Enhancement of Naringenin Biosynthesis from ... - ACS Publications

Subscriber access provided by UNIV OF DURHAM

Article

Enhancement of naringenin biosynthesis from tyrosine by metabolic engineering of Saccharomyces cerevisiae Xiaomei Lyu, Kuan Rei Ng, Jie Lin Lee, Rita Mark, and Wei Ning Chen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02507 • Publication Date (Web): 14 Jul 2017 Downloaded from http://pubs.acs.org on July 14, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 32

Journal of Agricultural and Food Chemistry

Enhancement of naringenin biosynthesis from tyrosine by metabolic engineering of Saccharomyces cerevisiae Xiaomei Lyu, Kuan Rei Ng, Jie Lin Lee, Rita Mark, Wei Ning Chen*

* Corresponding author: Wei Ning Chen Address: School of Chemical and Biomedical Engineering, College of Engineering, Nanyang Technological University, 62 Nanyang Drive, Singapore 637459, Singapore Tel: (+65)6316 2870 Email: [email protected]

1

ACS Paragon Plus Environment


1

Abstract

2

Flavonoids are an important class of plant polyphenols that possess a variety of health benefits. In this

3

work, S. cerevisiae was metabolically engineered to produce the flavonoid naringenin, using tyrosine

4

as the precursor. Our strategy to improve naringenin production was comprised of three modules. In

5

module 1, we employed a modified GAL system to overexpress the genes of the naringenin

6

biosynthesis pathway, and investigated their synergistic action. In module 2, we simultaneously

7

up-regulated acetyl-CoA production and down-regulated fatty acid biosynthesis in order to increase

8

the precursor supply, malonyl-CoA. In module 3, we engineered the tyrosine biosynthetic pathway to

9

eliminate the feedback inhibition of tyrosine, and also down-regulated competing pathways. It was

10

found that module 1 and 3 played important roles in improving naringenin production. We succeeded

11

in producing up to ~ 90 mg/L of naringenin in our final strain, which is a 20-fold increase as compared

12

to the parental strain.

13

Keywords: naringenin, Saccharomyces cerevisiae, metabolic engineering, tyrosine

14

2


Page 2 of 32

Page 3 of 32


15

Introduction

16

Plant flavonoids comprise a highly diverse family of over 9000 compounds derived from the

17

phenylpropanoid pathway. In recent years, they have attracted increasing interest due to their

18

multitude of health benefits such as antioxidant, anticancer and anti-inflammatory properties. Due to

19

this, the flavonoids have applications as high-value nutraceutical and pharmaceutical ingredients 1-4.

20

Traditionally, natural flavonoids are obtained via plant extraction. However, this production is highly

21

dependent on long breeding cycles and seasonal/regional limitations typical of plants. In addition,

22

downstream extraction processes of plant flavonoids are energy-intensive. The microbial synthesis of

23

flavonoids is a promising alternative, which has inherent advantages, such as amenability to

24

large-scale fermentation and the use of renewable feedstocks for production.

25

The flavonoid biosynthetic pathway, exclusive to plants thus far, has been well-characterized. It

26

starts from the conversion of phenylalanine/tyrosine to the compound 4-coumaroyl CoA which then

27

undergoes a condensation reaction with three molecules of malonyl-CoA in order to form the

28

tetrahydroxychalcone, naringenin chalcone. Naringenin chalcone is then isomerized to form the

29

flavanone, naringenin 5. Naringenin is the main branch point of the pathway. From here, the pathway

30

branches to produce several other major flavonoid classes: flavones, isoflavones, flavonols, catechins,

31

and anthocyanins. Accordingly, naringenin is the most essential flavonoid scaffold that precedes all

32

other flavonoids, and comes with its own set of reported health-beneficial effects, such as normalizing

33

lipid levels in diabetes and inhibiting proliferation of hepatitis C virus 6. The development of a

34

microbial platform strain capable of high-level production of naringenin is thereby highly important

35

and valuable for industrial production of flavonoids.

36

Currently, microbial production of naringenin has been reported in engineered Escherichia coli

37

and S. cerevisiae by using phenylpropanoid precursors such as p-coumaric acid. The highest 3



Page 4 of 32

38

naringenin production reported reached 474 mg/L from 2.6 mM coumaric acid in Escherichia coli 7,

39

and up to 28 mg/L from 1 mM coumaric acid in S. cerevisiae 8. Considering the substantial cost of the

40

precursors themselves, more economical approaches using simple feedstocks like glucose are being

41

pursued. In plants, the flavonoid biosynthetic pathway starts either from phenylalanine through

42

deamination by phenylalanine ammonia lyase (PAL) and cinnamate 4-hydroxylase (C4H), or from

43

tyrosine via deamination by tyrosine ammonia lyase (TAL). In 2012, Koopman et al. came up with the

44

de novo biosynthesis design via metabolic engineering based mainly on phenylalanine route. They

45

achieved approximately 200 µM (54 mg/L) of naringenin in shake-flask culture and 400 µM (108

46

mg/L) in batch bioreactor cultivation by using glucose 9. These above works proved the inherent

47

potential of S. cerevisiae for flavonoid production while also leaving room for a broader exploration,

48

such as the capacity of flavonoid biosynthesis from the alternative precursor, tyrosine. Moreover, since

49

secondary metabolite pathways usually involve complicated multi-step reactions and consist of

50

various catalytic elements such as substrates, enzymes and co-factors, comprehensive yet delicate

51

regulation of these multivariate elements is critical for metabolites accumulation and high yield. In the

52

case of heterologous flavonoid biosynthesis, metabolic engineering based on maximizing catalytic

53

capability of key enzymes as well as the regulation of altering key precursor fluxes (e.g. malonyl-CoA

54

and tyrosine) within multiple modules is currently lacking and worth exploring for improving

55

production.

56

In addition to improving enzyme activity and specificity as exhibited in previous studies

10, 11

,

57

gene expression via promoter control also plays an important role to ensure proper functioning of the

58

introduced heterologous pathway. Constitutive strong promoters are often selected for gene expression

59

in engineered yeast, which offer a high yield of protein but also result in increased metabolic burden on 4


Page 5 of 32


60

the host cell. In S. cerevisiae, GAL promoters are often the preferred choice due to their high strength

61

and galactose regulation characteristics, with the caveat of galactose being expensive which hinders

62

large-scale application. A glucose-regulated system by GAL80 deletion has been established in our

63

previous study

64

conditions. This design has been successfully applied for high-yield production of isoprenoids like

65

isoprene, carotenoids, and astaxanthin 13-15, and provides a feasible approach for increasing flux of the

66

core flavonoid biosynthetic pathway.

12

, in which the genes under control of PGAL can be expressed in glucose-limiting

67

Apart from enzyme capacity, sufficient precursor flux is another critical factor to achieve a

68

high-yield of the target product. In 2015, Jendresen et al. carried out a comparative study among 22

69

PAL and TAL gene properties and revealed some novel TALs with high activity and specificity 16.

