Productive Amyrin Synthases for efficient Î±-amyrin synthesis in

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Productive Amyrin Synthases for efficient #-amyrin synthesis in engineered Saccharomyces cerevisiae yuan yu, pengcheng chang, huan yu, huiyong ren, danning hong, zeyan li, Ying Wang, Hao Song, yi-xin huo, and Chun Li ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.8b00176 • Publication Date (Web): 14 Sep 2018 Downloaded from http://pubs.acs.org on September 19, 2018

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ACS Synthetic Biology

1

Productive Amyrin Synthases for efficient α-amyrin synthesis in engineered

2

Saccharomyces cerevisiae

3

Yuan Yu1, 2, 4, Pengcheng Chang2, Huan Yu3, Huiyong Ren3, Danning Hong3, Zeyan

4

Li3, Ying Wang2, Hao Song1, Yixin Huo3,* and Chun Li1, 2,*

5

1

6

Chemical Engineering and technology, Tianjin University, Tianjin, China

7

2

8

of Chemistry and Chemical Engineering, Beijing Institute of Technology, 5 Zhong

9

Guan Cun Nan road, Beijing, P. R. China

Key Laboratory of Systems Bioengineering (Ministry of Education), School of

Institute for Synthetic Biosystem/Department of Biochemical Engineering, School

10

3

11

Road, Beijing, P. R. China

12

4

13

Hai road, Tangshan, P. R. China

14

*

15

Abstract

16

α-amyrin is a plant-derived pentacyclic triterpenoid, with a lot of important

17

physiological and pharmacological activities. The formation of α-amyrin from

18

(3S)-2,3-oxidosqualene is catalyzed by α-amyrin synthase (α-AS), a member of the

19

oxidosqualene cyclase (OSC) protein family. However, α-amyrin is not yet

20

commercially developed due to its extremely low productivity in plant. The

21

engineered Saccharomyces cerevisiae with efficient α-amyrin production pathway

22

could be used as an alternative and sustainable solution to produce α-amyrin from

School of Life Sciences, Beijing Institute of Technology, 5 Zhong Guan Cun Nan

College of life sciences, North China University of Science and Technology, 21 Bo

Corresponding author E-mail: [email protected] and [email protected]

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

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renewable raw materials. To efficiently improve α-amyrin production in S. cerevisiae,

24

we identified two α-ASs, EjAS and MdOSC1 from Eriobotrya japonica and

25

Malus×domestica, respectively, through strict bioinformatics screening criteria and

26

phylogenetic analysis. The specific activities of purified EjAS and MdOSC1 were

27

0.0032 and 0.0293 µmol/min/mg, respectively. EjAS produced α-amyrin and

28

β-amyrin at a ratio of 17:3, MdOSC1 produced α-amyrin, β-amyrin and lupeol at a

29

ratio of 86:13:1, indicating MdOSC1 had significantly higher specific activity and

30

higher ratio of α-amyrin than EjAS. Furthermore, MdOSC1 was introduced into S.

31

cerevisiae combining with the increased supply of (3S)-2,3-oxidosqualene to achieve

32

the encouraging α-amyrin production, and the titer of α-amyrin achieved 11.97±0.61

33

mg/L, 5.8 folds of the maximum production reported.

34

Keywords:

35

engineering

α-amyrin;

oxidosqualene

cyclase;

synthetic

36


biology;

metabolic

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37

Terpenoids, the largest family of plant secondary metabolites, are widely

38

distributed in nature. Pentacyclic triterpenoids and their glycosylated derivatives

39

(saponins) have lots of important physiological and pharmacological activities

40

have been widely used in medicine, health care, industrial and agricultural production

41

2-4

42

isomers, α-amyrin and β-amyrin, differing only on the position of the methyl group on

43

the triterpene skeleton

44

oleanolic acid, respectively, which have similar physiological and pharmacological

45

activities, but ursane-type triterpene α-amyrin demonstrated potential antiproliferative

46

activity 7.

1

and

. Amyrins are important plant pentacyclic triterpenoids, including two important

5-6

. α-amyrin and β-amyrin are precursors of ursolic acid and

47

Traditional methods of obtaining amyrins were chemical extraction and separation.

48

The application of amyrins was seriously restricted by the slow growth of plants,

49

environmental concerns, high energy consumption and production cost 8. Synthetic

50

biology and metabolic engineering could redesign and construct the entire metabolic

51

pathway, assemble different genes into microbes to efficiently produce target products,

52

which could be employed to improve the productivity of amyrins to satisfy the market

53

demand

54

advantages, such as low cost, short cycle times, environmentally friendly, efficiency

55

and sustainability. Up to now, many successful cases of plant-derived natural products

56

synthesized with S. cerevisiae have been reported, including mono-, sesqui-, di- and

57

triterpenoids with important physiological and pharmacological activity 11-14.

58

9-10

. Compared with the traditional methods, biosynthesis has many

(3S)-2,3-oxidosqualene is the common precursor for the production of triterpenoids



Page 4 of 38

15-16

59

and sterols

. It could be cyclized by oxidosqualene cyclases (OSCs) to generate

60

triterpenoids in plants

61

The structures of the produced triterpenes and sterols depend on the specific OSC

62

involved. Over the years, lots of OSCs from different plants, animals and fungi have

63

been cloned and characterized, including β-amyrin synthase (β-AS), multifunctional

64

amyrin synthase, lupeol synthase, thalianol synthase, cycloartenol synthase and so on.

65

Most of the known amyrin synthases are β-AS, such as AeAS from Aralia elata

66

AsOXA1 from Medicago

67

synthases were multifunctional amyrin synthases with more than one product

68

(Table 1). For example, LjAMY2 from Lotus japonicus produced primarily lupeol and

69

β-amyrin as well as a number of other minor products 26, while SlTTS2 from Solanum

70

lycopersicum produced mixture of triterpenoids, including δ-amyrin, α-amyrin,

71

β-amyrin, taraxasterol, and et al 27.

17

or to generate sterols in most animals and fungi (Fig. 1) 18.

