1

Development of a Recombinant Escherichia coli strain for Overproduction of 1

Plant Pigment, Anthocyanin 2

Chin Giaw Lima,1, Lynn Wonga,b,1, Namita Bhanb, Hila Dvoraa, Peng 3 Xua,b, Sankaranarayanan Venkiteswarana, and Mattheos A.G. Koffasa,b† 4

5

aDepartment of Chemical and Biological Engineering, 6 University at Buffalo, the State University of New York 7

Buffalo, NY 14260 8 9

bDepartment of Chemical and Biological Engineering and Center 10 for Biotechnology and Interdisciplinary Studies, Rensselaer 11

Polytechnic Institute, Troy, NY 12180 12 13

Submitted for publication to Applied and Environmental 14 Microbiology 15

16

1C.G.L. and L.W. contributed equally to this work 17

† Corresponding author 18

19

Department of Chemical and Biological Engineering 20

Rensselaer Polytechnic Institute, Troy, NY 12180 21

CBIS 4005D, 22

Telephone: (518) 276-2220 23

Fax: (518)-276-3405 24

AEM Accepted Manuscript Posted Online 6 July 2015Appl. Environ. Microbiol. doi:10.1128/AEM.01448-15Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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E-mail: koffam@rpi.edu 25

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ABSTRACT 26

Anthocyanins are water-soluble colored pigments found in terrestrial plants that are responsible 27

for the red, blue and purple coloration of many flowers and fruits. In addition to the plethora of 28

health benefits associated with anthocyanins (cardioprotective, anti-inflammatory, antioxidant 29

and anti-aging properties), these compounds have attracted widespread attention due to their 30

promising potential as natural food colorants. Previously, we reported the biotransformation of 31

anthocyanin, specifically cyanidin 3-O-glucoside (C3G), from the substrate (+)-catechin in 32

Escherichia coli. In the present work, we set to systematically improve C3G titer by enhancing 33

substrate/precursor availability, balancing gene expression level and optimizing 34

cultivation/induction parameters. We first identified E. coli transporter proteins that are 35

responsible for the uptake of catechin and secretion of C3G. We then improved the expression of 36

the heterologous pathway enzymes anthocyanidin synthase (ANS) and 3-O-glycosyl transferase 37

(3GT) using a bicistronic expression cassette. Next, we augmented the intracellular availability 38

of the critical precursor UDP-glucose, which has been known as the rate-limiting precursor to 39

produce glucoside compounds. Further optimization of culture and induction conditions led to a 40

final titer of 350 mg/L C3G. We also developed a convenient colorimetric assay for easy 41

screening of C3G overproducers. The work reported here constitutes a promising foundation to 42

develop a cost-economic process for large-scale production of plant-derived anthocyanin from 43

recombinant microorganisms. 44

Keywords: anthocyanin / pigment / cyanidin-3-O-glucoside / metabolic engineering / Escherichia coli / 45

UDP-glucose 46

Abbreviations: 47

ANS, anthocyanidin synthase; 3GT, 3-O-glycosyl transferase; C3G, cyanidin-3-O-glucoside; UDP-glucose, 48 Uridine diphosphate-glucose 49

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1 Introduction 50

Plant natural products currently have extensive applications in the food and 51

pharmaceutical industries (1, 2). Among other things, their unique bioactivities and structures 52

allow them to serve as anti-microbial agents, therapeutic drugs, food condiments and natural 53

dyes (3–5). However, most of these resources remain largely untapped due to the limited 54

availability of these plants that is dependent upon regional and seasonal factors (6). Other major 55

obstacles to reaping the full benefit of natural products include low yields of the active 56

compounds in crude plant extracts, difficulty of purification, high cost and turnaround time of 57

chemical synthesis due to their complex chemical structures, and regioselectivity issues 58

associated with chemical synthesis (7). The engineering of recombinant microorganisms capable 59

of producing these heterologous compounds is a feasible solution to overcome these limitations. 60

