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Characterization and Functional Identification of a Gene Encoding Geranylgeranyl Diphosphate Synthase (GGPS) from Dunaliella bardawil Ming-Hua Liang, Ying-Jie Liang, Hong-Hao Jin, and Jian-Guo Jiang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b02732 • Publication Date (Web): 20 Aug 2015 Downloaded from http://pubs.acs.org on August 22, 2015

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Journal of Agricultural and Food Chemistry

1

Characterization

and

Functional

Identification

of

a

Gene

Encoding

2

Geranylgeranyl Diphosphate Synthase (GGPS) from Dunaliella bardawil

3 4

Ming-Hua Liang1, Ying-Jie Liang2, Hong-Hao Jin1, Jian-Guo Jiang 1*

5

1

College of Food Science and Engineering, South China University of Technology, Guangzhou, 510640, China

6

2

School of Biological Science & Engineering, South China University of Technology, Guangzhou, 510006, China

7

*Author (Jian-Guo Jiang) for correspondence (e-mail: [email protected]; phone: +86-20-87113849; fax:

8

+86-20-87113849).

9

1

ACS Paragon Plus Environment


10

Abstract

11

Geranylgeranyl diphosphate synthase (GGPS) catalyzes the biosynthesis of geranylgeranyl diphosphate, a key

12

precursor for carotenoid biosynthesis. In this study, a full-length cDNA encoding GGPS from Dunaliella bardawil

13

(DbGGPS) was isolated by rapid amplification of cDNA ends (RACE) for the first time. The full-length cDNA of

14

DbGGPS was 1,814 bp containing a 1,074-bp ORF encoding 357 amino acids with a calculated mass of 38.88

15

kDa. Analysis of DbGGPS genomic DNA revealed that it contained 10 exons and 9 introns. It was predicted that

16

DbGGPS possessed a 48 amino-acid transit peptide at its N-terminal. Bioinformatic analysis revealed that

17

DbGGPS was a member of polyprenyltransferases with five conserved domains and two highly conserved

18

aspartate-rich motifs. Using heterologous expression, carotenoid complementation assay and gene deletion

19

analysis, it was shown that the coding region of DbGGPS encodes a functional GGPS. This provides new gene

20

sources for carotenoid genetic engineering.

21 22

Keywords: Dunaliella bardawil; geranylgeranyl diphosphate synthase (geranylgeranyl pyrophosphate synthase);

23

functional expression; carotenoids.

2


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24

Introduction

25

Carotenoids are important natural pigments produced by many microorganisms (microalgae, fungi and bacteria)

26

and plants and used commercially as food colorants, animal feed supplements and, more recently, as

27

nutraceuticals for cosmetic and pharmaceutical purposes.1

28

In chloroplasts of plants and algae, the isoprenoid biosynthetic pathway is responsible for the synthesis of a

29

variety of products including carotenoids, steroids, prenyl side-chain of quinones and prenyl proteins (Figure 1).2,

30

3

31

dimethylallyl pyrophosphate (DMAPP), which are synthesized through either mevalonate (MVA) pathway in

32

fungi and the cytosol/endoplasmic reticulum of plants or the 2-C-methyl-D-erythritol-4-phosphate (MEP)

33

pathway in bacteria, algae and plastid of plants.3,

34

biosynthesis of geranylgeranyl diphosphate (GGPP), the C20 precursor of phytoene, via sequential adding three

35

IPP molecules to a DMAPP molecule under the catalysis of geranylgeranyl diphosphate synthase (GGPS)

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(geranylgeranyl pyrophosphate synthase, EC: 2.5.1.29).5 GGPP is one of the key precursors in the biosynthesis of

37

biologically significant isoprenoid compounds. The condensation of two GGPP molecules produces the first

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carotene, phytoene, catalyzed by phytoene synthase (PSY). Phytoene is desaturated by phytoene desaturases (PDS)

39

and ζ-carotene desaturases (ZDS) and isomerized by 15-cis-ζ-carotene isomerase (ZISO)

40

isomerase (CRTISO)

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(LycE) and lycopene β-cyclase (LycB) introduces ε- and β-ionone end groups, respectively, yielding α-carotene

42

and β-carotene.

Carotenoids are derived from the C5 isoprenoid precursors, isopentenyl pyrophosphate (IPP) and its isomer

7

4

Thereby, the early step of carotenogenic pathway is the

6

and carotenoid

to form the linear all trans-lycopene. The cyclation of lycopene by lycopene ε-cyclase

43

In the carotenogenic bacterium Erwinia uredovora, a chromosomal gene cluster capable of directing

44

carotenoid biosynthesis has been isolated and characterized.8 This gene cluster consists of crtE (encoding GGPS,

45

crtX (encoding xeaxanthin β-glucosidase), crtY (encoding lycopene cyclase), crtl (encoding phytoene desaturase),

46

crtB (encoding phytoene synthase), and crtZ (encoding β-carotene hydroxylase). Expression of these crt genes in

47

E. coli cell results in carotenoid production. Plasmids pACCRT-EB, pACCRT-EIB, pACCAR16∆crtX and

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pACCAR25∆crtX have been constructed and expressed in E. coli resulting in phytoene, lycopene, β-carotene and

49

zeaxanthin, respectively.9 GGPS enzymatic activity can be detected in E. coli cells expressing the GGPS gene in

50

two different ways. One was the direct measurement of GGPS activity in cell extracts and the other was the color

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carotenoids production when the GGPS gene was co-expressed with crtB, crtl, crtX, crtY and crtZ genes derived

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from Erwinia uredovora.2 3



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Many plant GGPS genes have been isolated, characterized and expressed from various sources, such as

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Arabidopsis thaliana,2 Jatropha curcas,10 Catharanthus roseus,11 Taxus canadensis,12 Corylus avellana,13

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Scoparia dulcis and Croton sublyratus.14 To our knowledge, few algal GGPS genes have been cloned

56

and identified their function. The chlorophyte Dunaliella bardawil can produce large amounts of β-carotene under

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high irradiance, nutrient depletion and high salt concentration.15 When exposed to stress conditions such as

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salinity, high irradiance, or nutrient starvation, β-carotene may be accumulated reaching up to 10% of the dry cell

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weight.15 The unique ability of D. bardawil to accumulate very large amounts of β-carotene under defined

60

experimental conditions makes it an excellent choice for studying the intermediate of carotenoids. PSY 16 and ZDS

61

17

62

from D. bardawil (DbGGPS) was first cloned and characterized. Then heterologous expression using pET32a

63

plasmid and functional complementation of DbGGPS using plasmids pACCRT-EIB and pACCAR16∆crtX in E.