19

,

20

, and GgbAS1 from Glycyrrhiza glabra 21. Other amyrin 22-25

72

A small number of amyrin synthase had a product preference for α-amyrin and

73

were named as α-amyrin synthase (α-AS). The examples of α-AS include ObAS2

74

from Sweet Basil 6, IaAS1 from Ilex asprella 28, CrAS from Catharanthus roseus 25,

75

MdOSC1 from Malus×domestica 29, and ATLUP2 from Arabidopsis thaliana 30. The

76

production routes of α-amyrin and β-amyrin were separated after the formation of

77

oleanyl cation. β-amyrin was formed after the transfer of a hydride directly from C-18

78

and C-12 (route a in Fig. 1). For α-amyrin, a methyl group of the oleanyl cation at

79

C-20 was shifted to C-19, forming taraxasteryl cation and further ursanyl cation,

80

followed by the transfer of a hydride to generate taraxerol and α-amyrin (route b in


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81

Fig. 1). However, the checkpoint for entering route α-amyrin or β-amyrin is unknown,

82

making it hard to predict the ratio of the two isomers. Mutations in individual amino

83

acids of amyrin synthase may lead to changes in their spatial structure, affecting the

84

enzyme specificity, activity and product type 31.

85

No amyrin synthase has yet been found to generate α-amyrin as the sole product.

86

The ratio of α-amyrin to β-amyrin produced by α-ASs was different. Only CrAS,

87

IaAS1 and MdOSC1 produced α-amyrin more than 80%. The reported maximum

88

α-amyrin production was 2.06 mg/L by CrAS in yeast, which was too low for

89

industrial production demand 25. To identify more α-AS efficiently produce α-amyrin

90

in S. cerevisiae, EjAS from Eriobotrya japonica was identified as a candidate through

91

strict bioinformatic screening criteria, which was annotated as putative mix amyrin

92

synthase. BmOSC from Bacopa monniera was the cut-off of our bioinformatic

93

screening as control. MdOSC1 was also identified to evaluate and compare enzyme

94

activities. Eriobotrya japonica is an ornamental, medicinal and edible plant which

95

contains many effective bioactive components in its fruits, flowers, leaves and roots 1,

96

32

97

except for EjAS cloned by Hui-Hua, Li 33. Apple (Malus×domestica) belongs to the

98

Rosaceae family, deciduous tree, which is rich in a variety of bioactive compounds

99

beneficial to human health. Lots of triterpenoids, especially ursolic acid and oleanolic

100

acid were main components of apple peels, which were catalyzed by CYP450 enzyme

101

with α-amyrin and β-amyrin as precursors, respectively 34. Bacopa monniera belongs

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to Scrophulariaceae, which has lots of active pharmaceutical ingredient, such as

. To our knowledge, there is no report of amyrin synthase from Eriobotrya japonica



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35-36

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glycoside, flavonoids, saponins and bacosides in its stolon, leaves and roots

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Phylogenetic analysis revealed that BmOSC is closely related with other plant OSCs,

105

especially mixed α-AS, suggesting that BmOSC may be involved in formation of

106

pentacyclic triterpenoids. In

107

order

to

characterize

their

ability

to

catalyze

the

.

cycling

of

108

(3S)-2,3-oxidosqualene to α-amyrin, we expressed candidate α-ASs in yeast and

109

compared their specific activities. MdOSC1 showed a significantly higher specific

110

activity and higher ratio of α-amyrin than EjAS, while BmOSC was not a productive

111

OSC. Therefore, MdOSC1 with the higher activity was introduced into S. cerevisiae

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to produce α-amyrin. The yield of α-amyrin achieved an inspiring level through

113

increasing the supply of (3S)-2,3-oxidosqualene by metabolic engineering.

114

Results and Discussion

115

Mining and sequence analysis of Oxidosqualene Cyclase (OSC) Dozens of OSCs have been cloned and characterized from different plants, most

116 117

of

which

are

β-ASs

(only

catalyzing

a

cyclization

118

(3S)-2,3-oxidosqualene) with β-amyrin as the sole product (Table S1). However, the

119

conversion from (3S)-2,3-oxidosqualene to α-amyrin needs both cyclization and

120

isomerization steps which were catalyzed by a single enzyme, α-AS. Only a few

121

α-ASs have been found until now. With the rapid development of molecular biology

122

and RNA-seq technology, more and more OSCs should have been discovered.

123

However, it is difficult to predict the final product types of some putative OSCs

124

accurately, because the sequences of OSC are very similar. To accurately predict the


reaction

from

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125

types of OSC, putative amyrin synthases based on the sequences of the known OSCs

126

were retrieved from the nucleotide collection database (nr/nt) and the Plant & Food

127

Research expressed sequence tag (EST) database. We selected several known α-ASs

128

and β-ASs as the reference sequences (Table S1). TBLASTN was performed using the

129

common conserved regions as query sequence to preliminarily screen candidate

130

OSCs.

131

To accurately predict the type of OSC, we compared the conserved sequences

132

between α-ASs and β-ASs. Several conserved amino acid regions close to the OSCs’

133

catalytic center were analyzed. We speculated that two conserved sequences,

134

MXCYCRXX and NXXXXLQSX, decided the type of amyrin synthase:

135

MXCYCRXT and NXXXXLQSP present in α-ASs, while MXCYCRXV and

136

NXXXXLQSK present in β-ASs. Therefore, one candidate named EjAS (Accession

137

number. JX173279.2) was identified as a putative α-AS. We chose BmOSC

138

(Accession number. HM769762.1) as a control, which did not meet any of our

139

screening criteria but was annotated as “putative β-AS” in a previous study 36.

140

To analyze the evolutionary relationship among EjAS, BmOSC and other 46

141

reported OSCs, a phylogenetic tree was generated on the basis of the amino acid

142

sequences of EjAS and BmOSC along with OSCs (Fig. 2). These OSCs were mainly

143

classified into four groups, which are β-AS, lupeol synthase, lanosterol synthase, and

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multifunctional synthase. Multifunctional synthase produced a variety of products and

145

was named after its main product. α-ASs were part of the multifunctional synthase

146

group. Most of the multifunctional synthase, β-AS and lupeol synthase were derived



147

from an ancestral or parental precursor and intercrossed with each other except α-AS.

148

It seemed that α-AS evolved from β-AS. Lanosterol synthase derived from protosteryl

149

cation and distinguished from multifunctional synthase and β-AS, consistent with

150

evolutionary of increasing diversification from the dammarenyl to the oleanyl cation

151

via the lupenyl cation. As shown in Fig. 3, EjAS and BmOSC consist of 761 and 764

152

amino acid residues, respectively, containing all the highly conserved motifs of

153

amyrin synthase. QXXXXW motifs may play the role of strengthening the enzyme

154

structure and stabilizing its carbocation intermediates. DCTAE motif may associate

155

with substrate binding. MXCYCR motif may be related to the product specificity 34.

