Dynamic engineering of 1-alkenes biosynthesis and secretion in yeast

Subscriber access provided by the University of Exeter

Article

Dynamic engineering of 1-alkenes biosynthesis and secretion in yeast Yongjin J. Zhou, Yating Hu, Zhiwei Zhu, Verena Siewers, and Jens Nielsen ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.7b00338 • Publication Date (Web): 28 Dec 2017 Downloaded from http://pubs.acs.org on December 29, 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 22 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

Dynamic engineering of 1-alkenes biosynthesis and

2

secretion in yeast

3

Yongjin J. Zhou1,2*, Yating Hu2,3, Zhiwei Zhu2,3, Verena Siewers2,3,

4

Jens Nielsen2,3,4*

5 6

1

7 8

2

9 10

3

11 12 13

4

Division of Biotechnology, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian 116023, PR China Department of Biology and Biological Engineering, Chalmers University of Technology, Kemivägen 10, SE-41296 Gothenburg, Sweden Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, SE-41296 Gothenburg, Sweden Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark

14 15

*Correspondence to: Jens Nielsen E-mail: [email protected]

16

Yongjin J. Zhou E-mail: [email protected]

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

18

Abstract

19

Microbial production of fatty acid-derived hydrocarbons offers a great opportunity

20

to sustainably supply biofuels and oleochemicals. One challenge is to achieve a high

21

production rate. Besides, low efficiency in secretion will cause high separation costs

22

and it is therefore desirable to have product secretion. Here, we engineered the

23

budding yeast Saccharomyces cerevisiae, to produce and secrete 1-alkenes by

24

manipulation of the fatty acid metabolism, enzyme selection, engineering the

25

electron transfer system and expressing a transporter. Furthermore, we

26

implemented a dynamic regulation strategy to control the expression of membrane

27

enzyme and transporter, which improved 1-alkene production and cell growth by

28

relieving the possible toxicity of overexpressed membrane proteins. With these

29

efforts, the engineered yeast cell factory produced 35.3 mg/L 1-alkenes with more

30

than 80% being secreted. This represents a 10-fold improvement compared with

31

earlier reported hydrocarbon production by S. cerevisiae.

32

Key words: Yeast cell factories; Dynamic regulation; Fatty acids; Hydrocarbons;

33

Metabolic engineering

34

Introduction

35

Microbial utilization of biomass for production of biofuels represents a sustainable

36

solution for provision of liquid transportation fuels with a reduced carbon footprint.1,

37

2

38

of surfactants and lubricants, and also have the potential to serve as “drop-in”

39

compatible fuels due to their high energy density.

40

The fatty acid biosynthesis pathway has attracted significant attention for

41

production of oleo-chemicals and biofuels. Among these, 1-alkenes can be

42

synthesized from activated fatty acids (fatty acyl-CoA/ACP) by polyketide synthases,3,

43

4

44

iron-dependent fatty acid decarboxylases.6, 7 All of these pathways were discovered

45

in bacteria and some of these enzymes have been heterologously expressed in

Long-chain linear 1-alkenes (α-olefins) are widely used as feedstock for production

or from free fatty acids (FFAs) by H2O2-dependent P450 enzymes5 and


Page 2 of 22

Page 3 of 22 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

Escherichia coli for 1-alkene production.8 It is also interesting to explore the potential

47

of 1-alkene production in the industrially widely applied Saccharomyces cerevisiae,

48

which has high robustness and tolerance towards harsh fermentation conditions as

49

well as its already wide use for bioethanol production.9, 10 However, compared to E.

50

coli, productivity and yield of fatty acid-derived hydrocarbons are much lower in

51

engineered S. cerevisiae,11-17 as well as in the oleaginous yeast Yarrowia lipolytica.18,

52

19

53

had a titer of 100 mg/L, while an engineered S. cerevisiae only produced < 5 mg/L

54

1-alkenes.13, 14, 17 This might be attributed to a more complex yeast metabolism, an

55

unsuitable cellular environment for the heterologous enzymes and/or the lack of

56

suitable cofactors. Furthermore, due to the hydrophobic nature of hydrocarbons

57

they may tend to accumulate in the lipid bi-layer of the cell membrane and hence

58

not be secreted to the extracellular medium, which is an obstacle for product

59

separation.

60

Here, we aim to engineer an ideal yeast cell factory for production and secretion of

61

1-alkene by pathway screening, engineering of the electron transfer system, overall

62

dynamic metabolic balancing and heterologous transporter expression (Figure 1).

63 64

Figure 1. Schematic overview of metabolic pathways engineered for the production of 1-alkenes in S.

65

cerevisiae. Multiple arrows indicate multiple steps and single arrows represent a single step. Fatty

66

acid consumption pathways were eliminated by deleting fatty acyl-CoA synthetase encoding genes

With regard to 1-alkene production, an engineered E. coli with FFA accumulation



Page 4 of 22

67

FAA1 and FAA4, and fatty acyl-CoA oxidase encoding gene POX1. 1-Alkenes can be synthesised from

68

free fatty acids catalysed by H2O2-dependent P450 enzyme OleT, nonheme iron oxidase UndA or

69

desaturase-like enzyme UndB. The NADH-dependent putidaredoxin (Pdx)-putidaredoxin reductase

70

(Pdr) system was introduced to handle the electron transfer involved in the UndB catalysed FFA

71

decarboxylation. Pdxox and Pdxred represent the oxidative and reductive state of Pdx, respectively.

