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Biotechnology and Biological Transformations

Significantly enhanced production of patchoulol in metabolically engineered Saccharomyces cerevisiae Bin Ma, Min Liu, Zhen-Hai Li, Xinyi Tao, Dong-Zhi Wei, and Feng-Qing Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b03456 • Publication Date (Web): 09 Jul 2019 Downloaded from pubs.acs.org on July 18, 2019

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Journal of Agricultural and Food Chemistry

1

Significantly enhanced production of patchoulol in metabolically engineered

2

Saccharomyces cerevisiae

3

Bin Ma#, Min Liu#, Zhen-Hai Li, Xinyi Tao, Dong-Zhi Wei, Feng-Qing Wang*

4 5

State key Lab of Bioreactor Engineering, Newworld Institute of Biotechnology, East

6

China University of Science and Technology, Shanghai 200237, China.

7 8

*Address

9

Email addresses for other authors:

correspondence to Feng-Qing Wang, [email protected]

10

Min Liu: [email protected]

11

Bin Ma: [email protected]

12

Zhen-Hai Li: [email protected]

13

Xinyi Tao: [email protected]

14

Dong-Zhi Wei: [email protected]

15 16

Bin Ma and Min Liu contributed equally to this work.

17 18

Declarations of interest: none

19 20 21 22

1

ACS Paragon Plus Environment


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Abstract

25

Patchoulol, a natural sesquiterpene compound, is widely used in perfumes and

26

cosmetics. Several strategies were adopted to enhance patchoulol production in

27

Saccharomyces cerevisiae: (i) farnesyl pyrophosphate (FPP) synthase and patchoulol

28

synthase were fused to increase the utilization of FPP precursor; (ii) expression of the

29

limiting genes of mevalonate pathway was enhanced; (iii) squalene synthase was

30

weakened by a glucose-inducible promoter of HXT1 (promoter for hexose transporter)

31

to reduce metabolic flux from FPP to ergosterol; (iv) farnesol biosynthesis was

32

inhibited to decrease the consumption of FPP. Glucose was used to balance the

33

trade-off between the competitive squalene and patchoulol pathways. The patchoulol

34

production was 59.2 ± 0.7 mg/L in flask shake, and final 466.8 ± 12.3 mg/L (20.5 ±

35

0.5 mg/g dry cell weight) combined with fermentation optimization, which was

36

7.8-fold higher than the reported maximum production. The work significantly

37

promoted the industrialization process of patchoulol production using bio-based

38

microbial platforms.

39 40

Keywords

41

Patchoulol; sesquiterpene; metabolic engineering; Saccharomyces cerevisiae;

42

terpenoids

43 44 2


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45 46

Introduction

47

Terpenoids are a large family of natural products with wide applications as the

48

precursors of pharmaceuticals, biofuels, and perfume ingredients, and so on.

49

Sesquiterpenoids, one of the largest subgroup of terpenoids, contain a large variety of

50

useful compounds.

51

pharmacological properties, such as neuroprotective, anti-inflammatory, and

52

anti-cancer activities. 5, 6 Typically, patchoulol represents 30-40 % of the total mass of

53

compounds in patchouli oil (with annual price ranging between 15 and 200 $/kg), an

54

essential oil commonly obtained from the leaves of Pogostemon cablin, which is

55

extensively used in the perfume and fragrance industry. 7 Patchoulol is an interesting

56

compound due to its potent pharmacological properties as well as its characteristic

57

aromas and flavors. 8, 9

58

Compared with plants, microorganisms exhibit several advantages in producing

59

terpenoids, such as land-saving, fast-growing and controllable culture conditions.

60

With the development of metabolic engineering and synthetic biology, many valuable

61

plant-derived terpenoids have been produced in microbial cell factories, such as

62

artemisinic acid,

63

Although naturally produced patchoulol can be obtained from plant materials,

64

patchoulol production using bio-based microbial platforms is an economical and

65

sustainable alternative. Until recently, based on the endogenous mevalonate (MVA)

66

pathway or 2-C-methyl-D-erythritol 4-phosphate (MEP)-pathway, heterologous

10

1-4

Patchoulol, a sesquiterpene alcohol, exhibits diverse

carotenoids,

11

ginsenosides,

12

and glycyrrhetinic acid.

2, 4

13

3



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production of patchoulol is accomplished by introducing patchoulol synthase gene in

68

Saccharomyces cerevisiae

69

moss Physcomitrella patens 5 and eukaryotic microalga Chlamydomonas reinhardtii. 6

70

Nevertheless, the highest titer obtained in C. glutamicum is only 60 mg/L in fed-batch

71

fermentation using complex medium supplemented with 40 g/L glucose monohydrate,

72

which is too low for industrial production.

73

required to achieve sufficient yields for industrial applications.

74

Of the available producing microorganisms, S. cerevisiae has emerged as the most

75

successful chassis due to a wide range of advantages. Unlike prokaryotes, S.

76

cerevisiae has multiple organelles providing various compartments and environments

77

for biosynthesis of terpenoids.

78

several advantages of ease of manipulation, ample engineering tools, depth of genetic

79

and physiological characterizations, high sugar catabolic rate, relatively fast growth

80

rate, GRAS status, and high tolerance against harsh industrial conditions.

81

cerevisiae thus has been developed as a platform microorganism for synthetic biology

82

and metabolic engineering.

83

In S. cerevisiae, farnesyl pyrophosphate (FPP) is formed by the condensation of two

84

molecules of isopentenyl pyrophosphate (IPP) and one molecule of dimethylallyl

85

pyrophosphate (DMAPP) generated through the MVA pathway, which is catalyzed by

86

the FPP synthase.

87

precursor of FPP by the catalysis of sesquiterpene synthases.

88

biosynthetic pathways have been widely investigated with the aim of controlling the

3

8, 14

and Corynebacterium glutamicum 9, as well as in the

4

9

Therefore, significant engineering is

As a model eukaryotic system, S. cerevisiae shows

1, 4

S.

