Improvement of Squalene Production from CO2 in Synechococcus

Apr 3, 2017 - (13, 19) Peptide sequences for the linkers: SF, short flexible linker (4 aa), ... since expression of the fusion protein(12) of IDI and ...
0 downloads 0 Views 2MB Size
Subscriber access provided by University of Newcastle, Australia

Letter 2

Improvement of squalene production from CO in Synechococcus elongatus PCC 7942 by metabolic engineering and scalable production in a photobioreactor Sun Young Choi, Jin-Young Wang, Ho Seok Kwak, Sun-Mi Lee, Youngsoon Um, Yunje Kim, Sang Jun Sim, Jong-il Choi, and Han Min Woo ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.7b00083 • Publication Date (Web): 03 Apr 2017 Downloaded from http://pubs.acs.org on April 4, 2017

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 free 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 accessible to all readers and 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.

ACS Synthetic Biology 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 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

1

Letter

2

Improvement of squalene production from CO2 in Synechococcus elongatus

3

PCC 7942 by metabolic engineering and scalable production in a

4

photobioreactor

5 6

Sun Young Choi1,2, Jin-Young Wang1, Ho Seok Kwak3, Sun-Mi Lee1,2, Youngsoon Um1,

7

Yunje Kim1, Sang Jun Sim2,3, Jong-il Choi4, Han Min Woo*,5

8 9

1

Clean Energy Research Center, Korea Institute of Science and Technology, Hwarangro 14-

10

gil 5, Seongbuk-gu, Seoul 02792, Republic of Korea

11

2

12

and Biological Engineering, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul 02841,

13

Republic of Korea

14

3

15

Seongbuk-gu, Seoul 02841, Republic of Korea

16

4

17

Yongbong-ro, Buk-gu, Gwangju 61186, Republic of Korea

18

5

19

Seobu-ro, Jangan-gu, Suwon 16419, Republic of Korea

Green School (Graduate School of Energy and Environment) and 3Department of Chemical

Department of Chemical and Biological Engineering, Korea University, 145 Anam-ro,

Department of Biotechnology and Bioengineering, Chonnam National University, 77

Department of Food Science and Biotechnology, Sungkyunkwan University (SKKU), 2066

20 21

*Corresponding author: E-mail address: [email protected]. (H.M. Woo). Phone: +82 31 290

22

7808

1 ACS Paragon Plus Environment

ACS Synthetic Biology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

23

ABSTRACT

24

The push-and-pull strategy for metabolic engineering was successfully demonstrated in

25

Synechococcus elongatus PCC 7942, a model photosynthetic bacterium, to produce squalene

26

from CO2. Squalene synthase (SQS) was fused to either a key enzyme (farnesyl diphosphate

27

synthase) of the methylerythritol phosphate pathway or the β-subunit of phycocyanin

28

(CpcB1). Engineered cyanobacteria with expression of a fusion CpcB1-SQS protein showed

29

a squalene production level (7.16 ± 0.05 mg/L/OD730) that was increased by 1.8-fold

30

compared to that of the control strain expressing SQS alone. To increase squalene production

31

further, the gene dosage for CpcB1·SQS protein expression was increased and the fusion

32

protein was expressed under a strong promoter, yielding 11.98 ± 0.49 mg/L/OD730 of

33

squalene, representing a 3.1-fold increase compared to the control. Subsequently, the best

34

squalene producer was cultivated in a scalable photobioreactor (6 L) with light optimization,

35

which produced 7.08 ± 0.5 mg/L/OD730 squalene (equivalent to 79.2 mg per g dry cell

36

weight). Further optimization for photo-bioprocessing and strain development will promote

37

the construction of a solar-to-chemical platform.

38 39 40 41

KEYWORDS: Synechococcus elongatus PCC 7942, protein engineering, metabolic

42

engineering, squalene, CO2 conversion, scalable production

43

2 ACS Paragon Plus Environment

Page 2 of 20

Page 3 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

44

ACS Synthetic Biology

A Graphical Table of Contents entry

45

3 ACS Paragon Plus Environment

ACS Synthetic Biology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

46

Squalene (C30H50)1, a triterpene intermediate for sterol and hopanoid biosynthesis, is widely

47

used in the food, personal care, and medical industries because of its various beneficial

48

functions.2,3 Since the feedstock supply for squalene production is limited and unstable

49

because of animal protection policies on the use of shark liver oil, and regional and seasonal

50

variations of plant oils, synthetic squalene has also been commercialized by the biotech

51

company Amyris, Inc. using an engineered yeast strain that is capable of converting sugar-

52

based carbon sources to squalene.4 Recently, the metabolic engineering of cyanobacteria has

53

shown the potential to overcome concerns related to greenhouse gas emissions and address

54

the demand of sustainable renewable energy and chemicals.5,6 Thus, the sustainable

55

production of squalene from CO2 is another promising platform that could lower the

56

production costs and provide a continuous supply from CO2. However, the key to engineering

57

a biosolar cell factory for the conversion of CO2 to squalene relies on achieving a high-level

58

conversion yield and productivity.