Extensively studied organisms such as Escherichia coli and Saccharomyces cerevisiae are 61

especially attractive for this task due to the ease and wide availability of tools for genetic 62

modification and process optimization (8, 9). Coupled with recent advances in the metabolic 63

engineering and synthetic biology of these model organisms, systematic approaches such as 64

computational modeling, genomics, proteomics and transcriptomics can be used to predict their 65

complex behavior (10, 11). This allows for the exploitation of microorganisms to function as 66

“micro-factories” to produce specific compounds that are of high-market value in various 67

industrial sectors (12). However, in order for a strain to be viable for industrial scale-up, the 68

engineered strain must be able to achieve high yields, high production rates and high final titers 69

of the desired compounds (13). 70

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In recent years, anthocyanins have drawn tremendous interest due to their potential as 72

natural food colorants (14). In the food industry, it is common practice to use artificial food 73

colorants (AFCs) to improve the appearance of products and make them more palatable to 74

consumers. However, there is increasing concern over the usage of AFCs as research has shown 75

possible health risks associated with their consumption. Particularly, consuming AFCs can cause 76

behavioral changes in children with and without attention-deficit/hyperactivity disorder 77

(ADHD), with the latter group exhibiting greater sensitivity to AFCs (15, 16). Due to growing 78

demand from health-conscious consumers for natural food colorants, anthocyanins can provide a 79

possible alternative to artificial food dyes. Apart from being natural, these polyphenols also 80

possess a huge range of health benefits, many of which are associated with their potent 81

antioxidant properties (17, 18). In fact, anthocyanins’ antioxidant properties are reported to be 82

equipotent to quercetin or catechin gallates, both of which are major flavonoids found in green 83

tea. The antioxidant properties of these compounds can be attributed to their common catechol 84

ring backbones that dramatically increase the molecule’s radical-scavenging ability (19). 85

Anthocyanins are naturally synthesized from flavanones starting with the conversion of 86

flavanones to dihydroflavonols by flavanone 3β-hydroxylase (FHT), which are then converted 87

by dihydroflavonol 4-reductase (DFR) to leucoanthocyanidins. Subsequently, catechins are 88

formed by the action of leucoanthocyanidin reductase (LAR). Anthocyanidin synthase (ANS), a 89

2-oxoglutarate and ferrous iron-dependent oxygenase then utilizes either catechins or 90

leucoanthocyanidins as substrates to form the unstable cyanidin intermediates before structure 91

stabilization, in which the intermediates are converted to anthocyanins by the activity of UDPG-92

flavonoid 3-O-glycosyl transferase (3GT) (Fig. 1a). 93

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Ten years ago, we reported the biosynthesis of natural anthocyanins from a recombinant 94

E. coli for the first time (20). We achieved cyanidin-3-O-glucoside (C3G) production of up to 95

110 mg/L with the optimization of media pH, gene expression cassette and the supply of UDP-96

glucose (21, 22). In this paper, we present the various engineering strategies to further improve 97

the titer of C3G. Towards the understanding of detailed mechanism of anthocyanin biosynthesis 98

in E. coli, we have identified transporter proteins facilitating the efficient transportation of 99

catechin and C3G to increase substrate availability and remove product inhibition. We then 100

balanced the expression of the two critical enzymes (ANS and 3GT) using a bicistronic 101

expression cassette. Combined with a convenient colorimetric assay (Fig. 1b), we rapidly 102

optimized the culture condition and induction parameters, leading to a C3G titer of 350 mg/L, 103

which is about 3.3-fold improvement over our previous efforts and is the highest production 104

level reported to date. This work, coupled with work that recently reported the high-yield, high-105

titer production of catechin in E. coli can lead to a commercially viable process for the 106

sustainable production of anthocyanins (23). 107

2 Materials and methods 108

2.1 Strains and Media 109

E. coli BL21* was used for recombinant protein production and plasmid cloning 110

materials were purchased from Invitrogen. DNA manipulations were performed according to 111

standard recombinant DNA procedures (24). Restriction enzymes and T4 DNA Ligase were 112

purchased from New England Biolabs. All PCR amplification and cloning reactions were 113

performed using Accuzyme DNA polymerase from Bioline. Plasmid DNA was prepared from 114

stock strains using a Zyppy Plasmid MiniPrep Kit, while fragment DNA was isolated by gel 115

extraction using a Zymoclean Gel DNA Recovery Kit (Zymo Research). Genomic DNA of E. 116