156

MdOSC1 was selected as the representative of α-AS. EjAS exhibits more similarity

157

with MdOSC1 (97%) than BmOSC (53%). As a result, EjAS and MdOSC1 belong to

158

α-AS, closer to β-AS than lupeol synthase. Phylogenetic analysis was consistent with

159

our previous prediction, but still need further verification.

160

Expression, purification and in vitro characterization of OSCs

161

E. coli was used to express OSCs due to its high yield of heterologous expression.

162

Table 2 summarized all strains constructed in this study. Gibson assembly was

163

performed to construct expression vectors of pET28a-EjAS, pET28a-MdOSC1 and

164

pET28a-BmOSC. Positive clones were identified by colony PCR (Fig. S1A-C).

165

Constructed plasmids were electroporated into E. coli Rosseta (DE3) and named as

166

strains YY9, YY10 and YY11, respectively. Gene expression was induced by

167

isopropyl-β-D-thiogalactoside (IPTG), cells were harvested and analyzed by

168

SDS/PAGE (Fig. S2A-C). The results indicated EjAS, MdOSC1 and BmOSC were


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169

successfully expressed with molecular weight of 88 kDa and the expression level

170

increased with the induction time. However, EjAS, MdOSC1 and BmOSC were

171

expressed as inclusion bodies in E. coli, probably due to the presence of multiple

172

glycosylation sites NXT/S in the protein. We denatured the protein with 8M urea and

173

purified the denatured protein with Ni-NTA spin column. Purified protein was

174

renatured by stepwise dialysis to remove urea. However, renatured protein formed the

175

inclusion body again after dialysis.

176

EjAS, MdOSC1 and BmOSC were expressed in S. cerevisiae to avoid the

177

inclusion body. Strains YY6, YY7 and YY8 harboring pESC-Trp-EjAS,

178

pESC-Trp-MdOSC1 and pESC-Trp-BmOSC plasmid (Fig. S1D-F) were induced by

179

galactose and analyzed by SDS/PAGE. No noticeable band was found with the

180

theoretical size due to low expression (Fig. S3A). Western blotting was performed to

181

further analyze EjAS, MdOSC1 and BmOSC expression in S. cerevisiae. The results

182

showed that all of the three genes were successfully expressed in S. cerevisiae with

183

molecular weight of 88 kDa (Fig. S3B). Although S. cerevisiae can express EjAS,

184

MdOSC1 and BmOSC with correct folded conformation, the expression was too low

185

to define enzyme kinetic parameters (Fig. S3A). Therefore, we purified the EjAS,

186

MdOSC1 and BmOSC from 1-L S. cerevisiae culture (Fig. S3C-F).

187

The enzyme activities of the purified proteins were further verified with

188

(3S)-2,3-oxidosqualene as substrate. (3S)-2,3-oxidosqualene was indeed different

189

with 2,3-oxidosqualene, the functional group of (3S)-2,3-oxidosqualene towards the

190

inside of the plane, while 2, 3-oxidosqualene does not have the optical rotation. Most



191

importantly, we did not get any product when we used 2,3-oxidosqualene as the

192

substrate to determine specific activities of OSCs in vitro. As shown in Fig. 4A,

193

MdOSC1 produced α-amyrin and β-amyrin with a ratio of approximately 5:1 ratio.

194

No product was detected for EjAS and BmAS, probably because the protein

195

expression or the enzyme activity was too low. EjAS, MdOSC1 and BmOSC were

196

further expressed in Pichia pastoris to define enzyme kinetic parameters in vitro.

197

Compared with E. Coli and S. cerevisiae expression systems, Pichia pastoris has

198

obvious advantages in folding, external division, post-translational modification and

199

glycosylation, which were widely used in heterologous protein expression 37. Positive

200

cloning of pGAPZa-EjAS, pGAPZa-BmOSC and pGAPZa-MdOSC1 were identified

201

by colony PCR (Fig. S1G-H). The plasmids were digested with AvrII and

202

electroporated into Pichia pastoris GS115 named as strains YY12, YY13 and YY14,

203

respectively. We purified EjAS, MdOSC1 and BmOSC from culture medium by

204

250mM imidazole. Enzyme activities of purified proteins were verified by

205

determining product concentrations (Fig. 4B). The specific activities of purified EjAS

206

and MdOSC1 were 0.0032 and 0.0293 µmol/min/mg, respectively. No product was

207

produced by BmOSC. As shown in Table 3, Km and kcat values of EjAS and

208

MdOSC1 for (3S)-2,3-oxidosqualene were determined, respectively. The kcat/Km

209

values of EjAS and MdOSC1 were 0.123 and 0.856 µM-1·min-1, respectively. The

210

difference in Km and kcat values was not significant between MdOSC1 and EtAS

211

(Euphorbia tirucalli β-amyrin synthase). Km and kcat values of EjAS were significant

212

higher and lower than EtAS, respectively.


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213

EjAS specific activity was significantly lower than MdOSC1, even though EjAS

214

exhibits 97% similarity with MdOSC1. The differences of amino acid residues may

215

cause changes in the high spatial structure of the enzyme, further affecting enzyme

216

activity. In order to further explore the reason for difference in enzyme activity, we

217

used SWISS-MODEL and PyMOL to analyze the structure of EjAS and MdOSC1,

218

especially the area close to activity center of substrate binding motif 484DCTAE488

219

and product specificity motif 256MFCYCR261. 432NTEMAPTLKQ441 sequence of

220

EjAS (432NAEMGPTLKK441 sequence of MdOSC1) has attracted our attention

221

because these amino acid residues were close to the enzyme activity center DCTAE,

222

causing the alpha helix becomes longer which may inhibit the substrate access to the

223

enzyme activity center. We also noticed the other two sequences 329NIAEP333 and

224

338WPLKK342 of EjAS (329NIVEP333 and 338WPFKK342 of MdOSC1), these

225

amino acid residues were involved in the folding of the two alpha helices near the

226

enzyme activity center and may interfere with enzyme activity (Fig. S4). Except for

227

these three regions, other different areas may also affect the spatial structure of the

228

enzyme and disturb enzyme activity, but still need further validation.

229

Verification of OSCs’ function and production of α-amyrin in engineered S.

230

cerevisiae

231

The synthetic precursor of α-amyrin is (3S)-2,3-oxidosqualene, derived from 38

232

MVA pathway

. MVA pathway in S. cerevisiae provide a steady stream of

233

(3S)-2,3-oxidosqualene.

