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Modular engineering of the flavin pathway in Escherichia coli for improved flavin mononucleotide and flavin adenine dinucleotide production Shuang Liu, Na Diao, Zhiwen Wang, Wenyu Lu, Ya-Jie Tang, and Tao Chen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b02646 • Publication Date (Web): 17 May 2019 Downloaded from http://pubs.acs.org on May 21, 2019

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Journal of Agricultural and Food Chemistry

1

Modular engineering of the flavin pathway in Escherichia

2

coli for improved flavin mononucleotide and flavin adenine

3

dinucleotide production

4

Shuang Liu∞,1, Na Diao∞,1, Zhiwen Wang1, Wenyu Lu1,*, Ya-Jie Tang2,3, Tao Chen1,*

5 6

1

7

Bioengineering (Ministry of Education); SynBio Research Platform, Collaborative

8

Innovation Center of Chemical Science and Engineering (Tianjin); School of

9

Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China.

Frontier Science Center for Synthetic Biology and Key Laboratory of Systems

10

2

11

266237, China

12

3

13

Laboratory of Industrial Microbiology, Hubei Provincial Cooperative Innovation

14

Center of Industrial Fermentation; Hubei University of Technology, Wuhan 430068,

15

China

State Key Laboratory of Microbial Technology, Shandong University, Qingdao

Key Laboratory of Fermentation Engineering (Ministry of Education); Hubei Key

ACS Paragon Plus Environment


16

ABSTRACT

17

In this work, modular engineering of Escherichia coli was peformed to improve flavin

18

production and the conversion ratio of riboflavin (RF) to FMN/FAD. The RF operon

19

and the bifunctional RF kinase/FAD synthetase were divided into two separated

20

modules. The two modules were expressed at different levels to produce RF:ribF

21

ratios ranging from 2:20 to 7:5. The best strain respectively produced 324.1 and 171.6

22

mg/L of FAD and FMN in shake flask fermentation, and the titers reached 1899.3 and

23

872.7 mg/L in a fed-batch process. Furthermore, error-prone PCR (epPCR) of the E.

24

coli ribF gene was performed. The highest FMN production of the best mutant

25

reached 586.1 mg/L in shake flask cultivation. Moreover, this mutant produced

26

1017.5 mg/L FMN with a greatly reduced proportion of FAD in fermenter culture. To

27

the best of our knowledge, this is the highest production of FAD and FMN in a

28

microbial fermentation process reported to date.

29

KEYWORDS: Flavins, Escherichia coli, modular engineering, Error-Prone PCR


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INTRODUCTION

31

Flavin is a general term for pteridine-based organic compounds derived from the

32

isoalloxazine ring. The central biological source of the flavin moiety is riboflavin (RF,

33

also known as vitamin B2). Intracellularly, RF is often phosphorylated into flavin

34

mononucleotide (FMN), and then attached to an adenosine diphosphate to form flavin

35

adenine dinucleotide (FAD). FMN and FAD are the active forms of RF, which play

36

key roles as co-factors for dehydrogenases and oxidoreductases. RF is an essential

37

nutrient for humans and animals because they cannot synthesize RF de novo. By

38

contrast, all plants and fungi, as well as most bacteria are capable of producing RF. At

39

the same time, all organisms, including animals, convert RF to the flavin coenzymes

40

FMN and FAD 1. About 70% of the industrial RF is used as a feed additive and the

41

other 30% is used as a food additive and for pharmaceutical applications 2. In some

42

cases, FMN has an advantage over RF due to its high water solubility (about 200

43

times higher than that of RF) and higher therapeutic potency in treating some diseases

44

3.

45

such as Friedreich ataxia 4 and chronic granulomatous disease 5.

46

Nowadays, commercial RF production is exclusively done using fermentation 2, and

47

the most widely used strains include the bacterium Bacillus subtilis, the mold Ashbya

48

gossypii, and the yeast Candida famata. In our previous study, E. coli was also

49

engineered for RF production, which afforded a titer of more than 10 g/l in fed-batch

50

cultivation 6. However, the production levels of FMN and FAD are much lower than

Recent studies have shown that FAD may be helpful in the treatment of diseases



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that of RF. Recently, the flavogenic yeast Candida famata has been engineered to

52

overproduce FMN

53

highly pure FMN via approaches such as cofactor trapping

54

compartmentalizing the final FMN biosynthesis step in the periplasm 11. The optimal

55

periplasmically engineered strain accumulated 70.8 mg/L FMN, which accounted for

56

92.4% of total excreted flavins. However, while the cofactor trapping approach led to

57

FMN production with high purity, it had a low yield and low titer.

58

All organisms1,

59

catalyzed by RF kinase (RFK, EC2.7.1.26) and the resulting FMN is further

60

adenylated to FAD by FMN adenylyl transferase (FMNAT/FAD synthetase, EC

61

2.7.7.2). Both reactions require ATP and generate ADP and pyrophosphate,

62

respectively. Several eukaryotes use two divided monofuntional RFK and FMNAT

63

for FMN and FAD synthesis respectively. For instance, the FMN1 and FAD1 genes of

64

S. cerevisiae encode RFK and FAD synthetase, respectively. By contrast, most

65

prokaryotes have a single bifunctional protein, which exhibits both RF kinase and

66

FAD synthetase activities. In E. coli, this bifunctional enzyme is encoded by the ribF

67

gene. In the prokaryotic bifunctional enzyme, the RFK activity occurs at the

68

C-terminal region of the protein, and the FMNAT activity is located at the N-terminus

69

of the protein 12-14.

