Directed Coevolution of Î²-Carotene Ketolase and Hydroxylase and Its

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Bioactive Constituents, Metabolites, and Functions

Directed co-evolution of #-carotene ketolase and hydroxylase and its application in temperature-regulated biosynthesis of astaxanthin Pingping Zhou, Min Li, Bin Shen, Zhen Yao, Qi Bian, Lidan Ye, and Hongwei Yu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05003 • Publication Date (Web): 04 Jan 2019 Downloaded from http://pubs.acs.org on January 10, 2019

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Journal of Agricultural and Food Chemistry

Directed co-evolution of β-carotene ketolase and hydroxylase and its application in temperature-regulated biosynthesis of astaxanthin Pingping Zhou2,3, Min Li2, Bin Shen2,Zhen Yao2, Qi Bian2, Lidan Ye1,2*, Hongwei Yu1,2 1Key

Laboratory of Biomass Chemical Engineering of Ministry of Education,

Zhejiang University, Hangzhou 310027, PR China 2Institute

of Bioengineering, College of Chemical and Biological Engineering,

Zhejiang University, Hangzhou 310027, PR China 3Joint

International Research Laboratory of Agriculture and Agri-Product Safety/Key

Laboratory of Prevention and Control of Biological Hazard Factors (Animal Origin) for Agrifood Safety and Quality, The Ministry of Education of China, Yangzhou University, Yangzhou, 225009, PR China

*Corresponding author: Lidan Ye ORCID iD: 0000-0002-6248-8457 Address: Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, 310027 Hangzhou, P.R. China. E-mail: [email protected] Tel/Fax: +86-571-88273997 1

ACS Paragon Plus Environment


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1

ABSTRACT

2

As an outstanding antioxidant with wide applications, biotechnological production of

3

astaxanthin has attracted increasing research interest. However, the astaxanthin titer

4

achieved to date is still rather low, attributing to the poor efficiency of β-carotene

5

ketolation and hydroxylation, as well as the adverse effect of astaxanthin

6

accumulation on cell growth. In order to address these problems, we constructed an

7

efficient astaxanthin-producing Saccharomyces cerevisiae strain by combining protein

8

engineering and dynamic metabolic regulation. Firstly, superior mutants of β-carotene

9

ketolase and β-carotene hydroxylase were obtained by directed co-evolution to

10

accelerate the conversion of β-carotene to astaxanthin. Subsequently, the

11

Gal4M9-based temperature-responsive regulation system was introduced to separate

12

astaxanthin production from cell growth. Finally, 235 mg/L of (3S, 3'S)-astaxanthin

13

was produced by two-stage high-density fermentation. This study demonstrates the

14

power of combining directed co-evolution and temperature-responsive regulation in

15

astaxanthin

16

biotechnological production of other value-added chemicals.

17

KEYWORDS:

18

astaxanthin, β-carotene hydroxylase, directed co-evolution, temperature-responsive

19

regulation, high-density fermentation

biosynthesis,

and

may

provide

methodological

reference

for

2


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20

INTRODUCTION

21

Astaxanthin as a tetraterpene with strong antioxidant activity has wide applications in

22

aquaculture, food, pharmaceutical and cosmetic industries 1. Currently, the majority of

23

commercial astaxanthin is obtained from chemical synthesis, while the rest is

24

extracted from Haematococcus pluvialis and Xanthophyllomyces dendrorhous

25

However, the biosafety concern with chemical routes and the high cost of the

26

extraction route limit the extensive application of astaxanthin 6. Moreover, among

27

these sources, only the algae-extracted astaxanthin is the bioactive (3S,

28

3'S)-stereoisomer. Alternatively, microbial chassis cells have been engineered for

29

fermentative production of astaxanthin by means of metabolic engineering techniques

30

7, 8.

31

production of the bioactive (3S, 3'S)-astaxanthin as confirmed in our previous work 9.

32

2-5.

In particular, heterologous expression of algal genes could lead to microbial

Although astaxanthin biosynthesis has been enabled in non-carotenogenic

33

bacteria

34

cloned from natural producers, the yield is rather unsatisfactory as compared with

35

other carotenoids

36

production as compared to astaxanthin implies the ketolation and hydroxylation of

37

β-carotene as the rate-limiting steps in the astaxanthin synthetic pathway

38

improvement of β-carotene ketolase and β-carotene hydroxylase is the key to further

39

enhancement of astaxanthin production. Although some positive mutants of the

40

β-carotene ketolase have been generated adopting a high-throughput screening

41

method based on the color difference between β-carotene and canthaxanthin

10-12

and yeast

17-19.

9, 13-16

by heterologous expression of carotenogenic genes

The far higher titer of β-carotene obtained in heterologous

14.

Hence,

9, 20, 21,

3



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42

the high concentrations of ketocarotenoids in those strains suggest β-carotene

43

hydroxylase as a remaining rate-limiting enzyme. To address this issue, β-carotene

44

hydroxylases from different species were screened and the expression level of

45

β-carotene hydroxylase was adjusted

46

β-carotene hydroxylase is rarely reported due to the lack of structure information and

47

a proper high-throughput screening method. In the astaxanthin biosynthetic pathway,

48

β-carotene is converted to astaxanthin as a result of sequential catalysis by β-carotene

49

ketolase and β-carotene hydroxylase 16, changing the colony color from yellow to red,

50

which provides the basis for development of a color-indicated high-throughput

51

screening method. Directed co-evolution of two or more enzymes in a cascade or

52

pathway has efficiently improved the catalytic performance of cellulases and

53

DXR/DXS/IDI in E. coli

54

β-carotene ketolase and β-carotene hydroxylase based on the distinct color difference

55

between β-carotene and astaxanthin, which may provide a possible solution to the

56

bottleneck in astaxanthin biosynthetic pathway.

