to Î±-Farnesene Using Metabolically Engineered - ACS Publications

Subscriber access provided by READING UNIV

Letter 2

Direct conversion of CO to #-farnesene using metabolically engineered Synechococcus elongatus PCC 7942 Hyun Jeong Lee, Jiwon Lee, Sun-Mi Lee, Youngsoon Um, Yunje Kim, Sang Jun Sim, Jong-il Choi, and Han Min Woo J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03625 • Publication Date (Web): 25 Oct 2017 Downloaded from http://pubs.acs.org on November 1, 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.

Journal of Agricultural and Food Chemistry 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 19

Journal of Agricultural and Food Chemistry

1

Letter

2

Direct conversion of CO2 to α-farnesene using metabolically engineered

3

Synechococcus elongatus PCC 7942

4 5

Hyun Jeong Lee1,2, Jiwon Lee3, Sun-Mi Lee3, Youngsoon Um3, Yunje Kim3, Sang Jun Sim4,

6

Jong-il Choi5, Han Min Woo1*

7 8

1

9

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

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

10

2

11

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

12

3

13

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

14

4

15

Seongbuk-gu, Seoul 02841, Republic of Korea

16

5

17

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

Institute of Biotechnology and Bioengineering, Sungkyunkwan University (SKKU), 2066

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

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

Department of Biotechnology and Bioengineering, Chonnam National University, 77

18 19

*Corresponding author at Department of Food Science and Biotechnology, Sungkyunkwan

20

University (SKKU), 2066 Seobu-ro, Jangan-gu, Suwon 16419, Republic of Korea; Tel.: +82

21

31 290 7810; E-mail address: [email protected] (H. M. Woo)

1 ACS Paragon Plus Environment


22

Abstract: Direct conversion of carbon dioxide (CO2) to value-added chemicals by

23

engineering of cyanobacteria has received attention as a sustainable strategy in food and

24

chemical industries. Herein, Synechococcus elongatus PCC 7942, a model cyanobacterium,

25

was engineered to produce α-farnesene from CO2. Due to the lack of farnesene synthase (FS)

26

activity in the wild-type cyanobacterium, we metabolically engineered S. elongatus PCC

27

7942 to express heterologous FS from either Norway spruce or apple fruit, resulting in

28

detectable peaks of α-farnesene. To enhance α-farnesene production, an optimized

29

methylerythritol phosphate (MEP) pathway was introduced in the farnesene-producing strain

30

to supply farnesyl diphosphate. Subsequent cyanobacterial culture with a dodecane overlay

31

resulted in photosynthetic production of α-farnesene (4.6 ± 0.4 mg/L in 7 days) from CO2. To

32

the best of our knowledge, this is the first report of the photosynthetic production of α-

33

farnesene from CO2 in the unicellular cyanobacterium S. elongatus PCC 7942.

34 35

Keywords: cyanobacteria, metabolic engineering, farnesene, CO2 conversion

36


Page 2 of 19

Page 3 of 19

37


INTRODUCTION

38

Solar-to-chemical and solar-to-fuel production from CO2 have been developed using

39

engineered microorganisms that assimilate CO2 and convert target chemicals and fuels using

40

solar energy.1 Besides integrated bio-electrochemical systems, photosynthetic cyanobacteria

41

have been metabolically engineered to re-direct CO2 to value-added chemicals as bio-solar

42

cell factories. In cyanobacteria, approximately 5% of photosynthetic carbon flux is

43

distributed to the terpenoid biosynthetic pathway.2 However, the corresponding free terpenes

44

are not accumulated. Thus, to re-direct the terpenoid biosynthesis flux to free terpenes in

45

cyanobacteria, a methylerythritol phosphate (MEP) pathway that initiates from central

46

intermediates (pyruvate and glyceraldehyde-3-phosphate) must be engineered, and the

47

cyanobacteria must express the corresponding heterologous terpene synthase gene. The

48

cyanobacterial terpene production platform has been developed through metabolic

49

engineering and synthetic biology, based on a number of isoprene units: hemiterpene (C5;

50

isoprene3,4; 57.4 mg/L/d), monoterpene (C10; limonene5; 0.05 mg/L/d), sesquiterpene (C15;

51

amorpha-4,11-diene6; 1.98 mg/L/d), diterpene (C20; manoyl oxide7; 0.45 mg/gDCW; volume

52

concentration data not presented), triterpene (C30; squalene8; 5.3 mg/L/d), and tetraterpene

53

(not reported yet).