59

The push-and-pull strategy of metabolic engineering has been successfully applied for

60

the production of isoprenoids7,8 and lipids9 in microbial hosts by supplying key intermediates

61

and increasing the rate of formation of the final product. In cyanobacteria, expression of the

62

fusion protein between the β-subunit of phycocyanin (CpcB) and phellandrene synthase

63

(PHLS) has resolved the limitation of the β-phellandrene formation rate by increasing the

64

fusion protein levels up to 20% of total protein.10 Subsequently, the β-phellandrene-

65

producing strains were engineered to co-express mevalonate pathway enzymes to supply

66

isopentenyl-diphosphate (IPP)/dimethylallyl-diphosphate (DMAPP) in Synechocystis sp.

67

PCC 6803, producing 12.4 mg/g dry cell weight β-phellandrene.11 Similarly, the combined

68

overexpression of 1-deoxy-D-xylulose-5-phosphate synthase (Dxs) and 4-hydroxy-3-

69

methylbut-2-enyl-diphosphate synthase (IspG) of Thermosynechococcus elongatus for 4 ACS Paragon Plus Environment

Page 4 of 20

Page 5 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

70

optimization of the methylerythritol phosphate (MEP) pathway with overexpression of the

71

fused enzyme of Saccharomyces cerevisiae IPP isomerase (IDI) with isoprene synthase (IspS)

72

increased the DMAPP/IPP ratio as well as the rate of IPP conversion to isoprene, yielding

73

1.26 g/L isoprene from CO2 in Synechococcus elongatus PCC 7942.12

74

The production of squalene from CO2 in cyanobacteria was achieved through the

75

metabolic engineering of S. elongatus PCC 7942 by overexpressing the MEP pathway genes

76

(dxs and idi) and expressing a key gene (ispA, encoding farnesyl diphosphate synthase [IspA])

77

in the OverMEP module, and by expressing the truncated squalene synthase (SQS) gene in

78

the SQS (or terpene synthase [TPS]) module, yielding strain SeSC33S (Fig. 1A).13 To

79

complete the push-and-pull strategy of metabolic engineering, we adopted a protein

80

engineering strategy to construct a fusion protein of SQS for improvement of squalene

81

production, based on the strain SeHL33 (a parental strain) using only the OverMEP module

82

(Table 1).

83

For squalene strain development, two fusion protein partners and three different flexible

84

linkers were selected for the protein engineering of SQS. First, a key enzyme of the MEP

85

pathway (IspA) was fused to the N-terminal of SQS via the peptide linkers (short linker [SF],

86

long linker [LF], and long linker 2 [LF2]), since expression of the fusion protein12 of IDI and

87

IspS via linkers was shown to improve isoprene production via artificial substrate channeling.

88

Then, the fused SQS genes were inserted into SyneBrick vectors14 and the plasmids were

89

transformed in strain SeHL33, yielding strains SeSC34S, SeSC35S, and SeSC36S.

90

Confirmation of the transformants was established by polymerase chain reaction (PCR) (Fig.

91

1A) using genomic DNAs and oligonucleotide primers (Table S2). As a result, the

92

recombinant strains with the IspA-SQS fusion protein showed several growth defects

93

compared to the control strain SeSC33S (with sole expression of SQS) (Fig. 1B). With

94

respect to squalene production, strain SeSC34S showed higher squalene contents (5.71 ± 0.17 5 ACS Paragon Plus Environment

ACS Synthetic Biology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

95

mg/L/OD730) than SeSC33S (3.77 ± 0.09 mg/L/OD730). However, lower production levels

96

were observed given that longer flexible linkers were used for establishing the fusion protein.

97

The squalene production level in strain SeSC36S was even lower than that of the control. In

98

the case of the strain expressing the fusion protein of IDI-IspS12, no differences in isoprene

99

production and growth rate were observed, regardless of the length and composition of the

100

linkers (flexible or helical linkers). Thus, expression of the IspA-SQS fusion protein may not

101

have a negative influence on the growth rate or squalene contents in the cell. However, the

102

structure of the IspA-SQS fusion protein due to the linkers used could influence the cellular

103

activities given that the growth rate and squalene content were lower when a longer linker

104

was used. A potential reason for this growth inhibition could be the accumulation of toxic

105

farnesyl diphosphate (FPP) in association with the expression of IspA-SQS. Previously, the

106

strain SeHL33 with the OverMEP module (expression of the dxs, idi, and ispA genes) alone

107

also exhibited growth inhibition, and the growth rate was relieved when TPS was used to pull

108

the accumulated FPP.13 In the present study, additional IspA activity from IspA-SQS

109

expression could have increased the local concentrations of toxic FPP, which would inhibit

110

cellular growth and reduce the squalene contents. Thus, the pull-and-push strategy for

111

squalene production with expression of a direct fusion protein of IspA-SQS was not

112

successful in this case.