72

FATP1, encoding long chain fatty acid transporter protein 1, was also expressed for exporting

73

1-alkenes.

74

Results and Discussion

75

Enzyme selection for 1-alkene production

76

Previous studies explored the P450 fatty acid decarboxylase OleT from

77

Jeotgalicoccus sp. ATCC8456 for 1-alkene production in S. cerevisiae, which enabled

78

the production of 0.2-3.7 mg/L 1-alkenes,13, 14 only about 5 % compared with an

79

engineered E. coli strain.20 Speculating that this enzyme works poorly in yeast we

80

searched for a more efficient pathway for 1-alkene production by using the fatty acid

81

over-producing strain YJZ06,12 which carries triple deletions of the fatty acyl-CoA

82

synthase genes FAA1/4 and the fatty acid oxidase gene POX1 (Figure 2A). Besides the

83

H2O2-dependent P450 enzyme OleT, we also evaluated the recently identified

84

non-heme iron oxidase UndA6 and two different variants of the desaturase-like

85

enzyme UndB.7 All these fatty acid decarboxylase genes were expressed under

86

control

87

UASTEF-UASCIT-UASCLB-TDH3p)12 and cloned into the pYX212 plasmid backbone.

88

Expression of OleT from Jeotgalicoccus enabled production of 0.92 mg/L of 1-alkenes,

89

while expression of PpUndA from Pseudomonas putida F1 resulted in a lower

90

1-alkene titer of 0.57 mg/L (Figure 2B). It was reported previously that more than 3

91

mg/L of medium chain 1-alkenes were produced by S. cerevisiae expressing PpUndA

92

under simple cultivation in vials.17 Here, the low 1-alkene production via expression

93

of PpUndA was attributed to the fact that PpUndA shows poor activity toward long

94

chain fatty acids (LCFAs)6 and the fatty acids of host strain YJZ06 are mainly LCFAs.12

95

Interestingly, the desaturase-like UndB enzymes were much more efficient in terms

96

of 1-alkene biosynthesis in S. cerevisiae (Figure 2B). Among the two enzymes

97

evaluated, PmUndB from Pseudomonas mendocina enabled production of 2.90 mg/L

of

the

enhanced

strong

promoter

eTDH3p


(the

version

of

Page 5 of 22 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


98

1-alkenes whereas expression of PfUndB from Pseudomonas fluorescens Pf-5

99

resulted in a titer of 5.76 mg/L (Figure 2B). This is different from findings in E. coli,

100

where PmUndB was found to be more efficient than PfUndB for 1-alkene

101

production.7 This performance variation might be owing to the differences in protein

102

expression level and/or in the cellular environment for enzyme activity.

103 104

Figure 2: Engineering 1-alkene biosynthesis in S. cerevisiae by enzyme evaluation and fatty acid

105

manipulation A) Schematic overview of the metabolic pathways used for production of 1-alkenes

106

from FFAs via three different routes. B) Evaluation of enzymes for total 1-alkene production in the

107

fatty acid overproducing strain YJZ06 that carries deletions of POX1, FAA1 and FAA4. C) The effect of

108

manipulating the production of FFAs on total 1-alkene biosynthesis in a strain expressing PfUndB. All

109

data represent the mean ± s.d. of three yeast clones.

110

We then investigated the effect of FFA accumulation on 1-alkene production (Figure

111

2C). The deletion of POX1 and/or FAA4 had a marginal effect on 1-alkene production,

112

while FAA1 deletion improved it by 36%. Furthermore, the triple deletion of POX1

113

and FAA1/4 led to a 2.8-fold higher 1-alkene titer (6.20 mg/L) compared with the

114

wild-type background. The 1-alkene titers were well correlated with the free fatty

115

acid levels (Figure 2C), which was also observed in previous studies using OleT for



3, 13

Page 6 of 22

116

1-alkene production

117

fatty acids were essential for 1-alkene production with using UndB.

118

Cofactor engineering for enhanced 1-alkene biosynthesis

119

1-Alkene biosynthesis from FFAs is an oxidation process involving an electron

120

transfer process. Several studies suggested that the lack of an efficient reduction

121

system could be one of the reasons for the low activities of fatty acid decarboxylases

122

both in vitro21 and in vivo.13 We therefore compared three electron transfer systems:

123

ferredoxin-ferredoxin reductase (Fd/Fnr) and flavodoxin-ferredoxin reductase

124

(Fld/Fnr)

125

putidaredoxin-putidaredoxin reductase from P. putida (Pdx/Pdr, encoded by camB

126

and camA, respectively) that uses NADH as cofactor (Figure 3A). Expression of the

127

ferredoxin- and flavodoxin-based electron transfer systems improved 1-alkene

128

production by 42% and 58%, respectively, while the putidaredoxin reductase system

129

increased 1-alkene production by 108% (Figure 3B). To facilitate electron

130

channelling,22 we fused the Pdx/Pdr system to PfUndB with different linkers.