25, 26,

12, 22-24,

whereas protein engineering of

inspiring us to conduct directed co-evolution of

57

Besides the catalytic performance of pathway enzymes, the conflict between the

58

heterologous pathway and the native metabolism of the chassis organism is another

59

factor restricting the biosynthesis efficiency. Stored in cell membrane, accumulation

60

of carotenoids often leads to growth inhibition

61

fermentation of astaxanthin is rarely reported. In the few reports on fermentative

62

production of astaxanthin, the cell density was also not high, with OD600 of only 60

63

and 120, respectively

29, 30.

27, 28.

In particular, high-density

When we tried to scale up astaxanthin biosynthesis of S. 4


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64

cerevisiae strain Yast-D03 which produced 8.10 mg/g dry cell weight (DCW) of

65

(3S,3'S)-astaxanthin in shake-flask cultures 9, difficulty was also encountered to

66

achieve high cell density, implying adverse effect of astaxanthin accumulation on

67

biomass. To decouple heterologous production from cell growth, we have developed a

68

temperature-dependent dynamic control strategy in S. cerevisiae based on the

69

modified GAL regulation system. After knocking out GAL80 encoding the Gal4

70

inhibitor, the expression of PGAL-driven genes is controlled solely by the

71

transcriptional activator Gal4. By replacing the wild-type Gal4 with the

72

temperature-sensitive mutant Gal4M9, we achieved two-stage fermentation of

73

lycopene in S. cerevisiae in response to temperature shift

74

regulation system in astaxanthin biosynthesis may help to minimize the conflict

75

between cell growth and astaxanthin accumulation, and thus make high-density

76

fermentation possible.

31.

The application of this

77

In the present study, directed co-evolution of H. pluvialis β-carotene hydroxylase

78

and β-carotene ketolase was first conducted to accelerate the conversion of β-carotene

79

to astaxanthin, so as to diminish the metabolic bottleneck in the astaxanthin

80

biosynthetic pathway, and then the Gal4M9-based temperature regulation system was

81

introduced to separate the production stage from the growth stage, so as to achieve

82

high-density fermentation of astaxanthin in S. cerevisiae.

83

MATERIALS AND METHODS

84

Strains and growth media

85

Escherichia coli DH5α as the host for propagation of plasmids was cultivated 5



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overnight in Luria-Bertani (LB) complete medium (0.5% Bacto yeast extract, 1%

87

Bacto-tryptone [Difco Laboratories], 1% NaCl, pH 7.0) at 37°C. 100 μg/mL of

88

ampicillin or 50 μg/mL of kanamycin was supplemented for selection of resistance

89

gene.

90

S. cerevisiae reference strain BY4741

32

was used as the host for construction of

91

recombinant yeast strains, and the detailed genotypes of these strains are listed in

92

Table 1. The YPD plate (20 g/L D-glucose, 20 g/L peptone, 10 g/L yeast extract, 2%

93

agar) (Sangon Biotech, Shanghai, China) containing 200 μg/mL of geneticin (G418)

94

was used for screening of recombinant strains harboring kanMX gene. For

95

investigation of carotenoids production, the recombinant strains were cultivated in

96

YPD broth supplemented with 0.52 mM of Fe2+. Synthetic drop out media (SD) were

97

used for cultivation of the selected transformants carrying plasmids with the

98

corresponding auxotroph marker, whereas synthetic complete plate supplemented

99

with 100 μg/mL 5-fluoroorotic acid was used for counter-selection of yeast strains

100

with KanMX-URA-PRB322ori marker excision.

101

Gene amplification and plasmids construction

102

All primers (purchased from Sangon Biotech, Shanghai, China) used for gene

103

amplification and plasmids construction are listed in Table 2. To assemble a highly

104

efficient astaxanthin biosynthetic pathway, the tHMG1 gene from S. cerevisiae

105

BY4741 and the positive crtE mutant created previously (crtE03M) were amplified

106

from pUMRI-10-LYC01

107

were amplified from pUMRI-13-crtYB-crtI

33.

crtI and crtYB of X. dendrorhous CGMCC As2.1557 9.

The codon-optimized OcrtZ 6


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108

(KP866869) and positive Obkt mutant ObktM of H. pluvialis Flotow N-212 were

109

cloned from pUMRI-11-OcrtZ-ObktM

110

were used for integration of the pathway genes into different loci 9, 34. Meanwhile, the

111

left and right homologous arms of YPLO62W were amplified from the genome DNA

112

of

113

YPL062WUP-F/YPL062WUP-R primers respectively and fused together by overlap

114

extension PCR to generate the “YPL062W left arm-SfiI-YPL062W right arm”

115

structure. Replacing the DPP1 homologous arms of pUMRI-11 with the above

116

constructed YPL062W homologous arms generated the pUMRI-20 plasmid. In

117

addition, p416XWP01/04-OcrtZ-ObktM plasmids containing bidirectional promoters

118

of different strength were constructed based on p416XWP-PGAL10-Obkt for validation

119

of the screening method in directed co-evolution 9. All recombinant plasmids are

120

listed in Table 3.