113

High-level expression of CpcB-PHLS has previously been achieved, which was reported

114

to lead to 100-fold yield improvement.10 Thus, native CpcB1 (a homologous protein of CpcB

115

of Synechocystis sp. PCC 6803) was fused to the N-terminal of SQS via the same peptide

116

linkers described above (SF, LF, LF2). The cpcB1 gene, encoding the β-subunit of

117

phycocyanin as an essential antenna for light harvesting, is highly expressed under the

118

activity of the strong endogenous cpcB1 promoter in S. elongatus PCC 7942.10 Thus, the trc

119

promoter of the SyneBrick vector was replaced with the strong endogenous cpcB1 promoter 6 ACS Paragon Plus Environment

Page 6 of 20

Page 7 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

120

to express the CpcB1-SQS fusion protein. As a result, three different recombinant strains

121

were constructed (SeSC37S, SeSC38S, and SeSC39S) (Fig. 1A). Unlike fusion IspA-SQS

122

proteins, CpcB1-SQS fusion proteins did not cause severe growth defects. SeSC37S showed

123

the least amount of growth defects compared to the control strain SeSC33S. Compared to the

124

squalene production levels of SeSC33S (3.77 ± 0.09 mg/L/OD730), the SeSC375, SeSC386,

125

and SeSC39S strains expressing the CpcB1-SQS fusion protein (SeSC37S, 7.15 ± 0.05

126

mg/L/OD730; SeSC38S, 6.23 ± 0.16 mg/L/OD730; SeSC39S, 6.93 ± 0.31 mg/L/OD730) showed

127

increased squalene production levels by 1.8-fold, 1.65-fold, and 1.8-fold, respectively (Fig.

128

1B). There were no significant differences between the production levels, regardless of the

129

length of the flexible linkers. Thus, SeSC37S with a fusion CpcB1-SF-SQS protein (7.16 ±

130

0.05 mg/L/OD730) was chosen for further improvement of squalene production.

131

Increasing the gene dosage of TPS that yields the final product and expressing it under a

132

strong constitutive promoter has been shown to help enhance production levels.9,15,16 Thus,

133

engineered strains (SeSC33SII, SeSC34SII, and SeSC35SII) with two-copy insertions of the

134

SQS gene on the chromosome were constructed to investigate whether or not additional gene

135

copies and replacement of the strong promoter could enhance the level of squalene

136

production from CO2 (Fig. 2A). The squalene content of strain SeSC33SII harboring two

137

gene copies (7.22 ± 0.17 mg/L/OD730) was 1.9-fold higher than that of the control strain

138

SeSC33S harboring one gene copy (3.77 ± 0.09 mg/L/OD730) (Fig. 2B). When the SQS gene

139

integrated at neutral site II (NSII) was replaced with the gene encoding a CpcB1-SF-SQS

140

fusion protein, the production level of SeSC34SII was also increased by 2.3-fold (8.94 ± 0.46

141

mg/L/OD730) compared to that of SeSC33S. Finally, the duplicated SQS gene strain with a

142

fusion protein (SeSC35SII) resulted in a squalene content of 11.98 ± 0.49 mg/L/OD730, which

143

represents an increase of 3.1-fold or 1.7-fold compared to the levels produced in strain

144

SeSC33S or SeSC37S (6.94 ± 0.31 mg/L/OD730), respectively. Therefore, both strategies of 7 ACS Paragon Plus Environment

ACS Synthetic Biology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

145

increasing gene dosage and replacement of a strong promoter resulted in improvement of

146

squalene production from CO2, which is consistent with the previous result of the high-level

147

expression of the fusion CpcB-PHLS protein in Synechocystis sp. PCC 6803.10 High-level

148

expression of the CpcB1-SF-SQS fusion protein could lead to high SQS activity for the

149

formation of squalene. To date, the SeSC35SII strain has shown the highest production level

150

of squalene from CO2 (11.98 ± 0.49 mg/L/OD730). Thus, the push-and-pull strategy with

151

protein engineering was successful to construct squalene-producing cyanobacteria.

152

For the scalable production of squalene, a 6-L CO2-fed bag-type photobioreactor17 that

153

has been optimized for scale-up production (6 L×n) was used, and the strains SeSC35SII and

154

SeSC33S (as a control) were cultivated with 5% CO2 under 100 µmol photons·m-2·s-1 (Fig.