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coli was isolated using PureLink Genomic DNA Mini Kit. Plasmid-bearing cultures were grown 117

in media containing the necessary antibiotics ampicillin (70 μg/mL), kanamycin (40 μg/mL), 118

chloramphenicol (20 μg/mL), and/or streptomycin (40 μg/mL) when needed. (+)-Catechin was 119

purchased from TCI America. 120

2.2 Plasmid and Strain Constructions 121

Plasmids pACYCDuet-1,pCDFDuet-1, pCOLADuet-1 and pETDuet-1 were purchased 122

from Novagen. Plasmids pCDF-3A, pET-pgm-galU, pCOLA-cmk-ndk were provided by MK 123

laboratory stocks. Gene ycjU and transporter proteins: tnaB, marA, tolC, acrAB and yadH were 124

isolated from E. coli K12 and separately cloned into the multiple cloning sites of pACYC 125

downstream of the T7 promoter. Positive transformants were verified by colony PCR, and 126

further verified by DNA-sequencing. 127

Target gene knockouts were carried out using the λ-Red recombinase/FLP system as 128

previously described (25) with a few modifications. Briefly, E. coli harboring pKD46 plasmid 129

was induced with 50 mM arabinose and grown at 30°C to the OD600 of 0.6 before being made 130

electrocompetent by washing three times with ice-cold 10% (v/v) glycerol. Concentrated 131

electrocompetent cells were then electroporated using a BioRad Genepulser X-Cell system with 132

0.75 μg of PCR-amplified FRT-flanked kanamycin cassette with 50bp homologous flanking 133

regions. Next, cells were incubated at 37°C for one hour and plated on kanamycin selection 134

plates. All deletions were verified by colony PCR. Positive mutants were then transformed with 135

pCP20 plasmid for the excision of antibiotic marker. A complete list of plasmids and strains can 136

be found in Table 1. 137

2.3 C3G Fermentations 138

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For E. coli strain BL21* harboring the genes that encode the anthocyanin biosynthetic 139

pathway in the pCDF plasmid, pre-inoculums were grown overnight in LB broth, then diluted to 140

OD600 of 0.1 in 50 mL of fresh LB for culturing in shake flasks. Cells were incubated at 37°C 141

until an OD600 of 0.8 before they were induced with 1 mM IPTG and grown for another 3 hours 142

at 30°C for protein overexpression. Next, cells were collected by centrifugation and re-suspended 143

in M9 medium (pH 5), which was one fifth of the initial LB volume containing the necessary 144

antibiotics, 1% glucose, 1 mM (+)-catechin, 1 mM IPTG, 0.1 mM 2-oxoglutarate, 2.5 mM 145

sodium ascorbate and 0.1 mM orotic acid. Incubation continued at 30°C for 48 hours prior to 146

analysis of the heterologous products. For HPLC analysis, 500 µl of fermentation samples were 147

centrifuged and 30 µl of the supernatant was injected into HPLC to quantify C3G. 148

For high C3G production, an optimized fermentation approach was followed in which 149

OD600 0.1 of overnight pre-inoculum culture was used to inoculate 100mL of LB in a shake flask 150

and incubated at 37°C until an OD600 of 3.2 was reached before being induced with 1 mM IPTG 151

and grown for another 3 hours at 30°C. Next, cells were concentrated to OD600 of 15 in 10mL of 152

M9 medium (pH 5), containing 1% glucose, 2.5 mM (+)-catechin, 5 mM IPTG, 0.1 mM 2-153

oxoglutarate, 2.5 mM sodium ascorbate and 0.1 mM orotic acid. Additional 1 mM catechin and 154

1% glucose were supplemented after 24 hours of fermentation. 155

2.4 HPLC Analysis 156

Cell-free culture medium from fermentations (30 µl) was analyzed by HPLC, using an 157