64

coli were carried out to identify the cDNA encoding for functional GGPS. This provides new gene sources for

65

carotene genetic engineering and is helpful for the understanding of the carotene biosynthesis.

66

Materials and methods

67

from D. bardawil, and PDS

18

from Dunaliella salina have been isolated, characterized. In this study, GGPS

Algal Strains and Cultivation Conditions. The green algae D. bardawil strain was obtained from the 19

68

Institute of Hydrobiology, Chinese Academy of Science. D. bardawil cells were grown in defined medium

69

containing 2 mol/L NaCl at 26 °C in a controlled chamber for 10-14 days and 8,000 lx provided by cool-white

70

fluorescent lamps, under a 16/8 h light/dark cycle with shaking at 96 rpm.

71

Bacterial strains and plasmids. Escherichia coli DH5α was used as the host for the multiplication of

72

plasmids. The plasmid pACCRT-EIB and pACCAR16∆crtX 8 was kindly provided by Norihiko Misawa professor,

73

Ishikawa Prefectural University, Japan.

74

Cloning of GGPS from D. bardawil. The total RNA was prepared from 8 mL of D. bardawil cells grown at

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the late log phase with Trizol reagent (Life Technology) according to the manufacture’s instruction. The reverse

76

transcription (RT) reaction was performed with the parameters set as 42°C for 60 min followed by 70°C for 5 min,

77

according to the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific). In order to clone the DbGGPS

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cDNA,

79

5’-DSCRATRWAVTYNGCHARVSC-3’) were designed on the basis of the two conserved amino acid regions

80

(upstream MRYSLLA and downstream AKYIGYR, Supplemental Figure S1) from the GGPS protein sequences

81

of several species (Dunaliella viridis, ADL16411.1; Chlamydomonas reinhardtii, XP_001703169.1; Volvox carteri

two

degenerated

primers

(5’-

ATGMGHTAYTCNBTDYTNGC-3’

4


and

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82

f. nagariensis, XP_002953468.1; Arabidopsis thaliana, NP_195399.1; Chrysanthemum boreale, AGU91431.1;

83

Nicotiana tabacum, ADD49734.1; Croton sublyratus, BAA86284.1; Adonis palaestina, AAV74395.1; Taxus

84

Canadensis, AAD16018.1; Cistus incanus subsp. Creticus, AAM21638.1), then the expressed sequence tag (EST)

85

of DbGGPS was acquired.

86

Then, based on the switching mechanism at 5’ end of the RNA transcript (SMART) technique, 5’-RACE

87

PCR was performed using a modified oligo (dT) primer, 5’ SMARTer II A Oligonucleotide primer and the

88

SMARTScribe™ Reverse Transcriptase (a variant of MMLV RT) (BD Clontech). On the basis of the obtained

89

EST of DbGGPS, two gene specific primers (5’-TCCTCTCCGTACACCTTGTGGTTGGTGG-3’ and 5’-

90

TGTCCCCTCCGACTAATTCACAGGCAGC-3’) were designed to carry out the 5’ RACE-PCR. The following

91

gradient PCR program was used: initial denaturalization at 95°C for 3 min, followed by 5 cycles of 94°C, 30 s,

92

72 °C,3 min; another 5 cycles of 94 °C, 30 s, 70 °C, 30 s, and 72°C, 3 min; a final 20 cycles of 94 °C, 30 s, 68 °C,

93

30 s, and 72 °C, 3 min; at last, 72 °C, 5 min. For nested PCR of 5’-RACE, the following PCR program was used:

94

initial denaturalization at 95°C for 3 min, followed by 25 cycles of 94°C, 30 s, 68 °C, 30s, and 72°C, 3 min; at last,

95

72 °C, 5 min.

96

3′-RACE was performed using the RNA PCR Kit (AMV) Ver.3.0 (TaKaRa) according to the protocol: RT

97

reaction was implemented to synthesize the first strand cDNA using Oligo dT-Adaptor Primer supplied; the first

98

PCR of 3′-RACE was primed by a specific upstream primer (5’-TGCGGACGACGTGACAGTGGA-3’) and

99

Oligo dT-Adaptor Primer supplied by the manufacturer. For nested PCR of 3’-RACE, the primers were used as

100

followed: another specific upstream primer (5’-CCTCGCGTTCCAGGTGGTTGA-3’) and Adaptor Primer

101

(5’-GTTTTCCCAGTCACGAC-3’). The PCR procedure was as the following: 1 cycle of 94 °C, 5 min; 30 cycles

102

of 94 °C, 30 s, annealing temperature, 30 s, and 72 °C, 1 min/kb.

103 104

All of the amplified fragments were cloned to pCR2.1 vector (Life Technology) and transformed into E. coli DH5α for multiplication, then sequenced before the further experiments.