155

3A). As a result, both recombinant strains showed similar growth rates, although they

156

exhibited slower growth than the wild-type. After isopropyl-β-D-1-thiogalactopyranoside

157

(IPTG) induction at Day 3, both strains produced squalene at similar levels at Day 8. The

158

final squalene content in SeSC35SII (5.60 ± 0.54 mg/L/OD730; equivalent to 54.3 mg

159

squalene per g dry cell weight) was higher than that in SeSC33S (4.01 ± 0.46 mg/L/OD730;

160

equivalent to 34.7 mg squalene per g dry cell weight) (Fig. 3B). To improve the production

161

levels for scalable production, the light intensity was increased to 200 µmol photons·m-2·s-1

162

to achieve better photosynthetic efficiency in the recombinant cyanobacteria. Both squalene-

163

producing strains and the wild-type showed better cell growth than obtained under the

164

previous culture condition. In addition, the strains SeSC35SII and SeSC33S produced 7.08 ±

165

0.5 mg/L/OD730 (equivalent to 79.2 mg per g dry cell weight) and 4.79 ± 0.46 mg/L/OD730

166

(equivalent to 57.5 mg per g dry cell weight) of squalene, respectively (Fig. 3C). Although

167

the squalene content in the scalable photobioreactor was a bit lower than that in the test bottle

168

culture, this is the first report to demonstrate the scale-up production of recombinant

169

cyanobacteria to produce squalene from CO2 as a sole carbon source. Nevertheless, many 8 ACS Paragon Plus Environment

Page 8 of 20

Page 9 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

170

factors still need to be considered for realizing the economically feasible mass production of

171

squalene, including potential contamination18 in the absence of antibiotics and optimizing the

172

photobioreactor operation and downstream processing conditions.

173

In summary, the push-and-pull strategy of metabolic engineering for cyanobacteria was

174

successfully applied for improvement of squalene production from CO2. Combined with

175

MEP pathway engineering, high-level protein expression of a CpcB1-SF-SQS fusion protein

176

led to improvement in squalene production by 2.3-fold. Subsequently, the engineered

177

cyanobacteria were cultivated in a scalable photobioreactor for mass production. Further

178

development of the photo-bioprocessing conditions with strain improvement will promote

179

establishment of an engineered biosolar cell factory for industrial-scale CO2 conversion.

180 181

METHODS

182

Chemicals and reagents. All chemicals were purchased from Sigma-Aldrich (St. Louis, St.

183

Louis, MO, USA) unless otherwise specified. Restriction enzymes, Phusion DNA polymerase,

184

and ligases were purchased from Fermentas (Thermo Fisher Scientific Inc., Waltham, MA,

185

USA).

186 187

Plasmid and strain construction. The plasmids and strains used in this study are listed in

188

Table 1. For fusion protein construction, SQS13,19 was modified to fuse with a protein partner

189

(E. coli IspA13 or CpcB1) and a linker. The cpcB1 operon (the upstream 500 bp of SYNPCC

190

7942_1047) and the cpcB1 (SYNPCC 7942_1047)-encoding DNA fragment were obtained

191

by PCR with the genomic DNA of S. elongatus PCC 7942. Three different linkers were used:

192

SF (amino acid sequence GGGS), LF (GSGGGGS), and LF2 [LS(GGGGS)4AAA].12,20 The

193

DNA fragment containing either the ispA gene-linker (SF) or the cpcB1 gene-linker (SF) was

194

inserted into the pSe2Bb1k-SQS plasmid (targeting NSII), yielding pSe2Bb1k-ispA-SF-SQS 9 ACS Paragon Plus Environment

ACS Synthetic Biology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

195

or pSe2k-cpcB1-SF-SQS, respectively. Similarly, each LF or LF2 linker was used to

196

construct the fusion SQS plasmid (Table 1). The plasmids were constructed using the Gibson

197

assembly cloning kit (E5510s, BioLabs, New England) with a pair of oligonucleotides (Table

198

S1) and pSe1Bb1k-SQS. DNA sequences of the constructed plasmids were correctly verified

199

by DNA sequencing. For the two-copy SQS gene expression, pSe3Bb1c-SQS and pSe3c-

200

cpcB1-SF-SQS were constructed from pSe3Bb1c-eyfp, targeting NSIII.

201

Transformation of S. elongatus PCC 7942 (SeHL3313, used as a parental strain for this

202

work, and SeSC33S13 as the control) with the plasmid was performed as described

203

previously21. In brief, the cyanobacterial strains were transformed by incubating cells at a

204

mid-log phase (OD730 of 1–16) with 100 ng of plasmid DNA for 24 h in the dark. The mixed

205

culture was then spread on BG-11 plates supplemented with appropriate antibiotics for

206

selection of successful recombination. The strains were confirmed by genomic DNA PCR to

207

verify chromosomal integration of the target DNAs into either the NSI, NSII, and/or NSIII

208

sites (Fig. 1 and Fig. 2), and the DNA sequences were also correctly verified using a pair of

209

oligonucleotides (Table S2). Recombinant strains are listed in Table 1 along with their

210

genotypes.