70

Recently, a novel approach called multivariate modular metabolic engineering

71

(MMME) has been widely used for the optimization of metabolic pathways and

72

strains

15,

3

7, 8

and FAD 9. Some reports concentrated on the production of 10

and by

are able to transform RF into FMN via specific phosphorylation

as well as genetic optimization of multigene systems in synthetic biology


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due to its standardized interchangeable parts

74

has been applied for the genetic engineering of modular multienzyme polyketide

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synthases 18, for improving N-acetylglucosamine production in Bacillus subtilis 19, as

76

well as advanced β-carotene production in Escherichia coli

77

establish E. coli strains that are capable of overproducing FMN and/or FAD with high

78

titer and high proportion among total flavins. For this goal, co-overexpression of the

79

RF operon and the ribF gene was performed, which endowed E. coli with the ability

80

of overproducing FAD and FMN. Then, knocking out of 6-phosphofructokinase I and

81

simultaneously blocking the Entner-Doudoroff pathway resulted in a significant

82

improvement of the titers of FAD and FMN. Afterwards, the RF operon and the ribF

83

gene were divided into two separated modules. Plasmids with different copy numbers

84

were constructed and/or integration of the RF operon into the genome was carried out

85

to regulate the expression strength of the RF operon and the ribF gene. The best result

86

was achieved by combining the low copy number RF operon expression plasmid

87

pLS01 and the high copy number plasmid p20C-ribF. The resulting strain respectively

88

produced 239 and 145 mg/L FAD and FMN in shake flask fermentation, as well as

89

1899.3 and 872.7 mg/L FAD and FMN in fed batch process. Finally, epPCR of the

90

ribF gene was performed to modify the bifunctional RFK/FMNAT. Several ribF

91

mutants that exhibited FMN overproduction without FAD accumulation or with a

92

small of proportion of FAD in the medium were successfully screened. A strain with

93

an optimal combination of ribF mutant and RF operon was obtained, and the

94

maximum FMN titer in shake flask fermentation reached 586.1 mg/L, which was

16, 17.

For instance, modular engineering


20.

This study sought to


95

further improved to 1017.5 mg/L FMN in a 5-L fermenter, representing arguably the

96

highest titer to date.

97

98

MATERIALS AND METHODS

99

Strains, plasmids, media and culture conditions

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The strains and plasmids used in this study are listed in Table 1. E. coli K-12 MG1655

101

was used to engineer the flavin producing strains. E. coli K-12 DH5α was selected as

102

the host to propagate vector DNA, and all E. coli cells were cultured at 30°C or 37°C

103

in LB medium (10 g/l peptone (Oxoid Limited, United Kingdom), 5 g/l yeast extract

104

(Oxoid Limited, United Kingdom), 10 g/l sodium chloride) with the addition of

105

appropriate antibiotics. MSY medium

106

shake-flasks and the fermentation approach was identical to a previous study 6. For

107

fed-batch fermentation, a single colony was transferred into LB medium with

108

appropriate antibiotics and cultured at 30°C and 220 rpm overnight. A 1 % (v/v)

109

aliquot of the resulting seed culture was transferred into a shake flask containing LB

110

medium and grown at 30°C and 220 rpm for 8-10 h, after which 10 % (v/v) of the

111

second seed culture was used to inoculate a 5 L bioreactor containing 2.5 L MSY

112

medium. The initial glucose titer was 10 g/L. Fed-batch fermentation was performed

113

at 30°C and 300-800 rpm with aeration at 1 vvm. A solution comprising 10 % (v/v)

114

ammonia was added automatically when the pH decreased below 7. Glucose and

115

yeast extract were supplemented to about 10 g/L and 5 g/L, respectively, once the

6, 21

was selected for riboflavin production in


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glucose titer fell under 1 g/L. For each strain, three parallel fermentations were

117

performed.

118

Plasmid construction and genome manipulation

119

All plasmids in this study were constructed by Circular Polymerase Extension

120

Cloning (CPEC)

121

sequence overlaps ready for plasmid construction were amplified by PCR, put

122

together for CPEC or Gibson assembly, and the resulting DNA used to transform

123

DH5α. Positive transformants were identified by colony PCR of the respective

124

dominant selectable marker. The native promoter region of the ribF gene, predicted

125

using the regulon DB website 24, was found to be located at the fragment from -84 to

126

-25 base upstream of the CDS. The gene ribF together with the 84 bp upstream (the

127

promoter Ptrc was introduced at the front of the PribF at the same time), were

128

amplified and inserted downstream of the RF operon on plasmid pLS01, resulting in

129

p5C-RF-ribF. The ratio of the expression strength of the ribF gene to the RF operon

130

was defined as 5:5 (abbreviated as ribF:RF Operon = 5:5). The ColE1 origin of

131

galK_Y145*_120/17, the spectinomycin resistance gene aadA of pTKRED, the ribF

132

gene on MG1655 genome together with its native promoter PribF (with a Ptrc

133

promoter sequence upstream as well), and the T1 terminator on pTKS/CS were

134

amplified and integrated together, resulted in the plasmid p20C-ribF. The amplified

135

ribF fragment was also inserted into pZY48, resulted in p5C-ribF. All the positive

136

clones were verified by colony PCR and sequenced by Genewiz (China).

22

or Gibson assembly 23. Briefly, DNA fragments sharing terminal



137

The genome editing technique used in this study was based on λ-Red recombineering

138

and double-strand DNA breaks triggered by mitochondrial homing endonuclease

139

I-SceI 25, according to a previous report 21. The donor DNA fragment was amplified

140

by overlap extension PCR. The primers used in this study are listed in the

141

Supplemental Tables S1 and S2.