105

Genomic DNA Isolation from D. bardawil and Manipulation. Genomic DNA extraction from D. bardawil

106

cells in the log or late log phase was performed according to the E.Z.N.A.HP Plant DNA Kit. Genome walking

107

was implemented with gene-specific primers to identify the genomic DNA of DbGGPS (as for the primers, data

108

not shown). The initial PCR was fulfilled using gene-specific primers complemented with 3’-UTR of DbGGPS

109

and genomic DNA as template. Subsequently, additional gene-specific primers were synthesized for genome

110

walking based on the initial PCR product. Genome walking was carried out by genome walking kit (TaKaRa). All 5



111

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the manipulations were on the basis of the user manual. Bioinformatics

112

analysis.

Sequence

analysis

was

performed

using

BLAST

software

113

(http://blast.ncbi.nlm.nih.gov/). Component analysis of ACL was calculated using DNAStar software 7.1.0.

114

Multiple alignments among similar enzymes were conducted using ClustalX 1.83. The open reading frame (ORF)

115

of DbGGPS was predicted by ORF Finder on NCBI (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). Physical and

116

chemical features of DbGGPS were analyzed by ProtParam tool (http://expasy.org/tools/protparam.html).

117

Subcellular localization was predicted by TargetP 1.1 Server (http://www.cbs.dtu.dk/services/TargetP/). Conserved

118

domains

119

(http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). Protein domains, families and functional sites were

120

predicted by Prosite (http://prosite.expasy.org/). The protein secondary structures were predicted by Phyre2

121

(http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index). The three-dimensional (3D) structure of DbGGPS

122

was

123

(http://bmm.cancerresearchuk.org/~3djigsaw/) and the results were visualized by RasMol software 2.7.2.1.1.

in

DbGGPS

automatically

were

detected

predicted

by

using

3D-JIGSAW

the

Conserved

Protein

Domains

Comparative

Search

Modeling

tool

Server

Construction of plasmid for DbGGPS expression in E. coli. The coding region of a cDNA of DbGGPS

124 125

was

amplified

by

PCR

using

specific

126

TCCGAATTCATGGCCGCCCATCAAATGCA-3')

127

GTGCTCGAGGTTTTGGCGGTAGCCAATGT -3') with XhoI site, respectively. The PCR product was purified,

128

digested with EcoRI and XhoI, and then ligated to expression vector pET32a, which was predigested with the

129

same restriction enzymes. The resulting recombinant plasmid, pET32a-DbGGPS was then sequenced to confirm

130

for the correction of the ORF. Subsequently, the expression plasmid pET32a-DbGGPS was transformed into the

131

host strain E. coli BL21 (DE3) for protein expression.

with

primers EcoRI

site

EcoRI-GGPS and

XhoI-GGPS

For Rev

(5'(5'-

132

A single colony of E. coli BL21 cells harboring the expression plasmid pET32a-DbGGPS was inoculated at

133

37°C in Luria-Bertani (LB) medium containing ampicillin (100 mg/L) and inoculated with shaking (200 rpm) at

134

37°C until the optical density OD600 reached about 0.6. Then, the protein expression was induced by addition of

135

IPTG (isopropyl-β-D-thiogalactoside) to a final concentration of 0.6 mM. The cultivation was continued for 4 h.

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The expression levels of the protein were assessed by analyzing total protein on SDS-PAGE followed by

137

Coomassie Brilliant Blue R250 staining. A single colony of E. coli strain BL21 cells harboring the expression

138

plasmid pET32a was used as control.

139

Functional complementation expression of DbGGPS in E. coli. The plasmid pACCRT-EIB contains the 6


Page 7 of 27


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gene cluster crtE, crtI and crtB, and the E. coli DH5α harboring the plasmid pACCRT-EIB can produce lycopene.

141

The plasmid pACCAR16∆crtX contains the gene cluster crtE, crtI, crtB and crtY, and the E. coli DH5α harboring

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the plasmid pACCAR16∆crtX can produce β-carotene.

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The plasmids pAC-IB∆crtE and pAC-IBY∆crtE, where the crtE encoding GGPS had been deleted, were

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constructed in this study and used as the control. The linearized pAC-IB∆crtE (or pAC-IBY∆crtE) was amplified

145

from pACCRT-EIB or pACCAR16∆crtX by using the primers 5’-TCGGGCTGTCCTTATAAACGGA-3’ and 5’-

146

TAAGGATGCTGCATGAGCCATTTC-3’. The plasmids pAC-IB∆crtE and pAC-IBY∆crtE were constructed by

147

incubation the linearized sequences with T4 DNA ligase (Takara) at 16°C for 30 min, respectively, and then

148

transformed in E. coli DH5α, respectively.

149

To test if DbGGPS encoded the anticipated functional protein, the plasmids pACCRT-DbGGPS-IB and

150

pACCRT-DbGGPS-IBY were constructed from the corresponding plasmids pACCRT-EIB and pACCAR16∆crtX,

151

where

152

(5’-ATAAGGACAGCCCGAATGGCCGCCCATCAAATGCAGC-3’

153

5’-CATGCAGCATCCTTACTAGTTTTGGCGGTAGCCAATG-3’) for amplifying the GGPS sequence was

154

designed with 15 bp extensions (5’) that are complementary to the ends of the linearized vector. The carotenoids

155

were extracted from the transformants mentioned above.

crtE

was

replaced

by

DbGGPS

using

In-Fusion

Enzyme

(Clontech).

The

primers and

156

Extraction of carotenoids from E. coli. For E. coli cells biomass Analysis, first E. coli cells (200 mL) were

157

cultivated in LB medium with shaking (200 rpm) at 37°C overnight. The absorbance values of the E. coli cultures

158

(using blank medium diluted into different proportion, 1:1, 1:2, 1:5，1:10, 1:15 ) were read at 600 nm in a

159

spectrophotometer, and a corresponding concentration of blank medium without E. coli was used as the control

160

sample. For E. coli biomass dry weight measurement, E. coli sample (100 mL) was harvested by centrifugation at

161

10,000 g for 2 min, washed twice with distilled water, and dried at 80°C to a constant weight (24 h). Then, the

162

relationship curve between E. coli OD600 and cell dry weight was obtained.