211 212

Cyanobacteria growth condition. For the production of squalene, S. elongatus PCC 7942

213

and its derivatives were cultivated at 30°C in 100-mL cultures under continuous fluorescent

214

light (100 µmol photons·m-2·s-1) in BG-11 medium supplemented with 10 mM MOPS (pH

215

8.0).13,22,23 CO2 gas (5% v/v) was supplied at constant flow rate of 10 mL/min into the

216

medium. Spectinomycin (10 µg/mL), kanamycin (5 µg/mL), and/or chloramphenicol 3

217

(µg/mL) was added to the BG-11 medium for selection pressure where appropriate. For the

218

induction, 1 mM IPTG was added to the culture medium at an OD730 of 1 after inoculation.

219

No growth inhibition has been reported with 1 mM IPTG.14 10 ACS Paragon Plus Environment

Page 10 of 20

Page 11 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

220 221

Gas chromatography-mass spectrometry (GC-MS) analysis for squalene quantification.

222

For the quantification of squalene, the extraction method was performed as described

223

previously.13 In brief, 50 mL of the culture was used for extraction. After centrifugation at

224

3000 ×g for 10 min, cell pellets were re-suspended with 2 mL of a mixture of chloroform and

225

methanol (1:2 ratio), and liquid-liquid extraction was conducted for 30 min at room

226

temperature. After additional centrifugation (16,000 ×g for 3 min), the supernatants (200 µL)

227

were collected and the samples supplemented with 20 µg/mL of 1-phenyloctadecane as an

228

internal standard were analyzed using GC-MS on a GC-MSD Agilent Technologies (Santa

229

Clara, CA, USA) system equipped with an Ultra-2 capillary column (31 m × 0.2 mm, film

230

thickness 0.11 mm; Agilent Technologies) under the following conditions: carrier gas, He

231

(0.8 mL/min); oven temperature: 120–290°C (increase rate 6°C/min); run time 24.33 min.

232

The electron ionization mass spectrometer was operated under the following conditions: scan

233

mode; electron energy, 70 eV. The electron ionization mode with ion-selected monitoring (69

234

m/z, 93 m/z) range was 35–50 m/z.

235 236

Squalene production in a scalable photobioreactor. For the large-scale culture, a

237

polypropylene-based cast polypropylene film scalable photobioreactor17 (10 cm in diameter

238

and 1.2 m in height; house-manufactured products; maximum 6 L) was used for the

239

production of squalene. The photobioreactor was disinfected by ultraviolet exposure for 24 h

240

until use. The exponentially growing cyanobacterial cells were inoculated into 4 L of the BG-

241

11 medium to start the culture at an OD730 of 0.5 at 20°C. The vessel in the form of the

242

photoreactor was set under the condition of continuous light (100 or 200 µmol photons·m-2·s-

243

1

) measured by a photometer (LI-250A, LI-COR Bioscience, USA) and 25°C. CO2 (5% v/v)

11 ACS Paragon Plus Environment

ACS Synthetic Biology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

244

was pumped into the cell medium at a flow rate of 160 mL/min. For induction of the

245

engineered strain, 0.5 mM IPTG was added to the culture medium at an OD730 of 1.

246 247

ASSOCIATED CONTENT

248

Supporting Information

249

The Supporting Information is available free of charge on the ACS Publications website at

250

DOI:

251 252

AUTHOR INFORMATION

253

Corresponding author

254

*Phone: +82-31-290-7808. E-mail: [email protected].

255 256

Notes

257

The authors declare no competing financial interest.

258 259

ACKNOWLEDGMENTS

260

This work was supported by Korea CCS R&D Center (KCRC) (2014M1A8A1049277) and

261

the National Research Foundation of Korea (2017R1A2B2002566) funded by the Korean

262

Government (2016, University-Institute Cooperation program). This work was also partially

263

supported by a Golden Seed Project (213008-05-1-WT911) grant funded by the Ministry of

264

Agriculture, Ministry of Oceans and Fisheries.