70

This led the way forward for high-yield production of relevant biochemicals, using tyrosine as a

71

precursor. In yeast, aromatic amino acid supply is subject to feedback inhibition of DAHP synthase

72

(ARO4 and ARO3) and chorismite mutase (ARO7). Luttik et al. showed that introduction of mutated

73

ARO4 and ARO7 alleviated this feedback inhibition, resulting in 200-fold yield improvement of

74

aromatic compounds

75

by-product phenylethanol, by knocking out the most active phenylpyruvate decarboxylase, and

76

obtained resultant 3-fold increase of naringenin from the precursor phenylalanine

77

phenylalanine and tyrosine biosynthetic pathways share the same genes which control upstream

78

feedback mechanism and downstream degradation, the above strategies to produce naringenin by

79

using the TAL gene, and tyrosine as the precursor, might be interesting.

17

. Based on this discovery, Jean-Marc et al. prevented formation of the

9

. Since

80

In addition to tyrosine/phenylalanine, malonyl-CoA is another essential precursor of flavonoids.

81

The supply of malonyl-CoA has been proved to be a major bottleneck of the phenylpropanoid 5



Page 6 of 32

82

pathway in E. coli due to the low basal levels of this metabolite. Improvement of flavonoid

83

production in E. coli has been achieved via introduction of heterologous pathways, modification of

84

Acetyl-CoA carboxylase (ACC1) and down-regulation of the competing pathways

85

such studies have been reported in yeast thus far.

18-21

, although no

86

In this work, the capacity for flavonoid biosynthesis via the tyrosine route in S. cerevisiae was

87

explored through modulating the regulation of two essential elements (precursor supply and promoter

88

control) mentioned above. For this purpose, the expanded naringenin biosynthetic pathway - inclusive

89

of precursors syntheses - was partitioned into three modules (Figure 1): the core flavonoid biosynthetic

90

pathway (M1), malonyl-coA biosynthetic pathway (M2) and tyrosine biosynthetic pathway (M3).

91

Ultimately, intra-modular engineering and integration of all modules were conducted towards

92

improving naringenin production.

93

Methods and materials

94

Strains, media and reagents

95

S. cerevisiae strain BY474122 was used as the host for DNA integration and pathway engineering. E.

96

coli Top10 (Novagen, USA) was used for DNA manipulation. Luria-Bertani broth (LB) medium

97

containing 50 µg/mL of kanamycin, 40 µg/mL of phleomycin or 100 µg/mL of ampicillin was used

98

for culturing E. coli carrying cloning vectors. YPD medium (1% yeast extract, 2% peptone and 2%

99

glucose), YPG medium (1% yeast extract, 2% peptone and 2% D-galactose), YPS medium (1% yeast

100

extract, 2% peptone and 2% sucrose), YPDg (1% yeast extract, 2% peptone and 1% glucose - 1%

101

glycerol), and YPSg (1% yeast extract, 2% peptone and 1% sucrose-1% glycerol), or these above

102

media supplemented with 40 µg/mL of phleomycin & 150 µg/mL of hygromycin were used for

103

cultivation of engineered yeast. SD-HIS (synthetic complete drop-out medium with 2% D-glucose 6


Page 7 of 32


104

and without histidine) and SD-MET (drop-out medium without methionine) were used for auxotroph

105

selection for genes knockout by using HIS/MET marker. YPD medium containing 200 µg/mL

106

geneticin (G418) and SD-URA (drop-out medium without uracil) were used for selection of yeast

107

strains with integrated genes by using pUMRI plasmid harboring recyclable KanMX-URA marker.

108

SD-FOA (SD medium with 0.1% w/v 5-fluoroorotic acid) was used for selection of yeast strains with

109

KanMX-URA-PRB322ori marker excision. The standard naringenin, antibiotics and chemicals were

110

purchased from Sigma (Sigma Aldrich, USA). pKS2µHyg-4CL-CHS was a gift from John A.

111

Morgan (Purdue University, USA) based on their work on phenylpropanoid pathway engineering 23.

112

Plasmid and strain construction

113

The recombinant plasmids for gene integration were constructed based on the pUMRI toolbox as

114

reported in the previous study 13. The foreign gene 4-coumarate:coenzyme A (CoA) ligase (4CL) from

115

Arabidopsis thaliana and chalcone synthase (CHS) from Hypericum androsaemum were obtained by

116

PCR from pKS2µHyg-4CL-CHS. ATP-citrate lyase (ACL) from Yarrowia lipolytica, TAL from

117

Flavobacterium johnsoniae and chalcone isomerase (CHI) from Medicago sativa were synthesized

118

by Sangon Biotech (Shanghai. China) according to the preferred codon usage of S. cerevisiae. The

119

other genes, GAL4 (DNA-binding transcription factor required for activating GAL genes),

120

ACS1(encoding Acetyl CoA Synthetase), IDH1(encoding isocitrate dehydrogenase), ARO1

121

(encoding pentafunctional AROM polypeptide), ARO2 (encoding bifunctional chorismate synthase),

122

ARO8 (encoding aromatic aminotransferase I), TYR1 (encoding prephenate dehydrogenase), ZWF1

123

(encoding glucose-6-phosphate dehydrogenase), and the DNA sequences designed for gene knockout

124

were amplified from the genome of S. cerevisiae. They were cloned into the corresponding pUMRI

7



Page 8 of 32

125

plasmids as listed in Table S1. The detailed information of plasmid construction was presented in

126

Supporting Information. All the primers were listed in Table S2.

127

The strains were constructed following the procedures of reiterative recombination as described 13

128

in our previous study

. The pUMRI derived plasmids were linearized with either SfiI or HpaI and

129

then integrated into the yeast genome by electroporation or chemical transformation to generate the

130

recombinant strains as listed in Table 1. Specifically, pUMRI-22-∆GAL80-CHS-4CL was used for

131

construction of Y-01.

132

construction of Y-02/Y-03/Y-11 based on Y-01. pUMRI-21-∆IDH1-ylACL-sub12 was used for

133

construction of Y-12/Y-13 based on Y-02/Y-11. pUMRI-22-PACC1::PHXT7/PACC1::PHXT7-ARO4K229L

134

was used for construction of Y-14/Y-22 based on Y-13/Y-02. PMRI-28-PPHA2::PHXT1 was used for

135

construction of Y-23 based on Y-22. PDC5, ARO10 and TRP2 were knocked out by homologous

136

recombination and used markers of HIS, G418, MET for auxotroph selection, to generate

137

Y-24/Y-25/Y-32. pUMRI-24-ARO4::ARO4K229L/-ARO1/ARO2/ARO8/TYR1/ZWF1 was used for

138

construction of Y-(35-40) based on Y-25. The genotypes of transformants were verified by PCR

139

using their specific primers (Table S2). pCEV-Ph-mCHI-fjTAL and/or pKS2µHyg-4CL-CHS were

140

transformed into the above strains and named as Y-n-P-P. The detailed information of engineered

141

strains was presented in Table 1.