Agilent 1100 series instrument and a reverse-phase ZORBAX SB-C18 column (4.6 X 150mm) 158

maintained at 25°C. 0.1% (v/v) Formic acid in acetonitrile and 0.1% (v/v) formic acid in water 159

were used as the mobile phases at a flow rate of 1.0 mL per minute. The mobile-phase 160

composition profile was as follows (percentile refers to v/v acetonitrile concentration): 10-50% 161

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for 10 min, 40-60% for 5 min and 60-10% for 2 min. C3G was quantified by monitoring 162

absorbance at 520nm. 163

2.5 Colorimetric Assay and C3G Purification 164

The red-colored C3G was measured with a microplate reader at an absorbance of 512 nm 165

and quantified using a standard curve. For simple purification, C3G-containing culture media 166

was extracted with an equal volume of ethyl acetate, vortexed vigorously for 1 min and 167

centrifuged to separate the polar and non-polar layers. 168

3 Results and discussions 169

3.1 Identifying C3G-associated E. coli Transporter Proteins 170

In plants, anthocyanins are synthesized via the flavonoid biosynthetic pathway in the 171

cytosol, which is a pH neutral environment (26). Prevacuolar compartments containing 172

anthocyanins are then mass-transported into the central vacuole by merging with the central 173

vacuole in the epidermal cells, and subsequently ruptured to release the colored compound (27). 174

For storage in the vacuole, anthocyanins must be stabilized under low pH conditions for 175

prolonged accumulation and prevention of degradation (28). Prokaryotic organisms such as E. 176

coli lack many organelles found in plants, especially the vacuole for storage of anthocyanins. In 177

a previous study, Yan et al. emulated the low pH environment in vacuole by lowering the pH of 178

M9 minimal medium to 5 for stable C3G production in E. coli (21). Next, we resolved to identify 179

native transport proteins responsible for transporting C3G across the cell membrane. The 180

rationale for this study was based on firstly, the claim that the intracellular accumulation of C3G 181

accounted for more than 50% of the total yield and secondly, the observation of cell culture 182

medium in which centrifuged cell pellets were more intensely colored than the extracellular 183

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medium (Fig. 2a). We postulated that there was either a transport limitation for C3G or that C3G 184

was taken up by the cells following sequestration to the intracellular medium (Fig. 2b). The 185

latter hypothesis was based on the comparison between C3G titers in the extracellular medium at 186

24 and 48 hours, in which the titers consistently decreased over time while the centrifuged cell 187

pellets turned more red (Fig.3: control). 188

Transport proteins are generally classified into three categories: uptake pumps, efflux 189

pumps and regulators. In microorganisms, they are responsible for conferring multiple-drug 190

resistance and protection against harmful substances such as detergents and disinfectants (29). In 191

this case, we were interested in identifying transport proteins that are specific for the efflux of 192

C3G and to a lesser extent, the uptake of catechin for the gram-negative E. coli strain BL21*. 193

Five transporter proteins consisting of four efflux pumps: acrAB, tolC, aaeB and yadH, one 194

uptake pump: tnaB and one regulator: marA, were selected based on prior reports on secondary 195

metabolite transporters in microorganisms (29, 30). 196

All the aforementioned transport proteins were overexpressed in the host strain harboring 197

both the heterologous anthocyanin pathway and UDP-glucose pathway expression plasmids. Out 198

of all the transporter proteins tested, only yadH overexpression improved the extracellular 199

production of C3G by 15% (Fig. 3a), indicating that C3G might be a substrate for the yadH 200

efflux pump. Further quantification of extracellular catechin and cyanidin revealed a 201

significantly lower substrate uptake by all transport proteins except yadH when compared to the 202

control strain. Specifically, overexpressing tolC led to the lowest consumption of catechin while 203

the amount of cyanidin intermediate in the extracellular medium was comparable to the control 204

strain (Fig. 3b). We postulated that the overexpression of tolC, an efflux pump for many 205

common hydrophobic molecules, might facilitate the secretion of catechin. As expected, tolC 206

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knockout in BL21* expressing the heterologous anthocyanin biosynthetic pathway (pCDF-3A) 207

was able to improve C3G production by 55% to 66 mg/L. Coupling of both tolC knockout and 208

yadH overexpression further improved the extracellular C3G production to 69 mg/L (Table 2: 209