12 ACS Paragon Plus Environment

Page 12 of 20

Page 13 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

REFERENCES (1) Thimmappa, R., Geisler, K., Louveau, T., O'Maille, P., and Osbourn, A. (2014) Triterpene biosynthesis in plants, Annu. Rev. Plant Biol. 65, 225-257. (2) Spanova, M., and Daum, G. (2011) Squalene - biochemistry, molecular biology, process biotechnology, and applications, Eur. J. Lipid Sci. Tech. 113, 1299-1320. (3) Reddy, L. H., and Couvreur, P. (2009) Squalene: A natural triterpene for use in disease management and therapy, Adv. Drug Deliv. Rev. 61, 1412-1426. (4) Fisher, K., Schofer, S. J., and Kanne, D. B. Squalane and isosqualane compositions and methods for preparing the same. U.S. Patent 8,586,814 B2, November 19, 2013. (5) Woo, H. M. (2017) Solar-to-chemical and solar-to-fuel production from CO2 by metabolically engineered microorganisms, Curr. Opin. Biotechnol. 45, 1-7. (6) Melis, A. (2009) Solar energy conversion efficiencies in photosynthesis: Minimizing the chlorophyll antennae to maximize efficiency, Plant Sci 177, 272-280. (7) Rodriguez, S., Denby, C. M., Van Vu, T., Baidoo, E. E., Wang, G., and Keasling, J. D. (2016) ATP citrate lyase mediated cytosolic acetyl-CoA biosynthesis increases mevalonate production in Saccharomyces cerevisiae, Microb. Cell Fact. 15, 48. (8) Kim, E. M., Eom, J. H., Um, Y., Kim, Y., and Woo, H. M. (2015) Microbial Synthesis of Myrcene by Metabolically Engineered Escherichia coli, J. Agric. Food Chem. 63, 46064612. (9) Tai, M., and Stephanopoulos, G. (2013) Engineering the push and pull of lipid biosynthesis in oleaginous yeast Yarrowia lipolytica for biofuel production, Metab. Eng. 15, 1-9. (10) Formighieri, C., and Melis, A. (2015) A phycocyanin.phellandrene synthase fusion enhances recombinant protein expression and beta-phellandrene (monoterpene) hydrocarbons production in Synechocystis (cyanobacteria), Metab. Eng. 32, 116-124. (11) Formighieri, C., and Melis, A. (2016) Sustainable heterologous production of terpene hydrocarbons in cyanobacteria, Photosynth. Res. 130, 123-135. (12) Gao, X., Gao, F., Liu, D., Zhang, H., Nie, X. Q., and Yang, C. (2016) Engineering the methylerythritol phosphate pathway in cyanobacteria for photosynthetic isoprene production from CO2, Energy Environ. Sci. 9, 1400-1411. (13) Choi, S. Y., Lee, H. J., Choi, J., Kim, J., Sim, S. J., Um, Y., Kim, Y., Lee, T. S., Keasling, J. D., and Woo, H. M. (2016) Photosynthetic conversion of CO2 to farnesyl diphosphate-derived phytochemicals (amorpha-4,11-diene and squalene) by engineered cyanobacteria, Biotechnol. Biofuels 9, 202. (14) Kim, W. J., Lee, S.-M., Um, Y., Sim, S. J., and Woo, H. M. (2017) Development of SyneBrick vectors as a synthetic biology platform for gene expression in Synechococcus elongatus PCC 7942, Front. Plant Sci. 8, 293. (15) Anthony, J. R., Anthony, L. C., Nowroozi, F., Kwon, G., Newman, J. D., and Keasling, J. D. (2009) Optimization of the mevalonate-based isoprenoid biosynthetic pathway in Escherichia coli for production of the anti-malarial drug precursor amorpha-4,11-diene, Metab. Eng. 11, 13-19. (16) Woo, H. M., Murray, G. W., Batth, T. S., Prasad, N., Adams, P. D., Keasling, J. D., Petzold, C. J., and Lee, T. S. (2013) Application of targeted proteomics and biological parts assembly in E. coli to optimize the biosynthesis of an anti-malarial drug precursor, amorpha4,11-diene, Chem. Eng. Sci. 103, 21-28. (17) Yoo, J. J., Choi, S. P., Kim, B. W., and Sim, S. J. (2011) Optimal design of scalable photo-bioreactor for phototropic culturing of Haematococcus pluvialis, Bioprocess Biosyst. Eng. 35, 309-315. 13 ACS Paragon Plus Environment

ACS Synthetic Biology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(18) Shaw, A. J., Lam, F. H., Hamilton, M., Consiglio, A., MacEwen, K., Brevnova, E. E., Greenhagen, E., LaTouf, W. G., South, C. R., van Dijken, H., and Stephanopoulos, G. (2016) Metabolic engineering of microbial competitive advantage for industrial fermentation processes, Science 353, 583-586. (19) Zhang, D., Jennings, S. M., Robinson, G. W., and Poulter, C. D. (1993) Yeast squalene synthase: expression, purification, and characterization of soluble recombinant enzyme, Arch. Biochem. Biophys. 304, 133-143. (20) Arai, R., Ueda, H., Kitayama, A., Kamiya, N., and Nagamune, T. (2001) Design of the linkers which effectively separate domains of a bifunctional fusion protein, Protein Eng. 14, 529-532. (21) Golden, S. S., Brusslan, J., and Haselkorn, R. (1987) Genetic engineering of the cyanobacterial chromosome, Methods Enzymol. 153, 215-231. (22) Lee, H. J., Choi, J., Lee, S.-M., Um, Y., Sim, S. J., Kim, Y., and Woo, H. M. (2017) Photosynthetic CO2 Conversion to Fatty Acid Ethyl Esters (FAEEs) Using Engineered Cyanobacteria, J. Agric. Food Chem. 65, 1087-1092. (23) Chwa, J. W., Kim, W. J., Sim, S. J., Um, Y., and Woo, H. M. (2016) Engineering of a modular and synthetic phosphoketolase pathway for photosynthetic production of acetone from CO2 in Synechococcus elongatus PCC 7942 under light and aerobic condition, Plant Biotechnol. J. 14, 1768-1776. (24) Zhang, C., Chen, X., Stephanopoulos, G., and Too, H. P. (2016) Efflux transporter engineering markedly improves amorphadiene production in Escherichia coli, Biotechnol. Bioeng. 113, 1755-1763.