121

Directed co-evolution of β-carotene ketolase and β-carotene hydroxylase

122

For construction of OcrtZ and ObktM random mutagenesis library, error-prone PCR

123

was

124

PCYC1-BST1-F/ADH1tR2 respectively with the plasmid p416XWP04-OcrtZ-ObktM

125

as template. p416XWP04-OcrtZ-ObktM was linearized by SalI/ BglII digestion. The

126

5’-end of the ObktM mutant cassette overlapped with SalI and BglII digested plasmid

127

p416XWP04, while the 3’-end overlapped with the OcrtZ mutant cassette. The 3’-end

128

of OcrtZ mutant cassette also overlapped with the vector p416XWP04.

129

Co-transformation of the two mutant libraries and the linearized p416XWP04 into the

strain

carried

BY4741

out

using

9, 16.

using

the

primer

A set of SfiI-linearized pUMRI plasmids

YPL062WD-F/YPL062WD-R

pair

qtHMG1R1/PCYC1-BST1-R

and

and

7



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130

β-carotene-producing

131

p416XWP04-ObktM-OcrtZ mutant libraries via homologous recombination. After

132

three-day cultivation on SD-URA- plate at 30°C, colonies that showed darker red

133

color, indicating improved activity of β-carotene ketolase and β-carotene hydroxylase,

134

were isolated and further confirmed by shake-flask culture, followed by DNA

135

sequencing (Sangon Biotech, Shanghai, China).

136

Real-time quantitative PCR analysis

137

Total RNA was extracted from yeast cells using RNAiso Plus Kit (TaKaRa, Dalian,

138

China) according to the manufacturer's protocol. Then genomic DNA elimination and

139

reverse transcription were conducted using a PrimeScript™ RT reagent Kit with

140

gDNA Eraser (TaKaRa, Dalian, China). Quantitative PCR was performed using TB

141

GreenTM Premix Ex Taq™ II (Tli RNaseH Plus) (TaKaRa, Dalian, China) on

142

BIO-RAD CFX ConnectTM Real-Time PCR Detection Systems (Bio-Rad, California,

143

USA). ACT1 was used as an internal control gene to normalize different samples. The

144

transcriptional level was calculated by using the 2−ΔΔCT method36.

145

Analysis of carotenoids

146

The carotenoids were extracted using acetone from HCl-heat-treated cells

147

Astaxanthin was quantified by HPLC (SHIMADZU LC-20 AT) equipped with an

148

Amethyst C18-H column (4.6×150 mm, 5 μm, Sepax Technologies, Inc.) and a

149

UV/VIS detector at 470 nm. Samples were eluted with a gradient program at a flow

150

rate of 1.0 mL/ min at 40°C. The proportion of solvent A (90 % acetonitrile, 10%

151

water) was gradually decreased from 100% to 10% while solvent B (60% methyl

strain

YXWP-7435

resulted

in

transformants

with

16.

8


Page 9 of 36


152

alcohol, 40% isopropyl alcohol ) increased from 0 to 90% during 0-15 min, and kept

153

at 10% solvent A and 90% solvent B for 15 min. Finally, the solvent B was decreased

154

from 90% to 0 within the last 5 min.

155

Fed-batch fermentation

156

Single colonies were picked into 5 mL YPD tubes and incubated overnight at 30ºC as

157

precultures, which was then inoculated (1% v/v) to 500 mL shake flasks containing

158

100 mL YPD medium and further incubated for 20 h at 30ºC. Two flasks of seed

159

culture were inoculated (10% v/v) into a 5-L bioreactor (Shanghai Baoxing

160

Bioengineering Equipment Co. Ltd, China) containing 2.3 L fermentation medium

161

which consisted of 20 g/L corn steep liquor (Yuan Peptide Biotechnology Co., Ltd,

162

Shanghai, China), 40 g/L glucose, 10 mL/L concentrated trace metal solution. The

163

concentrated trace element solution contained: EDTA, 15 g/L; ZnSO4·7H2O 5.75 g/L;

164

MnCl2·4H2O, 0.32 g/L; CuSO4, 0.50 g/L; CoCl2·6H2O, 0.47 g/L; Na2MoO4·2H2O,

165

0.48 g/L; CaCl2, 2.9 g/L; FeSO4·7H2O 2.8 g/L

166

Dongfeng Chemical Factory (Wuxi, China) and all other metal compounds were

167

obtained from Sangon Biotech (Shanghai, China).

37.