142

Cultivation in Shake-Flasks

143

Engineered strains were cultivated in 5 mL YPD medium (or supplemented with corresponding

144

antibiotics) at 30°C with shaking (200 rpm). Then about 2% of the overnight-grown seed culture was

145

inoculated into 50 mL fresh YPD/YPG/YPS/YPDg/YPSg media (or supplemented with

146

corresponding antibiotics) to an initial OD600 of 0.05 and incubated under the same conditions for

pUMRI-21-∆HO-fjTAL/

fjTAL-GAL4/fjTAL-ACS1

8


was used for

Page 9 of 32


147

another 72 h or 120 h. The concentration of glucose in medium was measured by coupled glucose

148

oxidase and peroxidase assay kit (Rsbio, China).

149

Quantification of naringenin with HPLC

150

After completion of the shake flask experiments, 2 mL of culture were harvested by centrifugation at

151

9600 g, washed twice, and then dried at 95 oC to a constant weight for measuring the dry cell weight.

152

Another 1 mL culture was centrifuged at the same condition for naringenin analysis. For analysis of

153

extracellular naringenin, the supernatant was added with the same volume of ethyl acetate, vortexed

154

vigorously for 30 s, and rotated at room temperature for 2 h. After centrifugation at 9600 g for 10

155

min, the upper organic layer was collected and evaporated to dryness. The resulting powder was

156

dissolved in 500 µL ethyl acetate and filtered for analysis and quantification. For analysis of

157

intracellular naringenin production, the cell pellet was washed twice with distilled water, resuspended

158

in 1 mL deionized water, and disrupted by glass beads for 120 s followed by the same extraction

159

process as for extracellular naringenin. The analysis of naringenin was performed by HPLC (Agilent

160

1100) equipped with a variable wavelength detector and C18 column (4.6 mm x 150 mm, RESTEK).

161

Samples were analyzed using a gradient method. The program started with 25% of solvent A

162

(methanol) and 75% of solvent B (water). The concentration of A subsequently increased to 75%

163

within 10 min, continued up to 100% at 20 min, and then held for 10 min. The solvent was returned

164

to 25% A over 2 min and held for 13 min. The flow rate was 0.5 mL/min and signal was detected at

165

280 nm.

166

Sample preparation for metabolites and fatty acid

167

The same volume of yeast cell cultures (1 mL for fatty acid analysis and 0.5 mL for metabolites

168

analysis) was collected at 48 h and centrifugated at 9600 g for 10 min. The cell pellet was washed

9



169

twice and resuspended in 1 mL of 0.9 NaCl solution and acidified with 200 µL of acetic acid. Five

170

microliters of heptadecanoic acid (10 mg/mL, dissolved in ethanol) and 10 µL of ribitol (2 mg/mL,

171

dissolved in water) were added as internal standard (IS) to correct for metabolites loss during sample

172

preparation. The sample was broken with glass beads using a Fast Prep bead grinder (MP

173

Biomedicals, Solon, OH, USA). Next, 3 mL of chloroform-methanol in ratio of 2:1 was added. These

174

samples were subsequently mixed by vigorous vortexing for half an hour and centrifuged at 9600 g

175

for 10 min. The upper aqueous layer containing intracellular metabolites, and the lower chloroform

176

layer containing lipids were collected separately and both evaporated to dryness.

177

GC-MS analysis

178

For fatty acid analysis, the dried lipid residue was firstly converted to fatty acid methyl esters by

179

transesterification reaction and then analyzed by GC-MS. Specifically, six hundred microliters of

180

boron trifluoride-methanol (FLUKA, 15716) was added to each sample and incubated at 95 oC for 30

181

min, and immediately added 600 µL of saturated NaCl to stop the reaction. The fatty acid methyl

182

esters were extracted by adding 600 µL of hexane and used for GC-MS analysis. The GC-MS system

183

(Agilent Technologies 7890A-5975C) was equipped with a DB-5MS capillary column (30 m×0.250

184

mm i.d.; 0.25 µm film thickness; Agilent J&W Scientific, Folsom, CA, USA). The injector

185

temperature and ion source temperature were set at 250 oC and 230 oC, respectively. The oven

186

temperature was as following: 80 oC for 1 min, ramped to 250 oC at the rate of 7 oC /min, 250 oC for

187

10 min. Data were acquired in a full scan mode from 35 to 600 m/z with a 0.3 s of scan time. Fatty

188

acids were identified using the NIST08 mass spectral library, based on mass spectral similarity, and

189

further confirmed by comparing their retention time with standards. Samples were normalized using

190

the IS, heptadecanoic acid, before comparison. 10


Page 10 of 32

Page 11 of 32


191

For metabolic profiling, the dried samples were dissolved in 50 µL of 20 mg/mL methoxyamine

192

hydrochloride in pyridine and kept at 37 oC for 1 h. Silylation was then performed by adding 100 µL

193

of N-methyl-N-(trimethylsilyl)-trifluoroacetamide (MSTFA) with 1% trimethylchlorosilane (TMCS),

194

incubated at 70 oC for 30 min, and used for GC-MS after centrifugation. It used the same GC-MS

195

system, scan mode as well as the data library, except the oven gradient: 75 °C for 4 min, ramped to

196

280 °C by 4 °C / min, and held for 2 min at 280 °C. Similarly, biochemicals were identified by using

197

the NIST08 mass spectral library and comparing with the standards. Samples were normalized using

198

the IS, ribitol, before comparison.

199

Results and discussion

200

Metabolic engineering of Module 1: Naringenin biosynthetic pathway

201

For construction of the naringenin biosynthetic pathway, TAL from Flavobacterium johnsoniae, 4CL

202

(4-coumarate CoA ligase) from Arabidopsis thaliana and CHS (chalcone synthase) from Hypericum

203

androsaemum were chosen as the target genes for over-expression due to their known high

204

activity/specificity

205

high expression of target genes under regulation of glucose, a GAL regulation system modified by

206

GAL80 deletion was adopted herein (Figure 2A). After three days of fermentation in YPG, the

207

extracellular naringenin production in Y-02 reached 4.3 mg/L, whereas no naringenin production was

208

detected in BY4741 (Figure S1). This demonstrated the successful de novo biosynthesis of naringenin

209

from tyrosine in Y-02. The production of naringenin even with absence of CHI indicates spontaneous

210

autocyclization of tetrahydroxychalcone under acidic conditions within YPG, also reported in

211

previous studies in E. coli 24, 25.