54

Specifically, α-farnesene (C15H24), which belongs to sesquiterpenes found in apple peels

55

and plays a role in plant defense, has been known as a precursor of high-performance

56

polymers9 and a promising bio-jet fuel candidate10. To produce α-farnesene, metabolic

57

engineering has been successfully applied to generate α-farnesene in microbial hosts such as

58

Escherichia coli11 (0.38 mg/g glycerol), Saccharomyces cerevisiae12 (0.57 mg/g glucose), or

59

Yarrowia lipolytica13 (6.5 mg/g glucose and fructose). The combination of heterologous gene

60

expression of farnesene synthase and the reconstitution of the terpenoid biosynthesis pathway

61

was key to increasing microbial α-farnesene production levels. Recently, the filamentous 3 ACS Paragon Plus Environment


62

cyanobacterium Anabaena sp. PCC 7129, harboring a plasmid (pFaS, the codon-optimized

63

farnesene synthase gene from Norway spruce), was constructed and yielded 305.4 µg/L

64

farnesene in 15 days by expressing only the heterologous farnesene synthase gene.14

65

Here, we report the metabolic engineering of unicellular Synechococcus elongatus

66

PCC 7942 to improve the photosynthetic production of α-farnesene from CO2. In this study,

67

both heterologous gene expression and optimization of the terpenoid biosynthesis pathway

68

led to substantial levels of α-farnesene production from CO2. This engineered strain could be

69

further optimized to accelerate the development of bio-solar cell factories to convert CO2 to

70

α-farnesene as a value-added bio-product (fragrance, surfactants, etc.) or α-farnesane, a

71

potential bio-jet fuel.

72 73

MATERIALS AND METHODS

74

Strains and plasmid construction. All bacterial strains and plasmids used in this study are

75

listed in Table 1. The E. coli strain DH5α was used for gene cloning and was grown in Luria-

76

Bertani medium (10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl) at 37°C. When

77

appropriate, the medium was supplemented with 50 µg/mL kanamycin and 100 µg/mL

78

spectinomycin. All plasmids were derived from SyneBrick expression plasmids (standard

79

vectors for chromosomal integration at neutral site I (NSI) or neutral site II (NSII),

80

pSe1Bb1s-GFP and pSe2Bb1k-GFP)15,16 using the BglBrick standard cloning method17 as a

81

synthetic platform for gene expression in S. elongatus PCC 7942. The α-farnesene synthase

82

genes from Picea abies L. Karst (Norway spruce; the NSFS gene)18 and Malus × domestica

83

Borkh. (apple fruit; the AFS gene; GenBank accession number AY182241)19 were codon-

84

optimized using Gene Designer 2.0 software (DNA2.0; ATUM, Menlo Park, CA) (see details

85

in the Supporting Information). They were then synthesized (Genscript Inc., Piscataway, NJ)

86

for heterologous expression in S. elongatus PCC 7942. The NSFS or AFS genes were also 4 ACS Paragon Plus Environment

Page 4 of 19

Page 5 of 19


87

cloned into pSe2Bb1k-GFP to construct pSe2Bb1k-NSFS and pSe2Bb1k-AFS for production

88

of α-farnesene.

89 90

Transformation of engineered S. elongatus PCC 7942. Transformation of S. elongatus

91

PCC 7942 was performed as described previously.20 The SyneBrick vectors constructed in

92

this study were transferred for chromosomal integration. First, SeHL01FS and SeHL02FS

93

were constructed with pSe2Bb1k-NSFS and pSe2Bb1k-AFS, respectively. Furthermore, four

94

engineered strains were constructed by transforming the two different farnesene synthase

95

strains with plasmids pSe1Bb1s-dxs or pSe1Bb1s-dxs-idi-ispA for the optimized MEP

96

pathway, as previously described.6 The genotypes of recombinant S. elongatus strains are

97

listed in Table 1. The strains were confirmed by PCR to verify chromosomal integration of

98

targets into either NSI or NSII (Figure 1). DNA sequences were also verified using Se1-fw

99

(5’-AAG CGC TCC GCA TGG ATC TG-3’) and Se1-rv (5’-CAA GGC AGC TTG GAA

100

GGG CG-3’) for NSI and Se2-fw (5’-GGC TAC GGT TCG TAA TGC CA-3’) and Se2-rv

101

(5’-GAG ATC AGG GCT GTA CTT AC-3’) for NSII.