CoCl2·6H2O was purchased from

168

The dissolved oxygen was maintained at 30% during fermentation by manually

169

adjusting the air flow rate in range of 1-3.0 vvm and the agitation speed of 300-700

170

rpm. pH was controlled at 5.5 by automatic addition of 50% ammonia hydroxide. A

171

temperature-regulated two-stage fed-batch fermentation process was adopted for

172

high-cell density fermentation (defined as volumetric dry biomass ≥30 g/L or OD600

173

≥120). In the first stage, the temperature was maintained at 30ºC to sustain fast cell 9



Page 10 of 36

174

growth, and 500 g/L glucose was fed into bioreactors to keep the glucose

175

concentration below 2 g/L. During this period, 50 mL of concentrated corn steep

176

liquor (400 g/L) was fed into the bioreactor every 8 h. At the second stage, the

177

temperature was changed to 24ºC to induce astaxanthin biosynthesis. After the

178

residual glucose was depleted, 400 g/L of ethanol was fed at a flow rate of 2 g/L/h-4

179

g/L/h to promote astaxanthin accumulation until harvest of the cells. The glucose

180

concentration in the fermentation broth was measured by a glucose assay kit (Rsbio,

181

Shanghai, China). The ethanol concentration was analyzed by a GC-9790 gas

182

chromatography system (Fuli, Wenling, China) equipped with a flame ionization

183

detector and an HP-FFAP column (30 m×0.25 mm, 0.25 µm film thickness, Agilent,

184

USA). The temperatures of oven, detector and injector were 120°C, 180°C and

185

180°C, respectively.

186

RESULTS AND DISCUSSION

187

Development of screening method for co-evolution of β-carotene ketolase and

188

β-carotene hydroxylase

189

In our previous protein engineering effort of H. pluvialis codon-optimized β-carotene

190

ketolase (OBKT) targeting at improved ketolation of β-carotene, a positive triple

191

mutant OBKTM (H165R/V264D/F298Y) was obtained9. However, overaccumulation

192

of echinenone and canthaxanthin due to insufficient activity of the codon-optimized

193

β-carotene hydroxylase (OCrtZ) remains a barrier to efficient astaxanthin production

194

in S. cerevisiae. The color difference between β-carotene and astaxanthin inspired us

195

to seek for simultaneous engineering of β-carotene ketolase and β-carotene 10


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196


hydroxylase by directed co-evolution.

197

To validate the feasibility of directed co-evolution of β-carotene ketolase and

198

β-carotene hydroxylase based on the color difference between β-carotene and

199

astaxanthin,

200

co-expressing the two enzymes with bidirectional promoters of different strength (Fig.

201

1A) were respectively transformed into strain YXWP-74 with moderate production of

202

β-carotene

203

p416XWP04-ObktM-OcrtZ with weak promoters tended to be yellow, whereas that

204

carrying p416XWP01-ObktM-OcrtZ with strong promoters showed deepened colony

205

color, showing consistence between the colony color and the expression/activity of

206

these two enzymes (Fig. 1B). HPLC analysis showed astaxanthin accumulation in the

207

strain expressing OBKTM and OCrtZ under strong promoters while β-carotene was

208

hardly converted under week promoters (Fig. 1C). Thus, the yellow-to-red

209

color-based screening system could be used for directed co-evolution of β-carotene

210

ketolase and β-carotene hydroxylase.

211

Directed co-evolution of β-carotene ketolase and β-carotene hydroxylase

212

To avoid color saturation and meanwhile facilitate easy distinguishing of positive

213

mutants with enhanced color intensity by visual inspection, the weak bidirectional

214

promoter PCYC1 and PBST1 was adopted to control the expression of OBKTM and

215

OCrtZ

216

p416XWP04-ObktM-OcrtZ as the template for construction of ObktM and OcrtZ

217

mutant libraries (Fig. 2). From 6000 colonies, 8 recombinant strains with darker color

p416XWP01-ObktM-OcrtZ

35

and


to avoid color saturation. As anticipated, the strain harboring

respectively.

Error-prone

PCR

was

carried

out

using

11



Page 12 of 36

218

were selected and verified by DNA sequencing (Fig. S1). The plasmids carrying the

219

positive mutants were then digested with BamHI and SalI or EcoRI and BglII to

220

obtain ObktM mutant segments and OcrtZ mutant segments and inserted into the same

221

sites of pUMRI-11-OcrtZ

222

integration into the previously constructed β-carotene hyper-producing strain

223

Ycarote-02(-) 9 for further confirmation.

16

or pUMRI-11-ObktM

9

respectively, followed by

224

Among these isolated mutants, some had mutations solely in OBKTM or OCrtZ,

225

whereas the other had mutations in both enzymes. Since canthaxanthin and

226

astaxanthin are similar in color

227

mutants

228

canthaxanthin/echinenone rather than astaxanthin may also be screened out, in

229

addition to those with both improved ketolation and hydroxylation activities.

230

Therefore, the method should be used with caution when superior β-carotene

231

hydroxylase mutant is the sole target. In order to achieve the best OBKTM and OCrtZ

232

mutants, all mutations were recreated and combined, and the performance of the

233

resulting mutants in astaxanthin biosynthesis was comparatively analyzed. The best

234

OCrtZ mutant OCrtZM1 carrying an L288R mutation increased the astaxanthin

235

production by about 33% as compared to the wild-type OCrtZ (Fig. 3). To our

236

knowledge, this is the first report on directed evolution of algal β-carotene

237

hydroxylase. To further confirm that the catalytic efficiency rather than the expression

238

level of OCrtZM1 mutant changed, real-time quantitative PCR was conducted to

239

investigate the transcriptional level of OcrtZM1. Similar trends were found in the

with

solely

38,

improved

using this colony-based colorimetric screening, ketolation

activity

which

produce

more

12


Page 13 of 36


240

transcriptional level of OcrtZM1 and OcrtZ (Fig. S2), ascribing the improvement in

241

astaxanthin production to the enhanced β-carotene hydroxylase activity. Lin et al

242

recently reported the creation of a β-carotene hydroxylase (Hpchyb) mutant K90R

243

with 1.34-fold activity improvement by site-directed mutagenesis

244

unmatch of the indicated site and the presented primers with the provided amino acid

245

sequence of the enzyme in the publication, as well as the lack of mutant design

246

principle, caused difficulty in repetition or extension of that work.