16, 23

. To eliminate the dependence on expensive galactose, while maintaining a

11



212

To increase the naringenin yield-cost ratio of carbon source used, various carbon sources were

213

tested: 2% glucose, 2% sucrose, 1% glucose-1% glycerol, 1% sucrose-1% glycerol. Naringenin was

214

successfully produced in all media tested (see Figure 2B). As shown in Figure 2B and Figure 2C, a

215

good balance between naringenin biosynthesis and cell growth was achieved within the two-stage

216

process during the first 3 days. In the first 24 h, cell growth was observed to be rapid while

217

comparatively little naringenin was produced, whereas in the subsequent 48 h, the majority of cellular

218

energy and resources were directed towards naringenin production. After 72 h of fermentation,

219

naringenin production as well cell growth entered the stationary phase. The highest production of

220

naringenin (5.2 mg/L) was obtained by using a 1% sucrose-1% glycerol combination after

221

approximately 72 h fermentation, which surpassed that of galactose media (Figure 2D). The

222

intracellular naringenin amounted to roughly 10% of the total naringenin production in all yeast

223

cultures. We speculated that the difference in naringenin production when YPD, YPS, YPDg, YPSg

224

media was used was related to the available glucose concentration in these media. This is because of

225

the difference in the way sucrose and glucose is broken down for assimilation for S. cerevisiae. In the

226

case of sucrose assimilation, in S. cerevisiae, sucrose is first converted to sucose-6P, and then broken

227

down into D-glucose-6P and D-fructose under the catalysis of SUC2 (sucrose hydrolyzing enzyme) 26.

228

D-fructose is then converted to D-fructose-6P and subsequently to D-glucose-6P under the catalysis of

229

PGI1 (phosphoglucoisomerase) 27, 28. Therefore, when compared with glucose medium, a much longer

230

time is required to convert all sucrose to glucose. Glucose released from sucrose would also be

231

promptly consumed and converted into other metabolites. This would result in a constant low glucose

232

concentration in the sucrose medium. This was in line with our results, as the glucose level in the YPS

233

culture broth was rather low or even below the detection limit throughout fermentation (Figure S2). 12


Page 12 of 32

Page 13 of 32


234

Furthermore, as the expression of our target genes was under the control of the GAL promoters, which

235

are only activated at low concentration of glucose in the modified GAL regulation system and

236

inhibited at high glucose concentration, the differences in available glucose concentration between

237

YPD and YPS might influence the expression levels of the pathway genes so as to cause the observed

238

differences in naringenin production.

239

In order to improve naringenin production, the genes of TAL/4CL/CHS/CHI (chalcone isomerase

240

from Medicago sativa) were over-expressed by using pCEV 29 and pKS2µHyg plasmid. We found that

241

naringenin production was improved to 8.8 mg/L, 10.3 mg/L, 6.8 mg/L and 15.8 mg/L by

242

overexpressing CHI, CHI-TAL, 4CL-CHS and CHI-TAL-4CL-CHS in Y-02, respectively (Figure 2E).

243

A slight decrease in cell growth was also observed (Figure S3). Naringenin yield was increased up to

244

70 % in Y-02 CHI (8.8 mg/L) as compared to Y-02 (5.2) mg/L, which proved that over-expressing

245

CHI compensated for the low efficiency of spontaneous isomerization of tetrahydroxychalcone into

246

naringenin at low pH. In contrast to single gene/two-gene overexpression, overexpressing multiple

247

limiting enzymes was also shown to have an additive effect that further increased the final product

248

yield. It is worth noting that when the transcriptional regulator for GAL promoter (GAL4) was

249

overexpressed in Y-02-P-P, naringenin production decreased instead (Figure 2E), demonstrating that

250

GAL4 baseline was already sufficient for activating the expression of genes under GAL promoters.

251

Metabolic engineering of Module 2: Malonyl-CoA biosynthetic pathway

252

Herein, the role of malonyl-CoA supply on flavonoid production in S. cerevisiae was explored by

253

employing two approaches as shown in module two (Figure 3A). The first strategy was up-regulation

254

of acetyl CoA biosynthesis. In S. cerevisiae, cytosolic acetyl-CoA is produced from the

255

pyruvate-acetaldehyde-acetate pathway, via the catalysis of cytoplasmic acetyl-CoA synthase (ACS). 13



Page 14 of 32

256

ACS is encoded by two genes, ACS1 and ACS2. Due to the superior kinetic parameters of ACS1

257

over ACS2 30, we chose ACS1 as the target enzyme in our study. Studies have demonstrated another

258

approach to produce cytosolic acetyl-CoA in human cells, plants and many fungi

259

cytosolic citrate transported from mitochondria by ATP-citrate lyase (ACL). However, this

260

mechanism is not present in S. cerevisiae. It is known that oleaginous microorganisms can produce

261

high amounts of lipids from malonyl-CoA and acetyl-CoA, but few studies focus on the role of ACL

262

in oleaginous microorganisms. Recently, Jean-Marc Nicaud et al. reported that inactivation of the

263

ACL from Yarrowia lipolytica, a classic oleaginous yeast, decreased 60% to 80% of its FA

264

synthesis33, thus confirming its essential role in FA synthesis. However, whether this gene could

265

improve FA or other cytosol acetyl-CoA-related production in non-oleaginous organisms remains

266

unknown. In this work, we introduce the heterologous acetyl-CoA pathway via ACL, by

267

over-expressing the ACL from Yarrowia lipolytica,

268

mainly responsible for down-regulation of citrate catabolism 34), and over-express the native ACS1.

269

We also attempted to down-regulate the competing fatty acids synthetic pathway by using the

270

inhibitor cerulenin in order to further redirect malonyl-CoA flux towards naringenin.

271

31, 32

, via the

knocking out IDH1 (isocitrate dehydrogenase,

Unlike the study on amorphadiene biosynthesis

35

, our results paradoxically showed that

272

over-expression of ACS1 had no influence on fatty acid production and also resulted in sharp

273

decrease in naringenin production (Figure 3B and 3C, Y-11). Introduction of heterologous

274

citrate-acetyl-CoA pathway resulted in 42% improvement in fatty acid productivity (based on

275

mass/dry weight ratio) but titer (based on mass/volume ratio) remained virtually unchanged (Figure

276

3B, Y-12). This showed that the heterologous citrate-cytosol acetyl-CoA pathway did indeed improve

277

the S. cerevisiae cytosolic acetyl-CoA production capability, but somehow also increased carbon and 14


Page 15 of 32


278

metabolic burden, decreasing cell growth and limiting the final production of fatty acids or

279

naringenin (Figure S4) (Figure 3C, Y-12). A previous report showed that, the production of fatty

280

acids reached up to ~ 300 mg/L in S. cerevisiae 34. The current level of malonyl-CoA production in

281

wild-type S. cerevisiae is probably adequate to produce a naringenin concentration of < 10 mg/L.

282

Furthermore, in order to enhance the catalytic ability of ACC1, we changed PACC1 to a strong

283

constitutive PHXT7 promoter (Figure 3B and 3C, Y-14), and the coenzyme biotin was added into the

284

media as supplementation (data not shown). However, no improvement was found for both fatty acid

285

and naringenin production.