Fusion). The effect of combining high intracellular concentration of substrate and effective 210

secretion of product was visible in the culture broth (Fig. S1: BL21*ΔtolC and 211

BL21*ΔtolCyadH). These data reaffirmed the complexity of the E. coli efflux system; both 212

catechin and C3G have very similar molecular backbones with minor differences in the C-ring 213

and the addition of a glucoside functional group, however they have very distinct routes of 214

efflux. Also, maintaining a high intracellular pool of substrates by knocking out a substrate-215

specific efflux pump can significantly enhance biotransformation efficiency (31). 216

3.2 Optimization of Expression Construct 217

In the past, a 17% improvement in C3G titers was achieved by using a fusion protein of 218

3GT and ANS when compared to a dual promoter system in a pCDF-duet plasmid (21). It was 219

hypothesized that the translational fusion of both catalytic proteins facilitated the transport of the 220

highly unstable intermediate cyanidin directly from ANS to 3GT and resulted in a higher C3G 221

production. As an alternative, a new expression cassette was proposed in which both ANS and 222

3GT were arranged in a bicistronic configuration driven by a single T7 promoter, with 3GT 223

placed upstream of ANS. An operon-based gene configuration allows the coordinated expression 224

of functionally-related proteins as both genes are transcribed and regulated together. For both 225

genes, the native ribosome binding site (RBS) from the pCDF vector and the distance between 226

the RBS and the start codon were retained to minimize the variation in translational efficiency. 227

By comparing solely the extracellular production prior to any further genetic modifications, a 228

titer of 94.2 mg/L was achieved, compared to 17.7 mg/L (2 promoters) and 20.7 mg/L (fusion 229

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protein, BL21* 3A), representing at least a 4.5-fold improvement (Table 2). It has been proved 230

that the translation initiation rate for a given mRNA is six-fold greater during transcription than 231

after its release, which could be explained by a co-transcriptional and co-translational 232

mechanism: longer “transcription distance” allows more time for ribosomes to occupy the RBS 233

region during transcription (32–34). As such, the relative expression of 3GT would be higher 234

than ANS because of the relatively longer “transcription distance”. Presumably, a stronger 235

expression of 3-GT could more efficiently drive the carbon flux towards the end product C3G 236

while preventing the accumulation of unstable cyanidin. 237

It was previously confirmed that UDP-glucose availability was the metabolic bottleneck 238

for high level C3G biosynthesis and previous efforts on improving this rate-limiting precursor by 239

simultaneous overexpression of pgm, galU, cmk and ndk in the host strain have yielded 240

promising results (22). When these UDP-glucose pathway enzymes were overexpressed, BL21* 241

3AO yielded 120 mg/L of C3G compared to 42 mg/L of BL21* 3A, representing a 2.9-fold 242

improvement (Table 2). 243

Surprisingly, coupling the new expression system with the overexpression of yadH and 244

deletion of tolC did not yield any significant improvement in extracellular C3G production 245

(Table 2: Operon). This data suggests that the transport of substrates and products may not be a 246

significant bottleneck in C3G overproducing strain when the pathway was expressed in the 247

operon format and UDP-glucose limitation was alleviated. 248

3.3 Augmentation of Intracellular UDP-glucose 249

In addition to pgm, galU, cmk and ndk, we sought to further improve UDP-glucose 250

availability to support high level of C3G production. Both phosphoglucomutases, pgm and ycjU 251

catalyze the same reversible reaction from glucose-6-phosphate to glucose-1-phosphate but act 252

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on different anomers, α and β respectively (35). It was of interest to compare the activity of both 253

enzymes in catalyzing the reversible reaction of glucose-6-phosphate to glucose-1-phosphate in 254

vivo. After 17 hours of cultivation, ycjU overexpression alone resulted in a 24% improvement 255

over wild type to 104.5 mg/L (Table 3). In combination with cmk and ndk, ycjU overexpression 256

resulted in C3G titer of 120.2 mg/L, or 12% higher than coexpression with pgm and galU. This 257

result suggested that galU is not a bottleneck enzyme in the UDP-glucose pathway. Further 258

overexpression of ycjU on top of pgm, galU, cmk and ndk did not yield any significant titer 259

improvement. 260

3.4 Colorimetric Screen and Assay for C3G Production. 261

One major advantage that anthocyanins possess compared to other flavonoids is their 262

associated color properties, especially at low pHs. Anthocyanins in red cabbage have been used 263

as a pH indicator, where they turn from red (very acidic) to purple (neutral) to greenish-yellow 264