14.

However, the

247

Meanwhile, mutant OBKTM29 (H165R/V264D/F298Y/M1T/N188D/L271R)

248

showed the best performance among all OBKTM mutants obtained by directed

249

co-evolution. After transformation of pUMRI-11-ObktM29-OcrtZ into Ycarote-02(-),

250

the canthaxanthin yield was further increased by about 51% and the β-carotene

251

accumulation was decreased by about 44% in comparison with those of the strain

252

integrated with pUMRI-11-ObktM-OcrtZ (Fig. 3). To investigate which mutation was

253

responsible for the improvement of β-carotene ketolase activity, mutants OBKTMM1T,

254

OBKTMN188D, OBKTML271R were generated based on OBKTM by introducing the

255

newly emerged site mutations. Interestingly, no obvious difference in carotenoids

256

production were observed between OBKTM and mutants OBKTMN188D or

257

OBKTML271R, while OBKTMM1T with mutation in the start codon, shifting the

258

translation initiation site to the 8th amino acid, gave an enhanced canthaxanthin

259

accumulation in spite of slightly lower activity as compared to OBKTM29, which

260

may be explained by the improved gene expression or changed protein structure due

261

to the truncation of N-terminal amino acids. 13



Page 14 of 36

262

When ObktM29 was integrated together with OcrtZM1 into the genome of

263

Ycarote-02(-), the resulting strain YPP-27 accumulated 5.70 mg/g DCW of

264

astaxanthin, exhibiting 39% improvement over the previously constructed strain

265

YPP-17 harboring ObktM and OcrtZ 9. Comparing to the diploid strain Yast-D03

266

harboring 2*2 copies of OBKTM and 2*3 copies of wild-type OCrtZ 9, the haploid

267

strain YPP-27 expressing only 1 copy each of the mutants OBKTM29 and OCrtZM1

268

produced 70% as much astaxanthin, demonstrating protein engineering as a potent

269

substitution to protein overexpression which has a risk of causing metabolic burden.

270

Construction of temperature-regulated astaxanthin biosynthetic pathway

271

As lipophilic compounds, carotenoids tend to accumulate in the cellular membranes,

272

causing burden for the host cells 39, 40. The host-unfriendly nature of carotenoids have

273

inspired us to develop a temperature-dependent dynamic control strategy based on

274

GAL regulation system to facilitate decoupling of production from growth 31. In order

275

to achieve high-density fermentation of astaxanthin in S. cerevisiae, this

276

temperature-responsive system was employed here to regulate the astaxanthin

277

biosynthetic pathway (Fig. 4). The temperature-sensitive Gal4M9 was integrated into

278

the GAL4/GAL80 double-knockout strain based on YPP-27 containing PGAL1/10-driven

279

astaxanthin synthetic pathway to construct a temperature-regulated yeast cell factory

280

for astaxanthin. The resulting strain Yast-TS8 showed scarcely any astaxanthin

281

accumulation (0.08 mg/g DCW) at 30°C, while accumulated astaxanthin to about 2.08

282

mg/g DCW in shaking-flask fermentation with temperature shift from 30°C to 24°C

283

(Table 4). This result further demonstrated the universality of the Gal4M9-based 14


Page 15 of 36


284

temperature regulation system in multi-gene pathways. Subsequently, the auxotrophic

285

marker MET13 was complemented in Yast-TS8 to generate the prototrophic haploid

286

strain Yast-TS9 which showed not only superior cell growth ability but also enhanced

287

astaxanthin accumulation (3.96 mg/g DCW) upon temperature shift from 30°C to

288

24°C. The result suggested that prototrophic strains were more robust and may

289

provide more cellular sources to support the target metabolic flux.

290

Accumulation of the intermediate canthaxanthin in Yast-TS9 indicated that the

291

insufficient hydroxylation efficiency of OCrtZM1 as compared to the ketolation

292

capability of OBKTM29. In order to drain the intermediate metabolites and further

293

pull the flux to astaxanthin biosynthesis, we constructed strain Yast-TS10 by

294

overexpressing one additional copy of OcrtZM1 in Yast-TS9. As a result, the

295

astaxanthin yield was improved to 5.02 mg/g DCW, which was 27% higher than

296

Yast-TS9. However, accumulation of the intermediate lycopene was still observed in

297

Yast-TS10 (Fig. S3), implying lycopene cyclization catalyzed by CrtYB as a

298

remaining rate-limiting step. Thus, Yast-TS11 was generated by overexpressing an

299

additional copy of crtYB in Yast-TS10, leading to production of 6.19 mg/g DCW of

300

astaxanthin upon temperature shift. Although the astaxanthin yield of Yast-TS11 is

301

slightly lower than that of the previously constructed diploid strain YastD-03 9, the

302

total number of gene copies in this haploid strain is much lower, implying less

303

metabolic burden and higher potential to be scaled up to high-density fermentation.