163

For extraction of carotenoids from E. coli, first E.coli samples (10 mL) were taken from each culture media

164

after mixing thoroughly. Cells were harvested by centrifugation at 12,000 g for 2 min. Then the cell pellets were

165

washed twice with distilled water, and mixed with 2 mL of acetone. The mixtures were shocked for 1 min and

166

incubated for 15 min at 55°C in the dark, then centrifuged for 20 min at 14,000 g to recover the supernatant with

167

the pigment until colorless. Acetone extracts were collected and subsequently filtered through a 0.2-µm

168

hydrophobic fluorophore membrane (Sigma-Aldrich). 7



169

Analysis of carotenoids. Carotenoid content was analyzed using the high-performance liquid

170

chromatography (HPLC) method. Lycopene (or β-carotene) was separated using an Agilent HPLC system

171

equipped with a Welch Ultimate XB-C18 column (250 mm×4.6 mm, 120 Å) with an isocratic solvent system

172

consisting of acetonitrile/methanol/ isopropanol (45: 10: 45) at a flow rate of 1.2 ml/min at a wavelength of 473

173

(or 453 nm). The standards used were lycopene (95%) and β-carotene (95%), which were obtained from Sigma.

174

Results

175

Isolation and Characterization of the DbGGPS cDNA. For the EST isolation of DbGGPS, according to the

176

multiple sequences alignment result (Supplemental Figure S1), a 768-bp fragment (Figure 2A) was isolated by

177

RT-PCR reaction, and BLAST results showed high homology to Dunaliella viridis GGPS mRNA (HM114366.1)

178

with 82% identity (632 identified nucleotides), which indicated the EST was from GGPS of D. bardawil.

179

Furthermore, RACE manipulations were undergone to obtain the 5′ and 3′ ends of DbGGPS cDNA. Two

180

fragments corresponding to the 5′ and 3′ ends with 556 bp (Figure 2B) and 804 bp (Figure 2C) in length were

181

recovered, respectively. Sequence assembly revealed a 1,814-bp cDNA of DbGGPS. ORF search found a

182

1,074-bp (Figure 2D) coding sequence (CDS) encoding 357 amino acids, which displayed 294 and 237 identical

183

amino acids (88 and 77 % homology, separately) with those of D. viridis and C. reinhardtii, respectively.

184

Analysis of the genomic structure of DbGGPS. The requried genomic sequence of DbGGPS was 5,636 bp.

185

It contained 4,765 bp coding region (from ATG to TAA) and 871 bp upstream of the coding region. Analysis of

186

the nucleotide sequence of the cloned genomic DNA fragment indicated that DbGGPS contained 10 exons

187

interrupted by 9 introns, which is much more complicated than other algal and plant GGPS genes (Figure 3). From

188

Figure 3, some algal GGPS genomic DNA were intron-rich (like GGPS from Auxenochlorella protothecoides,

189

Chlamydomonas reinhardtii and D. bardawil), while some were even no introns (like GGPS from Bathycoccus

190

prasinos and Ostreococcus tauri). And plant GGPS genomic DNA seemed to be intron-less or no introns. The

191

total length of the introns was 3,691 bp, about 3.44 fold of the total length of exons (1,074bp). The average

192

DbGGPS intron was about 410 bp in length. Each intron started with GT and ended with AG, belonging to the

193

most common style of exon.

194

Sequence Analysis. On the basis of the analysis of the nucleotide sequence, we demonstrated that the

195

DbGGPS full-length cDNA contained 1,814-bp nucleotides with a 1,074-bp-long putative ORF flanked by a

196

183-bp-long 5’ untranslated region upstream of the start codon and a 557-bp-long 3’ untranslated region after the

197

stop codon. The 1,074-bp putative ORF encoded a 357-amino-acid-long peptide. Analysis by ProtParam tool 8


Page 8 of 27

Page 9 of 27

198


revealed that the molecular weight of DbGGPS was 38.88 kDa, and the isoelectric point was 6.27.

199

The predicted amino acid sequence of DbGGPS has high similarity to other algal and plant GGPS (56~88 %).

200

Amino acid sequences of GGPSs from other algal and plant species were aligned according to the algorithm of the

201

ClustalW and the result was shown in Figure 4. Five domains (from I to V), found in the same relative locations as

202

described for prenyltransferases,20 were also identified in all the aligned sequences. Domains II and V contained

203

aspartate-rich motifs (ARMs, referred to as the first aspartate-rich motif (FARM, DD(X)4D) and the second

204

aspartate-rich motif (SARM, DD(X)2D)), which were proposed to be diphosphate-binding sites.20 Domain II

205

represented a longer region that contained highly conserved DD (Asp) and RR (Arg) dipeptides, DD(X)9RR

206

(Figure 4). The DDXXD motif in the domain V was the most conserved region and was proposed to be an allyl

207

isoprenoid binding site.21 Both DD(X)9RR and DDXXD motifs were important for the catalytic activity of

208

GGPS.21 In addition, two polyprenyl synthases signatures (at positions of 143~159 and 275~287 in DbGGPS

209

amino acid sequence) have been predicted by Prosite tool, and they were corresponding to ARMs. GGPS can be

210

classified into three types based on their amino acid sequences.11 Type I GGPS found in archaea have only two

211

amino-acids inserted in the FARM (DDXXD) and one aromatic amino acid residue at the fifth position upstream

212

from the FARM. Plant and eubacterial type II GGPS possess four amino-acids inserted in the FARM. However,

213

this type of GGPS lacks an aromatic amino-acid residue at the fifth position upstream from the FARM. Type-III

214

GGPS found in eukaryotes (except plants) contain two inserted amino-acids in the FARM and also lack an

215

aromatic amino-acid residue at the fifth position upstream from the FARM. Plant and algal GGPS belong to type

216

II GGPS as the FARM is DD(X)4D as shown in Figure 4.