102 103

Cyanobacterial growing conditions. Recombinant S. elongatus PCC 7942 used for the

104

production of α-farnesene was cultivated at 30°C under continuous fluorescent light

105

(100 µE/m2/s), measured using a LightScout Quantum meter (3415FXSE; Spectrum

106

Technologies, Aurora, IL). The cultivation was conducted using a sample volume of 100 mL

107

(Duran bottle with a three-port cap) in BG-11 medium supplemented with 10 mM MOPS at

108

pH 7.5, with 5% (v/v) CO2 gas (monitored by online gas analyzer) and 95% (v/v) filtered air,

109

supplied at a constant flow rate of 10 cc/min into the medium.6,15,21 In addition, 10 µg/mL

110

spectinomycin and 10 µg/mL kanamycin were supplemented for selection pressure. Next,

111

1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) was added into the culture medium 5 ACS Paragon Plus Environment


112

24 h after inoculation for induction. Samples were collected in 20% (v/v) dodecane overlay

113

for in situ α-farnesene extraction.6,21

114 115

Quantification of photosynthetic α-farnesene. To quantify the secreted α-farnesene,

116

samples (200 µL) from the dodecane layer in the culture were collected and diluted with ethyl

117

acetate (800 µL) containing 5 mg/L β-caryophyllene as an internal standard. Samples from

118

dodecane/ethyl acetate were analyzed using gas chromatography–mass spectrometry (GC-MS;

119

Agilent 6890N GC series, coupled with TOF-MS (LECO)). The sample solution (2 µL) was

120

injected in split mode (5:1) at an injector temperature of 250°C and was separated in an

121

Ultra-2 capillary column (17 m × 0.2 mm i.d., 0.11 µm film thickness; Agilent Technologies,

122

Santa Clara, CA). The oven temperature was 50°C for 1.50 min, ramped to 200°C at

123

20°C/min, then to 250°C at 10°C/min, and held for 3 min for a total run time of 17.00 min.

124

Helium (99.9999%) was used as the carrier gas (1.0 mL/min). The mass spectrometer was

125

operated at 70 eV, and the mass range was 50-650 m/z. The ion source temperature was

126

maintained at 220°C. Trans-β-farnesene (No. 73492; Sigma-Aldrich, St. Louis, MO) was

127

used as an authentic standard for quantitative analysis.

128 129

RESULTS AND DISCUSSION

130

The wild-type unicellular cyanobacterium S. elongatus PCC 7942 does not produce farnesene

131

due to the lacking activity of farnesene synthase and its corresponding gene. Thus, we

132

metabolically engineered S. elongatus PCC 7942 to convert CO2 directly to α-farnesene

133

(Figure 1).

134

First, two different farnesene synthase genes (NSFS and AFS) were codon-optimized and

135

chromosomally integrated into the wild type, yielding the strains SeHL01FS and SeHL02FS,

136

respectively. Instead of using an adsorption column for volatile α-farnesene, an in situ 6 ACS Paragon Plus Environment

Page 6 of 19

Page 7 of 19


137

dodecane overlay was applied to extract farnesene from the cultures (Figure 2). The dodecane

138

overlay has been chosen for not having a toxic effect on cyanobacterial cell growth and for

139

being widely used for isoprenoid extraction from microbial cultures.6,11 As a result,

140

photosynthetic α-farnesene was not detected in the dodecane layer of the cultures, of which

141

the strain SeHL01FS overexpressed the same farnesene synthase gene (NSFS) that was used

142

in the recombinant Anabaena sp. PCC 7129. However, α-farnesene was detected in the strain

143

SeHL02FS overexpressing the AFS gene (lower than limit of quantification, < 0.136 mg/L),

144

although the production level was significantly lower than that by the recombinant Anabaena

145

sp. PCC 7129. The reason could be the low level of farnesene synthase gene expression or