131

However, this endeavour resulted in a much lower 1-alkene production (Figure S1),

132

which might be owing to an impaired function of the membrane-bound enzyme

133

PfUndB after protein fusion. It is worth mentioning that the use of NADH as electron

134

donor by Pdx/Pdr may be responsible for the improved 1-alkene biosynthesis in S.

135

cerevisiae as this yeast has a much higher level (>7-fold) of NADH than NADPH.23

from

E.

. These results suggested that high levels of precursor free

coli,

which

use

NADPH

136


as

cofactor;

and

Page 7 of 22 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


137

Figure 3: Comparison of different electron transfer systems for total 1-alkene production in a FFA

138

overproducing strain expressing PfUndB. A) Schematic overview of the metabolic pathway including

139

electron transfer systems from E. coli (Fd/Fnr, Fld/Fnr) and P. putida (Pdx/Pdr), respectively. B) Total

140

1-alkene production in engineered strains. Data represent the mean ± s.d. of three yeast clones.

141 142

Dynamic regulation of UndB to improve 1-alkene production with relieved

143

metabolic burden

144

UndB was proposed to be a membrane-bound desaturase-like enzyme by

145

bioinformatics analysis (Figure 4A),7 and the enzyme may therefore also be involved

146

in export of the 1-alkenes as we found that the majority of the 1-alkenes produced

147

was secreted to the extracellular medium (Figure S2). Furthermore, fluorescence

148

microscopy analysis of the PfUndB-GFP carrying strain AE17 showed that this

149

enzyme predominantly localized to the membrane (Figure 4B). Initially, we used a

150

strong promoter, eTDH3p, for high-level expression of PfUndB, but this resulted in

151

reduced growth (strain AE24, Figure 4C), which might be due to eTDH3p-based

152

transcription being strongly upregulated in the glucose phase.24

153

High-level expression of a membrane protein might cause stress on cell growth.

154

Furthermore, cells showed a much lower fatty acid accumulation during the glucose

155

phase compared to the ethanol phase (Figure S4), and TDH3p-based transcription is

156

therefore not synchronized with FFA accumulation. We therefore fine-tuned the

157

expression of undB to balance cell growth and product formation. To reach this goal,

158

we designed a dynamic control system to separate cell growth and 1-alkene

159

production into two different phases by using the carbon source-dependent

160

promoter of GAL7 (GAL7p), which is repressed at high glucose levels and activated at

161

glucose depletion. Expression of PfundB under control of GAL7p, combined with the

162

deletion of the transcription factor Gal80 (strain AE38), resulted in a 100% higher

163

final titer of 1-alkenes compared to strain AE24 constitutively expressing PfUndB

164

under control of eTDH3p (Figure 4C). Strain AE24 produced more 1-alkenes in the

165

glucose phase compared to strain AE38. Both strains secreted the majority of the

166

1-alkenes produced (>60%) and the GAL7p driven expression of PfundB improved the



167

production of both extracellular and intracellular 1-alkenes (Figure S2). Furthermore,

168

strain AE38 showed better growth in the glucose phase resulting in a higher final

169

biomass yield and glucose consumption rate compared to strain AE24 (Figure 4C and

170

Figure S3). Furthermore, there was a much higher 1-alkene production (Figure 4C)

171

and lower specific FFA accumulation (Figure S4) in the cultures of strain AE38

172

compared to those of AE24. These results suggested that dynamic control could

173

relieve the toxicity of expressing the membrane protein PfUndB and improve

174

1-alkene production by separating cell growth and 1-alkene production into two

175

different phases, which could provide a feasible approach to improve the

176

performance of cell catalysts with toxic pathways or enzymes.

177 178

Figure 4: Dynamic control of membrane enzyme and transporter expression for 1-alkene production

179

and secretion. A) Schematic view of 1-alkene biosynthesis and secretion. B) Fluorescence microscopy

180

analysis of AE17 that carried an undB-gfp fusion gene. C) Dynamic control of UndB expression for

181

enhanced total 1-alkene biosynthesis. Expression of UndB under control of GAL7p (strain AE38) was

182

compared to expression under control of eTDH3p (strain AE24). D) Expression of the LCFA transporter

183

FATP1 for improving 1-alkene secretion and production. Strain AE39 contains FATP1, while AE38 was


Page 8 of 22

Page 9 of 22 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


184

used as control. E) Total 1-alkene profiles of AE38 and AE39 expressing FATP1 at 48 h and 70 h. All

185

data represent the mean ± s.d. of three yeast clones.