14 ACS Paragon Plus Environment

Page 14 of 20

Page 15 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

Table 1. Bacteria strains and plasmids used in this study. Strain or plasmid

Relevant characteristics

Strains E. coli DH5α24 S. elongatus PCC 7942 SeHL3313 SeSC33S13 SeSC34S SeSC35S SeSC36S SeSC37S SeSC38S SeSC39S SeSC33SII SeSC34SII SeSC35SII

F-(80d lacZ M15) (lacZYA-argF) U169 hsdR17(r– m+) recA1 endA1 relA1 deoR96 S. elongatus PCC 7942 S. elongatus NSI::Bb1s-dxs-idi-ispA S. elongatus NSI::Bb1s-dxs-idi-ispA NSII::Bb1k-SQS S. elongatus NSI::Bb1s-dxs-idi-ispA NSII::Bb1k-ispA·SF·SQS S. elongatus NSI::Bb1s-dxs-idi-ispA NSII::Bb1k-ispA·LF·SQS S. elongatus NSI::Bb1s-dxs-idi-ispA NSII::Bb1k-ispA·LF2·SQS S. elongatus NSI::Bb1s-dxs-idi-ispA NSII::k-PcpcB1-cpcB1·SF·SQS S. elongatus NSI::Bb1s-dxs-idi-ispA NSII::k-PcpcB1-cpcB1·LF·SQS S. elongatus NSI::Bb1s-dxs-idi-ispA NSII::k-PcpcB1-cpcB1·LF2·SQS S. elongatus NSI::Bb1s-dxs-idi-ispA NSII::Bb1k-SQS NSIII::Bb1c-SQS S. elongatus NSI::Bb1s-dxs-idi-ispA NSII::Bb1k-SQS NSIII::c-PcpcB1cpcB1·SF·SQS S. elongatus NSI::Bb1s-dxs-idi-ispA NSII:: k- PcpcB1-cpcB1·SF·SQS NSIII::cPcpcB1-cpcB1·SF·SQS

Plasmidsa pSe3Bb1c-eyfp14 pSe1Bb1s-dxr-idi-ispA13 pSe2Bb1k-SQS13 pSe3Bb1c-SQS pSe2Bb1k-ispA·SF·SQS pSe2Bb1k-ispA·LF·SQS pSe2Bb1k-ispA·LF2·SQS pSe2k-cpcB1·SF·SQS pSe2k-cpcB1·LF·SQS pSe2k-cpcB1·LF2·SQS pSe3c-cpcB1·SF·SQS

pUC, Cmr, LacI, Ptrc, BglBrick sites, NSIII targeting SyneBrick vector pUC, Spcr, LacI, Ptrc, dxr(se.co), idi(se.co), ispA(se.co), NSI target sites pUC, Kmr, LacI, Ptrc, SQS(se.co), NSII target sites pUC, Cmr, LacI, Ptrc, SQS(se.co), NSIII target sites pUC, Kmr, LacI, Ptrc, ispA·SF·SQS(se.co), fusion protein, NSII target sites pUC, Kmr, LacI, Ptrc, ispA-LF·SQS(se.co), fusion protein, NSII target sites pUC, Kmr, LacI, Ptrc, ispA·LF2·SQS(se.co), fusion protein, NSII target sites pUC, Kmr, PcpcB1, cpcB1·SF·SQS(se.co), fusion protein, NSII target sites pUC, Kmr, PcpcB1, cpcB1·LF·SQS(se.co), fusion protein, NSII target sites pUC, Kmr, PcpcB1, cpcB1·LF2·SQS(se.co), fusion protein, NSII target sites pUC, Cmr, PcpcB1, cpcB1·SF·SQS(se.co), fusion protein, NSIII target sites

a

Kmr, kanamycin resistance; Spcr, spectinomycin resistance; Cmr , chloramphenicol resistance; the dxs

gene (E. coli), encoding 1-deoxy-D-xylulose-5-phosphate synthase; the idi gene (E. coli), encoding 1deoxy-D-xylulose-5-phosphate reductase; the ispA gene (E. coli), encoding farnesyl diphosphate synthase (IspA); SQS (S. cerevisiae), encoding truncated squalene synthase; (se.co), codon-optimized for S. elongatus PCC 7942.13,19 Peptide sequences for the linkers: SF, short flexible linker (4 aa), GGGS12; LF, long flexible linker (7 aa), GSGGGGS12; LF2, long flexible linker 2 (25 aa), LS(GGGGS)4AAA20. Note that all strains and plasmids were constructed in this work unless otherwise cited.