142

Analytical methods

143

Cell growth was monitored by measuring the OD600 using a spectrophotometer. The

144

concentration of glucose was measured using a glucose analyzer as described before

145

21.

146

overproduce RF without the accumulation of FMN/FAD in the media, was identical

147

to a previous study

148

(FAD/FMN/RF) was determined by reverse-phase HPLC under the following

149

conditions: mobile phase, 10 mmol/l NaH2PO4, 30% methanol; flow rate 0.8 ml/min;

150

Column, BETASIL C18 250×4.6 Thermo-Fisher Scientific(USA); peak detection at

151

445 nm (UV detector). The FAD/FMN/RF reference standards were purchased from

152

Sigma-Aldrich (USA). The detection limit of flavin was 5 mg/L. Thus, lower

153

concentrations were regarded as no accumulation, unless stated otherwise. The HPLC

154

chromatograms of FAD/FMN/RF reference standard and the culture supernatant of

155

EF7 are shown in Supplemental Figure S1.

The measurement of RF in specific overproduction strains, which would

21.

The concentration of each component in the flavin mixture


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Error-Prone PCR and screening of mutants

157

Error-Prone PCR (epPCR) was carried out using an Error-Prone-PCR Kit

158

(101005-100, TIANDZ, P.R. China). The ribF gene was amplified by epPCR from

159

the genome of MG1655 using a pair of primers containing BamHI and SalI restriction

160

sites, respectively, and cloned into p20C using the two restriction sites, creating the

161

ribF mutant library. The resulting library was introduced into the host LS21 using

162

the heat shock method and the transformants were selected on spectinomycin plates.

163

A total of 287 single colonies were picked and individually fermented in 96 deep well

164

plates. After 24 h of fermentation, the broth was centrifuged and the supernatant was

165

analyzed by HPLC. For detection of the the RF kinase activity of the mutants, cells

166

were cultivated in shake flask as decried above. And then And then the cultures were

167

processed into cell-free extracts as previously described 21. The FMNAT activity was

168

measured accordingly to somewhere else14.

169

Quantitative real-time PCR (qRT-PCR) analysis

170

The expression level of RF operon to the ribF gene was measured by qRT-PCR. The

171

culture conditions for RNA extraction were consistent with the fermentation condition

172

for flavin production. Cells were harvested when the cell density (OD600 nm) was

173

corresponding to approximately 1.5-2.0 and then the total RNA of the E. coli strains

174

was extracted using the RNA Extraction Kit (Takara, Dalian, China). The quantity

175

and purity of the extracted RNA were determined by optical density measurements at

176

260 and 280 nm by a BD-2000 spectrophotometer (OSTC, China).



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The transcription strength (RNA level) of the ribC gene (representing for RF operon)

178

and the ribF gene were measured by qRT-PCR. The respective primers used for

179

qRT-PCR studies are listed in Supplemental Table S3. rrsA gene encoding 16S

180

ribosomal RNA was selected as internal standard. The cDNA templates used for

181

qRT-PCR were obtained via reverse transcription reaction using TransScript

182

First-Strand cDNA Synthesis SuperMix (TransGen Biotech, China). qRT-PCR was

183

carried out in a 96-well plate with a total reaction volume of approximately 20 μ l

184

utilizing TransStart Top Green qPCR SuperMix (TransGen Biotech, China). The

185

qRT-PCR process was carried out using a LightCycler 480 II Real-time PCR

186

instrument (Roche Applied Science, Germany). The qRT-PCR data were analyzed by

187

the comparative CT method26.

188

Detection of the concentration of intracellular flavins

189

Strains were cultured as described above to the cell density (OD600 nm) was

190

corresponding to approximately 2.0 and then cooled on an ice bath. After that cells

191

were harvested rapidly by centrifugation at 4 ℃ and washed in ice-cold deionized

192

water. Cells were resuspended in 5 ml deionized water and crushed by a grinding

193

instrument (Shanghaijingxin Experimental Technology, China ). The mixtures were

194

spun and the supernatants were dried using vacuum freeze drying. The dry powder

195

were dissolved in 1 ml deionized water and assayed by HPLC after filter. The dry

196

powder were dissolved in 1 ml deionized water and assayed by HPLC after filter. The

197

Dry cell weight (DCW) was calculated from the optical density at 600 nm


21

and the

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corresponding cell aqueous volumes were calculated according to the former reports27,

199

28.

200

intracellular flavins weights and cell aqueous volumes.

201

Calculations

202

RF is transformed into the coenzyme forms FMN and FAD by bifunctional enzyme

203

RFK/FMNAT. The reactions are listed as below:

204

riboflavin + ATP → ADP + FMN

205

ATP + FMN + H+ → FAD + diphosphate

206

No FAD/FMN accumulation was detected in the culture supernatants of the RF

207

overproducing strains (data not shown). Thus, it could be reasonably assumed that the

208

overproduction of FAD/FMN resulted from the overexpression of the enzyme

209

RFK/FMNAT. In order to measure the conversion ratio of RF (cr-RF) to FMN/FAD,

210

equivalent RF (e-RF) was defined as:

211

Me-RF = MRF + MFMN + MFAD (mmol/l)

212

[e-RF] = Mr(RF) * Me-RF (mg/L)

213

Where e-RF represents the total RF produced equivalently, a part of which was

214

further converted into FMN and FAD and the other was the actual RF in the

215

fermentation broth. Then, the cr-RF was defined as:

216

cr-RF = ([e-RF] – [RF]) / [e-RF]

217

where M represents the molar concentration, Mr represents the relative molecular

218

mass, [] represents concentration (mg/L).