286

For down-regulation of fatty acid biosynthesis, 1 ng/mL - 2 µg/mL of cerulenin was added in

287

media after 24 h of fermentation, and to maintain cell growth. Unfortunately, contrary to previous

288

reports of flavonoid regulation in E. coli

289

cerevisiae was found to decrease at all concentrations of cerulenin (Figure S5A). In addition, slight

290

inhibition to cell growth was also observed (Figure S5B). These results suggested that malonyl-CoA

291

may not be the bottleneck for flavonoids production, and other unknown regulatory mechanism of

292

malonyl-CoA on naringenin production is at play. Our results do however, confirm previous reports

293

that supply of the precursor malonyl-CoA, is the bottleneck for fatty acid biosynthesis.

294

Metabolic engineering of Module 3: Tyrosine biosynthetic pathway

295

In module 3, the tyrosine biosynthetic pathway was engineered to further improve naringenin

296

production (Figure 4A). We utilized the homologous recombination method, as shown in Figure 4B.

297

The feedback inhibition mechanism in the aromatic acid biosynthetic pathway, was deregulated in

298

Y-02 by the over-expression of mutated ARO4K229L. To redirect metabolic flux to tyrosine, various

299

genes involved in competing pathways were downregulated or knocked out. These included TRP2

10

, the production of naringenin in our engineered S.

15



300

(anthranilate synthase, catalyzes the initial step of tryptophan biosynthesis), PHA2 (prephenate

301

dehydratase, catalyzes the conversion of prephanate to phenylpyruvate), PDC5 and ARO10 (different

302

isozymes of phenylpyruvate decarboxylase). Additionally, other genes involved in the tyrosine

303

biosynthetic pathway was over expressed separately, to minimize potential limitation to naringenin

304

synthesis.

305

As a result, over-expression of mutated ARO4K229L led to 1.7-fold improvement of naringenin

306

production in Y-22 (8.9 mg/L) as compared to Y-02 (5.2 mg/L), and a 2.5-fold improvement in

307

tyrosine production (Figure 4C and 4D). This demonstrated that relieving the feedback inhibition for

308

accumulation of aromatic acids was effective. PHXT1 is a glucose-regulating promoter, with reportedly

309

low activity in glucose-limited conditions and have thus been widely applied to the downregulation

310

of genes

311

weaken expression of PHA2 and reduce metabolic flux towards unwanted phenylpyruvate. The

312

resultant Y-23 strain showed a 40% improvement in tyrosine production and 20% improvement for

313

naringenin (10.6 mg/L) as compared to the reference strain Y-22 (Figure 4C and 4D). To understand

314

the metabolic changes, we analyzed the intracellular metabolites. It was found that production of

315

phenylpyruvate was too low for detection, whereas hydroxy-phenylethanol production was improved

316

by 35% (Figure S6A). The production of phenylalanine showed no significant changes (Figure S6B).

317

Considering the complexity of metabolites, particularly amino acids - there might exist an

318

interconversion phenomenon, of similar metabolites in vivo, or from the complex media, in order to

319

make up for the loss of biosynthetic phenylamine, due to the down-regulation of PHA2.

320 321

Page 16 of 32

36, 37

. Herein, we employed PHXT1 to replacing its native promoter, PPHA2 (Figure 4B) to

With respect to our effort to down-regulate the by-product hydroxy-phenylethanol, it was found that the strain with a single deletion of PDC5 (Y-24) produced 12.0 mg/L of naringenin, which is 14% 16


Page 17 of 32


322

more than that of the reference strain Y-23 (10.6 mg/L). In the strain with the double knockout of

323

ARO10 and PDC5 (Y-25), naringenin production (21.7 mg/L) was improved by 4.2 folds as

324

compared to Y-02 (Figure 4C), and its intracellular tyrosine production was increased by 5.7 folds

325

(Figure 4D). In this double knockout strain, hydroxy-phenylethanol production was sharply

326

decreased below detection limits (Figure S6A). Taken together, our results confirmed the function of

327

phenylpyruvate decarboxylase on tyrosine degradation and unveiled effects of restraining

328

hydroxy-phenylethanol biosynthesis on tyrosine accumulation. In comparison to the regulation with

329

PHA2 and PDC5/ARO10, deletion of TRP2 produced no obvious changes in naringenin levels (data

330

not shown). The low tryptophan production, as found in our study, might be caused by

331

tryptophan-specific stimulation on ARO7

332

and Y-24 all exhibited a slight decrease, whereas Y-25 showed a slight improvement (Figure S7). We

333

speculate the growth improvement in Y-25 to be related to reduction of hydroxy-phenylethanol levels

334

(Figure S6A). This might have lessened the putative toxic effects of hydroxy-phenylethanol

335

accumulation, which causes damage to cell membranes, respiratory capacity, as well as impairs the

336

uptake of glucose and amino acids 39-41.

38

. With regards to cell growth comparisons, Y-22, Y-23

337

In order to completely eliminate the inhibition feedback of tyrosine on ARO4, a mutation in

338

K229L was introduced to the original ARO4 sequence in the Y-25 genome. This led to a 20%

339

improvement of naringenin (26.3 mg/L) (Figure 4E). However, no positive results were found in

340

other strains with over-expression of ARO1/ARO2/ARO8/TYR1 (Figure 4E). Considering that the

341

aromatic acids are derived from the pentose phosphate pathway, we also tried elimination of the

342

rate-limiting enzyme ZWF1 (glucose-6-phosphate dehydrogenase) in order to further increase

343

tyrosine flux, but with no avail. Finally, the key engineered strains in module 3 were transformed 17



344

with pKS2µHyg-4CL-CHS and pCEV-TAL-CHI, which was constructed in module 1, and led to a

345

3-fold improvement of naringenin production in almost all strains, amongst which Y-35 P-P was the

346

highest, attaining 84 mg/L (Figure 4F). The total naringenin production (extracellular and

347

intracellular), is estimated to be 90 mg/L, which is amongst the highest de novo microbial production

348

using shake flask fermentation (84 mg/L in E. coli

349

proved that bioreactor cultivation could contribute a lot to the improvement of flavonoid production 9.

350

Therefore, in order to continue improving naringenin yield via tyrosine route in S. cerevisiae, high

351

cell-density fermentation and process optimization will be investigated in our future work. Moreover,

352

comparative proteomic analysis as well as metabolomic profiling during different growth phases will

353

be carried out next, to shed light on the mechanism for the increased production of naringenin in our

354

engineered strains.

