: mobile phase, 75% (v/v)

213

acetonitrile/25% (v/v) water (pH 2.5, adjusted by orthophosphoric acid); column temperature,

214

28°C; flow rate, 1.0 mL/min. The elution was monitored using a fluorescence detector at an

215

emission wavelength of 500 nm and an excitation wavelength of 331 nm. A citrinin standard

216

(Sigma, USA) was used to verify the HPLC analysis.

toluene/ethyl

217

Transcriptome sequencing and analysis. Vegetative hyphae of M.purpureus YY-1 and

218

∆MptriA were harvested from 8-d liquid RM cultures. There were three biological replicates

219

for each strain. Transcriptome sequencing and analysis were performed by Novogene

220

Bioinformatics Technology Co., Ltd. (Beijing, China). Briefly, Total RNA was extracted using

221

TRIzol reagent according to the manufacturer’s protocol. RNA purity was checked using a

222

NanoPhotometer® spectrophotometer (Implen, USA). RNA concentration and integrity were

223

measured using the Qubit® RNA Assay Kit with a Qubit® 2.0 Fluorometer (Life Technologies,

224

USA) and the RNA Nano 6000 Assay Kit with a Bioanalyzer 2100 system (Agilent

225

Technologies, USA). Then, mRNA was purified from total RNA using poly-T oligo-attached

226

magnetic beads, and fragmentation was carried out using NEBNext First Strand Synthesis

227

Reaction Buffer. The obtained mRNA fragments were used as templates to synthesize

228

first-strand cDNA with random hexamer primers, and then, the second-strand cDNA was

229

synthesized using DNA polymerase I and RNase H. After adenylation of the 3’ ends of the

11

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

230

DNA fragments, NEBNext Adaptor was ligated to prepare the samples for hybridization.

231

cDNA that were 150-200 bp in length were preferentially size-selected and purified with the

232

AMPure XP system (Beckman Coulter, USA). After PCR amplification, the library

233

preparations were sequenced on an Illumina HiseqTM 2000 sequencer (Illumina, USA), and

234

125 bp/150-bp paired-end reads were generated. The index of the reference genome was built

235

using Bowtie v2.2.3, and paired-end clean reads were aligned to the reference genome using

236

TopHat v2.0.12. Gene expression was calculated using the FPKM (expected fragments per kb

237

of transcript per million mapped reads) method with HTSeq v0.6.1, and differentially

238

expressed genes were selected based on P-value