146

low levels of farnesyl diphosphate (FPP) pools, which are crucial factors for high-level

147

conversion of FPP to farnesene. The level of overexpression of the target gene in S. elongatus

148

PCC 7942 using the SyneBrick vector16 could be lower than in Anabaena sp. PCC 7129 due

149

to the chromosomal gene integration of the NSFS or AFS genes, instead of gene expression in

150

a high-copy plasmid (pZR1188)14. Nonetheless, a plasmid-bearing host has instability issues

151

due to long-term expression of the heterologous genes.22 Also, the strain SeHL01FS and

152

SeHL02FS showed growth inhibitions due to possible depletion of the FPP levels (Figure 3).

153

Thus, we focused on metabolic engineering to increase pools of FPP, the key intermediate for

154

improving the production level of α-farnesene.

155

Previously, FPP-derived phytochemicals, including amorpha-4,11-diene (19.8 mg/L)6 and

156

squalene (11.98 mg/L)6,8, have been successfully overproduced from CO2 in engineered S.

157

elongatus PCC 7942 constructed from the parental strain (SeHL33; OverMEP module), in

158

which the MEP pathway was optimized. Two different parental strains were used for the

159

production improvement: the MEP-basic strain, SeHL11 and the MEP-optimized strain,

160

SeHL33. The dxs gene is overexpressed in SeHL11, and the combinatorial expressions of the

161

dxs, idi, and ispA genes are allowed in SeHL33 (Table 1). As a result, four strains 7 ACS Paragon Plus Environment


162

(SeHL11FS, SeHL12FS, SeHL31FS, and SeHL32FS) were cultivated with a dodecane

163

overlay (Figure 3). MEP-basic strains with heterologous NSFS or ASF genes (SeHL11FS and

164

SeHL12FS, respectively) failed to produce quantifiable levels of α-farnesene. However,

165

SeHL31FS and SeHL32FS, for which the MEP-optimized strain (SeHL33) was used as a

166

parental strain, produced farnesene from CO2 (0.4 ± 0.01 mg/L and 4.6 ± 0.4 mg/L in 7 days,

167

respectively). Indeed, the OverMEP module increased the FPP pools and alleviated the

168

growth inhibitions. Subsequently, the farnesene productions were significantly enhanced with

169

the insertion of the NSFS or ASF genes. The heterologous expression of the AFS gene in

170

SeHL33 resulted in higher farnesene production than that of the NSFS gene. None of the

171

engineered strains was shown for α-farnesene accumulation inside a cell.

172

Each α-farnesene synthase (AFS and NSFS) used in this study has the RR(x8)W motif, a

173

characteristic that plays a role in the reaction mechanism of two sesquiterpene synthases that

174

yield an acyclic product, i.e. α-farnesene in this study (see details in the Supporting

175

Information; Figure S1). An absolutely conserved aspartate-rich motif (DDxxD) was also

176

present in both enzymes. However, the AFS and NSFS genes have been classified as the

177

TPBS-b23 and TPS-d18, respectively, based on the phylogenetic analysis of terpene synthases

178

(by sharing a 31.7% identity between AFS and NSFS). The difference in α-farnesene

179

production levels between the AFS and NSFS in S. elongatus PCC 7942 remains unclear

180

owing to missing information on the evolutionary relationship of the terpene synthases, such

181

as the specific selective pressure to maintain native α-farnesene production.23,24

182

In this study, the best photosynthetic farnesene producer was the strain SeHL32FS, which

183

overexpressed the ASF gene in SeHL33. Compared to the farnesene production level of the

184

recombinant Anabaena sp. PCC 7129 (305.4 µg/L farnesene in 15 days, equivalent to

185

20.4 µg/L/d)14, the production level by SeHL32FS was an order higher (4.6 ± 0.4 mg/L in 7

186

days, equivalent to 600 µg/L/d). In addition, the specific farnesene productivity of SeHL32FS 8 ACS Paragon Plus Environment

Page 8 of 19

Page 9 of 19


187

(232.2 ± 17.0 µg/L/OD730/d) during the first 3 days was 3.4-fold higher than the maximum

188

specific productivity of the recombinant Anabaena sp. PCC 7129 (69.1 µg/L/OD730/d) during

189

the same culturing period. The main reasons for the success could be the selection of the

190

terpene synthase (the AFS gene) and an increased supply of the FPP pool in engineered

191

cyanobacteria.