The intracellular flavins concentrations were calculated by the corresponding



219

The proportion of flavin was defined as:

220

i % = [ i ] / [Flavins] * 100%

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[Flavins] = [RF] + [FMN] + [FAD]

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RESULTS

223

Overexpression of the ribF gene in E. coli

224

It has been proved that metabolically engineered E. coli is an excellent RF producer 6,

225

which indicates that this cell chassis also has the potential to be developed into an

226

overproducer of FMN and/or FAD. RF is synthesized from GTP and ribulose

227

5-phosphate in vivo (Figure 1). Next, RF is converted into FMN by RF kinase (RFK,

228

EC2.7.1.26), and then FMN adenylyl transferase (FMNAT/FAD synthetase, EC

229

2.7.7.2) adenylates FMN to FAD. In E. coli, this process depends on a bifunctional

230

protein, the RFK/FAD synthetase encoded by the ribF gene. The constitutive

231

expression plasmid pLS01 bearing an artificial RF operon which contains the genes of

232

the riboflavin synthesis pathway (ribA, ribB, ribD, ribE, ribC) from E. coli was

233

constructed in our previous research

234

amplified and inserted downstream of the RF operon in pLS01, resulting in

235

p5C-RF-ribF. The plasmid was introduced into E. coli MG1655 to construct the strain

236

EF1. As shown in Figure 2A, the final titers of FAD/FMN/RF produced by EF1 were

237

81.4±0.5/72.4±1.0/80.9±0.6 mg/L in flask fermentation, with a cr-RF value of 0.55.

238

Interestingly, the WT MG1655 bearing the plasmid pLS01 (strain RF01T6), produced

239

55 mg/L RF. However, the e-RF of EF1 reached 179.6 mg/L, which was much higher

i = RF, FMN, FAD

6, 21.

For convenience, the ribF gene was


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than the RF titer of RF01T. Thus, the overexpression of the ribF gene greatly

241

increased the capacity of E. coli for the production of FAD/FMN, and also facilitated

242

RF synthesis. It seems to be contradictory that overexpression of the downstream

243

gene (ribF) stimulates RF production. It is indistinct whether FMN/FAD may have

244

some regulatory influence on RF operon expression or activity of corresponding

245

enzymes or not. Interestingly, in another case, it was also reported that up-regulation

246

of the downstream genes hemD or hemF has a positive correlation on the

247

5-aminolevulinic acid accumulation in E. coli 29.

248

Previous research has shown that a knockout of the pfkA gene and the ED pathway

249

(edd and eda genes) could efficiently increase RF production in E. coli 6. It is

250

therefore reasonable to assume that the deletion of these genes would contribute to

251

producing flavins. In order to confirm this presumption, the plasmid p5C-RF-ribF was

252

introduced into the strains LS02 (ΔpfkA, Δedd and Δeda mutant), resulting in strain

253

EF4. As expected, the final production of FAD/FMN/RF of EF4 (Figure 2B) reached

254

239.0±1.9 / 145.0±0.9 / 160.4±1.2 mg/L, respectively, which was much higher than

255

the corresponding values of EF1. This revealed that the knockout of the pfkA gene and

256

simultaneously blocking the ED pathway greatly contributed to the increase of flavin

257

production. Thus, the pfkA and ED double knockout strain LS02 was selected as the

258

host for further study.

259

Modular engineering of the RF operon and the ribF gene

260

Although the total flavin production was significantly improved in strain EF4, the



261

cr-RF value of this strain was only a bit higher than that of strain EF1 (0.59 vs 0.55).

262

In order to further facilitate the conversion of RF into FMN/FAD, modular metabolic

263

engineering was carried out. The synthetic operon responsible for RF synthesis was

264

termed as Module 1, while the bifunctional RFKs/FMNAT responsible for the

265

synthesis of FMN and FAD was termed as Module 2. Generally, diversifying the

266

expression strength of modules can be realized by various procedures, such as

267

modifying the promoter and/or RBS strengths, as well as using plasmids with

268

different copy numbers. In this study, the promoters and RBS of both modules were

269

not modified, and the expression levels of the modules were varied by using plasmids

270

with different copy numbers or integrating Module 1 into the genome. In our previous

271

study, we found that using a high copy number plasmid for RF operon overexpression

272

imposed an unacceptably high metabolic burden on the host 6, which resulted in a

273

high plasmid loss rate. Thus, in Module 1, the RF operon was overexpressed using the

274

low copy number plasmids pLS01 or p5C-RF-ribF. Additionally, two copies of the

275

RF operon were integrated into the genome of LS02 at the ldhA and yahI gene loci,

276

respectively, and the mutant strain harbouring 2 copies of the RF operon was named

277

LS21. In Module 2, the low copy number plasmids p5C-RF-ribF or p5C-ribF and the

278

high copy number plasmid p20C-ribF were utilized for the overexpression of the ribF

279

gene. Combinatorial expression was accomplished by changing hosts and plasmids

280

(EF3 ~ EF8). Due to the fact that both the RF operon and the ribF gene have the same

281

promoter and RBS sequence, the expression level ratio of RF operon to ribF could be

282

controlled semi-quantitatively with reasonable predictability, varying between


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283

approximately 7:5 and 2:20. Meanwhile, the actual expression level of RF operon to

284

the ribF gene was quantified by qRT-PCR. The quantitative result was basically

285

consistent with the predictive value (Figure. 3).