10

and 54 mg/L in S. cerevisiae 9). It has been

355

In conclusion, metabolic engineering on precursor supply and promoter control was proposed

356

and conducted towards improving naringenin production in S. cerevisiae in this study. Module 1

357

exhibited the potential of “modified glucose-regulated system by GAL80 knock-out” in flavonoid

358

production for achieving high product accumulation and cell growth balance, with cheap

359

glucose-limiting media, as well as synergistic effect of overexpression genes TAL, 4CL, CHS and

360

CHI. In module 2, 42% of improvement of fatty acid productivity was obtained by introduction of

361

ylACL which confirmed the role of ylACL to convert citrate to acetyl-CoA, in S. cerevisiae. The

362

large disparities in production of fatty acids and naringenin indicates that, while malonyl-CoA supply

363

is a limiting step for fatty acid biosynthesis, it is not the case for naringenin production. In module 3,

364

the improvement of naringenin (2.4-fold) by deletion of PDC5 & ARO10, and down-regulation of

365

PHA2, confirmed the benefits of restraining competing pathways. This also demonstrates the 18


Page 18 of 32

Page 19 of 32


366

important roles of PDC5 and ARO10 in synthesis of hydroxy-phenylethanol. From the comparison of

367

module 2 and module 3, it suggests phenylalanine/tyrosine is the limiting precursor for flavonoids

368

production in S. cerevisiae, and not malonyl-CoA. As a cumulative result of these engineering

369

strategies, a final maximum extracellular naringenin titer of 84 mg/L in shake flask fermentation was

370

achieved, which demonstrated the high capacity of S. cerevisiae for flavonoids biosynthesis, from the

371

precursor tyrosine.

372

Our study uncovers further details regarding the relationship between metabolic flux regulation

373

and flavonoids production, whilst simultaneously demonstrating that metabolic engineering approach

374

on precursor supply and genes control is required to fully maximize yield of important metabolites in

375

microbial cell factories. Metabolic engineering approaches, combined with further protein

376

engineering, downstream fermentation engineering and biorefinery techniques could in future push

377

production levels of flavonoids up to industrially applicable levels while still remaining

378

cost-effective and sustainable via this microbial cell factory platform.

19



Associated content Supporting information Some experimental results (Figure S1-S7), plasmids constructed in this study (Table S1), primers (Table S2), plasmid construction details (supplementary method).

Author information Corresponding Author * Tel: (+65)6316 2870. Email: [email protected]

Funding This work was supported by Nanyang Technological University Singapore (iFood Research grant).

Acknowledgment We thank Prof. John A. Morgan for kindly providing us the plasmid of pKS2µHyg-4CL-CHS. Notes The authors declare no competing financial interest

20


Page 20 of 32

Page 21 of 32


References 1.

Si, H. Y.; Li, D. P.; Wang, T. M.; Zhang, H. L.; Ren, F. Y.; Xu, Z. G.; Zhao, Y. Y., Improving the Anti-Tumor Effect

of Genistein with a Biocompatible Superparamagnetic Drug Delivery System. J. Nanosci. Nanotechnol. 2010, 10, 2325-2331. 2.

Wang, B. F.; Wang, J. S.; Lu, J. F.; Kao, T. H.; Chen, B. H., Antiproliferation Effect and Mechanism of Prostate

Cancer Cell Lines as Affected by Isoflavones from Soybean Cake. J. Agr. Food Chem. 2009, 57, 2221-2232. 3.

Birt, D. F.; Hendrich, S.; Wang, W. Q., Dietary agents in cancer prevention: flavonoids and isoflavonoids.

Pharmacol. Therapeut. 2001, 90, 157-177. 4.

F'guyer, S.; Afaq, F.; Mukhtar, H., Photochemoprevention of skin cancer by botanical agents. Photodermatol. Photo.

2003, 19, 56-72. 5.

Winkel-Shirley, B., Flavonoid biosynthesis. A colorful model for genetics, biochemistry, cell biology, and

biotechnology. Plant Physiol. 2001, 126, 485-493. 6.

Goldwasser, J.; Cohen, P. Y.; Yang, E.; Balaguer, P.; Yarmush, M. L.; Nahmias, Y., Transcriptional Regulation of

Human and Rat Hepatic Lipid Metabolism by the Grapefruit Flavonoid Naringenin: Role of PPAR alpha, PPAR gamma and LXR alpha. Plos One 2010, 5. 7.

Xu, P.; Ranganathan, S.; Fowler, Z. L.; Maranas, C. D.; Koffas, M. A. G., Genome-scale metabolic network

modeling results in minimal interventions that cooperatively force carbon flux towards malonyl-CoA. Metab. Eng. 2011, 13, 578-587. 8.

Yan, Y. J.; Kohli, A.; Koffas, M. A. G., Biosynthesis of natural flavanones in Saccharomyces cerevisiae. Appl.

Environ. Microb. 2005, 71, 5610-5613. 9.

Koopman, F.; Beekwilder, J.; Crimi, B.; van Houwelingen, A.; Hall, R. D.; Bosch, D.; van Maris, A. J. A.; Pronk, J.

T.; Daran, J. M., De novo production of the flavonoid naringenin in engineered Saccharomyces cerevisiae. Microb. Cell Fact. 2012, 11. 10. Santos, C. N. S.; Koffas, M.; Stephanopoulos, G., Optimization of a heterologous pathway for the production of flavonoids from glucose. Metab. Eng. 2011, 13, 392-400. 11. Wang, Y. C.; Halls, C.; Zhang, J.; Matsuno, M.; Zhang, Y. S.; Yu, O., Stepwise increase of resveratrol biosynthesis in yeast Saccharomyces cerevisiae by metabolic engineering. Metab. Eng. 2011, 13, 455-463. 12. Xie, W. P.; Liu, M.; Lv, X. M.; Lu, W. Q.; Gu, J. L.; Yu, H. W., Construction of a Controllable beta-Carotene Biosynthetic Pathway by Decentralized Assembly Strategy in Saccharomyces cerevisiae. Biotechnol. Bioeng. 2014, 111, 125-133. 13. Lv, X. M.; Wang, F.; Zhou, P. P.; Ye, L. D.; Xie, W. P.; Xu, H. M.; Yu, H. W., Dual regulation of cytoplasmic and mitochondrial acetyl-CoA utilization for improved isoprene production in Saccharomyces cerevisiae. Nat. Commun. 2016, 7. 14. Zhou, P.; Ye, L.; Xie, W.; Lv, X.; Yu, H., Highly efficient biosynthesis of astaxanthin in Saccharomyces cerevisiae by integration and tuning of algal crtZ and bkt. Appl. Microbiol. Biotechnol. 2015, 99, 8419-28. 15. Xie, W. P.; Lv, X. M.; Ye, L. D.; Zhou, P. P.; Yu, H. W., Construction of lycopene-overproducing Saccharomyces cerevisiae by combining directed evolution and metabolic engineering. Metab. Eng. 2015, 30, 69-78. 16. Jendresen, C. B.; Stahlhut, S. G.; Li, M. J.; Gaspar, P.; Siedler, S.; Forster, J.; Maury, J.; Borodina, I.; Nielsen, A. T., Highly Active and Specific Tyrosine Ammonia-Lyases from Diverse Origins Enable Enhanced Production of Aromatic Compounds in Bacteria and Saccharomyces cerevisiae. Appl. Environ. Microb. 2015, 81, 4458-4476. 17. Luttik, M. A. H.; Vuralhan, Z.; Suir, E.; Braus, G. H.; Pronk, J. T.; Daran, J. M., Alleviation of feedback inhibition in Saccharomyces cerevisiae aromatic amino acid biosynthesis: Quantification of metabolic impact. Metab. Eng. 2008, 10, 141-153. 21