304

Two-stage fed-batch fermentation of astaxanthin

305

In order to maximize astaxanthin production, two-stage fed-batch fermentation with 15



Page 16 of 36

306

temperature shift was conducted to obtain high cell density. The temperature was held

307

at 30°C in the first stage for rapid growth of the cells. After the biomass reached

308

OD600=120 indicating the strain entered the mid-log phase, the culture temperature

309

was changed to 24°C to initiate astaxanthin synthesis. The time for temperature shift

310

was set at the mid log phase instead of the end of the log phase, considering the time

311

required for Gal4 synthesis, subsequent transcription and translation of the astaxanthin

312

biosynthetic pathway genes, and finally synthesis of the metabolic intermediates and

313

the final product astaxanthin. Fermentation of Yast-TS11 achieved a high OD600 (up

314

to 258) and an astaxanthin titer of 107 mg/L in 121 h, however, with canthaxanthin

315

(160 mg/L) accumulated as the dominant component of carotenoids in the

316

fermentation broth (data not shown), indicating insufficient hydroxylation efficiency.

317

Further integration of an additional OcrtZM1 copy created Yast-TS14, which showed

318

slightly improved astaxanthin production in shake-flask fermentation as compared to

319

Yast-TS11 (Table 4), but significantly outperformed Yast-TS11 in fed-batch

320

fermentation. The accumulation of canthaxanthin was largely diminished (50 mg/L),

321

and the astaxanthin titer reached 235 mg/L in 83 h (Fig. 5), higher than the best

322

astaxanthin-producing yeast ever reported (218 mg/L achieved in 140 h)30.

323

To sum up, this study suggests the potential of directed co-evolution in

324

engineering of rate-limiting pathway enzymes that lack direct screening features, and

325

demonstrates the universality of temperature-responsive regulation system in

326

high-density fermentation, providing methodological reference for biotechnological

327

production of astaxanthin and other value-added chemicals. 16


Page 17 of 36


328

ABBREVIATIONS USED

329

DCW, dry cell weight; LB, Luria-Bertani; YPD, yeast extract peptone dextrose

330

medium; G418, geneticin; SD, synthetic drop out medium; HPLC, high-performance

331

liquid chromatography.

332

CONFLICT OF INTEREST

333

The authors declare that they have no competing interests.

334

ACKNOWLEDGMENT

335

This work was financially supported by Zhejiang Provincial Natural Science

336

Foundation of China (Grant No. LY18B060001), and the Natural Science Foundation

337

of China (Grant Nos. 21576234 and 21776244).

338

SUPPORTING INFORMATION DESCRIPTION

339

Supporting Information. Fig. S1, the results of OCrtZ and OBKTM directed

340

co-evolution; Fig. S2, transcriptional levels of OcrtZM1 and OcrtZ in Y-carot02(-)

341

during shake-flask fermentation; Fig. S3, HPLC analysis of carotenoids in Yast-TS10

342

(A) and Yast-TS11 (B). The Supporting information is available free of charge on the

343

ACS Publications website.

17



Page 18 of 36

344

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temperature-responsive yeast cell factory using engineered Gal4 as a protein switch.

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

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39. Yamashita, E., Astaxanthin as a medical food. Funct. Foods. Health. Dis. 2013,

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41. Xie, W.; Liu, M.; Lv, X.; Lu, W.; Gu, J.; Yu, H., Construction of a controllable

462

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

464 465

23



Page 24 of 36

466

Figure captions

467

Fig. 1 Pre-experiment to evaluate the color-indicated high-throughput screening

468

method for directed co-evolution of β-carotene ketolase and β-carotene hydroxylase.

469

A. Structure of p416XWP01-ObktM-OcrtZ and p416XWP04-ObktM-OcrtZ plasmids;

470

B. Color difference of the β-carotene producing yeast strain YXWP-74 after

471

transformed

472

respectively. C. HPLC analysis of carotenoids in YXWP-74 after transformed with

473

p416XWP01-ObktM-OcrtZ or p416XWP04-ObktM-OcrtZ. 01 indicated introduction

474

of p416XWP01-ObktM-OcrtZ with strong promoters PGAL1 and PGAL10. 04 indicated

475

introduction of p416XWP04-ObktM-OcrtZ with weak promoters PCYC1 and PBST1.

476

Fig. 2 The flow chart of color-based high-throughput screening for directed

477

co-evolution of β-carotene ketolase and β-carotene hydroxylase. Error-prone PCR was

478

carried out for construction of OcrtZ and ObktM mutant libraries, followed by

479

co-transformation of the two mutant libraries with SalI/BglII-linearized p416XWP04

480

into the β-carotene producing strain YXWP-74. The colonies exhibiting deepened

481

color were screened out from the library.

482

Fig. 3 The results of OBKTM and OCrtZ directed co-evolution. The carotenoids

483

production of Ycarote-02(-) expressing OCrtZ and OBKTM was set to 1. OCrtZ

484

mutants together with OBKTM or second-round mutants of OBKTM together with

485

wild-type OCrtZ were transformed into the β-carotene hyper-producing strain

486

Ycarote-02(-) respectively. OBKTM: OBKT(H165R/V264D/F298Y).

487

Fig. 4 Overview of Gal4M9-mediated temperature-responsive regulation of

with


and


24


Page 25 of 36


488

astaxanthin metabolic pathway. HMG-CoA: hydroxyl methylglutaryl coenzyme A;

489

FPP: farnesyl pyrophosphate; GGPP: geranylgeranyl pyrophosphate; tHMG1,

490

CrtE03M: positive mutant of geranylgeranyl pyrophosphate synthase (GGPPS);

491

CrtYB: bifunctional phytoene synthase and lycopene cyclase; CrtI: phytoene

492

desaturase; OCrtZM1: positive mutant of β-carotene hydroxylase; OBKTM29:

493

positive mutant of β-carotene ketolase.