286

As shown in Figure 3, the cr-RF of this series of strains increased at first and then

287

decreased with the decreasing RF:ribF expression ratio, apart from EF6. The cr-RF of

288

EF7 reached 0.78, which was the highest among the six strains. EF7 exhibited the

289

highest FAD titer as well. Interestingly, EF3 produced the highest titer of total flavins

290

among the six mutants but had the lowest cr-RF value (0.55) yet. The intracellular

291

contents of flavins of EF3-EF8 strains (Table. 2) showed that these strains had the

292

same order of magnitude of flavins concentration except for the RF titer of EF5. EF3

293

possessed the highest RF titer among these strains with a lower FMN/FAD titer. Thus,

294

we suspect that for EF3 strain, a large amount of RF was secreted from the cells to the

295

medium instead of being converted into FMN and FAD.

296

In view of its high cr-RF, EF7 was selected for a further scale-up culture in a 5 L

297

bioreactor using an intermittent fed-batch method. As shown in Figure 4, the final

298

production of FAD and FMN reached 1899.3 and 872.7 mg/L, respectively, with a

299

cr-RF of 0.73. The yield of total flavins was about 58 mg/g glucose in fed-batch

300

cultivation.

301

Error-prone PCR of the ribF gene and screening of improved mutants

302

Error-prone PCR (epPCR) of the ribF gene was carried out to modify the bifunctional

303

RFK/FMNAT, which was expected to yield strains harboring mutant ribF that



304

preferentially overproduce FMN or FAD with a high cr-RF. To highlight the

305

effectiveness of epPCR, the high copy number plasmid p20C was selected as the

306

vector for the expression of the ribF library. LS21 was picked as the host to ensure

307

stable and reliable screening results. The RF:ribF ratio in this system was 2:20.

308

The epPCR library of the ribF gene was generated by restriction enzyme digestion

309

and ligation, and then directly used to transform the host LS21. The transformants

310

were cultured in 96 deep well plates and the products in the supernatant detected by

311

HPLC. The screening results showed that more than half of the mutants (150 among

312

the 287 screened colonies with more obvious yellow color) lost the ability to

313

overproduce FMN and FAD. The other 137 mutants could overproduce FMN and

314

FAD simultaneously or showed FMN production without FAD accumulation with an

315

extensive range of cr-RF values (0.06-0.8). We acquired 15 mutants which could

316

accumulate FMN without FAD in the culture supernatant. The cr-RF values of these

317

15 mutants ranged from 0.06 to 0.56. FMNAT activity in cell-free extracts of EF8

318

was about 3.87±0.16 nmol/min/mg protein. While the FMNAT activity in cell-free

319

extracts of the mutants were hardly detectable. Unexpectedly, we failed to obtain

320

mutants which could overproduce FAD without the accumulation of FMN. Five of the

321

mutant strains which could overproduce FMN without FAD, together with another six

322

mutants with high cr-RF values ( ≥ 0.76) were selected for further fermentation in

323

shake flasks. The fermentation results of these strains in shake flasks are shown in

324

Table 3. Similar to the results of mutants screened in 96 deep well plate culture,

325

several mutants which could overproduce FMN without FAD accumulation in the


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326

medium were identified successfully. However, the flavin titers and cr-RF values of

327

these mutants were still low, and the best strain (No 8) only produced 152.8±5.5 mg/L

328

FMN.

329

According to the optimal ratio of RF:ribF (5:20), plasmids harboring these ribF

330

mutants were extracted and introduced into LS02T. Two of the resulting strains

331

exhibited both high FMN production and high cr-RF without detectable FAD

332

accumulation in shake flask cultivation. Some of the other mutants could overproduce

333

FMN with a lower proportion of FAD in the broth. The flavin production and cr-RF

334

values of the two best mutants, as well as the related mutant sites are listed in Table 4.

335

The nuclease sequences of the mutant ribF genes are listed in supplemental Table S4.

336

Strain EF12, which had the highest FMN production, was further cultured in a 5 L

337

bioreactor using a fed-batch fermentation process. As shown in Figure 5, the FMN

338

titer of EF12 reached 1017.5±12.7 mg/L. Moreover, the cr-RF value was 0.5 and the

339

yield of total flavins was about 76 mg/g glucose. Interestingly, the cr-RF of EF12 in

340

the fed-batch process was much lower than in shake flask (0.5 vs 0.71), which

341

indicated that different fermentation processes had a great influence on the cr-RF

342

value. Thus, optimization of fermentation condition is pivotal to achieve

343

simultaneously high flavin titers and cr-RF values for the mutants. We are still

344

working on this problem, but it is beyond the scope of this study.

345



Page 18 of 40

346

DISCUSSION

347

In this study, we achieved the efficient production of FAD/FMN in E. coli via

348

modular metabolic engineering in conjunction with the modification of the

349

bacterium’s native bifunctional RFKs/FMNAT. The industrial production of RF is

350

now conducted exclusively using fermentative synthesis utilizing microorganisms 2.

351

Even though RF can be directly converted into FMN and then into FAD by the

352

enzyme, the titer of FMN and FAD produced by microbial fermentation was rather

353

low. We therefore tried to engineer E. coli for high production of FMN and FAD. The

354

fermentation results of the strain EF1 bearing the plasmid p5C-RF-ribF proved the

355

capacity of E. coli to produce FMN and FAD. The further improved strain EF4

356

exhibited increased flavin production compared to EF1, which indicated that the

357

downregulation of glycolysis (ΔpfkA) and blocking of the Entner-Doudoroff pathway

358

(Δedd, Δeda) efficiently increased the total flavin production.