234

post-translational modification in yeast are suitable for the expression of heterologous

Furthermore,

the

complete


membrane

system

and


235

enzyme. Therefore, S. cerevisiae was employed to characterize the function of OSCs

236

and produce α-amyrin in vivo. Overexpression genes of MVA pathway could improve

237

the supply for (3S)-2,3-oxidosqualene and increase the terpenoid production 39-41. In a

238

previous study, high yield of β-amyrin has been obtained in S. cerevisiae by the

239

heterologous expression of GgbAS1 from Glycyrrhiza glabra 42.

240

Here, similar strategies were employed to synthesize α-amyrin using EjAS and

241

MdOSC1 to verify their functions (Fig. 5). Four key genes in MVA pathway were

242

overexpressed to enhance the metabolic flux towards (3S)-2,3-oxidosqualene (Fig.

243

S5), including squalene monooxygenase ERG1 from C.albicans, HMG synthase

244

tHMG1, FPP synthase ERG20 and squalene synthase ERG9 from S. cerevisiae.

245

However, ERG7 catalyzed the cyclization of (3S)-2,3-oxidosqualene to lanosterol in S.

246

cerevisiae, further produced ergosterol, which was the main component of the cell

247

membrane. Excessive supply of (3S)-2,3-oxidosqualene were probably used to

248

synthesize lanosterol rather than α-amyrin. In order to improve α-amyrin production,

249

CRISPR/dCas9 system was employed to repress the expression of ERG7 and enable

250

regulation of metabolic flux towards biosynthesis of α-amyrin.

251

For strain YY2, tHMG1, ERG20, ERG9, and ERG1 expression cassettes with

252

different promoter and terminator were integrated to δ-site (Fig. S5A). We amplify the

253

full length of dCas9 gene with SV40 NLS at 5’ region and Myc-tag at 3’ region,

254

which was assembled with PGAL1 and TCYC1. HO-site was used to integrate dCas9

255

expression cassette (Fig. S5B), pRS42K-mCherry-gRNA harboring reporter gene and

256

sgRNA target to mCherry was employed to evaluate the CRISPR/dCas9 system.


Page 12 of 38

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257

Colony PCR and western blotting results showed strain YY2 were successfully

258

constructed and dCas9 was successfully expressed after induced by galactose (Fig.

259

S6A-B). Fluorescence intensities from mCherry showed that the expression of

260

mCherry was significantly reduced in strain expressing dCas9 compared with

261

non-induced control strain (Fig. S6C). This result indicated that the dCas9 system can

262

regulate gene expression in strain YY2. We also tested the growth kinetics of the

263

strains and no impairment of growth rates relative to control strain (data not shown),

264

indicating cell growth was not affected by regulating heterogeneous genes using the

265

dCas9 system.

266

Next, we assembled tHMG1, ERG20, ERG9, and ERG1 together with EjAS and

267

MdOSC1 expression cassettes into δ-site of S. cerevisiae, respectively, as well as

268

integrated dCas9 into HO-site (Fig. S5C). We also constructed pRS42K-YYgRNA

269

harboring sgRNA for down-regulation of ERG7. Colony PCR showed strains YY3

270

and YY4 were successfully constructed (Fig. S6D). GC-MS results showed that a new

271

compound produced by strain YY4 harboring MdOSC1 compared with WT strain

272

INVSc1 (Fig. 4C), which was identified as α-amyrin by retention times and mass

273

spectra (Fig. 4D), but the yield was only 0.12±0.02 mg/L (Fig. 6A). By contrast, no

274

product was identified in strains YY3 harboring EjAS (Fig. 6B).

275

To make more (3S)-2,3-oxidosqualene metabolic flux toward the product, we used

276

CRISPR/dCas9 to repress the expression of ERG7, but the result was unsatisfactory.

277

Lanosterol was improved significantly (14.87±1.04 mg/L in strain YY4 and

278

10.68±0.85 mg/L in strain YY3, respectively) compared with the WT strain INVSc1



279

(2.57±0.21 mg/L) (Fig. 6C), suggesting most of the increased (3S)-2,3-oxidosqualene

280

was used to synthesize lanosterol rather than α-amyrin. qRT-PCR was performed to

281

further evaluate the transcript level of ERG7, as shown in Fig. S6E, ERG7 in strain

282

YY4 was 4-fold lower than that in YYOS strain, which was integrated tHMG1,

283

ERG20, ERG9, ERG1 and MdOSC1 expression cassettes at δ-site, indicating the

284

transcript level of ERG7 was successfully repressed. Taken together, strain YY4

285

produced more lanosterol rather than α-amyrin, this is probably due to low k(cat)/K(m)

286

of the MdOSC1, suggesting that the enzyme activities of α-AS might be a

287

rate-limiting for the production α-amyrin by engineered yeast.

288

Enabling alpha amyrin production by high-copy plasmids

289

In order to increase the production of α-amyrin in vivo, we used high copy

290

plasmid to replace the genomic integration strategy. Positive cloning of

291

pESC-Trp-EjAS and pESC-Trp-MdOSC1 were identified by colony PCR, sequencing

292

corrected plasmids were transformed into S. cerevisiae and induced by galactose. As

293

shown in Fig. 4E, GC-MS was performed and a new compound produced by the

294

strain YY6 harboring EjAS compared with the control strain (S. cerevisiae carrying

295

the empty vector), which was identified as α-amyrin by retention times and mass

296

spectra with α-amyrin standards. The yield of α-amyrin in strain YY6 achieved

297

0.16±0.02 mg/L. MdOSC1 expressing transgenic strain YY7 produced two new

298

compounds, which were identified as α-amyrin and β-amyrin, respectively. The yield

299

of α-amyrin in strain YY7 achieved 2.26±0.11 mg/L. These results demonstrated that

300

EjAS encodes α-AS, MdOSC1 encodes for a multifunctional amyrin synthase with a


Page 14 of 38

Page 15 of 38 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


301

product preference for a-amyrin.