192

Furthermore, specific farnesene production rates of SeHL32FS were calculated during 7

193

days of growth (three different periods) (see details in Supporting Information; Figure S2).

194

The production rates continued to increase during the growth period to 480.3 µg/L/OD730/d

195

from days 3-5 and to 625.2 µg/L/OD730/d from days 5-7. The increased productivities differ

196

from the results of recombinant Anabaena sp. PCC 712914, where the rates decreased rapidly.

197

Despite the cyanobacterial lifestyles of Anabaena sp. and S. elongatus being significantly

198

different, the expression of chromosomally-integrated target genes in S. elongatus PCC 7942

199

could be more stable than plasmid-borne genes in Anabaena sp. PCC 7129.

200

We demonstrated direct conversion of CO2 to α-farnesene using metabolically engineered

201

cyanobacteria at the highest rate reported so far (4.6 ± 0.4 mg/L in 7 days). The OverMEP

202

module for MEP pathway optimization has again proven to increase the carbon flux toward

203

FPP. Combined with the expression of any terpene synthases, FPP-derived chemical

204

production can be achieved directly from CO2 in S. elongatus PCC 7942. Combined with the

205

pathway optimization, the activity of farnesene synthase can be improved by increasing gene

206

dosages8 or expressing the gene with fusion partners.3,8,25 Further optimization of our

207

cyanobacterial strains is necessary to enable a higher production of farnesene from CO2

208

through the recent programmable RNA-guided genome engineering.26,27

209 210

Funding



211

This work was supported by Korea CCS R&D Center (KCRC) (2017M1A8A1072034) and

212

Basic Science Research Program (2017R1A2B2002566, 2017R1A6A3A01011460) through

213

the National Research Foundation of Korea, funded by the Korean Government (Ministry of

214

Science and ICT). In addition, this work was partially supported by the Golden Seed Project

215

(213008-05-1-WT911) grant, funded by the Ministry of Agriculture and the Ministry of

216

Oceans and Fisheries.

217 218

Acknowledgements

219

The authors thank M. S. Jaeyeon Choi at the Korean Institute of Science and Technology for

220

technical support.

221 222

Supporting Information

223

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

224

DOI:

225

Codon-optimized DNA sequence for each target gene and its original GenBank accession

226

number. Figure S1. Protein sequence alignment of NSFS and AFS. Figure S2. Specific

227

productivity of α-farnesene in the cyanobacterial culture.

228 229

Notes

230

The authors declare that they have no conflict of interest.