Sesquiterpene products are generated from the prenylated 15

Isoprenoid

4


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89

pathway flux for product of interest. Many strategies have been proposed to engineer

90

S. cerevisiae toward the synthesis of terpene molecules, the major objectives of which

91

include the enhancement of precursor supplies and the optimization of metabolic

92

pathways by genome and pathway engineering. The general strategies of metabolic

93

engineering for synthesis of terpenes in S. cerevisiae are as follows: to enhance the

94

MVA pathway fluxes by overexpressing the key genes; to downregulate competitive

95

pathways by replacement of the native promoter with HXT1 (glucose inducible

96

promoter), MET3 (methionine repressible promoter), or CTR3 (copper repressible

97

promoter); to knock out or inhibit some negative regulators or uncharacterized targets;

98

to strengthen the terpene synthases or other rate-limiting enzymes associated with

99

terpene synthesis, either by overexpression or protein engineering. 1-4

100

In the present study, several metabolic engineering strategies were conducted to

101

enhance the production of patchoulol in S. cerevisiae. First, ERG20 (encoding FPP

102

synthase) and PTS (encoding patchoulol synthase) were fused to increase the

103

utilization of FPP precursor; second, the limiting genes tHMGR (encoding truncated

104

hydroxymethylglutary-CoA reductase), IDI1 (encoding isopentenyl diphosphate

105

δ-isomerase), and upc2-1 (encoding an activated allele of the UPC2 transcription

106

factor, UPC2-1) were integrated into the genome to enhance the flux of MVA

107

pathway; third, ERG9 (encoding squalene synthase) was weakened by a glucose

108

inducible promoter of HXT1 to reduce metabolic flux from FPP to ergosterol; fourth,

109

farnesol biosynthetic pathway was inhibited by knocking out DPP1 (encoding

110

phosphatidate phosphatase) and LPP1 (encoding phosphatidate phosphatase) to 5



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111

decrease the consumption of FPP. The main optimizing strategy and the biosynthesis

112

pathway of patchoulol in S. cerevisiae are shown in Figure 1. To develop a

113

cost-effective production process, a carbon source controlled three-stage fermentation

114

was conducted. Glucose was used to control the switch time between the competitive

115

squalene and patchoulol pathways, and to balance the trade-off between the two

116

pathways. The strategy achieved the production of patchoulol with a titer of 466.8 ±

117

12.3 in a 5 L bioreactor, which was 7.8-fold higher than the reported maximum

118

production (60 mg/L).

119

Materials and Methods

120

Strains, media and cell cultivation

121

S. cerevisiae BY4741 derived from S288c was used as the parent strain. YPD medium

122

(1% yeast extract, 2% peptone, and 2% glucose), YPDL medium (1% yeast extract,

123

2% peptone, 2% lactic acid, and 3% glycerol), and YPDG medium (1% yeast extract,

124

2% peptone, 1% glucose, and 1% glycerol) were used to cultivate yeast cells.

125

Escherichia coli DH5α (Invitrogen) was used for the construction of plasmids.

126

Recombinant E. coli was cultivated at 37℃ in Luria-Bertani (LB) medium

127

supplemented with 100 mg/L ampicillin (Sangon Biotech, China). Synthetic complete

128

medium minus the corresponding amino acids was used for the selection of

129

auxotrophic marker. The medium for fed-batch fermentation was composed of 25 g/L

130

glucose, 15 g/L (NH4)2SO4, 8 g/L KH2PO4, 3 g/L MgSO4, 0.72 g/L ZnSO4·7H2O, 10

131

mL/L trace metal solution, and 12 mL/L vitamin solution. To make cells grow better,

132

leucine (1 g/L), histidine (1 g/L), and methionine (1 g/L) were added in the medium. 6


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133

Feeding solution I was composed of 500 g/L glucose, 20 g/L peptone, and 10 g/L

134

yeast extract. Feeding solution II was composed of 250 g/L glucose, 250 g/L glycerol,

135

20 g/L peptone and 10 g/L yeast extract.

136

Construction of plasmids and strains

137

All the primers and plasmids used for plasmid construction are listed in Table 1 and

138

Table 2, respectively. The codon-optimized gene encoding PTS (GenBank ID:

139

AY508730) was synthesized by RUIMIANBIO (Shanghai, China). The genes of

140

tHMGR, IDI1, and upc2-1 were PCR-amplified from the genome of S. cerevisiae

141

4741. The fusion of ERG20 and PTS with a Gly-Ser-Gly (GSG) tag was constructed

142

according to a previous work,

143

gene tHMGR was amplified using primers tHMGR-F and tHMGR-R. The gene IDI1

144

was amplified using primers IDI1-F and IDI1-R. Genes tHMGR and IDI1 were

145

introduced into pESC-URA by BamH I/Sal I and EcoR I/Spe I, generating plasmid

146

pESCU-tHMGR-IDI.

147

UPC21-F/UPC21-UP-R and UPC21-R/UPC21-DN-F firstly. Then, the two parts were

148

separately

149

UPC21-R/UPC21-OL-F. Finally, the two parts were overlapping amplified using

150

primers UPC21-F and UPC21-R. The gene upc2-1 was introduced into pESC-URA

151

by Not I and Spe I, generating plasmid pESCU-UPC21. The gene PTS was amplified

152

using primers PTS-F and PTS-R. The genes tHMGR and PTS were introduced into

153

pESC-URA by BamH I/Sal I and EcoR I/Spe I, generating plasmid pESCU-tPTS. The

154

gene FPTS was amplified using primers FPTS-UF/FPTS-UR FPTS-DF/PTS-R, and

The

amplified

8

which generated a fusion protein called FPTS. The

gene

using

upc2-1

primers

was

amplified

using

UPC21-F/UPC21-OL-R

primers

and

7



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two parts were separately amplified using primers FPTS-UF/FPTS-OL-R and

156

FPTS-OL-F/PTS-R, and two parts were overlapping amplified using primers

157

FPTS-UF and PTS-R. The gene FPTS was introduced into pESC-URA by EcoR I and

158

Spe I, generating plasmid pESCU-FPTS. The genes tHMGR and FPTS were

159

introduced into pESC-URA by BamH I/Sal I and EcoR I/Spe I, generating plasmid

160

pESCU-tFPTS.

161

The gRNA-expressing plasmid p426-SNR52p-gRNA.CAN1.Y-SUP4t was purchased

162

from Addgene Inc (ID: 43803). The gRNA-expressing plasmid targeting GAL80 was

163

constructed by using p426-SNR52p-gRNA.CAN1.Y-SUP4t as the template. The

164

gRNA fragment was amplified using primers g80-F and g80-R. Then, seamless

165

cloning was carried out using this fragment, generating gRNA-expressing plasmid

166

pSCM-g80. With the same method, the gRNA-expressing plasmids targeting GAL1-7,

167

PERG9, DPP1 and LPP1 were constructed using primers g17-F/g17-R, gE9p-F/gE9p-R,

168

gDPP1-F/gDPP1-R and gLPP1-F/gLPP1-R, respectively.