302

To increase the supply of metabolic precursors, ERG20, ERG9, ERG1, and OSCs

303

expression cassettes were together assembled into pYYG plasmid (a truncated

304

plasmid based on pESC). Positive clone was picked out by colony PCR (Fig. S1I). As

305

shown in Fig. 4E, GC-MS results showed that α-amyrin and β-amyrin were produced

306

by strain YY15, indicating EjAS indeed encodes a multifunctional amyrin synthase

307

with a ratio of 17 (α-amyrin) to 3 (β-amyrin). The yield of α-amyrin in strain YY15

308

achieved 0.92±0.05 mg/L, about 6 times higher than that in strain YY6. The yield of

309

α-amyrin produced in strain YY16 achieved 11.97±0.61 mg/L, which is significantly

310

higher than strain YY15 (Fig. 6D). This is consistent with the result of the enzyme

311

activity in vitro. Surprisingly, besides α-amyrin and β-amyrin, a small quantity of

312

lupeol was also detected in strain YY16 expressing MdOSC1. The ratio of α-amyrin,

313

β-amyrin and lupeol is determined as 86:13:1, which is very similar with the ratio of

314

MdOSC3 products previously

315

discovered for the first time in S. cerevisiae. In addition, lanosterol was also

316

significantly increased in strains YY15 (7.22±0.59 mg/L) and YY16 (6.84±0.54 mg/L)

317

with

318

(3S)-2,3-oxidosqualene was indeed significantly improved. However, most excessive

319

supply of (3S)-2,3-oxidosqualene was used for lanosterol biosynthesis but not

320

α-amyrin. The triterpenoid yields of each strain and the comparison with the

321

lanosterol were summarized in Fig. 6.

322

overexpression

of

34

. The production of lupeol by MdOSC1 was

MVA

pathway

genes

(Fig.

6C),

indicating

pYYG plasmid was constructed by ourselves with Gibson assembly, most of



Page 16 of 38

323

non-essential sequences was removed to minimize plasmid size as much as possible,

324

which has many benefits such as facilitated genetic engineering operation, easy to

325

transformation, more stable and more plasmid copies. There was only one GAL1

326

promoter in the pYYG plasmid which could be induced by galactose, significantly

327

improving the transcription and expression of genes. When glucose was used as the

328

carbon source, transcriptional inhibitory factor Gal 80 and transcriptional activator

329

Gal 4 combined together to form the Gal 4/Gal 80 complex, inhibiting the

330

transcription of the GAL1 promoter. When galactose was present as the only carbon

331

source in the medium, it could induce the disassociation of Gal 80 and Gal 4,

332

activating the transcription of GAL1 promoter

333

control the expression of dCas9 for the integrated strains, which was expressed only

334

when galactose was the sole carbon source, preventing the impact of the other protein

335

expression.

336

43

. We also used GAL1 promoter to

Up to now, the reported maximum α-amyrin production was 2.06 mg/L using 25

, however, Huang

44

337

CrAS from Catharanthus roseus by Yu

338

production was lower than 0.1 mg/L using CrAS, differing by only one amino acid.

339

Significant difference in yield might be due to the K8 and E8 of CrAS, but still need

340

to be further verified. MdOSC1 has been characterized in Nicotiana benthamiana

341

leaves and Pichia methanolica by Brendolise 29. But it was never used for producing

342

α-amyrin in S. cerevisiae. In this study, an accurate method for predicting OSC types

343

was established through strict bioinformatics screening criteria. EjAS and BmOSC

344

were first characterized in vivo and in vitro. MdOSC1 was introduced into S.


reported α-amyrin

Page 17 of 38 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


345

cerevisiae to produce α-amyrin with the titer achieved 11.97±0.61 mg/L through

346

increasing the supply of (3S)-2,3-oxidosqualene by metabolic engineering, 5.8 folds

347

of the maximum production reported. β-amyrin was also produced and increased as a

348

by-product because no amyrin synthase has yet been found to generate α-amyrin as

349

the sole product, seriously impact enzyme activity and specificity, resulting in low

350

yield of α-amyrin. To further improve the production of α-amyrin, we are committed

351

to the transformation of α-AS into the specific producing α-amyrin by protein

352

engineering in the future. In the alternative, we could also directly convert β-AS with

353

high enzyme activity and specificity into α-AS, producing α-amyrin as the sole

354

product and achieving higher yield of α-amyrin.

355

Materials and Methods

356

Phylogenetic analysis

357

Phylogenetic analysis based on amino acid sequence was generated by

358

GENEIOUS 10.2.3 to investigate the phylogenetic relationship of EjAS, MdOSC1

359

and BmOSC with other OSCs, these OSCs were generally grouped into three main

360

branches, including multifunctional amyrin synthases, β-ASs and lupeol synthases. In

361

addition to the plant-derived OSCs, we also selected some lanosterol synthases as

362

matching objects. We used different colors to represent different functions of the

363

enzyme. The selected typical representative of OSCs were listed in Table S1.

364

Construction of expression plasmids

365

OE-PCR was employed to amplify the full length of EjAS, MdOSC1 and BmOSC



Page 18 of 38

366

gene with 6×His-Tag at 3’ region. Codon optimization for yeast was performed with

367

online tools (http://www.jcat.de). PCR primers used in this study were list in Table S2.

368

Gibson assembly was performed to construct expression vectors of pET28a-EjAS,

369

pET28a-MdOSC1,

pET28a-BmOSC,

370

pESC-Trp-BmOSC,

pYYG-Trp-OEOS-EjAS,

371

pYYG-Trp-OEOS-BmOSC in E. coli (Fig. S7A-D). PCR production and vectors

372

backbone were gel purified and estimated concentration, mixed the target gene and

373

backbone in proportion, 50 ℃ for 60 min, followed by transformation of E. coli

374

(Top10) competent cells. Positive clone was picked out by colony PCR and plasmids

375

were

376

pET28a-MdOSC1, pET28a-BmOSC were transformed into E. coli Rosseta (DE3),

377

respectively. pESC-Trp-EjAS, pESC-Trp-MdOSC1 and pESC-Trp-BmOSC were

378

transformed into S. cerevisiae (Invsc1) with LiAC transformation, respectively. The

379

Pichia pastoris expression vectors of pGAPZa-EjAS, pGAPZa-MdOSC1 and

380

pGAPZa-BmOSC was constructed by FastDigest enzyme and T4 ligase (Thermo

381

Fisher). EjAS and BmOSC gene were amplified by PCR with KpnI and NotI sites in

382

sense primer and anti-sense primer, while MdOSC1 with KpnI and XbaI sites in

383

primers. The PCR product and pGAPZa plasmid were digested with the

384

corresponding FastDigest enzymes, gel purified and ligated with T4 ligase. PCRs

385

were conducted with a 30-cycle program: 98 °C for 1 min, 56 °C for 20 min, 72°C for

386

2 min, and a final extension at 72 °C for 5 min. PrimerSTAR DNA polymerase

387

(Takara) was used according to the manufacturer’s protocol. Positive clone was

extracted

for

sequencing.

pESC-Trp-EjAS,

pESC-Trp-MdOSC1,

pYYG-Trp-OEOS-MdOSC1

Successfully

constructed


and

pET28a-EjAS,

Page 19 of 38 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


388

picked out by colony PCR and plasmids were extracted for sequencing. Successfully

389

constructed plasmids were digested with AvrII to obtain linearized DNA, gel purified

390

and transformed into Pichia pastoris with electroporation. The transformants were

391

plated onto YPDS plates containing 100 µg/ml Zeocin, and then incubated at 30 °C

392

for 3 days.