231


Page 10 of 19

Page 11 of 19


232

References

233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279

(1) Woo, H. M., Solar-to-chemical and solar-to-fuel production from CO2 by metabolically engineered microorganisms. Curr. Opin. Biotechnol. 2017, 45, 1-7. (2) Melis, A., Carbon partitioning in photosynthesis. Curr. Opin. Chem. Biol. 2013, 17, 453456. (3) Gao, X.; Gao, F.; Liu, D.; Zhang, H.; Nie, X.; Yang, C., Engineering the methylerythritol phosphate pathway in cyanobacteria for photosynthetic isoprene production from CO2. Energy Environ. Sci. 2016, 9, 1400-1411. (4) Lindberg, P.; Park, S.; Melis, A., Engineering a platform for photosynthetic isoprene production in cyanobacteria, using Synechocystis as the model organism. Metab. Eng. 2010, 12, 70-79. (5) Kiyota, H.; Okudac, Y.; Ito, M.; Hirai, M. Y.; Ikeuchi, M., Engineering of cyanobacteria for the photosynthetic production of limonene from CO2. J. Biotechnol. 2014, 185, 1-7. (6) Choi, S. Y.; Lee, H. J.; Choi, J.; Kim, J.; Sim, S. J.; Um, Y.; Kim, Y.; Lee, T. S.; Keasling, J. D.; Woo, H. M., Photosynthetic conversion of CO2 to farnesyl diphosphatederived phytochemicals (amorpha-4,11-diene and squalene) by engineered cyanobacteria. Biotechnol. Biofuels 2016, 9, 202. (7) Englund, E.; Andersen-Ranberg, J.; Miao, R.; Hamberger, B.; Lindberg, P., Metabolic Engineering of Synechocystis sp. PCC 6803 for Production of the Plant Diterpenoid Manoyl Oxide. ACS Synth. Biol. 2015, 4, 1270-1278. (8) Choi, S. Y.; Wang, J. Y.; Kwak, H. S.; Lee, S. M.; Um, Y.; Kim, Y.; Sim, S. J.; Choi, J. I.; Woo, H. M., Improvement of Squalene Production from CO2 in Synechococcus elongatus PCC 7942 by Metabolic Engineering and Scalable Production in a Photobioreactor. ACS Synth. Biol. 2017, 6, 1289-1295. (9) Yoo, T.; Chao, H.; Henning, S., Farnesene-based polymers and liquid optically clear adhesive compositions incorporating the same, WO 2017003573 A1. 2017. (10) Renninger, N. S.; Sahm, H.; McPhee, D. J., Fuel Composition Comprising Farnesene and Farnesane Derivatives and Method of Making and Using Same, US 20080098645 A1. 2008. (11) Wang, C.; Yoon, S. H.; Jang, H. J.; Chung, Y. R.; Kim, J. Y.; Choi, E. S.; Kim, S. W., Metabolic engineering of Escherichia coli for alpha-farnesene production. Metab. Eng. 2011, 13, 648-655. (12) Tippmann, S.; Ferreira, R.; Siewers, V.; Nielsen, J.; Chen, Y., Effects of acetoacetylCoA synthase expression on production of farnesene in Saccharomyces cerevisiae. J Ind. Microbiol. Biotechnol. 2017, 44, 911-922. (13) Yang, X.; Nambou, K.; Wei, L.; Hua, Q., Heterologous production of alpha-farnesene in metabolically engineered strains of Yarrowia lipolytica. Bioresour. Technol. 2016, 216, 1040-1048. (14) Halfmann, C.; Gu, L.; Gibbons, W.; Zhou, R., Genetically engineering cyanobacteria to convert CO2, water, and light into the long-chain hydrocarbon farnesene. App. Microbiol. Biotechnol. 2014, 98, 9869-9877. (15) Chwa, J. W.; Kim, W. J.; Sim, S. J.; Um, Y.; Woo, H. M., 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. 2016, 14, 1768-1776. (16) Kim, W. J.; Lee, S. M.; Um, Y.; Sim, S. J.; Woo, H. M., Development of SyneBrick Vectors As a Synthetic Biology Platform for Gene Expression in Synechococcus elongatus PCC 7942. Front Plant. Sci. 2017, 8, 293. 11 ACS Paragon Plus Environment