169

The donors targeting GAL80 were amplified using primers gal80-UP-F/gal80-UP-R

170

and gal80-DN-F/gal80-DN-R. The donors targeting GAL1-7 were amplified using

171

primers GAL17-UP-F/GAL17-UP-R and GAL17-DN-F/GAL17-DN-R. The donors

172

targeting PERG9 were amplified using primers ERG9-Up-F/ERG9-Up-R and

173

ERG9-Dn-F/ERG9-Dn-R. The donors targeting DPP1 were amplified using primers

174

DPP1-UF/DPP1-UR and DPP1-DF/DPP1-DR. The donors targeting LPP1 were

175

amplified

LPP1-UF/LPP1-UR

and

176

TCYC1-tHMGR-PGAL1-PGAL10-IDI1-TADH1 cassette

was

using

primers

LPP1-DF/LPP1-DR. amplified

using

The

primers 8


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80-TADH1-F/80-CYC-R. The PGAL10-upc2-1-TADH1 cassette and promoter HXT1 were

178

amplified using primers GAL17-UPC21-F/GAL17-UPC21-R and HXT1pro-F/

179

HXT1pro-R, respectively. Plasmids of p414-TEF1p-Cas9-CYC1t (ID: 43802) and

180

p415-GalL-Cas9-CYC1t (ID: 43804) containing cas9 cassette were purchased from

181

Addgene Inc. The modification of p415-GalL-Cas9-CYC1t was conducted to replace

182

the promoter of Cas9 with PTEF1 as follows. First, plasmid p414-TEF1p-Cas9-CYC1t

183

was digested with Swa I and Spe I, and the fragment containing promoter PTEF1 was

184

collected. Plasmid p415-GalL-Cas9-CYC1t was digested with Swa I and Spe I, and

185

the fragment containing Cas9 was collected. Second, the two fragments were ligated

186

to generate plasmid pTCL, which was transformed into yeast using the Frozen-EZ

187

Yeast Transformation II kit (ZYMO RESEARCH, USA). The plasmid pTCL was

188

introduced into BY4741 to create strain 47419. The DNA assembly was conducted as

189

follows:

190

TCYC1-tHMGR-PGAL1-PGAL10-IDI1-TADH1 cassette and pSCM-g80. Second, cells were

191

plated on a SD-Leu-Ura (synthetic defined medium with glucose as carbon source

192

plus leucine and uracil omitted) plate to screen for leu2 and ura3 auxotrophic mutants.

193

Third, in order to drop out the gRNA-expressing plasmid pSCM-g80, the confirmed

194

mutant was cultured in liquid YPD medium and then streaked onto a SD-Leu-5-FOA

195

(synthetic defined medium with glucose as carbon source and 5-fluoroorotic acid

196

added, leucine omitted) plate to confirm the losing of the gRNA-expressing plasmid.

197

The confirmed recombinant yeast was named REL001. With the same method, the

198

recombinant yeasts REL002, REL003 and REL004 were also constructed. The

First,

47419

was

transformed

with

donors

targeting

GAL80,

9



Page 10 of 33

199

primers used for DNA assembly are listed in Table 1. Prime STAR HS DNA

200

polymerase (Takara, China) and T4 DNA ligase (Fermentas, USA) were applied for

201

DNA amplification and ligation. PCR product and Plasmid DNA were purified using

202

AxyPrep DNA gel extraction kit and Axygen plasmid mini prep kit (Axygen

203

Biosciences, Hangzhou, China).

204

Cultivation in shaking flask

205

The recombinant yeasts were precultured in 5 mL YPD at 30℃, 220 rpm for 24 h.

206

Precultures were inoculated to 50 mL YPGD or YPGL in 250 mL flasks at an initial

207

OD600 of 0.05 and were grown under the same condition. An overlay of 10 mL

208

dodecane was added to the flasks after 12 h.

209

Analysis of patchoulol, squalene, farnesol, and glucose

210

Samples from organic layer were centrifuged for 10 min at 6,000 g to determine the

211

level of sesquiterpenes during the course of fermentation. The patchoulol and farnesol

212

were analyzed by Gas Chromatography-Mass Spectrometer (GC-MS, Agilent 6890N

213

GC couple with 59751 MSD) using the HP-5 column (30 m X 0.25 mm, 0.25 μm film

214

thickness, Agilent, USA). Sample (1 μL) was injected in splitless mode. A constant

215

flow of 1.2 mL/min nitrogen was used as carrier gas. The injector temperature was

216

250℃. The initial oven temperature was 80℃. After 1 min, the oven temperature was

217

increased to 120℃ at the rate of 10℃/min, subsequently increased to 160℃ at the rate

218

of 3℃/min, and further to 270℃ at the rate of 10℃/min and was held for 5 min. The

219

patchoulol standard (P115183, Aladdin, Shanghai, China) and farnesol standard

220

(F113776, Aladdin, Shanghai, China) were used to identify the substances. Squalene 10


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221

in the cells was extracted using ethyl acetate and analyzed by GC-MS as described

222

above. Sample (1 μL) was injected and the split ratio was set to 5:1. A constant flow

223

of 1 mL/min nitrogen was used as carrier gas. The injector temperature was 280℃.

224

The initial oven temperature was 90℃. After 0.5 min, the oven temperature was

225

increased to 170℃ at the rate of 20℃/min and was held for 0.5 min. Subsequently, it

226

was increased to 190℃ at the rate of 10℃/min and was held for 0.5 min. Finally, the

227

oven temperature was increased to 280℃ at the rate of 20℃/min and was held for 10

228

min. The standard compound squalene (Sigma, Aldrich, St. Louis, MO) was dissolved

229

in ethyl acetate for standard curve preparation. Samples from water layer were

230

centrifuged for 10 min at 6,000 g to determine ethanol concentration. Ethanol was

231

analyzed by GC (Agilent 7820) using the DB-WAX column (30 m X 0.25 mm, 0.25

232

μm film thickness, Agilent, USA). A constant flow of 1 mL/min nitrogen was used as

233

carrier gas. The injector temperature was 180℃. The oven temperature was kept

234

120℃ for 10 min. The standard compound of ethanol (Titan, shanghai, China) was

235

dissolved in water and used for standard curve preparation. Glucose and glycerol

236

concentrations in the medium were determined using assay kits (ID: 361500 and

237

361320, Rsbio, China), respectively. All values were average of three independent

238

experiments. The statistical significance was calculated using Student’s t-test. A

239

p-value of ≤ 0.05 was considered statistically significant.