186

Expressing a transporter for improved 1-alkene production and secretion

187

Downstream extraction and purification usually greatly raises the overall cost of

188

production, and if the product is intracellular there is a need for cell disruption and

189

extraction by using environmentally unfriendly solvents.25, 26 Engineering product

190

secretion is therefore attractive for improving the process economy. Furthermore,

191

this may also relieve product inhibition and toxicity. Previous studies successfully

192

improved terpene production by using resistance-nodulation-cell division (RND)27 or

193

ATP binding cassette (ABC)25, 28 transporters. Here, we alternatively explored the

194

long chain fatty acid transporter protein 1 (FATP1) from Homo sapiens29 to improve

195

1-alkene production, based on the similar hydrophobic property between LCFAs and

196

1-alkenes. In order to alleviate the toxicity of membrane protein FATP1 expression in

197

the glucose phase, we expressed FATP1 using HXT6p,30 which was expected to be

198

highly expressed after glucose depletion when 1-alkene production is initiated.

199

Genome integration of a FATP1 expression cassette (strain AE39) had a marginal

200

effect on cell growth and fatty acid production (Figure S5), but significantly improved

201

the extracellular and total 1-alkene production after 70 h of cultivation by 40% and

202

37%, respectively, compared with the control strain AE38 (Figure 4D). There was

203

little difference in the 1-alkene profiles between AE38 and AE39, but slightly more

204

C17 1-alkenes were accumulated at 70 h compared with at 48 h (Figure 4E). The

205

FATP1 expressing strain AE39 clearly showed the highest 1-alkene secretion resulting

206

in an extracellular final titer of 29.3 mg/L corresponding to 83% of the total

207

1-alkenes produced at 48 h. Although yeasts have been extensively explored for

208

production of fatty acid derived hydrocarbons, the titers are still very low and most

209

products accumulate intracellularly, which limits future industrial application. Here,

210

our S. cerevisiae strain AE39 exhibited the highest reported production of 1-alkenes

211

from free fatty acids in yeast, and this represents a 10-fold improvement compared

212

to previously engineered S. cerevisiae cell factories11-15 (Table 1). Moreover, product



Page 10 of 22

213

secretion provides great benefits for industrial production as it will decrease the

214

separation costs.

215

In summary, we engineered a yeast cell factory for production and secretion of

216

1-alkenes by cofactor engineering, transporter engineering and dynamic enzyme

217

control, which enabled the highest reported titers of 1-alkenes by a eukaryotic cell

218

factory.

219 220

Table 1: Fatty acid-derived hydrocarbons in engineered eukaryotic cell factories Host

Product

Total (mg/L)

Extracellular (mg/L)

Ref.

S. cerevisiae Aspergillus carbonarius Y. lipolytica S. cerevisiae

Alkanes

0.1-3.2

0

11-13, 15

Alkanes

1.0

N.D.

31

N.D. 0

18

N. D

17

29.3

This study

S. cerevisiae S. cerevisiae 221

Alkanes 23.5 1-Alkenes 3.7 Medium chain 3.0 1-alkenes 1-Alkenes 35.3

N.D., not detected

222


14

Page 11 of 22 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


223

Methods

224

Yeast strains, plasmids and reagents. The plasmids and strains used in this study are listed

225

in the Tables S1 and S2, respectively. The primers (Table S3) were ordered from

226

Sigma-Aldrich. Genes oleT, PpundA, PfundB and PmundB (Table S7) were codon-optimized

227

for yeast expression and synthesized by Genscript or Invitrogen. PrimeStar DNA polymerase

228

was purchased from TaKaRa. Zymoprep™ Yeast Plasmid Miniprep II was supplied by Zymo

229

Research Corp. Restriction enzymes, DNA gel purification and plasmid extraction kits were

230

purchased from Thermo Fisher Scientific. Analytical standards for quantification of 1-alkenes

231

and fatty acid methyl esters (FAMEs) were supplied by Sigma-Aldrich.

232

Strain cultivation. Yeast strains were normally cultivated in YPD media consisting of 10 g/L

233

yeast extract (Merck Millipore), 20 g/L peptone (Difco), and 20 g/L glucose (Merck Millipore).

234

Strains containing URA3 and/or HIS3 based plasmids/cassettes were selected on synthetic

235

complete media without uracil or L-histidine (SC-URA, SC-HIS or SC-URA-HIS), which

236

consisted of 6.7 g/L yeast nitrogen base (YNB) without amino acids (Formedium), 20 g/L

237

glucose (Merck Millipore) and 0.77 g/L complete supplement mixture without corresponding

238

nutrition (CSM-URA, CSM-HIS or CSM-HIS-URA, Formedium). The URA3 maker was removed

239

and selected against on SC+5-FOA plates, which contained 6.7 g/L YNB, 0.77 g/L complete

240

supplement mixture and 0.8 g/L 5-fluoroorotic acid. Strains containing the amdSYM32

241

cassette were selected on SM+AC media (5 g/L (NH4)2SO4, 3 g/L KH2PO4, 0.5 g/L MgSO4∙7H2O,

242

6.6 g/L K2SO4, 0.6 g/L acetamide, 20 g/L glucose, trace metal and vitamin solutions33

243

supplemented with 40 mg/L histidine and/or 60 mg/L uracil if needed). Strains containing

244

the kanMX cassette were selected on YPD plates containing 200 mg/L G418 (Formedium).