393

Construction of high-production strain

394

Yeast promoters (PADH1, PTYS1, PGPM1, and PALA1) and terminators (TADH1, TTYS1,

395

TGPM1, and TALA1) were amplified by PCR from the genomic DNA of S. cerevisiae.

396

Squalene monooxygenase (ERG1) gene from C. albicans were obtained by OE-PCR.

397

The genes of tHMG1, ERG20 and ERG9 were amplified from S. cerevisiae.

398

Expression cassettes were assembled by OE-PCR in the form of “promoter–gene–

399

terminator”. The genotypes of strains are shown in Table 2 and primers are shown in

400

Table S2. δ-site of S. cerevisiae was used to integrate because δ-site copies in the

401

genome was greater than 100

402

passaged 50 times in the absence of selective stress and the introduced exogenous

403

gene can still exist after multicopy passaged 46. Yeast transformations were done using

404

lithium acetate and PEG3350. The integrated strains YY2, YY3 and YY4 were

405

constructed via homologous recombination mediated DNA assembler (Fig. S5), all of

406

expression cassettes were designed with flanking overlap homology sequences that

407

can direct assembly into δ-site of S. cerevisiae. The expression cassettes and

408

additional selection marker cassette PTEF1-HIS3-TADH1 or PTEF1-Trp-TCYC1 were

409

co-transformed and integrated into δ-site of S. cerevisiae. Yeast genome DNA was

45

. The integrated genotype can still be stable after



Page 20 of 38

410

extracted and PCR was performed to screen positive clones. The dCas9 were

411

integrated similarly as description above and the additional selection marker cassette

412

PTEF1-URA-TADH1 were co-transformed and integrated into HO-site.

413

We used online software for gRNA designs (http://crispr.dbcls.jp/) and analysis

414

(http://rna.tbi.univie.ac.at/). Gibson assembly was performed to constructed

415

pRS42K-mCherry-sgRNA

416

pRS42K-mCherry-sgRNA contains mCherry and gRNA target to mCherry, while

417

pRS42K-YYgRNA contains gRNA target to ERG7. The strains harboring

418

corresponding sgRNA were induced by galactose to express dCas9, fluorescence

419

intensities were measured to evaluate whether the system works and products were

420

assessed by GC following the protocol above.

421

qRT-PCR Analysis

and

pRS42K-YYgRNA

plasmid,

422

Yeast cells were collected at 48 hours after induced by galactose, total RNA was

423

isolated and purified with Yeast RNA Kit (Omega). RNA concentration was quantified

424

by NanoDrop 2000c (Thermo Scientific, Waltham, MA). The total RNA samples were

425

used for cDNA synthesis by PrimeScript™ RT Master Mix (Takara). qRT-PCR was

426

performed at the LightCycler Real-Time PCR 96 system (Roche) using SYBR Premix

427

Ex Taq II (Takara) following the manufacturer’s instructions. The housekeeping gene

428

ACT1 was selected as reference gene. Primers used for qRT-PCR analysis were shown

429

in Table S2.

430

Expression and purification of EjAS, MdOSC1 and BmOSC in E. coli Rosseta

431

(DE3)


Page 21 of 38 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


432

E. coli Rosseta (DE3) harboring corrected plasmid of pET28a-EjAS,

433

pET28a-MdOSC1 and

434

rpm until an absorbance of OD600≈0.3 to 0.5, IPTG was added to a final

435

concentration of 0.1 mM, 16℃, 220rmp induced expression, we optimized the time of

436

induction to obtain EjAS, MdOSC1 and BmOSC as much as possible. Cells were

437

harvested after induced expression by centrifugation, ultrasonic cracking cells, the

438

supernatant and the precipitate were separated by centrifugation, added 5×loding

439

buffer (SDS/PAGE) and boiled for 10 min, SDS/PAGE performed to analyze the

440

protein expression. Since EjAS, MdOSC1and BmOSC was mainly expressed in the

441

form of inclusion bodies, 8M urea was used to dissolve inclusion bodies and Ni-NTA

442

spin column was used to purify protein. Centrifuge dissolved inclusion bodies at

443

12,000 rmp for 30 min to pellet the cellular debris and collect supernatant. Equilibrate

444

a Ni-NTA spin column with 600 µl Tris-HCL buffer containing 8M urea and

445

centrifuge for 2 min at 2000rmp. Load up to 600 µl of the cleared protein containing

446

the 6×His-tag onto pre-equilibrated Ni-NTA spin column. Centrifuge and collect the

447

flow-through. Wash the Ni-NTA spin column with washing buffer containing 50mM

448

imidazole, repeat 2 times. Elute the protein twice with 250mM (pH7.9) imidazole

449

concentrations and collect the eluate. Purification results were analyzed by

450

SDS/PAGE on 10% gels. Denatured protein was renatured by stepwise dialysis.

451

Expression and purification EjAS, MdOSC1 and BmOSC in S. cerevisiae and

452

Pichia pastoris

453

pET28a-BmOSC was cultured in LB medium, 37℃, 220

S. cerevisiae strains (Invsc1) harboring recombinant plasmid pESC-Trp-EjAS,



Page 22 of 38

454

pESC-Trp-MdOSC1 and pESC-Trp-BmOSC were respectively cultured in SD-Trp

455

selective medium containing 2% glucose, 30℃, 200 rpm for 2 days. Yeast cells were

456

harvested by centrifugation, resuspended in 20mL ddH2O and centrifuged 5 min, 4000

457

rpm. Cells were resuspended in 200 mL of SD-Trp medium containing 2% galactose,

458

cultured at 30℃, 200 rpm to induce expression of protein. Harvested yeast cells at 24,

459

48 and 72 hours, for each time point, determined the OD600 of each sample, 10 mL of

460

culture was removed to perform SDS/PAGE same as described above and western

461

blotting. Next, we use expanded the cells with 1-L SD-Trp medium to obtain large

462

quantity protein, cells were harvested by centrifugation, resuspended in 20mL binding

463

buffer. Ni-NTA spin column was used to purify EjAS, MdOSC1 and BmOSC by

464

following the protocol above but without 8M urea in buffer.