240

Fed-batch fermentation

241

The fed-batch fermentation was carried out in a 5 L bioreactor (5SJA-AUTO, BLBIO,

242

China) containing 2.8 L fermentation medium. The seed culture was prepared by 11



Page 12 of 33

243

inoculating the colonies into a 500 ml flask containing 100 ml YPD and culturing at

244

220 rpm and 30 oC for 20 h to an OD600 of 4-8. Four flasks of seed cultures were

245

transferred to the bioreactor. The temperature of fermentation was kept at 30℃. The

246

agitation speed was kept at 200-500 rpm with an airflow rate of 1 vvm to 2 vvm. The

247

pH was controlled at 5.5 by automated addition of 5 M ammonia hydroxide.

248

Moreover, 280 mL dodecane was added after 16 h of fermentation.

249

Results and Discussion

250

Engineering S. cerevisiae for the production of patchoulol

251

Generally, introduction of a biosynthetic pathway containing heterologous genes is

252

the first step for the production of pharmaceuticals, chemicals, and biofuels with a

253

bio-based microbial platform, moss or microalga. 5, 6, 8, 9 A patchoulol synthase (PTS)

254

from Pogostemon cablin could catalyze the conversion of FPP to the product of

255

patchoulol and other different by-products. 8, 15 Previous work has shown that coupled

256

farnesyl diphosphate synthase (ERG20) of yeast and patchoulol synthase (PTS) could

257

reduce the loss of FPP intermediate and increase the production of patchoulol.

258

order to test the capability of codon-optimized PTS and the fusion strategy for

259

patchoulol production, PTS and ERG20 were fused to generate a coupled enzyme of

260

FPTS. PTS and FPTS were introduced into BY4741 by a high-copy plasmid

261

pESC-URA containing tHMGR, generating the recombinant strains 4741-tPTS and

262

4741-tFPTS, respectively. GC-MS analysis indicated that patchoulol was successfully

263

synthesized in strains 4741-tPTS and 4741-tFPTS, as shown in Figure 2. A and B.

264

The production of patchoulol by 4741-tPTS was determined to be 1.0 ± 0.1 mg/L and

8

In

12


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265

the fusion strategy led to one-fold increase to 2.0 ± 0.1 mg/L in 4741-tFPTS with

266

galactose as carbon source (Figure 2. C). Thus, this strategy of fusing FPTS to PTS

267

could take advantage of the FPP pool and redirect the flux toward patchoulol

268

production in yeast.

269

Enhancing patchoulol production by repressing the limiting steps

270

In the present work, to enhance the FPP supply, several genes responsible for FPP

271

synthesis were upregulated to increase the metabolic flux to patchoulol. All

272

modifications in the genome were conducted utilizing the CRISPR/Cas9 system and

273

homologous recombination in S. cerevisiae. The HMG-CoA reductase was the major

274

rate-limiting enzyme of MVA pathway in yeast. IDI1 is an essential single-copy gene

275

that encodes isopentenyl diphosphate isomerase.

276

transcriptional regulator to GAL promoters when induced by glucose, tHMGR and

277

IDI1 were introduced into GAL80 loci to knock out the GAL80 gene, generating

278

engineered strain REL001. Disruption of GAL80 gene could switch the regulatory

279

sugar from galactose to glucose and eliminate the dependency of gene expression on

280

galactose induction, which could avoid the utilization of high-cost galactose, making

281

it more suitable for industrial purposes.

282

proteins, in some cases, overexpression of positive regulators is an effective method

283

for metabolic engineering. As a representative example, overexpression of UPC2-1,

284

an active mutant of UPC2 transcription factor involved in increasing the expression of

285

genes for sterol uptake and MVA pathway, could result in significantly improved

286

production of artemisinin precursor and bisabolene.

17

16

Since GAL80 is a negative

Besides the modification of the regulated

4, 10

Thus, we introduced upc2-1 13



Page 14 of 33

287

gene into GAL1-7 loci in strain REL001, generating strain REL002.

288

The plasmid pESCU-FPTS expressing FPTS was transformed into REL001 and

289

REL002, generating strains REL001-FPTS and REL002-FPTS, respectively. As

290

expected, GC analysis showed that the production of patchoulol was significantly

291

enhanced. Production of patchoulol in strain REL001-FPTS was 10.7 ± 0.1 mg/L (2.8

292

± 0.1 mg/g dry cell weight (DCW)), while increasing up to 18.9 ± 2.1 mg/L (4.0 ± 0.4

293

mg/g DCW) in strain REL002-FPTS with glucose as carbon source (Table 3).

294

Production of squalene in strains REL001-FPTS and REL002-FPTS was also

295

determined, reaching 314.8 ± 8.8 mg/L and 361.6 ± 11.9 mg/L, respectively. The

296

results demonstrated that the throughput capacity of MVA pathway towards FPP was

297

strengthened by overexpressing the tHMGR, IDI1 and upc2-1 genes.

298

Enhancing patchoulol production by repressing the competitive step

299

In S. cerevisiae, MVA pathway is the only pathway for the biosynthesis of isoprenoid

300

precursors, which can originally lead to the formation of ergosterol as the major

301

product in yeast cells. In the case of ergosterol biosynthesis, FPP, direct precursor for

302

patchoulol, is converted to squalene by squalene synthase (encoded by ERG9 gene).

303

Squalene can be converted to ergosterol, which is essential for yeast growth. Since

304

yeast cells are unable to assimilate exogeneous ergosterol during aerobic growth,

305

ERG9 gene cannot be deleted completely. In order to enhance the availability of FPP

306

for synthesis of patchoulol, the native promoter of ERG9 gene was replaced with a

307

sugar-responsive promoter HXT1 herein, generating strain REL003. The plasmid

308

pESCU-FPTS was transformed into REL003, generating strain REL003-FPTS. As 14


Page 15 of 33


309

shown in Figure 3, repression of ERG9 at the late stage reduced the growth of yeast

310

cells because of the decreased flux towards ergosterol. Production of patchoulol in

311

strain REL003-FPTS increased up to 59.2 ± 0.7 mg/L (Table 3). Meanwhile, the titer

312

of squalene decreased to 6.5 ± 0.3 mg/L. In terms of DCW, the production of

313

patchoulol in REL003-FPTS was almost 8-fold higher (31.3 ± 0.3 mg/g DCW) than

314

that of strain REL002-FPTS (4.0 ± 0.4 mg/g DCW).