245

Shake flask batch fermentations for production of alkenes, were carried out in minimal

246

medium containing 5 g/L (NH4)2SO4, 14.4 g/L KH2PO4, 0.5 g/L MgSO4∙7H2O, 30 g/L glucose,

247

trace metal and vitamin solutions33 supplemented with 40 mg/L histidine and/or 60 mg/L

248

uracil if needed. Cultures were inoculated, from 24 h precultures, at an initial OD600 of 0.1

249

using 15 mL minimal medium in 100 mL unbaffled shake flasks and cultivated at 200 rpm,

250

30 °C for 72 h.

251

Genetic engineering. All episomal vectors or genome-integrated pathways (Figure S6) were

252

constructed by the modular pathway engineering (MOPE) strategy.34 Briefly, genes,

253

promoters, and terminators were amplified from the yeast genome or the custom

254

synthesized templates (Table S4). Then, gene expression modules, consisting of a promoter,

255

a structural gene, a terminator, and the promoter of the next module for homologous

256

recombination, were assembled by one-pot fusion PCR (Table S5).34 The modules were gel



257

purified and transformed to the S. cerevisiae (Table S6). Deletion of GAL80 was performed

258

by using an amdSYM gene as selection marker. The deletion cassettes were constructed by

259

fusing 200-600 nucleotide homologous arms with the amdSYM expression module32 and the

260

clones were selected on a SM+AC media. The genomic manipulations were verified with

261

colony PCR by using the genomic DNA, which was prepared by a quick extraction method as

262

previously described.35

263

Product extraction and quantification. Intracellular 1-alkenes were extracted by a

264

microwave-based method modified from our previous study.36 Cell pellets were collected

265

from 5 mL cell cultures and subjected to freezer drying for 48 h. Freeze-dried cells were

266

mixed with 4 mL of chloroform-methanol (2:1, v/v) in an extraction glass tube containing 0.5

267

mg/L hexadecane as internal standard. The extraction tubes were vigorously vortexed and

268

then place in the microwave reaction vessel (12 cm X 3 cm I.D., 0.5 cm thickness; Milestone

269

Start D, Sorisole Bergamo, Italy) that contained 30 mL of Mili-Q water inside and then sealed

270

with a TFM screw cap. The vessel was heated using a microwave digestion system equipped

271

with a PRO-24 medium pressure high throughput rotor (Milestone). The temperature

272

programing of microwave extraction was ramped to 60 °C (from room temperature, using

273

800 W for 24 vessels) within 6 min and kept constant for 10 min. After the samples were

274

cooled down to room temperature, 1 mL of NaCl (0.73% w/v) was added and then the

275

samples were vortexed vigorously. Thereafter, the samples were centrifuged at 1000 x g for

276

10 min allowing for phase separation and the organic phase was transferred into a new

277

clean extraction tube. The extracted sample was then pre-concentrated by drying under

278

vacuum, and re-suspended with 200 µL n-hexane.

279

The extracellular 1-alkenes were extracted from the supernatant by using n-hexane. 2 mL

280

supernatant were added to 2 mL hexane containing 1 mg/L hexadecane and the mixtures

281

were shaken for 30 min by using a vortex mixer (1000 rpm). The samples were centrifuged at

282

2000 x g for 10 min allowing for phase separation and the organic phase was transferred into

283

a new clean extraction tube for analysis. The intracellular and extracellular extraction

284

solvents were analysed by a GC-FID (Focus GC with a flame ionization detector (FID), Thermo

285

Fisher Scientific) equipped with a Zebron ZB-5MS GUARDIAN capillary column (30 m x 0.25

286

mm x 0.25 µm, Phenomenex). The GC program for 1-alkenes was as follows: initial

287

temperature of 50 °C, hold for 5 min; then ramp to 310 °C at a rate of 10 °C per min and hold

288

for 6 min. The temperature of inlet and detection were kept at 250 °C and 300 °C,

289

respectively. The flow rate of the carrier gas (nitrogen) was set to 1.0 mL per minute, and

290

data were analyzed by using the Xcalibur software.


Page 12 of 22

Page 13 of 22 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


291

Free fatty acids were simultaneously extracted and methylated by dichloromethane

292

containing methyl iodide as methyl donor12, 37. Cell cultures from shake flask were mixed

293

well and 100 µl cell cultures were diluted 2-fold with water, then 10 µl base catalyst 40%

294

tetrabutylammonium was added immediately followed by addition of 200 µl

295

dichloromethane containing 200 mM methyl iodide as methyl donor and 100 mg/L

296

pentadecanoic acid as an internal standard. The mixtures were shaken for 30 min at 1400

297

rpm by using a vortex mixer, and then centrifuged at 3500 g to promote phase separation.