465

Sequencing

corrected

recombinant

Pichia

pastoris

GS115

integrating

466

pGAPZa-EjAS, pGAPZa-MdOSC1 and pGAPZa-BmOSC were cultured in YPD

467

medium. Use 0.1 ml of the overnight culture to inoculate 50 ml of YPD in a 250 ml

468

flask, 30°C, 250 rpm. Determine the optimal time to harvest and transfer the

469

supernatant to a separate flask. Precipitated with ice-cold acetone, resuspended in

470

20mL binding buffer. Ni-NTA spin column was used to purify EjAS, MdOSC1 and

471

BmOSC by following the protocol above.

472

SDS/PAGE and western blotting for estimation of protein expression

473

5×SDS/PAGE sample buffer (0.25 mM Tris/HCl pH 6.8, 10% SDS, 5%

474

β-mercaptoethanol, 10% glycerol, 0.2 mg/mL bromophenol blue) was mixed with

475

purified protein, boiled for 5 min, 40µL sample was applied to 10% polyacrylamide


Page 23 of 38 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


476

gels, 80 mA for 120 min. For the western blotting procedure, yeast cells were broken

477

by glass beads, the supernatant was collected and separated on SDS/PAGE,

478

electrotransfered onto a 0.22 µm PVDF membrane at 180mA for 90min, the

479

membrane were blocked in PBS with 5% skimmed milk for 4h, then incubated with

480

the anti-His antibody (1:10000 dilution) at 4℃ overnight. After washing for three

481

times with PBST buffer, the goat anti-mouse IgG antibody (1:10000 dilution) was

482

added and incubated for 2h at 25℃, washed for three times with PBST buffer,

483

visualized with ECL kit. Signal detection and imaging were performed with Bio-Rad

484

ChemiDoc MP.

485

Functional characterization by GC-MS

486

Yeast cells harboring recombinant plasmid or integrated with OSC were collected

487

at 48 hours after induced by galactose, resuspended in 10mL 20% KOH/50% EtOH

488

and boiled for 10 min, the production was extracted with hexane, the hexane phase

489

was separated and rotary evaporated to dryness. The crude sample was

490

trimethylsilylated with N-methyl-N (trimethylsilyl) trifluoroacetamide and pyridine at

491

80℃ for 30 min. GC-MS was carried out on a Shimadzu GCMS-QP2010 Plus

492

(Shimadzu) with a DB-5MS column (Agilent). The column initial temperature was 80℃

493

for 1 min, raised to 280℃ for 5 min at a rate of 40℃/min and then raised to 300℃

494

for 10 min at a rate of 20℃/min. Injector and detector temperatures were 300℃ and

495

250℃ respectively. The MS scan range acquired 45-450 m/z, helium flow rate was

496

1.0mL/min, 1µL sample was injected with 10:1 split stream mode.

497

Analysis of enzyme specific activities in vitro



498

The kinetic parameters relating to enzyme activity were measured in reaction

499

buffer (20 mM Tris/HCl, pH 7.9, containing 300 mM NaCl, and 0.1% Triton X-100)

500

47

501

enzymes (EjAS, MdOSC1 and BmOSC) (250 µg) was mixed with reaction buffer,

502

30 °C, 30 min. The reaction was stopped by heating at 100 °C for 1 min. 10mL 20%

503

KOH/50% EtOH was added and boiled for 10 min, the production was extracted with

504

hexane, the hexane phase was separated and rotary evaporated to dryness, then

505

trimethylsilylated. Processed samples were subjected to GC analysis to measure the

506

quantities of production. GC operated by following the protocol above.

507

Supporting Information

508

Figures S1-S5 and Tables S1-S2, including colony PCR, expression and purification

509

of the OSCs, genome integration strategy, evaluation of CRISPR/dCas9 system, and

510

plasmids map

511

Author Contributions

512

Yuan Yu, Ying Wang, Yixin Huo, and Chun Li developed the research plan. Yuan Yu,

513

Pengcheng Chang, Huan Yu, Huiyong Ren, Danning Hong performed the experiments.

514

Yuan Yu, Pengcheng Chang, Zeyan Li collected and analyzed data. Yuan Yu, Yixin

515

Huo, and Chun Li wrote the manuscript. All authors commented on and revised the

516

manuscript.

517

Notes

518

The authors declare no competing financial interest.

519

Acknowledgments

. (3S)-2,3-oxidosqualene (200 µg), BSA (1 mg/mL), DTT (1 mM) and purified


Page 24 of 38

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520

The authors would like to acknowledge the fund support from National Natural

521

Science Foundation of China (No.21676026, No.21425624, No.21606018, and

522

No.21736002).

523



524

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647 648


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Page 29 of 38 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


649

Table 1. Percentage of α-amyrin produced by different OSC Name

Production

Percentage of α-amyrin

Source

Reference

MdOSC1 CrAS IaAS1 OEA ObAS2 PSM ATLUP2 SlTTS2

αA, βA, LP αA, βA αA, βA αA, βA, ψT, B αA, βA αA, βA αA, βA αA, βA, δA, ψT, T, M αA, βA, LP αA, βA, LP

> 80% > 80% ≥ 80% 80% >a-amyrin > 50% 80% >a-amyrin > 50% 80% >a-amyrin > 50% 80% >a-amyrin > 50% < 30%

Malus×domestica Catharanthus roseus Ilex asprella Olea europaea Ocimum basilicum Pisum sativum Arabidopsis thaliana Solanum lycopersicum

28 24 27 21 6 22 29 26

< 30% < 30%

Kandelia candel Lotus japonicus

23 25

KcMS LjAMY2 650 651

αA: α-amyrin; βA: β-amyrin; LP: Lupeol; ψT: ψ-taraxasterol; T: taraxasterol; δA: δ-amyrin; B: butyrospermol; M: multiflorenol.