315

Promoter HXT1 (promoter for hexose transporter) was high-glucose induced and

316

low-glucose repressed, which was weaker than native ERG9 promoter in low-glucose

317

condition.

318

promoters was repressed by glucose and was induced by glucose-limiting conditions.

319

At the beginning of culture, expression of ERG9 gene controlled by HXT1 was high

320

due to the abundant glucose. 11, 18 When glucose was exhausted, expression of ERG9

321

was repressed and patchoulol pathway was highly expressed. Therefore, balancing the

322

trade-off between the two pathways was important to improve patchoulol production.

323

Nevertheless, the repressed expression of ERG9 resulted in lower squalene production

324

and cell density of REL003-FPTS, compared with REL002-FPTS (Table 3 and Figure

325

3). It was indicated that early repression of ERG9 or induction of the patchoulol

326

pathway might be toxic to the cells. Similar results were also found for α-santalene

327

and β-carotene. 11

18

In yeast, for a GAL80 disrupted regulation system, the activity of GAL

328

Genes DPP1 and LPP1, encoding two phosphatases, were considered to be

329

responsible for conversion of FPP to farnesol. Previous report indicated that deletion

330

of DPP1 and LPP1 could increase the production of sesquiterpene.

19

Thus, DPP1 15



Page 16 of 33

331

and LPP1 were knocked out in REL003, generating strain REL004. The plasmid

332

pESCU-FPTS was transformed into REL004, generating strain REL004-FPTS.

333

Compared with farnesol titer in REL003-FPTS (44.3 ± 1.3 mg/L), the titer of

334

farnesol was decreased significantly in REL004-FPTS (13.9 ± 0.5 mg/L). However,

335

the production of patchoulol did not increase as anticipated (Table 3). Surprisingly, it

336

was a bit lower in REL004-FPTS, compared with that of REL003-FPTS (Table 3).

337

The similar result was also observed in the previous study.

338

previously tried to decrease the flux toward farnesol by deleting the LPP1 and DPP1

339

genes for biosynthesis of patchoulol in yeast, which did not increase the

340

sesquiterpene production in the ERG9-repressed strain. 8 Beside LPP1 and DPP1 for

341

farnesol and squalene synthase (ERG9) for squalene, FPP is also the substrate for

342

several other enzymes, including hexaprenyl diphosphate synthetase (COQ1), heme

343

A farnesyltransferase (COX10), and the cis-prenyltransferases (RER2 and SRT1).

344

Thus, we speculated that the FPP flux might redirect from LPP1 and DPP1 to other

345

enzymes, but not to PTS for patchoulol production.

8

Albertsen et al have

8

346

Optimizing fermentation process to improve the patchoulol production

347

When glucose-inducible HXT1 was combined with the glucose-repressible modified

348

GAL regulation system, glucose concentration could decide the expression level of the

349

HXT1-controlled squalene pathway and the GAL promoter-controlled patchoulol

350

pathway. To develop a cost-effective production process, carbon source controlled

351

three-stage fermentation was conducted in a 5 L fermenter. Glucose was used to

352

control the switch time between the squalene and patchoulol pathways. To explore the 16


Page 17 of 33


353

highest performance of the recombinant yeast REL003-FPTS, a three-stage fed-batch

354

fermentation with patchoulol capture using a two-phase dodecane-culture was

355

employed, as shown in Figure 4.A.

356

At the first stage, feeding-medium I (1 L) with high glucose concentration was fed at

357

12 h to achieve rapid cell growth. The OD600 reached 42.7 and the production of

358

patchoulol was 6.36 ± 0.7 mg/g DCW at the end of the stage 1. Previous report

359

indicated that by reducing glucose in media and adding glycerol as carbon source, the

360

repression of GAL promoter could be alleviated and more cellular resources could be

361

devoted to the transcription of exogenous genes controlled by GAL promoters.

362

Moreover, the switch time from squalene pathway to the assembled pathway could be

363

earlier when compared to using glucose as a sole carbon source. 18 At the second stage,

364

feeding-medium II (500 mL) with lower concentration of glucose and glycerol was

365

added at 60 h. As shown in Figure 4.B, the production of patchoulol was quickly

366

improved (reached 203 ± 15.3 mg/L, 9.97 mg/g ± 0.8 DCW), while the cell growth

367

rate was decreased. Glycerol concentration was increased from 9.8 ± 0.2 g/L (60 h)

368

to 29.3 ± 0.5 g/L (72 h), and was almost unchanged until the end of fermentation.

369

Ethanol is a commonly used carbon source for the production of terpenoids in yeast,

370

such as amorpha-4,11-diene (precursor of artemisinin)

371

which can increase the titer and yield of terpenoids on biomass tremendously in

372

comparison to glucose. 14 At the third stage, ethanol was fed into medium at 96 h and

373

dominated as the sole carbon source for patchoulol production until the end of

374

fermentation. Finally, 466.8 ± 12.3 mg/L patchoulol was obtained (with volumetric

10

and protopanoxadiol

11

20,

17



Page 18 of 33

375

productivity of 66.7 ± 1.8 mg/L/d), and the final content of patchoulol reached 20.5 ±

376

0.5 mg/g DCW. The titer, yield, and volumetric productivity of patchoulol obtained in

377

this work was compared to those obtained in previous reports, such as in S. cerevisiae,

378

C. glutamicum and C. reinhardtii, as shown in Table 4. Our results indicated that the

379

titer, yield, and volumetric productivity of patchoulol obtained in this work were the

380

highest. Combined with fermentation optimization, the final titer of patchoulol was

381

7.8-fold higher than the reported maximum production (60 mg/L). 9 Nevertheless, low

382

OD600 (up to 50) was obtained in the high-density fermentation (Figure 4), which

383

might be due to the repressed expression of ERG9 and lower squalene production. To

384

our limited knowledge, this is the highest titer of patchoulol obtained in microbial cell

385

factories. The work significantly promotes the industrialization process of patchoulol

386

production using bio-based microbial platforms.

387 388

Acknowledgments

389

This work was financially supported by the National Natural Science Foundation of

390

China (No. 31500043), the Natural Science Foundation of Shanghai (No.

391

19ZR1473000), the General Project of Beijing Municipal Education Commission (No.

392

SQKM201311417004), the Fundamental Research Funds for the Central Universities

393

(No. 22221818014), and the Open Funding Project of the State Key Laboratory of

394

Bioreactor Engineering.