298

160 µl dichloromethane layer was transferred into a GC vial with glass insert, and

299

evaporated 4 hours to dryness. The extracted methyl esters were re-suspended in 160 µl

300

hexane and then analysed by GC-FID. The GC program was as follows: initial temperature of

301

40 °C, hold for 2 min; ramp to 130 °C at a rate of 30 °C per minute, then raised to 280 °C at a

302

rate of 10 °C per min and hold for 3 minutes. The temperature of inlet and detection were

303

kept at 250 °C and 300 °C, respectively. Final quantification was performed using the

304

Xcalibur software.

305

Fluorescence microscopy analysis. For UndB localization analysis, the C-termini of the

306

proteins were fused to a green fluorescent protein (GFP). The encoding genes were

307

transformed into the yeast strain YJZ304. The cells were cultivated in SC-URA or minimal

308

media for 48 h at 30 °C, 200 rpm. 3 µL of the cell cultures were dropped onto microscope

309

slides and then viewed with a LEICA DMI4000B microscope (Leica Microsystems CMS

310

GmbH).

311

SUPPORTING INFORMATION

312

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

313

website at DOI: xxx

314

Strains, plasmids, and gene sequences used in this study; and other supporting

315

figures described in the text (PDF)

316 317

AUTHOR INFORMATION

318 319 320 321 322 323 324 325

Corresponding Author Yongjin J. Zhou: Tel/Fax: +86 (0)411 84771060, E-mail: [email protected] Jens Nielsen: Tel: +46 (0)31 772 3804, Fax: +46 (0)31 772 3801, Email: [email protected] ORCID Yongjin J. Zhou: 0000-0002-2369-3079. Zhiwei Zhu: 0000-0002-2925-5758



326 327 328

Jens Nielsen: 0000-0002-9955-6003

329

Y.J.Z and J.N. conceived the study and designed the experiments. Y.J.Z performed all

330

experiments and analyzed the data. Y.H. and Z.Z participated in the genetic

331

engineering and product quantification. V.S. assisted with data analysis and

332

interpretation. Y.J.Z and J.N. wrote the manuscript. All authors revised and approved

333

the manuscript.

334

Notes

335

The authors declare no competing financial interest

Author Contributions

336 337 338

Acknowledgements

339

The authors acknowledge funding from the Novo Nordisk Foundation, the Knut and

340

Alice Wallenberg Foundation, FORMAS and Vetenskapsrådet (to J.N.), and research

341

grant from Dalian Institute of Chemicals Physics, CAS (Grant: DICP DMTO201701 to

342

Y.J.Z.).

343 344

References

345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363

[1] Pfleger, B. F., Gossing, M., and Nielsen, J. (2015) Metabolic engineering strategies for microbial synthesis of oleochemicals, Metab. Eng. 29, 1-11. [2] Becker, J., and Wittmann, C. (2015) Advanced biotechnology: metabolically engineered cells for the bio-based production of chemicals and fuels, materials, and health-care products, Angew Chem. Int. Ed. 54, 3328-3350. [3] Liu, Q., Wu, K., Cheng, Y., Lu, L., Xiao, E., Zhang, Y., Deng, Z., and Liu, T. (2015) Engineering an iterative polyketide pathway in Escherichia coli results in single-form alkene and alkane overproduction, Metab. Eng. 28, 82-90. [4] Mendez-Perez, D., Begemann, M. B., and Pfleger, B. F. (2011) Modular synthase-encoding gene involved in alpha-olefin biosynthesis in Synechococcus sp. strain PCC 7002, Appl Environ Microb 77, 4264-4267. [5] Rude, M. A., Baron, T. S., Brubaker, S., Alibhai, M., Del Cardayre, S. B., and Schirmer, A. (2011) Terminal olefin (1-alkene) biosynthesis by a novel P450 fatty acid decarboxylase from Jeotgalicoccus species, Appl Environ Microb 77, 1718-1727. [6] Rui, Z., Li, X., Zhu, X., Liu, J., Domigan, B., Barr, I., Cate, J. H., and Zhang, W. (2014) Microbial biosynthesis of medium-chain 1-alkenes by a nonheme iron oxidase, Proc. Natl. Acad. Sci. U S A 111, 18237-18242. [7] Rui, Z., Harris, N. C., Zhu, X. J., Huang, W., and Zhang, W. J. (2015) Discovery of a Family of Desaturase-Like Enzymes for 1-Alkene Biosynthesis, ACS Catal. 5, 7091-7094.