359

Interestingly, EF1 produced significantly more RF than RF01T (80.9 vs 55 mg/L),

360

with a much higher e-RF value (179.6 mg/L), which indicated that the overexpression

361

of the ribF gene promoted RF synthesis. Generally, downregulation of the

362

bifunctional RFK/FMNAT was beneficial for RF production. For instance,

363

introducing a mutant ribC gene with reduced RFK/FAD-synthetase activity

364

mutant ribR gene with decreased monofunctional RFK activity

365

overproduction of RF in Bacillus subtilis. These mutations resulted in decreased

366

accumulation of FMN and released the repression of the RF operon at high

367

concentrations of FMN via a riboswitch (RFN element)


32, 33.

31

30

and a

led to the

In E. coli, decreased

Page 19 of 40


368

expression levels of flavokinase by modulating the RBS of ribF also greatly

369

contributed to RF overproduction

370

RFK/FMNAT also led to increased RF production in the wild-type host. We suspect

371

that increased expression of RFK/FMNAT accelerated the conversion of RF into

372

FMN and FAD, which further promoted the synthesis of RF.

373

However, when the LS02T strain that overproduces riboflavin was used as the host,

374

overexpression of the ribF gene (EF4) resulted in a decrease of the RF titer (160.4 vs

375

605 mg/L), and even the e-RF (394.5 mg/L) of EF4 was still lower than the RF titer of

376

LS02T 6. This indicated that overexpression of the ribF gene in a RF overproducing

377

host obstructed its synthesis. However, these results are rather puzzling, since the only

378

difference between the two sets of strains was the knockout of pfkA, edd and eda. The

379

fermentation results showed that the glucose consumption rate of EF4 was lower than

380

that of EF1 and the biomass (OD600) of EF4 was also less than that of EF1, which

381

indicated that the downregulation of glycolysis and blocking the Entner-Doudoroff

382

pathway hindered cell growth. This may be due to two aspects of cellular metabolism.

383

On the one hand, downregulation of glycolysis would directly affect the utilization of

384

glucose. On the other hand, EF4 produced more FMN and FAD than EF1, which

385

means that more ATP was needed for FAD and FMN synthesis in EF4. A previous

386

report has shown that improving the intracellular energy availability was beneficial to

387

riboflavin production

388

synthesis would hinder RF production.

389

EF1 and EF4 displayed similar cr-RF values (0.55 and 0.59, respectively), which may

34.

21.

However, we found that the overexpression of

Thus, excessive consumption of ATP for FMN and FAD



390

be due to the same RF:ribF ratio. To improve the flavin production and cr-RF further,

391

modular engineering was carried out. RF operon and the bifunctional RFKs/FMNAT

392

were termed as Module 1 and Module 2 respectively. The strains EF3 to EF8 were

393

constructed to achieve various expression strengths of Module 1 and Module 2. EF7

394

had both the highest cr-RF (0.78) and the highest FAD titer. The results indicated that

395

an appropriate RF:ribF ratio could significantly facilitate the conversion of RF into

396

FMN/FAD. In previous studies, the cr-RF was about 0.08-0.62 7. We suspected that

397

the various cr-RF values also resulted from the different overexpression strength of

398

RFKs in their work (integration of the FMN1 gene with copy numbers from 1 to 8).

399

Furthermore, the maximum FMN titer in a previous study reached 231 mg/L at 40 h

400

of a batch culture, but decreased to about 160 mg/L after 63 h 8.

401

Both the RF:ribF ratio and the actual expression strength of the modules affected the

402

flavin production. For instance, EF3, EF4 and EF5 had a consistent expression

403

strength of the ribF gene but different RF:ribF ratios. With the decreased expression

404

of the RF operon, the flavin production decreased at the same time, but the cr-RF

405

increased. EF4 and EF7 had the same expression strength of the RF operon but

406

different expression strengths of the ribF gene, analogously to EF5 and EF8. The

407

titers of FAD and FMN of EF7 were higher than those of EF4 due to the increased

408

expression strength of the ribF gene. Interestingly, EF3 with a RF:ribF ration of 7:5

409

had the highest total flavin yield but the lowest cr-RF, which indicated that the

410

expression of Module 2 is the limiting factor for FMN/FAD production in this strain.

411

EF6 had the highest expression strengths of both the RF operon and ribF, but


Page 20 of 40

Page 21 of 40


412

produced the lowest flavin yield. The fermentation results showed that it took EF6 48

413

h to completely consume the glucose, while this time was only around 18-24 h for the

414

other mutants. This indicated that EF6 suffered from a heavy metabolic burden due to

415

strong overexpression, which affected the production of flavins further.

416

In fed-batch cultivation, EF7 produced 1899.3 mg/L FAD, which to our best

417

knowledge represents the highest production achieved through microbial fermentation

418

reported to date. In previous work, product degradation was found to affect FAD

419

production. The engineered strain under optimized conditions produced 451 mg/L

420

FAD at 40 h, but this value decreased sharply with the rapid increase of RF titer 9. By

421

contrast, the degradation of FAD was not observed in our work.

422

RF is transformed into FMN and FAD in reactions catalyzed by the bifunctional

423

RFKs/FMNAT encoded by the ribF gene. In order to engineer strains for the

424

production of either FMN or FAD individually, epPCR of the ribF gene was carried

425

out to modify the bifunctional RFKs/FMNAT. Among the 287 mutants screened, 15

426

overproduced FMN without the accumulation of FAD in the culture supernatant. The

427

highest FMN titer reached 152.8 mg/L, with a cr-RF of 0.59, indicating that the

428

FMNAT activity of the mutated bifunctional enzymes in these strains was probably

429

inactivated. It has been reported that partial deletions or point mutations in the

430

N-terminal domain of ribF selectively affect the FMNAT activity of the enzyme

431

However, we failed to obtain mutants which could overproduce FAD without the

432

accumulation of FMN. In a previous report about the engineering of Candida famata

433

for FAD overproduction (the highest FAD titer reached 451 mg/l), the strain also


35.