395

References

396

1.

Bian, G.; Deng, Z.; Liu, T., Strategies for terpenoid overproduction and new 18


Page 19 of 33


397

terpenoid discovery. Curr. Opin. Biotechnol. 2017, 48, 234-241.

398

2.

399

biology for engineering isoprenoid production in yeast. Curr. Opin. Chem. Biol. 2017,

400

40, 47-56.

401

3.

402

engineering of Saccharomyces cerevisiae. Crit. Rev. Biotechnol. 2017, 37, 974-989.

403

4.

404

Saccharomyces cerevisiae: New tools and their applications. Metab. Eng. 2018, 50,

405

85-108.

406

5.

407

the moss Physcomitrella patens to produce the sesquiterpenoids patchoulol and

408

alpha/beta-santalene. Front. Plant Sci. 2014, 5, 636.

409

6.

410

Hubner, W.; Huser, T.; Kruse, O., Efficient phototrophic production of a high-value

411

sesquiterpenoid from the eukaryotic microalga Chlamydomonas reinhardtii. Metab.

412

Eng. 2016, 38, 331-343.

413

7.

414

supercritical carbon dioxide. J. Chem. Eng. Data 2007, 52, 235-238.

415

8.

416

Mortensen, U. H., Diversion of flux toward sesquiterpene production in

417

Saccharomyces cerevisiae by fusion of host and heterologous enzymes. Appl. Environ.

418

Microbiol. 2011, 77, 1033-1040.

Vickers, C. E.; Williams, T. C.; Peng, B.; Cherry, J., Recent advances in synthetic

Paramasivan, K.; Mutturi, S., Progress in terpene synthesis strategies through

Lian, J.; Mishra, S.; Zhao, H., Recent advances in metabolic engineering of

Zhan, X.; Zhang, Y. H.; Chen, D. F.; Simonsen, H. T., Metabolic engineering of

Lauersen, K. J.; Baier, T.; Wichmann, J.; Wordenweber, R.; Mussgnug, J. H.;

Hybertson, B. M., Solubility of the sesquiterpene alcohol patchoulol in

Albertsen, L.; Chen, Y.; Bach, L. S.; Rattleff, S.; Maury, J.; Brix, S.; Nielsen, J.;

19



Page 20 of 33

419

9.

420

M.; Peters-Wendisch, P.; Kruse, O.; Wendisch, V. F., Patchoulol Production with

421

Metabolically Engineered Corynebacterium glutamicum. Genes 2018, 9, 1-15.

422

10. Westfall, P. J.; Pitera, D. J.; Lenihan, J. R.; Eng, D.; Woolard, F. X.; Regentin, R.;

423

Horning, T.; Tsuruta, H.; Melis, D. J.; Owens, A.; Fickes, S.; Diola, D.; Benjamin, K.

424

R.; Keasling, J. D.; Leavell, M. D.; McPhee, D. J.; Renninger, N. S.; Newman, J. D.;

425

Paddon, C. J., Production of amorphadiene in yeast, and its conversion to

426

dihydroartemisinic acid, precursor to the antimalarial agent artemisinin. P. Natl. Acad.

427

Sci. 2012, 109, 111-118.

428

11. Xie, W. P.; Ye, L. D.; Lv, X. M.; Xu, H. M.; Yu, H. W., Sequential control of

429

biosynthetic pathways for balanced utilization of metabolic intermediates in

430

Saccharomyces cerevisiae. Metab. Eng. 2015, 28, 8-18.

431

12. Wang, P.; Wei, W.; Ye, W.; Li, X.; Zhao, W.; Yang, C.; Li, C.; Yan, X.; Zhou, Z.,

432

Synthesizing ginsenoside Rh2 in Saccharomyces cerevisiae cell factory at

433

high-efficiency. Cell Discov. 2019, 5, 5-18.

434

13. Zhu, M.; Wang, C. X.; Sun, W. T.; Zhou, A. Q.; Wang, Y.; Zhang, G. L.; Zhou,

435

X. H.; Huo, Y. X.; Li, C., Boosting 11-oxo-beta-amyrin and glycyrrhetinic acid

436

synthesis in Saccharomyces cerevisiae via pairing novel oxidation and reduction

437

system from legume plants. Metab. Eng. 2018, 45, 43-50.

438

14. Gruchattka, E.; Kayser, O., In Vivo Validation of In Silico Predicted Metabolic

439

Engineering Strategies in Yeast: Disruption of alpha-Ketoglutarate Dehydrogenase

440

and Expression of ATP-Citrate Lyase for Terpenoid Production. PloS one 2015, 10.

Henke, N. A.; Wichmann, J.; Baier, T.; Frohwitter, J.; Lauersen, K. J.; Risse, J.

20


Page 21 of 33


441

15. Asadollahi, M. A.; Maury, J.; Moller, K.; Nielsen, K. F.; Schalk, M.; Clark, A.;

442

Nielsen, J., Production of plant sesquiterpenes in Saccharomyces cerevisiae: effect of

443

ERG9 repression on sesquiterpene biosynthesis. Biotechnol. Bioeng. 2008, 99,

444

666-677.

445

16. Zhao, Y.; Fan, J.; Wang, C.; Feng, X.; Li, C., Enhancing oleanolic acid

446

production in engineered Saccharomyces cerevisiae. Bioresour. Technol. 2018, 257,

447

339-343.

448

17. Wang, F.; Lv, X.; Xie, W.; Zhou, P.; Zhu, Y.; Yao, Z.; Yang, C.; Yang, X.; Ye,

449

L.; Yu, H., Combining Gal4p-mediated expression enhancement and directed

450

evolution of isoprene synthase to improve isoprene production in Saccharomyces

451

cerevisiae. Metab. Eng. 2017, 39, 257-266.

452

18. Xie, W. P.; Liu, M.; Lv, X. M.; Lu, W. Q.; Gu, J. L.; Yu, H. W., Construction of

453

a Controllable beta-Carotene Biosynthetic Pathway by Decentralized Assembly

454

Strategy in Saccharomyces cerevisiae. Biotechnol. Bioeng. 2014, 111, 125-133.

455

19. Zhang, C.; Liu, J.; Zhao, F.; Lu, C.; Zhao, G. R.; Lu, W., Production of

456

sesquiterpenoid zerumbone from metabolic engineered Saccharomyces cerevisiae.

457

Metab. Eng. 2018, 49, 28-35.