Page 14 of 22

Page 15 of 22 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


364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405

[8] Herman, N. A., and Zhang, W. (2016) Enzymes for fatty acid-based hydrocarbon biosynthesis, Curr. Opin. Chem. Biol. 35, 22-28. [9] Mattanovich, D., Sauer, M., and Gasser, B. (2014) Yeast biotechnology: teaching the old dog new tricks, Microb. Cell Fact. 13, 34. [10] Caspeta, L., Chen, Y., Ghiaci, P., Feizi, A., Buskov, S., Hallstrom, B. M., Petranovic, D., and Nielsen, J. (2014) Altered sterol composition renders yeast thermotolerant, Science 346, 75-78. [11] Buijs, N. A., Zhou, Y. J., Siewers, V., and Nielsen, J. (2015) Long-chain alkane production by the yeast Saccharomyces cerevisiae, Biotechnol. Bioeng. 112, 1275-1279. [12] Zhou, Y. J., Buijs, N. A., Zhu, Z., Qin, J., Siewers, V., and Nielsen, J. (2016) Production of fatty acid-derived oleochemicals and biofuels by synthetic yeast cell factories, Nat. Commun. 7, 11709. [13] Zhou, Y. J., Buijs, N. A., Zhu, Z., Gomez, D. O., Boonsombuti, A., Siewers, V., and Nielsen, J. (2016) Harnessing yeast peroxisomes for biosynthesis of fatty-acid-derived biofuels and chemicals with relieved side-pathway competition, J. Am. Chem. Soc. 138, 15368-15377. [14] Chen, B., Lee, D. Y., and Chang, M. W. (2015) Combinatorial metabolic engineering of Saccharomyces cerevisiae for terminal alkene production, Metab. Eng. 31, 53-61. [15] Foo, J. L., Susanto, A. V., Keasling, J. D., Leong, S. S., and Chang, M. W. (2017) Whole-cell biocatalytic and de novo production of alkanes from free fatty acids in Saccharomyces cerevisiae, Biotechnol. Bioeng. 114, 232-237. [16] Kang, M. K., Zhou, Y. J., Buijs, N. A., and Nielsen, J. (2017) Functional screening of aldehyde decarbonylases for long-chain alkane production by Saccharomyces cerevisiae, Microb. Cell Fact. 16, 74. [17] Zhu, Z., Zhou, Y. J., Kang, M. K., Krivoruchko, A., Buijs, N. A., and Nielsen, J. (2017) Enabling the synthesis of medium chain alkanes and 1-alkenes in yeast, Metab. Eng. 44, 81-88. [18] Xu, P., Qiao, K. J., Ahn, W. S., and Stephanopoulos, G. (2016) Engineering Yarrowia lipolytica as a platform for synthesis of drop-in transportation fuels and oleochemicals, Proc. Natl. Acad. Sci. U S A 113, 10848-10853. [19] Blazeck, J., Liu, L., Knight, R., and Alper, H. S. (2013) Heterologous production of pentane in the oleaginous yeast Yarrowia lipolytica, J. Biotechnol. 165, 184-194. [20] Liu, Y., Wang, C., Yan, J., Zhang, W., Guan, W., Lu, X., and Li, S. (2014) Hydrogen peroxide-independent production of alpha-alkenes by OleTJE P450 fatty acid decarboxylase, Biotechnol. Biofuels 7, 28. [21] Dennig, A., Kuhn, M., Tassoti, S., Thiessenhusen, A., Gilch, S., Bulter, T., Haas, T., Hall, M., and Faber, K. (2015) Oxidative decarboxylation of short-chain fatty acids to 1-alkenes, Angew Chem. Int. Ed. 54, 8819-8822. [22] Sibbesen, O., De Voss, J. J., and Montellano, P. R. (1996) Putidaredoxin reductase-putidaredoxin-cytochrome p450cam triple fusion protein. Construction of a self-sufficient Escherichia coli catalytic system, The Journal of biological chemistry 271, 22462-22469.