280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315

(17) Lee, T. S.; Krupa, R. A.; Zhang, F.; Hajimorad, M.; Holtz, W. J.; Prasad, N.; Lee, S.; Keasling, J. D., BglBrick vectors and datasheets: a synthetic biology platform for gene expression. J. Biol. Eng. 2011, 5, 1. (18) Martin, D. M.; Faldt, J.; Bohlmann, J., Functional characterization of nine Norway Spruce TPS genes and evolution of gymnosperm terpene synthases of the TPS-d subfamily. Plant Physiol. 2004, 135, 1908-1927. (19) Pechous, S. W.; Whitaker, B. D., Cloning and functional expression of an ( E, E)alpha-farnesene synthase cDNA from peel tissue of apple fruit. Planta 2004, 219, 84-94. (20) Golden, S. S.; Brusslan, J.; Haselkorn, R., Genetic engineering of the cyanobacterial chromosome. Methods Enzymol. 1986, 153, 215-231. (21) Bentley, F. K.; Melis, A., Diffusion-based process for carbon dioxide uptake and isoprene emission in gaseous/aqueous two-phase photobioreactors by photosynthetic microorganisms. Biotechnol. Bioeng. 2012, 109, 100-109. (22) Tyo, K. E.; Ajikumar, P. K.; Stephanopoulos, G., Stabilized gene duplication enables long-term selection-free heterologous pathway expression. Nat. Biotechnol. 2009, 27, 760765. (23) Nieuwenhuizen, N. J.; Green, S. A.; Chen, X.; Bailleul, E. J.; Matich, A. J.; Wang, M. Y.; Atkinson, R. G., Functional genomics reveals that a compact terpene synthase gene family can account for terpene volatile production in apple. Plant Physiol. 2013, 161, 787804. (24) Bohlmann, J.; Meyer-Gauen, G.; Croteau, R., Plant terpenoid synthases: molecular biology and phylogenetic analysis. Proc. Natl. Acad. Sci. U. S. A. 1998, 95, 4126-4133. (25) Formighieri, C.; Melis, A., A phycocyanin.phellandrene synthase fusion enhances recombinant protein expression and beta-phellandrene (monoterpene) hydrocarbons production in Synechocystis (cyanobacteria). Metab. Eng. 2015, 32, 116-124. (26) Gordon, G. C.; Korosh, T. C.; Cameron, J. C.; Markley, A. L.; Begemann, M. B.; Pfleger, B. F., CRISPR interference as a titratable, trans-acting regulatory tool for metabolic engineering in the cyanobacterium Synechococcus sp. strain PCC 7002. Metab. Eng. 2016, 38, 170-179. (27) Wendt, K. E.; Ungerer, J.; Cobb, R. E.; Zhao, H.; Pakrasi, H. B., CRISPR/Cas9 mediated targeted mutagenesis of the fast growing cyanobacterium Synechococcus elongatus UTEX 2973. Microb. Cell Fact. 2016, 15, 115. (28) Choi, S. Y.; Park, B.; Choi, I. G.; Sim, S. J.; Lee, S. M.; Um, Y.; Woo, H. M., Transcriptome landscape of Synechococcus elongatus PCC 7942 for nitrogen starvation responses using RNA-seq. Sci. Rep. 2016, 6, 30584.

316


Page 12 of 19

Page 13 of 19


317

Figure Legends

318

Figure 1. Scheme of photosynthetic farnesene production from CO2 in engineered S.

319

elongatus PCC 7942. (A) A schematic pathway for farnesene production by expressing

320

heterologous farnesene synthase in the MEP-optimized recombinant S. elongatus PCC 7942

321

(SeHL33). The strain SeHL33 overexpressing the key genes (dxs, idi, and ispA) of the MEP

322

has been developed to supply the pool of FPP6, which is shown in black. FPP is converted to

323

farnesene by heterologous farnesene synthase (shown in red). (B) Schematic diagrams of the

324

construction of S. elongatus PCC 7942 strains that produce farnesene. The dxs gene or the

325

dxs-idi-ispA genes have been introduced to neutral site I (NSI) for supplying the FPP

326

intermediate from CO2 [OverMEP module]. Subsequently, two different heterologous

327

farnesene synthase genes (NSFS or AFS, from Picea abies L. Karst18 or Malus × domestica

328

Borkh19, respectively) were introduced to neutral site II (NSII) of S. elongatus genomic DNA

329

[FS module]. A gel image shows colony-PCR results verifying recombinant S. elongatus

330

strains using a pair of Se1-fw/Se1-rv and Se2-fw/Se2-rv for the NSI and NSII integrations,

331

respectively. The DNA sequences were also verified. The target size of each PCR product for

332

wild-type or mutant cyanobacteria was: wild type (1.6 kb), SeHL11FS (6.4 kb), SeHL12FS

333

(6.4 kb), SeHL31FS (7.9 kb), and SeHL32FS (7.9 kb) at NSI and wild type (2.8 kb),

334

SeHL01FS (6.8 kb), SeHL02FS (6.7 kb), SeHL11FS (6.8 kb), SeHL12FS (6.7 kb),

335

SeHL31FS (6.8 kb), and SeHL32FS (6.7 kb) at NSII. The genotypes of the recombinant

336

strains SeHL01FS, SeHL02FS, SeHL11FS, SeHL12FS, SeHL31FS, and SeHL32FS are

337

presented in Table 1. Abbreviations of the corresponding enzymes are: Dxs (Ec), 1-deoxy-D-

338

xylulose-5-phosphate synthase of E. coli; Idi, isopentenyl diphosphate isomerase of E. coli;

339

IspA (Ec), farnesyl diphosphate synthase (IspA) of E. coli; FS (Apple fruit), farnesene