18. Miyahisa, I.; Kaneko, M.; Funa, N.; Kawasaki, H.; Kojima, H.; Ohnishi, Y.; Horinouchi, S., Efficient production of (2S)-flavanones by Escherichia coli containing an artificial biosynthetic gene cluster. Appl. Microbiol. Biot. 2005, 68, 498-504. 19. Zha, W. J.; Rubin-Pitel, S. B.; Shao, Z. Y.; Zhao, H. M., Improving cellular malonyl-CoA level in Escherichia coli via metabolic engineering. Metab. Eng. 2009, 11, 192-198. 20. Leonard, E.; Lim, K. H.; Saw, P. N.; Koffas, M. A. G., Engineering central metabolic pathways for high-level flavonoid production in Escherichia coli. Appl. Environ. Microb. 2007, 73, 3877-3886. 21. Fowler, Z. L.; Gikandi, W. W.; Koffas, M. A. G., Increased Malonyl Coenzyme A Biosynthesis by Tuning the Escherichia coli Metabolic Network and Its Application to Flavanone Production. Appl. Environ. Microb. 2009, 75, 5831-5839. 22. Winzeler, E. A.; Shoemaker, D. D.; Astromoff, A.; Liang, H.; Anderson, K.; Andre, B.; Bangham, R.; Benito, R.; Boeke, J. D.; Bussey, H.; Chu, A. M.; Connelly, C.; Davis, K.; Dietrich, F.; Dow, S. W.; EL Bakkoury, M.; Foury, F.; Friend, S. H.; Gentalen, E.; Giaever, G.; Hegemann, J. H.; Jones, T.; Laub, M.; Liao, H.; Liebundguth, N.; Lockhart, D. J.; Lucau-Danila, A.; Lussier, M.; M'Rabet, N.; Menard, P.; Mittmann, M.; Pai, C.; Rebischung, C.; Revuelta, J. L.; Riles, L.; Roberts, C. J.; Ross-MacDonald, P.; Scherens, B.; Snyder, M.; Sookhai-Mahadeo, S.; Storms, R. K.; Veronneau, S.; Voet, M.; Volckaert, G.; Ward, T. R.; Wysocki, R.; Yen, G. S.; Yu, K. X.; Zimmermann, K.; Philippsen, P.; Johnston, M.; Davis, R. W., Functional characterization of the S-cerevisiae genome by gene deletion and parallel analysis. Science 1999, 285, 901-906. 23. Jiang, H. X.; Wood, K. V.; Morgan, J. A., Metabolic engineering of the phenylpropanoid pathway in Saccharomyces cerevisiae. Appl. Environ. Microb. 2005, 71, 2962-2969. 24. Watts, K. T.; Lee, P. C.; Schmidt-Dannert, C., Exploring recombinant flavonoid biosynthesis in metabolically engineered Escherichia coli. Chembiochem 2004, 5, 500-507. 25. Hwang, E. I.; Kaneko, M.; Ohnishi, Y.; Horinouchi, S., Production of plant-specific flavanones by Escherichia coli containing an artificial gene cluster. Appl. Environ. Microb. 2003, 69, 2699-2706. 26. Gascon, S.; Neumann, N. P.; Lampen, J. O., Comparative study of the properties of the purified internal and external invertases from yeast. J. Biol. Chem. 1968, 243, 1573-7. 27. Aguilera, A.; Zimmermann, F. K., Isolation and Molecular Analysis of the Phosphoglucose Isomerase Structural Gene of Saccharomyces-Cerevisiae. Mol. Gen. Genet. 1986, 202, 83-89. 28. Maitra, P. K.; Lobo, Z., Genetic Studies with a Phosphoglucose Isomerase Mutant of Saccharomyces-Cerevisiae. Mol. Gen. Genet. 1977, 156, 55-60. 29. Vickers, C. E.; Bydder, S. F.; Zhou, Y. C.; Nielsen, L. K., Dual gene expression cassette vectors with antibiotic selection markers for engineering in Saccharomyces cerevisiae. Microb. Cell Fact. 2013, 12. 30. vandenBerg, M. A.; deJongGubbels, P.; Kortland, C. J.; vanDijken, J. P.; Pronk, J. T.; Steensma, H. Y., The two acetyl-coenzyme A synthetases of Saccharomyces cerevisiae differ with respect to kinetic properties and transcriptional regulation. J. Biol. Chem. 1996, 271, 28953-28959. 31. Hynes, M. J.; Murray, S. L., ATP-Citrate Lyase Is Required for Production of Cytosolic Acetyl Coenzyme A and Development in Aspergillus nidulans. Eukaryot. Cell 2010, 9, 1039-1048. 32. Fatland, B. L.; Ke, J. S.; Anderson, M. D.; Mentzen, W. I.; Cui, L. W.; Allred, C. C.; Johnston, J. L.; Nikolau, B. J.; Wurtele, E. S., Molecular characterization of a heteromeric ATP-citrate lyase that generates cytosolic acetyl-coenzyme A in Arabidopsis. Plant Physiol. 2002, 130, 740-756. 33. Dulermo, T.; Lazar, Z.; Dulermo, R.; Rakicka, M.; Haddouche, R.; Nicaud, J. M., Analysis of ATP-citrate lyase and malic enzyme mutants of Yarrowia lipolytica points out the importance of mannitol metabolism in fatty acid synthesis. Bba-Mol. Cell Biol. L. 2015, 1851, 1107-1117. 34. Tang, X. L.; Feng, H. X.; Chen, W. N., Metabolic engineering for enhanced fatty acids synthesis in Saccharomyces 22


Page 22 of 32

Page 23 of 32


cerevisiae. Metab. Eng. 2013, 16, 95-102. 35. Shiba, Y.; Paradise, E. M.; Kirby, J.; Ro, D. K.; Keasing, J. D., Engineering of the pyruvate dehydrogenase bypass in Saccharomyces cerevisiae for high-level production of isoprenoids. Metab. Eng. 2007, 9, 160-168. 36. Mosley, A. L.; Lakshmanan, J.; Aryal, B. K.; Ozcan, S., Glucose-mediated phosphorylation converts the transcription factor Rgt1 from a repressor to an activator. J. Biol. Chem. 2003, 278, 10322-10327. 37. Xie, W. P.; Ye, L. D.; Lv, X. M.; Xu, H. M.; Yu, H. W., Sequential control of biosynthetic pathways for balanced utilization of metabolic intermediates in Saccharomyces cerevisiae. Metab. Eng. 2015, 28, 8-18. 38. Brown, J. F.; Dawes, I. W., Regulation of chorismate mutase in Saccharomyces cerevisiae. Mol. Gen. Genet. 1990, 220, 283-8. 39. Stark, D.; Zala, D.; Münch, T.; Sonnleitner, B.; Marison, I. W.; von Stockar, U., Inhibition aspects of the bioconversion of l-phenylalanine to 2-phenylethanol by Saccharomyces cerevisiae. Enzyme Microb. Tech. 2003, 32, 212-223. 40. Ingram, L. O. N.; Buttke, T. M., Effects of Alcohols on Micro-Organisms. Adv. Microb. Physiol. 1985, 25, 253-300. 41. Lester, G., Inhibition of Growth, Synthesis, and Permeability in Neurospora crassa by Phenethyl Alcohol. J. Bacteriol. 1965, 90, 29-37.