15 ACS Paragon Plus Environment

ACS Synthetic Biology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 1. Engineering of S. elongatus PCC 7942 to improve the photosynthetic squalene production from CO2 by protein engineering. (A) The metabolic pathway for squalene production from CO2 was engineered by overexpressing the dxs, idi, and ipsA genes from E.

coli (OverMEP module at neutral site I [NSI]) and expressing the SQS gene from S. cerevisiae (SQS module at neutral site II [NSII]).13 The engineered strain was named SeSC33S (a parental strain for this work). For protein engineering, the SQS modules were engineered with two different fusion partners (IspA or CpcB1) and three different linker domains (SF, LF, LF2) and introduced to the strain SeHL33 lacking the SQS gene at NSII. Six strains (SeSc34S, SeSc35S, SeSc36S, SeSc37S, SeSc38S, and SeSc39S) were constructed and confirmed by colony PCR using a pair of primers (1F/1R and 2F/2R; Table S1). The corresponding DNA fragments (NSI and NSII) are shown in gel images with white arrows. The DNA sequences were also verified to be correct. (B) Growth (OD730) and squalene production (mg/L/OD730) from the engineered strains are shown. No squalene is formed in the wild-type.13 Strain SeSc33S was used as a control. Solid symbols represent strains with the IspA-SQS fusion protein (SeSC34S, SeSC35S, SeSC36S), and open symbols represent strains with the CpcB1-SQS fusion protein (SeSC37S, SeSC38S, SeSC39S). The genotypes of the recombinant strains are described in Table 1. All data are the mean ± standard deviation from triplicate cultures. Abbreviations: dxs, 1-deoxy-D-xylulose-5phosphate synthase gene of E. coli; idi, isopentenyl diphosphate isomerase gene of E. coli;

ispA, farnesyl diphosphate synthase (IspA) gene of E. coli; SQS (Sc), squalene synthase gene of S. cerevisiae; G3P, glyceraldehyde-3-phosphate; DXP, 1-deoxy-D-xylulose-5-phosphate; MEP, 2-C-methyl-D-erythritol-4-phosphate; CDP-ME, 4-diphosphocytidyl-2-C-methyl-Derythritol; CDP-MEP, 4-diphosphocytidyl-2C-methyl-D-erythritol-2-phosphate; MEcPP, 2Cmethyl-D-erythritol-2,4-cyclodiphosphate;

HMBPP,

(E)-4-hydroxy-3-methylbut-2-enyl-

diphosphate; IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; FPP, 16 ACS Paragon Plus Environment

Page 16 of 20

Page 17 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

farnesyl diphosphate; PcpcB1, promoter region of the upstream 500 bp of the cpcB1 operon from S. elongatus PCC 7942; cpcB1, encoding the phycocyanin β-subunit protein from S.

elongatus PCC 7942; SF, short flexible linker; LF, long flexible linker; LF2, long flexible linker 2; WT, wild type.

Figure 2. Overexpression of duplicated squalene synthase gene copies in S. elongatus PCC 7942 to enhance squalene contents. (A) Scheme of combinatorial overexpression with the fused squalene synthase protein (a CpcB1-[SF]-SQS fusion protein). Four strains (SeSc33SII, SeSc34SII, SeSc37S, and SeSc35SII) were constructed and confirmed by colony PCR using a pair of primers (2F/2R and 3F/3R; Table S1). The corresponding DNA fragments (NSI and NSII) are shown in gel images with white arrows. The genotypes of the recombinant strains are described in Table 1. WT and NC stand for the wild type and negative control, respectively. (B) Growth (OD730) and squalene production (mg/L/OD730) from engineered strains are shown. Squalene contents in the strain with overexpression of one copy of the gene (grey bars) or overexpression of two copies of the gene (black bars) are shown. All data are the mean ± standard deviation from triplicate cultures.

Figure 3. Scalable production of squalene using a photobioreactor. (A) Schematic diagram and photographic images of the scalable production system with engineered cyanobacteria to produce squalene from 5% (v/v) CO2 and 95% (v/v) air. The 6-L V-shaped cylindrical bioreactor was optimized and used for scalable production.17 (B and C) The growth (OD730) and photosynthetic squalene production (mg/L/OD730) of the wild type as a control (black squares) and engineered S. elongatus PCC 7942 strains SeSC33S (red circles; grey bars) and SeSC35SII (blue triangles; black bars) under a light intensity of 100 or 200 µmol photon·m2

·s-1. All data are the mean ± standard deviation from duplicate cultures. 17 ACS Paragon Plus Environment

ACS Synthetic Biology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 1

18 ACS Paragon Plus Environment

Page 18 of 20

Page 19 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

Figure 2

19 ACS Paragon Plus Environment

ACS Synthetic Biology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 3

20 ACS Paragon Plus Environment

Page 20 of 20