494

Fig. 5 High cell-density fermentation of Yast-TS14 for astaxanthin production. A.

495

Feeding proﬁle in the two-stage feeding process. Feeding rates are displayed as

496

milliliter feeding solution per hour and the time point for switching the feeding

497

solution from glucose to ethanol is indicated by black arrow. B. Time courses of cell

498

growth, carotenoids production and carbon sources of Yast-TS14 during

499

temperature-regulated high density fermentation. The time point for temperature shift

500

from 30°C to 24°C is indicated by the blue arrow.

501

25



Page 26 of 36

Table 1 The strains used in this study strain

genotype/ description

reference

BY4741

MATa, his3Δ1,leu2Δ0 , met15Δ0, ura3Δ0

32

YXWP-74

BY4741,

41

Δdpp1::TADH1-crtE-PGAL10-PGAL1-tHMG1-TCYC1,

Δho::TADH1-crtYB-PGAL10-PGAL1-crtI-TCYC1, Δty4::TADH1-crtYB-PGAL10-PGAL1-crtI-TCYC1, Δgal80::LEU2 Y-carot02(-)

BY4741, Δgal80::LEU2,

9

Δho::TADH1-crtE03M-PGAL10-PGAL1-crtI-TCYC1-TPGK1-crtYB-PGAL2-PG AL7-tHMG1-TTPS1,

Δgal1-7::TADH1-crtYB-PGAL10-PGAL1-crtI-TCYC1, marker recycling YPP-17

Ycarot-02(-), Δdpp1::TADH1-OcrtZ-PGAL10-PGAL1-ObktM-TCYC1

This study

YPP-27

Ycarot-02(-), Δdpp1::TADH1-OcrtZM1-PGAL10-PGAL1-ObktM29-TCYC1

This study

Yast-TS8

YPP27, gal4::HIS3, Δlpp1::PACT1-GAL4M9-TADH1

This study

Yast-TS9

Yast-TS8, met15::MET15

This study

Yast-TS10

Yast-TS9, ΔTy4::TADH1-OcrtZM1-PGAL10-PGAL1-MCS2-TCYC1

This study

Yast-TS11

Yast-TS9, ΔTy4::TADH1-OcrtZM1-PGAL10-PGAL1-crtYB-TCYC1

This study

Yast-TS14

Yast-TS11, Δypl062w::TADH1-OcrtZM1-PGAL10-PGAL1-MCS2-TCYC1

This study

26


Page 27 of 36


Table 2 Primers used in this study primer name

sequence (5’- 3’)*

qtHMG1R1

ATAGGGACCTAGACTTCAGGTTG

PCYC1-BST1-R

GGTGATAATATTACAGCCAGTTCATTTGGCGAGCGTTG

PCYC1-BST1-F

ACGCTCGCCAAATGAACTGGCTGTAATATTATCACCTT

ADH1tR2

CAACCTTGATTGGAGACTTGAC

PBTS1R

GCGGAATTCAACGAAATGAAGATTTTTGTATG

PCYC1-R1

GCGGGATCCTATTAATTTAGTGTGTGTATTTGTG

Obkt-F1(BamHI)

CAGGGATCCATGCACGTTGCTTCTGCT

Obkt-R(SalI)

GGAAGTCGACTTAAGCCAAAGCTGGAACCA

OcrtZ-F1(EcoRI） GCGGAATTCATGTTGTCTAAGTTGCAATC OcrtZ-R1(BglII)