23



Page 24 of 32

Figure captions Figure 1 Schematic representation of modular metabolic engineering used for naringenin biosynthesis in this study. M1 (module 1) presents naringenin biosynthetic pathway from tyrosine, M2 (module 2) presents malonyl-CoA biosynthetic pathway, M3 (module 3) presents tyrosine biosynthetic pathway. TAL, tyrosine ammonia-lyase; 4CL, 4-coumarate CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase. Figure 2 Metabolic engineering of module 1. A, Schematic diagram of core flavonoids pathway engineering by using a modified GAL regulation for gene expression. B, Extracellular naringenin production of Y-02 in optimized media with different carbon sources during five days of fermentation. Carbon sources include 2% glucose, 2% sucrose, 1% glucose-1% glycerol and 1% sucrose-1% glycerol. C, Growth curve of Y-02 in optimized media with different carbon sources during five days of fermentation, including 2% glucose, 2% sucrose, 1% glucose-1% glycerol and 1% sucrose-1% glycerol. D, Extracellular and intracellular naringenin production in Y-02 within different carbon sources in 72 h. D, dextrose/glucose; S, sucrose; Dg, dextrose and glycerol in 1:1; Sg, sucrose and glycerol in 1:1; G, galactose. E, Extracellular and intracellular naringenin production of recombinant strains in 72 h in YPSg media. Y-02 CHI: Y-02 harboring pCEV-ph-CHI; Y-02 CHI-TAL:

Y-02

harboring

pCEV-ph-CHI-TAL;

Y-02

4CL-CHS:

Y-02

harboring

pKS2µHyg-4CL-CHS; Y-02 P-P: Y-02 harboring pCEV-ph-CHI-TAL and pKS2µHyg-4CL-CHS; Y-03 P-P: Y-03 harboring pCEV-ph-CHI-TAL and pKS2µHyg-4CL-CHS. Error bars represent s.d. from three independent experiments. Figure 3 Metabolic engineering of module 2. A, Schematic diagram of engineering in malonyl-CoA biosynthetic pathway. The green arrows represent up-regulation with the native malonyl-CoA 24


Page 25 of 32


biosynthetic pathway, the red arrow represents introduction of heterologous acetyl-CoA biosynthetic pathway via ACL, the blue bar represents the restrain step for fatty acid biosynthesis. B, The effect of pathway regulation on intracellular total fatty acid biosynthesis in recombinant strains after 48 h culturing in YPSg. The production in Y-02 was used as reference for normalization (defined as 1). The blue column indicates the relative value according to mass-to-volume. The orange column indicates the relative value according to mass-to-cell dry weight. C, Extracellular production of naringenin in recombinant strains after 72 h culturing in YPSg, according to mass-to-volume (blue column) and mass-to-cell dry weight (orange column). Y-11, over-expression of ACS1 in Y-02; Y-12, introduction of ylACL-sub12 and knock-out of ADH1 in Y-02; Y-13, combination of strategies used for construction of Y-11 and Y-12 in Y-02; Y-14, replacement of PACC1 with strong PHXT7 in Y-13. Error bars represent s.d. from three independent experiments. Figure 4 Metabolic engineering of module 3. A, Schematic diagram of engineering in tyrosine biosynthetic pathway. The red arrows represent up-regulation with the tyrosine biosynthetic pathway from PEP (phosphoenolpyruvate) and E4P (D-erythrose 4-phosphate). Blue bars represent down-regulation or knock-out of the competing pathways to tyrosine. Blue cross represents elimination of feedback inhibition. B, Method for gene knock-out or integration or mutation on the genome. C, Extracellular production of naringenin in engineered strains after 72 h culturing in YPSg. Y-22, over-expression of mutant ARO4K229L in Y-02; Y-23, down-regulation of PHA2 via promoter replacement in Y-22; Y-24, knock-out of PDC5 in Y-23; Y-25, knock-out of ARO10 in Y-24. D, Production changes of intracellular tyrosine in engineered strains after 48 h culturing in YPSg. The tyrosine production in Y-02 was used as reference for normalization (defined as 1). E, Extracellular production of naringenin in recombinant strains with native genes over-expression. Y-25, modified 25



Page 26 of 32

with k229L mutation of ARO4 on the genome, was used as the control. F, Extracellular production of naringenin

in

the

main

engineered

strains

transformed

with

pCEV-ph-CHI-TAL and

pKS2µHyg-4CL-CHS. Y-35, Y-25 with ARO4K229L mutation on the genome. Error bars represent s.d. from three independent experiments.

26


Page 27 of 32


Table 1 List of the main strains in this study Strains

Genotype

Source

BY4741 MATa, his3△1, leu2△0 , met15△0, ura3△0

Ref.22

Y-01

BY4741, △GAL80::PGAL2-4CL-PGAL7-CHS

This study

Y-02

Y-01, △HO::PGAL1-TAL

This study

Y-03

Y-01, △HO::PGAL1-TAL-PGAL10-GAL4

This study

Y-11

Y-01, △HO::PGAL1-TAL-PGAL10-ACS1

This study

Y-12

Y-02, △IDH1-PGAL1-ylACL-sub1-PGAL10-ylACL-sub2 This study

Y-13

Y-11, △IDH1-PGAL1-ylACL-sub1-PGAL10-ylACL-sub2 This study

Y-14

Y-13, △PACC1::PHXT7

This study

Y-22

Y-02, △PACC1::PHXT7-PTEF1-ARO4K229L

This study

Y-23

Y-22, △PPHA2::PHXT1

This study

Y-24

Y-23, △PDC5::HIS

This study

Y-25

Y-24, △ARO10::G418

This study

Y-32

Y-25, △TRP2::MET

This study

Y-35

Y-25, ARO4::ARO4K229L

This study

Y-36

Y-35, PTEF1-ARO1

This study

Y-37

Y-35, PTEF1-ARO2

This study

Y-38

Y-35, PTEF1-ARO8

This study

Y-39

Y-35, PTEF1-TYR1

This study

Y-40

Y-35, PTEF1-ZWF1

This study

27



Figure 1

28


Page 28 of 32

Page 29 of 32


Figure 2

29



Figure 3 A

B

C

30


Page 30 of 32

Page 31 of 32


Figure 4

31



Graphic for table of contents

32


Page 32 of 32

Enhancement of Naringenin Biosynthesis from ... - ACS Publications

Recommend Documents