217

Conserved domains analysis using NCBI Conserved Domain Database (Supplemental Figure S2) showed the

218

deduced protein DbGGPS was conserved in substrate binding sites, active sites, catalytic center, and two

219

aspartate-rich regions. However, there was little homology among the N-terminal of other GGPSs as these regions

220

designed plastidial transit peptides which amino acid sequences varied much.22 The predicted DbGGPS transit

221

peptide was identified by TargetP, showing that DbGGPS had a 48-amino acid transit peptide at its N-terminal to

222

target to the plastids.

223

Protein Structure Prediction. The protein secondary structure was predicted by Phyre2. As shown

224

(Supplemental Figure S3), there were 70% α-helix regions, no β-sheet regions, 19% disordered regions and 9%

225

transmembrane- (TM-) helix in DbGGPS. The schematic view of transmembrane protein structure prediction was

226

shown in Figure 5A. 1~40-amino acid signal peptide at the N-terminal of DbGGPS was predicted, which was very 9



227

close to the prediction of TargetP tool. Two TM-helix regions were at the positions 170~185 and 242~257 in

228

DbGGPS amino acid sequence, which were corresponding to two yellow arrows in the 3D-structure of DbGGPS

229

predicted by 3D-JIGSAW (Figure 5B).

230

Heterologous expression of DbGGPS in E. coli BL21 (DE3). In order to express DbGGPS, the gene was

231

cloned into the plasmid pET32a to construct pET32a-DbGGPS. Upon induction by IPTG, DbGGPS was expressed

232

as a major protein product in the total cellular protein (Figure 6). SDS-PAGE patterns of total cellular protein,

233

visualized by Coomassie Brilliant Blue R250 staining, showed the recombinant protein (pET32a-DbGGPPS)

234

expression was achieved at 4 h after IPTG induction. The molecular weight of the expressed recombinant protein

235

was estimated to be about 58 kDa fused with a ~20 kDa of two His·Tags, one Trx·Tag and one S·Tag. Therefore,

236

the size of expressed DbGGPS protein was predicted to be 38~39 kDa, which was in good agreement with that

237

predicted by bioinformatics method (38.88 kDa). It was also found that the recombinant protein was expressed in

238

insoluble fraction of the total bacterial cultures as inclusion body. The expression and purification of the

239

DbGGPPS protein would be a preparation for investigating its detailed function, and facilitate the future research

240

in D. bardawil.

241

Functional complementation of DbGGPS in the E. coli transformants. Functional activity of expressed

242

GGPS was investigated by genetic complementation with the carotenogenic crt gene cluster. Carotenoids are

243

produced in E. coli harbouring a crt cluster gene from E. uredovora. Replacements of a crt gene with an unknown

244

gene with the same activity, can be used to determine the function of the gene. Herein, the DbGGPS gene was

245

used to replace crtE to construct the plasmids pACCRT-DbGGPS-IB and pACCRT-DbGGPS-IBY, and the

246

plasmids pAC-IB∆crtE and pAC-IBY∆crtE were used as the control, respectively. All the plasmids were

247

transformed into E. coli DH5α, respectively. The characteristic of the plasmids and the transformants has been

248

listed in Table 1. The pink color of carotenoid was observed in the transformants E. coli DH5α/pACCRT-EIB and

249

E. coli DH5α/pACCRT-DbGGPS-IB, and the carotenoids of the transformants were lycopene (Figure 7A). And

250

the yellow color of carotenoid was observed in the transformants E. coli DH5α/ pACCAR16∆crtX and E. coli

251

DH5α/pACCRT-DbGGPS-IBY, and the carotenoid productions of the transformants were β-carotene (Figure 7B).

252

E. coli cells do not produce lycopene or β-carotene but can produce IPP and DMAPP, which are the essential

253

precursors of carotenoids.23 So the tiny pink color was seen in E. coli DH5α/pAC-IB∆crtE. But no carotenoids

254

could be detected in the transformants E. coli DH5α/pAC-IB∆crtE and E. coli DH5α/pAC-IBY∆crtE by HPLC

255

(Table 1, Figure 7C and 7D). All these results suggested that DbGGPS can substitute the crtE gene, and the coding 10


Page 10 of 27

Page 11 of 27


256

region of a cDNA of DbGGPS encodes a functional GGPS. However, the lycopene production in E. coli

257

DH5α/pACCRT-DbGGPS-IB was decreased by 77.86% of that in the E. coli DH5α/pACCRT-EIB (Table 1,

258

Figure 7C). And the β-carotene production in the E. coli DH5α/pACCRT-DbGGPS-IBY was decreased by 89.80%

259

of that in the E. coli DH5α/ pACCAR16∆crtX (Table 1, Figure 7D). These indicated that that the function of

260

DbGGPS expressed in E. coli was not effective as crtE from Erwinia uredovora.

261

Discussion

262

Traditionally, GGPS enzymatic activity can be detected in E. coli cell extracts or color complementation assay.2

263

The gene encoding GGPS was constructed into a prokaryotic expression vector, like pBluescrip II SK- 13, 24 and

264

pQE30

265

gene (encoding GGPS) was deleted, were used to co-transform into E. coli. And the E. coli transformant can produce

266

carotenoid. One example was that carotenoid production was observed in E. coli harboring pACCAR25∆crtE

267

from Erwinia uredovora and plasmid carrying C. forskohlii GGPS.24 In this study, just one plasmid carrying the

268

Erwinia uredovora crt gene cluster which crtE gene (encoding GGPS) was replaced with DbGGPS was used to

269

transform into E. coli. And the transformant still can produce corresponding carotenoids. Although we found that E.