CCTTAGATCTTTATCTCTTAGACCAGTCCA

OBKTM(M1T)-F

CAGGGATCCACGCACGTTGCTTCTGCTTTGA

OBKTM(N188D) -F

CCCAGACTTCCACAAGGGTGACCCAGGTTTGGTTCCATGG

OBKTM(N188D) -R

CATGGAACCAAACCTGGGTCACCCTTGTGGAAGTCTGGGT

OBKTM(K271R) -F

ATGGCTTGGTTCAGAGCTAGGACTTCTGAAGCTAGTGACG

OBKTM(K271R) -R

GTCACTAGCTTCAGAAGTCCTAGCTCTGAACCAAGCCATA

MET15-F1

AAGTTCTCGTCGAATGCTAGGTC

MET15-R1

GGTGTTGACACCTTCTCCGC

GAL4-HisF1

GCCTTTTTCTGTTTTATGAGCTACTAGTACACTCTATATTTTT

GAL4-HisXF1

CTATCCGTAATCATGGTCGGCTACATAAGAACACCTTTGG

GAL4-CyF

AACCCAGAATCCCCTATACTAA

GAL4-HISR1

AAAATATAGAGTGTACTAGTAGCTCATAAAACAGAAAAAGGC

GAL4-His-XR1

CCAAAGGTGTTCTTATGTAGCCGACCATGATTACGGATAG

GAL4-XinF1

AATTGGATCTCCCAAGAGTA

YPL062WD-F

AAAGCTGGAGCTGGCCTTGTCACCGACCATGTGGGCAAAT

YPL062WD-R

TTAGGAGGTGCAGTGGTAGTGGCCTTTATGGCCGAGCTTTCATAAACT TGTTG

YPL062WUP-F

CAACAAGTTTATGAAAGCTCGGCCATAAAGGCCACTACCACTGCACC TCCTAA

YPL062WUP-R

TAATAGCGAAGAGGCCTACAGCCCTTACGTGAGGGGCAGT

PYPL062W-F

ATTTGCCCACATGGTCGGTGACAAGGCCAGCTCCAGCTTT

PYPL062W-R

ACTGCCCCTCACGTAAGGGCTGTAGGCCTCTTCGCTATTA

*The underlined bases represent the restriction site

27



Page 28 of 36

Table 3 Plasmids used in this study plasmid name

description

pUMRI-11

loxp-KanMX-URA3-pbr322ori-loxp, TADH1-MCS1-PGAL10-PGAL1-MCS2-TCYC1, DPP1

reference

NCBI: KM216413

homologous arm

pUMRI-16

loxp-KanMX-URA3-pbr322ori-loxp,

9

TADH1-MCS1-PGAL10-PGAL1-MCS2-TCYC1, Ty4 homologous arm loxp-KanMX-URA3-pbr322ori-loxp, PUMRI-PACT1-GAL4M9

31

TADH1-GAL4M9-PACT1-MCS2-TCYC1, LPP1 homologous arm

pUMRI-10-LYC01

TTPS1-tHMG1-PGAL7-PGAL2-crtYB11M-TPGK1-TCYC1-

33

crtI-PGAL1-PGAL10-crtE03M-TADH1, HO homologous arm pUMRI-13-crtYB-crtI

TADH1-crtYB-PGAL10-PGAL1-crtI-TCYC1, GAL1-7

9

homologous arm pUMRI-11-OcrtZ

TADH1- OcrtZ -PGAL10-PGAL1- MCS2-TCYC1

16

pUMRI-11-ObktM

TADH1- MCS1 -PGAL10-PGAL1-ObktM-TCYC1

9

pUMRI-11-OcrtZ-ObktM

TADH1- OcrtZ -PGAL10-PGAL1-ObktM-TCYC1

9

pUMRI-11-OcrtZM1-ObktM

TADH1-OcrtZM1-PGAL10-PGAL1-ObktM-TCYC1,

This study

DPP1 homologous arm pUMRI-11-OcrtZ-ObktM29

TADH1-OcrtZ-PGAL10-PGAL1-ObktM29-TCYC1,

This study

DPP1 homologous arm pUMRI-11-OcrtZM1-ObktM29

TADH1-OcrtZM1-PGAL10-PGAL1-ObktM29-TCYC1,

This study

DPP1 homologous arm pUMRI-16-OcrtZM1

TADH1-OcrtZM1-PGAL10-PGAL1-MCS2-TCYC1, Ty4

This study

homologous arm pUMRI-16-OcrtZM1-crtYB

TADH1-OcrtZM1-PGAL10-PGAL1-crtYB-TCYC1, Ty4

This study

28


Page 29 of 36


homologous arm pUMRI-20

loxp-KanMX-URA3-pbr322ori-loxp,

This study

TADH1-MCS1-PGAL10-PGAL1-MCS2-TCYC1, YPL062W homologous arm pUMRI-20-OcrtZM1

TADH1- OcrtZM1-PGAL10-PGAL1-MCS2-TCYC1,

This study

YPL062W homologous arm p416XWP-PGAL10-Obkt

CEN/ARS, URA3, PGAL10-Obkt-TADH1

p416XWP01-OcrtZ-ObktM

CEN/ARS, URA3,

9

This study

TCYC1-Obkt-PGAL1-PGAL10-OcrtZ-TADH1 p416XWP04-OcrtZ-ObktM

CEN/ARS, URA3,

This study

TCYC1-Obkt-PCYC1-PBTS1-OcrtZ-TADH1

29



Page 30 of 36

Table 4 Comparison of biomass and astaxanthin production with or without temperature shift. All strains were cultured in YPD medium with 0.52 mM of Fe2+ for 84 h constantly at 30°C or with temperature shift from 30°C to 24°C at 23 h. Data represent the average values calculated from triplicate experiments. strain

30°C

30°C /24°C

biomass

astaxanthin

astaxanthin

biomass

astaxanthin

astaxanthin

(g/L)

(mg/g

(mg/L)

(g/L)

(mg/g

(mg/L)

DCW)

DCW)

YPP-27

4.40±0.35

5.70±0.18

25.04±0.18

4.25±0.07

2.87±0.12

12.21±0.69

Yast-TS8

5.63±0.25

0.08±0.03

0.44±0.21

4.73±0.18

2.08±0.11

9.82±0.87

Yast-TS9

8.83±0.04

0.14±0.01

1.22±0.02

6.68±0.04

3.96±0.04

26.44±0.44

Yast-TS10

8.85±0.07

0.22±0.05

1.95±0.047

6.18±0.11

5.02±0.09

30.99±1.07

Yast-TS11

8.90±0.07

0.20±0.02

1.74±0.18

6.48±0.11

6.19±0.07

40.06±1.13

Yast-TS14

9.10±0.14

0.15±0.04

1.41±0.36

7.12±0.11

6.25±0.14

44.56±1.69

30


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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Table of Contents Graphics

36


Directed Coevolution of Î²-Carotene Ketolase and Hydroxylase and Its

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