340

synthase of Malus × domestica Borkh.; G3P, glyceraldehyde 3-phosphate; DXP, 1-deoxy-D-



MEP,

2-C-methyl-D-erythritol-4-phosphate;

Page 14 of 19

341

xylulose-5-phosphate;

342

diphosphocytidyl-2-C-methyl-D-erythritol; CDP-MEP,

343

erythritol-2-phosphate; MEcPP, 2C-methyl-D-erythritol-2,4-cyclodiphosphate; HMBPP, (E)-

344

4-hydroxy-3-methylbut-2-enyl-diphosphate;

345

dimethylallyl diphosphate; and FPP, farnesyl diphosphate.

IPP,

CDP-ME,

4-

4-diphosphocytidyl-2C-methyl-D-

isopentenyl

diphosphate;

DMAPP,

346 347

Figure 2. Measurement of extracellular farnesene from engineered S. elongatus PCC 7942

348

strains. The recombinant strain was cultivated and measured at OD730 under 5% (v/v) CO2

349

bubbling with a 20% (v/v) dodecane overlay. Photographic images and schematic diagram

350

are show in the upper panel. The samples from the dodecane overlay were analyzed by GC-

351

MS for quantification of farnesene. Authentic standard (β-farnesene) and internal standard

352

(IS; β-caryophyllene) were used. Retention times and relative abundances are shown for both

353

the standard and the sample.

354 355

Figure 3. Photosynthetic farnesene produced from CO2 in engineered S. elongatus PCC

356

7942. Engineered S. elongatus PCC 7942 strains were cultivated under 5% (v/v) CO2

357

bubbling in the presence of a 20% (v/v) dodecane overlay. Measurements of cell growth and

358

farnesene production (mg/L) were performed for the recombinant strains. The genotypes of

359

the recombinant strains are presented in Table 1. All data are expressed as mean ± standard

360

deviation from cultures run in triplicate. N.D., not detected; LOD, 0.045 mg/L; LOQ,

361

0.136 mg/L.


Page 15 of 19


Table 1. Bacterial strains and plasmids used in this study.

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

Relevant characteristics

References

Strains E. coli HIT-DH5α S. elongatus PCC 7942

F-(80d lacZ M15) (lacZYA-argF) U169 hsdR17(r– m+) recA1 endA1 relA1 deoR96 Wild type (ATCC 33912)

RBC Bioscience ATCC

SeHL11

S. elongatus PCC 7942 NSI::Bb1s-dxs

SeHL33

S. elongatus PCC 7942 NSI::Bb1s-dxs-idi-ispA

SeHL01FS

S. elongatus PCC 7942 NSII::Bb1k-NSFS

This study

SeHL02FS

S. elongatus PCC 7942 NSII::Bb1k-AFS

This study

SeHL11FS SeHL12FS SeHL31FS SeHL32FS

S. elongatus PCC 7942 NSI::Bb1s-dxs, NSII::Bb1k-NSFS S. elongatus PCC 7942 NSI::Bb1s-dxs, NSII::Bb1k-AFS S. elongatus PCC 7942 NSI::Bb1s-dxs-idi-ispA, NSII::Bb1k-NSFS S. elongatus PCC 7942 NSI::Bb1s-dxs-idi-ispA, NSII::Bb1k-AFS

This study 28

This study This study This study This study

Plasmid pUC, Kmr, LacI, Ptrc, BglBrick sites, NSII target 15 sites, SyneBrick Vector pUC, Kmr, LacI, Ptrc, NSII target sites, pSe2Bb1k-NSFS the farnesene synthase gene originated from Picea This study abies L. Karst18; the NSFS gene(se.co) pUC, Kmr, LacI, Ptrc, NSII target sites, the farnesene synthase gene (AFS) originated from This study pSe2Bb1k-AFS Malus × domestica Borkh19.; the AFS gene(se.co) Note: (se.co) represents that the gene sequence is codon-optimized to S. elongatus PCC 7942. pSe2Bb1k-GFP



Figure 1.


Page 16 of 19

Page 17 of 19


Figure 2.



Figure 3.


Page 18 of 19

Page 19 of 19


Graphic for table of contents


to Î±-Farnesene Using Metabolically Engineered - ACS Publications

Recommend Documents