458

20. Zhao, F. L.; Du, Y. H.; Bai, P.; Liu, J. J.; Lu, W. Y.; Yuan, Y. J., Enhancing

459

Saccharomyces cerevisiae reactive oxygen species and ethanol stress tolerance for

460

high-level production of protopanoxadiol. Bioresour. Technol. 2017, 227, 308-316.

461

21



Page 22 of 33

Figure captions Figure 1 Scheme for biosynthesis pathway of patchoulol in S. cerevisiae. Green arrow represents the transformation step for patchoulol catalyzed by patchoulol synthase. IPP, DMAPP, GPP and FPP are defined as isopentenyl pyrophosphate, dimethylallyl pyrophosphate, geranyl pyrophosphate and farnesyl pyrophosphate, respectively.

ERG10,

acetyl-CoA

C-acetyltransferase;

ERG13,

hydroxymethylglutaryl-CoA synthase; tHMGR, truncated hydroxymethylglutary-CoA reductase; ERG12, mevalonate kinase; ERG8, phosphomevalonate kinase; ERG19, diphosphomevalonate decarboxylase; IDI1, isopentenyl diphosphate δ-isomerase; ERG20,

farnesyl

diphosphate

synthase;

ERG9,

squalene

synthase;

DPP1,

phosphatidate phosphatase; LPP1, phosphatidate phosphatase. Figure 2 GC-MS analysis of patchoulol production in engineered strains. (A) The retention time of patchoulol standard and product of engineered yeast strain. The peak of patchoulol produced by strain 4741-tFPTS corresponds to the authentic patchoulol standard; (B) Mass spectra of patchoulol produced by strain 4741-tFPTS and authentic patchoulol standard; (C) Patchoulol production (mg/L) by strains 4741-tPTS and 4741-tFPTS. All values were average of three independent experiments. Error bars represent standard deviations. Figure 3 Growth curves of engineered strains REL001-FPTS, REL002-FPTS, REL003-FPTS and REL004-FPTS. All values were average of three independent experiments. Error bars represent standard deviations.

22


Page 23 of 33


Figure 4 Fermentation performance of strain REL003-FPTS in a 5L bioreactor. (A) Feeding strategy of fermentation process; (B) Time courses of cell growth and patchoulol production of strain REL003-FPTS.

23



Page 24 of 33

Table 1 Primers used in the work Primer name

Sequence (5’ to 3’)