270

coli harboring the plasmid with crtI, crtB, crtY (or no crtY) and DbGGPS can produce fewer corresponding

271

carotenoids than that harboring pACCRT-EIB and pACCAR16∆crtX, DbGGPS has been verified to encoded a

272

functional protein and played an important role in carotenoid pathway flux.

11

. Then the resulting plasmid and the plasmid carrying the Erwinia uredovora crt gene cluster which crtE

273

As for the reason why fewer carotenoids were produced in E. coli harboring the plasmid with crtI, crtB,

274

crtY (or no crtY) and DbGGPS, maybe the SD sequence and promotor of the gene crtE cannot identify the

275

expression of DbGGPS effectively. Codon optimization of DbGGPS or the eukaryotic expression system may

276

solve this problem. (Over)Expression of genes involved in carotenoid biosynthesis has been studied in detail in E.

277

coli.25-27 Recently, (over)expression of genes involved in carotenoid biosynthesis in yeast and algae have been

278

paid attention. It was reported that expression of the PSY from Dunaliella salina in Chlamydomonas reinhardtii

279

under the control of the RBCS2 and HSP70A promoters caused the stable production of the corresponding PSY

280

transcript and a significant increase in the content of carotenoids.28 As for the overexpression of GGPS, it seemed

281

to have a limitations of the carotenoid pathway.29 It was reported that maximum astaxanthin biosynthesis could

282

be achieved by an engineered Xanthophyllomyces dendrorhous overexpressed the enzyme GGPS in combination

283

with advantageous cultivation conditions like dim light illumination of and enhanced air supply.29

11



Page 12 of 27

284

On the other hand, as a very first step toward the genetic manipulation of terpenoid pathway, overexpression

285

of GGPS is rarely manipulated alone. Among the genes involved in the carotenoid biosynthetic pathway, GGPS is

286

a key gene that produces the general key precursor for carotenoid and a potential target for metabolic engineering.

287

Besides

288

including steroids, diterpenes and quinones (Figure 1).30 It was reported that the expressions of three key enzymes

289

of the general isoprenoid pathway, IPI, FPS and GGPS, an increase in the final carotenoid accumulation in Mucor

290

circinelloides.31 GGPS catalyses the ultimate non-carotenoid specific step in the pathway, participating not only in

291

the synthesis of the carotenoids, but also in that of the quinones and the prenyl groups of several proteins. It seems

292

more important for enhancement of the IPP and DMAP supply. So genetic manipulation of GGPS usually couple

293

with the enzyme IPI. The introduction of GGPS and IPI from Blakeslea trispora increased the β-carotene content

294

in E. coli from 0.5 to 0.95 mg/g dry weight.32 Another report was shown that genetic engineering of the complete

295

carotenoid pathway (expression of the genes of 3-hydroxymethyl-3-glutaryl coenzyme A reductase,

296

geranylgeranyl pyrophosphate synthase, phytoene synthase/lycopene cyclase, and astaxanthin synthase) obtained

297

an extremely high astaxanthin accumulation in Xanthophyllomyces dendrorhous. Maybe it could be also achieved

298

in algae like Chlamydomonas and Dunaliella.

carotenoids,

FPP

and

GGPP

are

essential

precursors

for

a

variety

of

products

299

In summary, we have successfully cloned the GGPS gene from the unicellular alga, Dunaliella bardawil.

300

GGPS catalyzed the production of GGPP, a key precursor for carotenoids biosynthesis. The successful isolation

301

and verified activity of the DbGGPS gene will enable us to regulate an important step involved in carotenoids

302

biosynthesis by genetic engineering in the future. And a new way by replacing the crt gene from Erwinia

303

uredovora in the plasmid (pACCRT-EIB or pACCAR16∆crtX or pACCAR25∆crtX) with an unknown gene was

304

provided to determine the function of the unknown gene involved in carotenoids biosynthesis.

305

Acknowledgements

306

This project was supported by the National Natural Foundation of China (31171631), Guangdong Province

307

Science and technology plan project (2011B031200005), and Guangdong Provincial Bureau of ocean and fishery

308

science and technology to promote a special (A201301C04).

309 310

Supplementary Material

311

Supplemental Figure S1 Multiple sequences alignment of GGPS from other algae and plants.

12


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312

Supplemental Figure S2 Conserved domains in DbGGPS and protein similarity detected by NCBI Blastp. There

313

was different homology among the N-terminal of other GGPSs (as shown in blue frame).

314

Supplemental Figure S3 The protein secondary structure of DbGGPS predicted by Phyre2.

315

These materials are available free of charge via the Internet at http://pubs.acs.org.

13



316

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317

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(3) Ye, Z.-W.; Jiang, J.-G.; Wu, G.-H., Biosynthesis and regulation of carotenoids in Dunaliella: progresses and prospects. Biotechnol. Adv. 2008, 26, 352-360. (4) Rohmer, M., The discovery of a mevalonate-independent pathway for isoprenoid biosynthesis in bacteria, algae and higher plants†. Nat. Prod. Rep. 1999, 16, 565-574. (5) Hirschberg, J., Carotenoid biosynthesis in flowering plants. Current opinion in plant biology 2001, 4, 210-218. (6) Chen, Y.; Li, F.; Wurtzel, E. T., Isolation and characterization of the Z-ISO gene encoding a missing component of carotenoid biosynthesis in plants. Plant Physiol. 2010, 153, 66-79.

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(10) Lin, J.; Jin, Y.; Zhou, X.; Wang, J., Molecular cloning and functional analysis of the gene encoding geranylgeranyl diphosphate synthase from Jatropha curcas. Afr. J. Biotechnol. 2010, 9, 3342-3351.

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in Cells Induced for Taxol Production. Arch. Biochem. Biophys. 1998, 360, 62-74.