652 653 654 655 656 657 658 659 660 661 662 663 664 665



666

667

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Table 2. Strain genotypes and products in this study Strain

Base strain

Genotype and [plasmid]

YY1 YY2

S. cerevisiae S. cerevisiae

YY3

S. cerevisiae

YY4

S. cerevisiae

YY6 YY7 YY8 YY9

S. cerevisiae S. cerevisiae S. cerevisiae E. coli

YY10 YY11 YY12 YY13 YY14 YY15 YY16

E. coli E. coli P. pastoris P. pastoris P. pastoris S. cerevisiae S. cerevisiae

MATa/MATα his3∆1 leu2 trp1-289 ura3-52 YY1Delta::PADH1-tHMG1_PALA1-ERG20_PGPM1-ER G9_PTYS1-ERG1_HIS HO:: PGAL1-dCas9_URA [pRS42K-mCherry-gRNA] YY1Delta::PADH1-tHMG1_PALA1-ERG20_PGPM1-ER G9_PTYS1-ERG1_PFBA1- EjAS_TRP HO::PGAL1-dCas9-URA [pRS42K-YYgRNA] YY1Delta::PADH1-tHMG1_PALA1-ERG20_PGPM1-ER G9_PTYS1-ERG1_PFBA1- MdOSC1_TRP HO::PGAL1-dCas9-URA [pRS42K-YYgRNA] YY1[pESC-Trp-EjAS] As YY6, but plasmid is pESC-Trp-MdOSC1 As YY6, but plasmid is pESC-Trp-BmOSC F- ompT hsdSB(rB- mB-) gal dcm (DE3) pRARE2 (CamR) [pET28a-EjAS] As YY9, but plasmid is pET28a-MdOSC1 As YY9, but plasmid is pET28a-BmOSC his4 , Mut+ [pGAPZa-EjAS] As YY12, but plasmid is pGAPZa-MdOSC1 As YY12, but plasmid is pGAPZa-BmOSC YY1[pYYG-Trp-OEOS-EjAS] As YY15, but plasmid is pYYG-Trp-OEOS-MdOSC1

αA: α-amyrin; βA: β-amyrin; LP: Lupeol.

668 669 670 671 672


Production

αA

αA αA βA

αA βA αA βA αA βA αA βA LP

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673

Table 3. Kinetic data for purified EjAS, MdOSC1 and EtAS Name MdOSC1 EjAS EtAS

674 675

Km (µM) 50.07 116.91 33.8±0.53

kcat (min-1)

kcat/Km (µM-1·min-1)

Specific activity (µmol-1·min-1·mg-1)

43.4 14.38 46.4±0.68

0.856 0.123 1.37±0.21

0.0293 0.0032 0.352±0.0118

Kinetic data for EtAS was determined by Ryousuke Ito, et al.

676 677 678 679 680 681 682 683 684 685 686 687 688



Page 32 of 38

Bacopa monniera

Eriobotrya japonica

(3S)-2,3-Oxidosqualene

Malus×domestica Glucose tHMG1 Acetyl-CoA

Mevalonate

HMG-CoA ERG20

ERG20 IPP

FPP

GPP DMAPP ERG9

ERG1 (3S)-2,3-Oxidosqualene

Squalene

α-amyrin Abstract Graphic


Page 33 of 38


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

Fig. 1. Triterpene and sterol biosynthesis. (3S)-2,3-oxidosqualene is cyclized by different oxidosqualene cyclases (OSCs) to protosteryl cation and dammarenyl cation, further produced triterpene and sterol



Fig. 2. Phylogenetic tree on the basis of the amino acid sequences of EjAS, MdOSC1 and BmOSC along with other OSCs. The tree was built by the neighbor-joining method. Protein distances were calculated with PROTDIST and the JonesTaylor-Thornton matrix of the PHYLIP package. Three OSCs involved in this study were marked by solid line boxes ACS Paragon Plus Environment

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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

Fig. 3. Multiple OSCs sequence alignment with CLUSTAL O (1.2.4) DCTAE as well as the M (W/L/F) CYCR sequences are shown with yellow and green backgrounds. QXXXXW motif were shown with yellow background and underlined An * (asterisk) indicates positions which have a single and fully conserved residue A : (colon) indicates conservation between groups of strongly similar properties A . (period) indicates conservation between groups of weakly similar properties



A

B

C

E

D

Fig. 4. GC-MS chromatographs of engineered yeast strains A. Characterization of EjAS, MdOSC1 and BmOSC purified from S. cerevisiae in vitro. α-amyrin and β-amyrin were produced by MdOSC1. Control was enzyme-free reaction mix. No product was produced by EjAS and BmOSC. B. Characterization of EjAS, MdOSC1 and BmOSC expressed from Pichia pastoris in vitro. α-amyrin and β-amyrin were produced by EjAS and MdOSC1. Control was enzyme-free reaction mix. No product was produced by BmOSC. C. Characterization of EjAS and MdOSC1 in vivo with genomic integration. α-amyrin was produced in strain YY4 harboring MdOSC1. WT was strain INVSc1. No production was identified in strains YY3. D. EI-MS patterns of trimethylsilylated triterpenoids identified in engineered yeast strains and EI-MS spectra of TMS derivatives of α-amyrin, β-amyrin and lupeol. E. Characterization of EjAS and MdOSC1 in vivo with plasmids. Vector control strain was transformed with empty vector (pESC-Trp). α-amyrin and β-amyrin were produced in strain YY15 harboring EjAS. α-amyrin, β-amyrin and lupeol were produced in strain YY16 harboring MdOSC1. ACS Paragon Plus Environment

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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

Fig. 5. Biosynthetic pathway of α-amyrin in S. cerevisiae. tHMG1, ERG20, ERG9, ERG1 and αAS were integrated into δ-site along with dCas9 into the HO-site for genetically integrated strains ERG20, ERG9, ERG1 and αAS were assembled into pESC-Trp plasmid for plasmid-containing strains IPP: isopentenyl diphosphate; DMAPP: isomer dimethylallyl diphosphate; GPP: geranyl diphosphate; FPP: farnesyl pyrophosphate



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A

B

C

D

Fig. 6. The relative amount of triterpenoid production accumulated in engineered yeast strains. A. Triterpenoid production in engineered yeast strains expressing MdOSC1. B. Triterpenoid production in engineered yeast strains expressing EjAS. C. Comparison of the lanosterol and triterpenoid production in engineered yeast strains. D. The total production and percentage of α-amyrin, β-amyrin and lupeol in engineered yeast strains.


Productive Amyrin Synthases for efficient Î±-amyrin synthesis in

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