FPTS-UF

GCGAATTCATGGCTTCAGAAAAAGAAATT

FPTS-UR

TCCGGACCCTTTGCTTCTCTTGTAAACTTTG

FPTS-DF

GGGTCCGGAATGGAATTGTATGCTCAATCA

FPTS-OL-F

GTTTACAAGAGAAGCAAAGGGTCCGGAATGGAATTGTATGCTCAA TCAGTTGGTG

FPTS-OL-R

TTGAGCATACAATTCCATTCCGGACCCTTTGCTTCTCTTGTAAACTT TGTTCAAGAACGC

PTS-F

TCGAATTCATGATGGAATTGTATGCTCAATCAG

PTS-R

CGACTAGTTTAATATGGAACTGGATGCAG

tHMGR-F

TACTCGAGTTAGGATTTAATGCAGGTGACG

tHMGR-R

CCCGGATCCAAAAATGGACCAATTGGTGAAAACTGAAG

UPC21-F

TCACTAAAGGGCGGCCGCATGAGCGAAGTCGGTATACAG

UPC21-UP-R

AAATGTTGCTGTTTCTGTTCATGTTTC

UPC21-OL-R

AACACTGCAGAGGGCGTTGATGGAGAAATGTTGCTGTTTCTGTTCATGTTTC

UPC21-DN-F

TCCATCAACGCCCTCTGCAGTGTT

UPC21-OL-F

GAAACATGAACAGAAACAGCAACATTTCTCCATCAACGCCCTCTGCAGTGTT

UPC2-1-R

CCTTGTAATCCATCGATACTAGTTCATAACGAAAAATCAGAGAAATT

IDI1-F

TCGAATTCATGACTGCCGACAACAATAGTATGCC

IDI1-R

GCACTAGTTTATAGCATTCTATGAATTTGCCTGTCATTTTCCAC

g80-F

TAAGGCTGCTGCTGAACGTGGTTTAAGAGCTATGCTGGAAACAG

g80-R

CACGTTCAGCAGCAGCCTTAGATCATTTATCTTTCACTGCGGAG

g17-F

TAGTGGATTGTAACGTCTATGTTTAAGAGCTATGCTGGAAACAG

g17-R

ATAGACGTTACAATCCACTAGATCATTTATCTTTCACTGCGGAG

gE9p-F

CCACTGCACTTTGCATCGGAGTTTAAGAGCTATGCTGGAAACAG

gE9p-R

TCCGATGCAAAGTGCAGTGGGATCATTTATCTTTCACTGCGGAG

gDPP1-F

AGTGAAAGCTTTGCAGGACTGTTTAAGAGCTATGCTGGAAACAG

gDPP1-R

AGTCCTGCAAAGCTTTCACTGATCATTTATCTTTCACTGCGGAG

gLPP1-F

TATGTACCTAACGAACTCGTGTTTAAGAGCTATGCTGGAAACAG

gLPP1-R

ACGAGTTCGTTAGGTACATAGATCATTTATCTTTCACTGCGGAG

gal80-UP-F

ATTGGGTGCCTCTATGATGGGTAT

gal80-UP-R

ATAGCATGAGGTCGCTCCAATTCAGGGAAAGAACGGGAAACCAACTATCG

80-TADH1-F

CGATAGTTGGTTTCCCGTTCTTTCCCTGAATTGGAGCGACCTCATGCTAT

80-CYC-R

GGGGGCCAAGCACAGGGCAAGACTTCGAGCGTCCCAAAACCTTCTC

gal80-DN-F

GAGAAGGTTTTGGGACGCTCGAAGTCTTGCCCTGTGCTTGGCCCCC

gal80-DN-R

GCCATTCATCGTGTTGTTTTGGC

Gal80-F

GGATTGCGCTTGCCTTTGTA

GAL17-UP-F

CCATCGATAACGACACCGACAAT

GAL17-UP-R

ATAGCATGAGGTCGCTCCAATTCAGTTGTCGACTTGAACGGAGTGACAAT

GAL17-UPC21-F

ATTGTCACTCCGTTCAAGTCGACAACTGAATTGGAGCGACCTCATGCTAT

GAL17-UPC21-R

AGTGTTACTACTCGTTATTATTGCGTCTGCGTTTCAGGAACGCGACCGGT

GAL17-DN-F

ACCGGTCGCGTTCCTGAAACGCAGACGCAATAATAACGAGTAGTAACACTTTTATAGT

24


Page 25 of 33


GAL17-DN-R

CCTCCTCGCGCTTGTCTACTAAA

ERG9-Up-F

AGCCTCAGTACGCTGGTAC

ERG9-Up-R

GGAATTATATTCCAGATGAGACCTGCATCCCAGAACCCACCGGGACACGC

HXT1pro-F

GCGTGTCCCGGTGGGTTCTGGGATGCAGGTCTCATCTGGAATATAATTCC

HXT1pro-R

ATGCCAATTGTAATAGCTTTCCCATGATTTTACGTATATCAACTAGTTGACGATTATGA

ERG9-Dn-F

TCAACTAGTTGATATACGTAAAATCATGGGAAAGCTATTACAATTGGCAT

ERG9-Dn-R

CTAAGATGTAGTCGGCCATACC

DPP1-UF

GATTCAACCGGCTCTTTGTCAACAG

DPP1-UR

ATCTAGGGTCCACTAACATACGCGCTTAATCTTGACGTGCAAGGGCCTGC

DPP1-DF

GCAGGCCCTTGCACGTCAAGATTAAGCGCGTATGTTAGTGGACCCTAGAT

DPP1-DR

ACTAGTACTCGATTTCTGGCGCAGC

LPP1-UF

GGCAACCTTGGAGAATGGATCTTGT

LPP1-UR

ACATCAACGCCTAAGGAAACTCGTCGCCGATCAAGCTTCATTCTCAGGTA

LPP1-DF

TACCTGAGAATGAAGCTTGATCGGCGACGAGTTTCCTTAGGCGTTGATGT

LPP1-DR

ACGTCTCCCAATCATGGTTTCATGG

25



Page 26 of 33

Table 2 Plasmids and Strains used in this study Name Plasmids

Description

Source

pESC-URA

2μ, URA3

Stored

in

the lab pESCU-tPTS

Cloning

TCYC1-tHMGR-PGAL1-PGAL10-PTS-TADH1

This study

cassettes into pESC-URA pESCU-tFPTS

Cloning

TCYC1-tHMGR-PGAL1-PGAL10-FPTS-TADH1

This study

cassettes into pESC-URA pESCU-FPTS

Cloning

PGAL10-FPTS-TADH1

cassettes

into

This study

TCYC1-tHMGR-PGAL1-PGAL10-IDI1-TADH1

This study

pESC-URA pESCU-tHMGR-IDI

Cloning

cassettes into pESC-URA pESCU-UPC21

Cloning

PGAL10-upc2-1-TADH1

cassettes

into

This study

pESC-URA p414-TEF1p-Cas9-CYC1t

Containing cas9 cassette

Addgene

p415-GalL-Cas9-CYC1t

Containing cas9 cassette

Addgene

pTCL

Containing PTEF1-cas9-TCYC1 cassette

This study

p426-SNR52p-gRNA.CAN1.Y-SUP4

gRNA-expressing plasmid

Addgene

pSCM-g80

gRNA-expressing plasmid which targets GAL80

This study

pSCM-g17

gRNA-expressing plasmid which targets GAL1-7

This study

pSCM-gE9p

gRNA-expressing plasmid which targets PERG9

This study

pSCM-gDPP1

gRNA-expressing plasmid which targets DPP1

This study

pSCM-gLPP1

gRNA-expressing plasmid which targets LPP1

This study

MATa, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0

Stored

t

Strains BY4741

in

the lab 4741-tPTS

BY4741, pESCU-tPTS

This study

4741-tFPTS

BY4741, pESCU-tFPTS

This study

REL001

BY4741,

ΔGAL80

::

This study

TCYC1-tHMGR-PGAL1-PGAL10-IDI1-TADH1 REL002

REL001, ΔGAL17 :: PGAL10-upc2-1-TADH1

This study

REL003

REL002, ΔPERG9::: PHXT1

This study

REL004

REL003, ΔDPP1, ΔLPP1

This study

REL001-FTPS

REL001, pESCU-FPTS

This study

REL002-FPTS

REL002, pESCU-FPTS

This study

REL003-FPTS

REL003, pESCU-FPTS

This study

REL004-FPTS

REL004, pESCU-FPTS

This study

26


Page 27 of 33


Table 3 The patchoulol and squalene production in recombinant yeasts

Strain

Patchoulol (mg/L)a

Patchoulol (mg/g DCW)b

Squalene (mg/L) c

REL001-FTPS

10.7 ± 0.1

2.8 ± 0.1

314.8 ± 8.8

REL002-FTPS

18.9 ± 2.1

4.0 ± 0.4

361.6 ± 11.9

REL003-FTPS

59.2 ± 0.7

31.3 ± 0.3

6.5 ± 0.3

REL004-FTPS

41.4 ± 1.5

22.5 ± 0.8

4.1 ± 0.2

a & c:

Patchoulol and squalene were measured at the 7th day of fermentation at shake

flask. a &b& c:

All values containing standard deviations were average of three independent

experiments.

27



Page 28 of 33

Table 4 Patchoulol production in different hosts

Host

Titer of shake flask (mg/L)

Titer of fed-batch (mg/L)

Yield (mg/g dry weight)

Volumetric productivity

References

(mg/L/d)

S. cerevisiae

59.2

466.8

20.5

66.7

This work

C. glutamicum

0.46

60

NA

18

9

C. reinhardtii

1.03

NA

0.922

NA

6

S. cerevisiae

23

40.9

NA

NA

8

NA: Not available

28


Page 29 of 33


Figure 1

29



Page 30 of 33

Figure 2 A

B

C

30


Page 31 of 33


Figure 3

31



Page 32 of 33

Figure 4 A

B

32


Page 33 of 33


Cell Density (OD600) Patchoulol (mg/L) Ethanol (g/L) Squalene (mg/L)

40

400

16

16

30

300

20

200

10

Cell Density (OD600)

0 0

24

48

72

96

120

144

Squalene (mg/L)

20

Ethanol (g/L)

20

Patchoulol (mg/L)

500

50

12

12

8

8

100

4

4

0

0

0

168

Time (h)

TOC Graphic

33


Saccharomyces cerevisiae - American Chemical Society

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