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encoding geranylgeranyl diphosphate synthase from hazel (Corylus avellana L. Gasaway). Mol. Biol. Rep. 2010,

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(14) Sitthithaworn, W.; Kojima, N.; Viroonchatapan, E.; SUH, D.-Y.; Iwanami, N.; Hayashi, T.; Noji, M.;

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Saito, K.; Niwa, Y.; Sankawa, U., Geranylgeranyl diphosphate synthase from Scoparia dulcis and Croton

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sublyratus. Plastid localization and conversion to a farnesyl diphosphate synthase by mutagenesis. Chem. Pharm.

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Bull. 2001, 49, 197-202.

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(15) Ben‐Amotz, A.; Katz, A.; Avron, M., Accumulation of β-carotene in halotolerant alge: purification and

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characterization of β-carotene-rich globules from Dunaliella bardawil (chlorophyceae). J.Phycol. 1982, 18,

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529-537.

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(16) Lao, Y.-M.; Xiao, L.; Jiang, J.-G.; Zhou, S.-S., In silico analysis of phytoene synthase and its promoter

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reveals hints for regulation mechanisms of carotenogenesis in Duanliella bardawil. Bioinformatics 2011, 27,

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(17) Ye, Z.-W.; Liu, G.-N.; Jiang, J.-G., Structural and phylogenetic analysis of a novel ζ‐carotene

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desaturase from Dunaliella bardawil, a unicellular alga that accumulates large amounts of β-carotene. Limnol.

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Oceanogr. 2011, 56, 133-138.

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(18) Zhu, Y.-H.; Jiang, J.-G.; Yan, Y.; Chen, X.-W., Isolation and characterization of phytoene desaturase

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cDNA involved in the β-carotene biosynthetic pathway in Dunaliella salina. J. Agric. Food Chem. 2005, 53,

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5593-5597.

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(19) Ben-Amotz, A.; Avron, M., The biotechnology of cultivating the halotolerant alga Dunaliella. Trends Biotechnol. 1990, 8, 121-126. (20) Sandmann, G., Carotenoid biosynthesis in microorganisms and plants. In EJB Reviews 1994, Springer: 1995; pp 129-146.

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(21) Carattoli, A.; Romano, N.; Ballario, P.; Morelli, G.; Macino, G., The Neurospora crassa carotenoid

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biosynthetic gene (albino 3) reveals highly conserved regions among prenyltransferases. J. Biol. Chem. 1991, 266,

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(22) Liao, Z.; Chen, M.; Gong, Y.; Guo, L.; Tan, Q.; Feng, X.; Sun, X.; Tan, F.; Tang, K., A new

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geranylgeranyl diphosphate synthase gene from Ginkgo biloba, which intermediates the biosynthesis of the key 15



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precursor for ginkgolides. Mitochondrial DNA 2004, 15, 153-158. (23) Rohmer, M.; Knani, M.; Simonin, P.; Sutter, B.; Sahm, H., Isoprenoid biosynthesis in bacteria: a novel pathway for the early steps leading to isopentenyl diphosphate. Biochem. J 1993, 295, 517-524. (24) Engprasert, S.; Taura, F.; Kawamukai, M.; Shoyama, Y., Molecular cloning and functional expression of geranylgeranyl pyrophosphate synthase from Coleus forskohlii Briq. BMC Plant Biol. 2004, 4, 18.

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(25) Albrecht, M.; Misawa, N.; Sandmann, G., Metabolic engineering of the terpenoid biosynthetic pathway

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of Escherichia coli for production of the carotenoids β-carotene and zeaxanthin. Biotechnol. Lett. 1999, 21,

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791-795.

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(26) Yang, J.; Guo, L., Biosynthesis of β-carotene in engineered E. coli using the MEP and MVA pathways. Microb. Cell Fact. 2014, 13, 160. (27) Li, X.-R.; Tian, G.-Q.; Shen, H.-J.; Liu, J.-Z., Metabolic engineering of Escherichia coli to produce zeaxanthin. J. Ind. Microbiol. Biotechnol. 2014, 1-10.

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(28) Couso, I.; Vila, M.; Rodriguez, H.; Vargas, M.; León, R., Overexpression of an exogenous phytoene

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synthase gene in the unicellular alga Chlamydomonas reinhardtii leads to an increase in the content of carotenoids.

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Biotechnol. Prog. 2011, 27, 54-60.

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(29) Breitenbach, J.; Visser, H.; Verdoes, J. C.; van Ooyen, A. J.; Sandmann, G., Engineering of

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geranylgeranyl pyrophosphate synthase levels and physiological conditions for enhanced carotenoid and

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astaxanthin synthesis in Xanthophyllomyces dendrorhous. Biotechnol. Lett. 2011, 33, 755-761.

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(30) Hemmi, H.; Noike, M.; Nakayama, T.; Nishino, T., An alternative mechanism of product chain‐length determination in type III geranylgeranyl diphosphate synthase. Eur. J. Biochem. 2003, 270, 2186-2194.

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(31) Csernetics, Á.; Nagy, G.; Iturriaga, E. A.; Szekeres, A.; Eslava, A. P.; Vágvölgyi, C.; Papp, T.,

395

Expression of three isoprenoid biosynthesis genes and their effects on the carotenoid production of the

396

zygomycete Mucor circinelloides. Fungal Genet.Biol. 2011, 48, 696-703.

397

(32) Sun, J.; Sun, X.-X.; Tang, P.-W.; Yuan, Q.-P., Molecular cloning and functional expression of two key

398

carotene synthetic genes derived from Blakeslea trispora into E. coli for increased β-carotene production.

399

Biotechnol. Lett. 2012, 34, 2077-2082.