406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449

[23] Heux, S., Cachon, R., and Dequin, S. (2006) Cofactor engineering in Saccharomyces cerevisiae: Expression of a H2O-forming NADH oxidase and impact on redox metabolism, Metab. Eng. 8, 303-314. [24] Peng, B., Williams, T. C., Henry, M., Nielsen, L. K., and Vickers, C. E. (2015) Controlling heterologous gene expression in yeast cell factories on different carbon substrates and across the diauxic shift: a comparison of yeast promoter activities, Microb. Cell Fact. 14, 91. [25] Lee, J. J., Chen, L., Cao, B., and Chen, W. N. (2016) Engineering Rhodosporidium toruloides with a membrane transporter facilitates production and separation of carotenoids and lipids in a bi-phasic culture, Appl. Microbiol. Biotechnol. 100, 869-877. [26] Jin, G., Yang, F., Hu, C., Shen, H., and Zhao, Z. K. (2012) Enzyme-assisted extraction of lipids directly from the culture of the oleaginous yeast Rhodosporidium toruloides, Bioresour. Technol. 111, 378-382. [27] Dunlop, M. J., Dossani, Z. Y., Szmidt, H. L., Chu, H. C., Lee, T. S., Keasling, J. D., Hadi, M. Z., and Mukhopadhyay, A. (2011) Engineering microbial biofuel tolerance and export using efflux pumps, Mol. Syst. Biol. 7. [28] Doshi, R., Nguyen, T., and Chang, G. (2013) Transporter-mediated biofuel secretion, Proc. Natl. Acad. Sci. U S A 110, 7642-7627. [29] Schaffer, J. E., and Lodish, H. F. (1994) Expression cloning and characterization of a novel adipocyte long chain fatty acid transport protein, Cell 79, 427-436. [30] Weinhandl, K., Winkler, M., Glieder, A., and Camattari, A. (2014) Carbon source dependent promoters in yeasts, Microb. Cell Fact. 13, 5. [31] Sinha, M., Weyda, I., Sorensen, A., Bruno, K. S., and Ahring, B. K. (2017) Alkane biosynthesis by Aspergillus carbonarius ITEM 5010 through heterologous expression of Synechococcus elongatus acyl-ACP/CoA reductase and aldehyde deformylating oxygenase genes, AMB Express 7, 18. [32] Solis-Escalante, D., Kuijpers, N. G., Bongaerts, N., Bolat, I., Bosman, L., Pronk, J. T., Daran, J. M., and Daran-Lapujade, P. (2013) amdSYM, a new dominant recyclable marker cassette for Saccharomyces cerevisiae, FEMS Yeast Res. 13, 126-139. [33] Verduyn, C., Postma, E., Scheffers, W. A., and Van Dijken, J. P. (1992) Effect of benzoic acid on metabolic fluxes in yeasts: a continuous-culture study on the regulation of respiration and alcoholic fermentation, Yeast 8, 501-517. [34] Zhou, Y. J., Gao, W., Rong, Q., Jin, G., Chu, H., Liu, W., Yang, W., Zhu, Z., Li, G., Zhu, G., Huang, L., and Zhao, Z. K. (2012) Modular pathway engineering of diterpenoid synthases and the mevalonic acid pathway for miltiradiene production, J. Am. Chem. Soc. 134, 3234-3241. [35] Looke, M., Kristjuhan, K., and Kristjuhan, A. (2011) Extraction of genomic DNA from yeasts for PCR-based applications, Biotechniques 50, 325-328. [36] Khoomrung, S., Chumnanpuen, P., Jansa-Ard, S., Stahlman, M., Nookaew, I., Boren, J., and Nielsen, J. (2013) Rapid quantification of yeast lipid using microwave-assisted total lipid extraction and HPLC-CAD, Anal. Chem. 85, 4912-4919. [37] Haushalter, R. W., Kim, W., Chavkin, T. A., The, L., Garber, M. E., Nhan, M., Adams, P. D., Petzold, C. J., Katz, L., and Keasling, J. D. (2014) Production of anteiso-branched fatty


Page 16 of 22

Page 17 of 22 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


450 451

acids in Escherichia coli; next generation biofuels with improved cold-flow properties, Metab. Eng. 26, 111-118.

452


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

Page 18 of 22

Figure for Table of Contents

Dynamic engineering

Suger Free fatty acid

1-Alkene

1-Alkene


Page 19 of 22 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


&]PµŒ í

%LRPDVV GHULYHG VXJDU

$FHW\O &R$ 1$'+

POX1

)DWW\ DF\O &R$ FAA1,4

)UHH IDWW\ DFLG 1$' 1$'+

$ONHQH

3GU

8QG%

$ONHQH


1-Alkenes (mg/L)

B A Acetyl-CoA POX1

OleT

Fatty acyl-CoA

OleT

C Free fatty acid

UndB

1-Alkene

UndA PmUndB PfUndB

Strain AE1

FAA1,4

H2O2

7 6 5 4 3 2 1 0

O2 + 2H+

UndA

8 7 6 5 4 3 2 1 0 pox1Δ faa4Δ faa1Δ

AE3

AE2

1-Alkenes

AE16

1.2

FAAs

Strain

1.0 0.8 0.6

0.4 0.2 0.0

‒ ‒ ‒

+ ‒ ‒

AE11 AE12


+ + ‒

+ ‒ +

AE14 AE15

+ + + AE16

FFAs (g/L)

Figure 2

Page 20 of 22

1-Alkenes (mg/L)

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


Page 21 of 22

Figure 3 Glucose

A

B

10

NADH

8

Acetyl-CoA

Free fatty acid NADPH Fnr

Fd/Fldred

NADP+

Fd/Fldox

UndB

Pdxred

NADH Pdr

Pdxox

NAD+

1-Alkenes (mg/L)

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


6 4 2 0 Control

Strain

AE16

Fd/Fnr AE22

1-Alkene


Fld/Fnr AE23

Pdx/Pnr AE21


B

Free fatty acid

C

(Intra) 1-alkene

UndB

10 μm

1-Alkenes (mg/L)

A

1-Alkene-AE38 1-Alkene-AE24 OD600-AE38 OD600-AE24

30 25

16 14 12 10 8 6 4 2 0

20 15 10

5 0 0 10 20 30 40 50 60 70 80 Time (h)

(Extra) 1-alkene

D

E

Extracellular-AE39 Extracellular-AE38 Total-AE39 Total-AE38

40 35 30 25 20 15 10 5 0

OD600

Figure 4

1-Alkenes (mg/L)

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

Page 22 of 22

48 h

48 h 1-Tridecene 1,7-Pentadecadiene

70 h

70 h

1-Pentadecene 1,8-Heptadecadiene 1-Heptadecene

0

10

20

30 40 50 Time (h)

60

70

AE38


AE39

Dynamic engineering of 1-alkenes biosynthesis and secretion in yeast

Recommend Documents