434

accumulated 87-154 mg/L FMN in the culture supernatant 9. It may be difficult to

435

obtain mutants that can overproduce FAD without the accumulation of FMN through

436

epPCR of the ribF gene encoding the bifunctional enzyme, and genes encoding

437

monofunctional RFK and FMNAT, such as FMN1 and FAD1 from Saccharomyces

438

cerevisiae, could be used as independent modules for increased proportion of FAD in

439

future work.

440

Although mutants overproducing FMN without FAD accumulation were obtained via

441

epPCR of the ribF gene, all of them exhibited lower total flavin production compared

442

to the control strain (EF8) harboring the intact ribF (table 3). The sequencing results

443

showed that all the mutant sites were located in the N-terminal region. Previous

444

studies have shown that the dimer-of-trimers quaternary structure of the bifunctional

445

RFK/FMNAT resulted in active site residues of one module inﬂuencing the activity of

446

the other

447

led to the inactivation of RFK, and thereby also further influenced the FMNAT

448

activity.

449

In this work, modular metabolic engineering of the flavin synthesis pathway and

450

epPCR of the ribF gene were performed to engineer E. coli for FMN and FAD

451

production with high titer and high cr-RF. To our best knowledge, this is the first

452

report on the fermentative production of both FAD and FMN at a scale of 1 g/L.

453

ASSOCIATED CONTENT

454

Supporting Information

13, 14.

Thus, we suspect that the mutations located at the N-terminal region


Page 22 of 40

Page 23 of 40


455

Primers used in this study and HPLC chromatograms of FAD/FMN/RF.

456

AUTHOR INFORMATION

457

Corresponding Authors

458

*Email: [email protected]

459

*Email: [email protected]

460

ORCID

461

Author Contributions

462

∞ S.L. and N.D. contributed equally to this work. S.L. and T.C. conceived the

463

experimental design. S.L. and N.D. performed the experimental work. S.L, N.D. and

464

T.C. conceived and designed the manuscript. S.L and N.D. drafted the manuscript.

465

T.C., Z.W, W.L and Y.T. helped in analyzing the results and revising the manuscript.

466

T.C. and W.L. supervised the project. All authors discussed the project.

467

Funding

468

This research was supported by the National Natural Science Foundation of China

469

(NSFC-21621004, NSFC-21776208 and NSFC-21576200).

470

Notes

471

The authors declare no competing financial interests.

472

ABBREVIATIONS RF

riboflavin

FMN

flavin mononucleotide



FAD

flavin adenine dinucleotide

RFK

riboflavin kinase

FMNAT

FMN adenylyl transferase/FAD synthetase

RF:ribF

Expression strength ration of the RF operon to the ribF gene

e-RF

equivalent riboflavin

cr-RF

conversion ratio of riboflavin

epPCR

Error-prone PCR


Page 24 of 40

Page 25 of 40

474


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475 476

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Page 30 of 40

578

Figures captions

579

Figure 1. Metabolic pathway for the production of flavins in the engineered E. coli.

580

Dashed lines indicate multiple enzymatic steps. A cross indicates the deletion of the

581

corresponding gene. The abbreviations of genes and corresponding enzymes are as

582

follows: pgi, phosphoglucose isomerase; pfkA, 6-phosphofructokinase I; pfkB,

583

6-phosphofructokinase

584

6-phosphogluconolactonase; edd, phosphogluconate dehydratase; eda, multifunctional

585

2-keto-3-deoxygluconate 6-phosphate aldolase, 2-keto-4-hydroxyglutarate aldolase

586

and

587

decarboxylating;

588

3,4-dihydroxy-2-butanone-4-phosphate

589

diaminohydroxyphosphoribosylaminopyrimidine

590

5-amino-6-(5-phosphoribosylamino)uracil

591

6,7-dimethyl-8-ribityllumazine synthase; ribC, riboflavin synthase; ribF, bifunctional

592

riboflavin kinase/FMN adenylyltransferase. Other non-standard abbreviations: FBP,

593

fructose 1,6-bisphosphate; GAP, D-glyceraldehyde 3-phosphate; TCA, tricarboxylic

594

acid cycle; glycerate-3P, 3-phospho-D-glycerate; PRPP, 5-phospho-a-D-ribose

595

1-diphosphate; DHPB, 3,4-dihydroxy-2-butanone-4-P;GTP, guanosine-triphosphate;

596

DARPP,

597

5-amino-6-(5′-phosphoribitylamino)

598

5-amino-6-(5-phospho-D-ribitylamino) uracil; ArP, 5-amino-6-(D-ribitylamino)uracil;

oxaloacetate

II;

zwf,

glucose-6-phosphate-1-dehydrogenase;

decarboxylase; ribA,

gnd,

GTP

6-phosphogluconate cyclohydrolase synthase;

pgl,

dehydrogenase, II;

ribB,

ribD,

fused deaminase/

reductase;

2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone uracil;


5'-phosphate;

ribE,

ARPP, ArPP,

Page 31 of 40


599

DRL,

600

riboflavin-5'-phosphate; FAD, flavin adenine dinucleotide.

601

Figure 2. Time-course profiles of FAD/FMN/RF production, OD600 and glucose

602

consumption of the engineered strains EF1 and EF4.