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400


FIGURE CAPTION

401 402

Figure 1 Isoprenoid biosynthetic pathway in the chloroplasts of plants and algae. IPP and DMAPP are synthesized

403

using MVA and MEP pathways. MEP, 2-C-methyl-D-erythritol-4-phosphate; IPP, isopentenyl pyrophosphate;

404

DMAPP, dimethylallyl pyrophosphate; GPP, geranyl diphosphate; FPP, farnesyl pyrophosphate; GGPP,

405

geranylgeranyl diphosphate; IPI, isopentenyl pyrophosphate isomerase; GGPS, geranylgeranyl diphosphate

406

synthase; PSY, phytoene synthase; PDS, phytoene desaturases; ZDS, ζ-carotene desaturases; ZISO,

407

15-cis-ζ-carotene isomerase; CRTISO, carotenoid isomerase; LycB, lycopene β-cyclase. CrtB, crtE, crtI and crtY

408

are encoding the corresponding genes in bacteria.

409 410

Figure 2 Isolation of DbGGPS cDNA. A. DbGGPS EST; B. 5’-end of the DbGGPS cDNA isolated by 5’ RACE;

411

C. 3′-end of the DbGGPS cDNA isolated by 3’ RACE; D. Full-length DbGGPS ORF. Lane M, DNA ladder; lane 1,

412

the first PCR products; lane 2, the second PCR products.

413 414

Figure 3 Distribution of exons and introns in the genomic DNA of GGPS. The sequences are used for analysis as

415

followed: GGPS from microalgae, DbGGPS (in this study); ApGGPS, Auxenochlorella protothecoides

416

(NW_011934251.1); BpGGPS, Bathycoccus prasinos (NC_024002.1) ; CrGGPS, Chlamydomonas reinhardtii

417

(NW_001843980.1); OtGGPS, Ostreococcus tauri (NC_014437.1); GGPS from plants, AtGGPS, Arabidopsis

418

thaliana (CP002687.1); MtGGPS, Medicago truncatula, (AC137839.19); PtGGPS, Populus trichocarpa

419

(NC_008475.2).

420 421

Figure 4 Alignment of amino acid sequences of DbGGPS and other algal and plant GGPSs. The following

422

sequences were used for comparison: DbGGPS, GGPS from D. bardawil in this study; DvGGPS, D. viridis

423

(HM114366.1); CrGGPS, Chlamydomonas reinhardtii (XP_001703169.1); VcGGPS, Volvox carteri f. nagariensis

424

(XM_002953422.1); AtGGPS, Arabidopsis thaliana (NM_119845.3); ApGGPS, Adonis palaestina (AY661706.1);

425

CsGGPS, Croton sublyratus (AB034249.1); CiGGPS, Cistus incanus (AF492022.1); CbGGPS, Chrysanthemum

426

boreale (KC202428.1); NtGGPS, Nicotiana tabacum (GQ911583.1); TcGGPS, Taxus canadensis (AF081514.1).

427

The conserved domains were boxed in black and numbered (I, II, III, IV and V). The highly conserved ARM in

428

the II and V domains of the GGPSs were boxed in red. 17



429 430

Figure 5 Schematic representations of transmembrane protein structure prediction and 3D-model of the DbGGPS.

431

A. transmembrane protein structure prediction of DbGGPS; B. 3D-model of the DbGGPS. Comparative modeling

432

was performed using 3D-JIGSAW basing on homologues of known structures automatically. The structures were

433

visualized using RasMol version 2.7.2.1.1. The α-helix region of the putative protein is indicated with pink

434

ribbons. Two TM-helix regions were indicated with yellow arrows. The loop regions are also indicated in the

435

schematics.

436 437

Figure 6 Expression of DbGGPS in pET32a vector analyzed by SDS-PAGE. M, molecular mass standards; lane 1,

438

control (E. coli transformed with pET32a); lane 2, total protein extract of E. coli transformed with pET32a-GGPS

439

after 4 hours of IPTG induction; lane 3, soluble cytoplasmic fraction of E. coli transformed with pET32a-GGPS

440

treated with IPTG; and lane 4, insoluble fraction of E. coli transformed with pET32a-GGPS treated with IPTG.

441 442

Figure 7 Carotenoid production of E. coli harboring different plasmids. A. Lycopene production of E. coli

443

harboring different plasmids: 1, pAC-IB∆crtE; 2, pACCRT-EIB; 3, pACCRT-DbGGPS-IB; B. β-Carotene

444

production of E. coli harboring different plasmids: 4, pAC-IBY∆crtE; 5, pACCAR16∆crtX; 6,

445

pACCRT-DbGGPS-IBY; C. E.coli cell extracts of lycopene production were analyzed by HPLC at a wavelength

446

of 473 nm. Peak 1: lycopene; D. E.coli cell extracts of β-carotene production were analyzed by HPLC at a

447

wavelength of 453 nm. Peak 2: β-carotene; E. The absorption spectrum of lycopene. The absorbance maximum of

448

lycopene is at a wavelength of 473 nm; F. The absorption spectrum of β-carotene. The absorbance maximum of

449

β-carotene is at a wavelength of 453 nm.

450

18


Page 18 of 27

Page 19 of 27


Table 1. The characteristic of the plasmids and the transformants.

Plasmids

Genes

Strain colors

HPLC results

Carotenoid productions (µg/g cell dry weight)

pACCRT-EIB

crtE, crtI, crtB

Pink

Lycopene

2385±130

pAC-IB∆crtE

crtI, crtB

Nearly no pink

Not detected

—

pACCRT-DbGGPS-IB

DbGGPS, crtI, crtB

Light pink

Lycopene

528±36

pACCAR16∆crtX

crtE, crtI, crtB, crtY

Yellow

β-Carotene

1049±73

pAC-IBY∆crtE

crtI, crtB, crtY

Nearly white

Not detected

—

pACCRT-DbGGPS-IBY

DbGGPS, crtI, crtB, crtY

Pale yellow

β-Carotene

107±17

19



Figure 1

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Figure 2

21



Figure 3

22


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Figure 4

23



Figure 5

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Figure 6

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Figure 7

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