603

Figure 3. Flavin production, the expression level of RF operon to the ribF gene and

604

the cr-RF of strains EF3 to EF8. p* represents the predicted ratios; q# represents the

605

ratios acquired by qRT-PCR

606

Figure 4 Time-profiles of glucose consumption, biomass (OD600) and flavin

607

production of EF7 in fed-batch culture in a 5-liter fermenter.

608

Figure 5 Time-profiles of glucose consumption, biomass (OD600) and FMN

609

production of strain EF7 in a 5-liter fermenter in fed-batch fermentation mode.

6,7-dimethyl-8-(1-D-ribityl)lumazine;

RF,


riboflavin;

FMN,


Page 32 of 40

Tables Table 1. Strains and plasmids used in this study Strain or plasmid

Description

Reference

DH5α

Host for cloning, CGSC#: 12384

6

MG1655

Wide type, CGSC#: 7740

6

LS01

MG1655, ΔpfkA

6

LS02

MG1655, ΔpfkA, Δedd, Δeda

6

LS02T

LS02 harboring pLS01

6

LS21

MG1655,

Strains

ΔpfkA,

Δedd,

Δeda,

RF This study

operon::ldhA, RF operon::yahI EF1

MG1655 harboring p5C-RF-ribF

This study

EF3

LS21 harboring p5C-RF-ribF

This study

EF4

LS02 harboring p5C-RF-ribF

This study

EF5

LS21 harboring p5C-ribF

This study

EF6

LS21 harboring pLS01 and p20C-ribF

This study

EF7

LS02 harboring pLS01 and p20C-ribF

This study

EF8

LS21 harboring p20C-ribF

This study

EF9

LS21 harboring p20C-ribFM-1

This study

EF10

LS21 harboring p20C-ribFM-2

This study

EF11

LS02 harboring pLS01 and p20C-ribFM-1

This study


Page 33 of 40


EF12

LS02 harboring pLS01 and p20C-ribFM-2

This study

pSC101 origin, Spcr, Red recombinase

25

Plasmids pTKRED

expression plasmid, lac-inducible expression pTKS/CS

p15A origin, Cmr, Tetr, I-SceI restriction

25

sites galK_Y145*_120/17 ColE1 origin, Ampr

36

p20C

ColE1 origin, Spcr, Ptrc promoter

This study

pLS01

pSC101 origin, Cmr, RF operon

6

pZY48

pSC101 origin, Cmr,

6

p5C-RF-ribF

pSC101 origin, Cmr, RF operon, ribF

This study

p5C-ribF

pSC101 origin, Cmr, ribF

This study

P20C-ribF

ColE1 origin, Spcr, ribF

This study

P20C-ribFM-1

ColE1 origin, Spcr, ribFM-1

This study

P20C-ribFM-2

ColE1 origin, Spcr, ribFM-2

This study

Table 2. Concentration of intracellular flavins of EF3-EF8 Strain

FAD (mg/L)

FMN (mg/L)

RF (mg/L)

EF3

N/A*

48.4±1.2

357.33±2.5

EF4

14.7±0.5

59.6±1.3

321.4.8±2.9

EF5

10.9±0.5

24.5±0.9

40.2±0.9

EF6

167.5±2.4

219.5±3.5

189.2±2.6



Page 34 of 40

EF7

20.4±0.4

64.6±1.7

115.5±4.8

EF8

111.3±3.8

27.7±1.2

N/A*

* N/A represents the corresponding concentration exceeds the detectable range

Table 3. Flavin production and cr-RF values of selected mutants screened by epPCR in shake flask fermentation.

No.

FAD (mg/L)

FMN (mg/L)

RF (mg/L)

e-RF (mg/L) cr-RF

1

100.9±4.3

120±4.1

44±1.4

191.3±2.4

0.77±0.01

2

156.1±4

99±2.2

44.8±0.9

201.2±4.6

0.78±0

3

177.7±6.2

133.8±2.7

51.9±1.7

247.5±6.9

0.79±0

4

214±7.3

118.6±6

43.6±2

244±6.5

0.82±0.01

5

42.4±2

131.6±5.4

50.9±3.4

179.8±0.1

0.72±0.02

6

50.8±3.9

163.8±4.7

46.4±1.1

205.8±4.6

0.77±0.01

7

0±

148.2±4.1

60±4.1

182.3±7.5

0.67±0.01

8

0±

152.8±5.5

89.2±3.4

215.3±8

0.59±0

9

0±

114.5±4

157.8±5.3

252.3±8.5

0.37±0

10

0±

70.8±3.5

110.3±5.2

168.7±8

0.35±0

11

0±

99.3±3.8

179.8±5.6

261.7±2.4

0.31±0.01


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Table 4 Flavin production, cr-RF, flavin proportion in shake flask fermentation and the ribF mutation sites of strains EF9 to EF12. Strain FAD

FMN

RF

(mg/L)

(mg/L)

EF11

557.7±18.3

EF12

586.1±7.5

(mg/L)

cr-RF

FAD%

FMN%

RF%

140.7±2.0

0.77±0.01

n.d.

80±1

20±1

194.2±1.9

0.71±0

n.d.

75±0

25±0

Strain

Mutation

Mutation site

EF11

ribFM-1

52 Val, 53 Met deletion

EF12

ribFM-2

Asp 66 Val, Lys 78 Glu, Ser 109 Ile, Lys 119 Gln, Gln 295 Leu



Figures

Figure 1.


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Figure 2.

Figure 3.



Figure 4


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Figure 5



TOC graphic


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Modular engineering of the flavin pathway in ... - ACS Publications

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