(basic). One other industrially important molecule with similar colorimetric property that has 265

long been used in the metabolic engineering and synthetic biology fields is lycopene. This 266

hydrophobic molecule commonly found in tomatoes can be monitored colorimetrically by 267

measuring its absorbance at 470nm (36). However, unlike anthocyanins, the absolute lycopene 268

quantification is more tedious as it requires the extraction of cell culture with organic solvents. 269

In order to demonstrate that the color intensity of C3G, under acidic conditions, is 270

proportional to the production, C3G standards were measured at an absorbance of 512nm. As 271

expected, a positive linear trend was observed (Fig. S2A, B). To assess the accuracy of the 272

calibration curve, a set of fermentations varying in culture conditions was constructed in a 24-273

well plate. After 2 days of fermentation, cell-free culture broths were measured by both 274

spectrophotometer and HPLC. A good correlation was observed between the actual and 275

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predicted production (Fig. S2C). We have demonstrated that this C3G colorimetric assay could 276

be used as a medium-throughput method for identifying overproducers with the added benefit of 277

product quantification. Unlike the lycopene screening assay in which plate-based photometric 278

screening is used, one disadvantage of the C3G colorimetric assay is the necessary cultivation of 279

individual colonies, which drives up assay time while decreasing throughput. Upon plating 280

transformants on agar plates supplemented with all necessary substrates, inducers and co-factors, 281

no color accumulation was observed both intra- and extracellularly after 48 hours of incubation 282

(data not shown). Regardless, cultivating C3G-producing strains in liquid culture is useful for 283

quick assays for studying and optimizing media and fermentation conditions. The current assay 284

time of 48 hours can be significantly reduced with more extensive engineering of the reporter 285

strain as well as improving media and culture conditions. 286

Utilizing this convenient colorimetric assay, we took a sequential approach to optimize 287

substrate, inducer and cofactor concentration, and culture pH (Fig. S3). After incubating the 288

cultures for 48 hours, the culture conditions of wells containing higher C3G were noted. Two 289

notable modifications to fermentation conditions were derived from this colorimetric assay: an 290

increase in initial IPTG concentration to 5 mM instead of 1 mM, and an increase in 291

biotransformation OD600 in M9 to 15 instead of OD600 of 4. 292

To the best of our knowledge, this is the first study reporting the use of anthocyanins for 293

“microtiter plate-based photometric screening” and optimization. Similar to lycopene, the C3G 294

colorimetric assay can be used as a screening molecule for desirable phenotypes in applications 295

such as combinatorial engineering (37, 38). The calibration curve generated could be utilized for 296

rapid C3G concentration quantification without the need for complex and time-consuming 297

analytical tools. 298

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3.5 Optimization of Culture Conditions 299

Additional modifications in shake flask fermentation, specifically substrate concentration, 300

carbon source concentration and induction at OD600 were implemented to further elevate C3G 301

titers. With the improved genetic construct utilizing an operon system, HPLC and glucose 302

analysis showed that the initial catechin (1mM) and glucose (1%, w/v) supplemented in M9 303

medium was rapidly exhausted after 24 hours (data not shown). Subsequent shake flask 304

fermentations optimizing substrate concentration suggested a concentration of 2.5 mM catechin 305

was necessary to maintain substrate-driving force. Glucose concentration on the other hand was 306

maintained at 1%, as a decline in overall C3G production was observed with both lower (0.5%) 307

and higher (up to 3%) initial glucose concentrations (data not shown). This effect can possibly be 308

attributed to the inverse correlation between glucose concentration and IPTG-induced protein 309

expression. In an IPTG-inducible T7 promoter system, optimal transcriptional initiation requires 310

both IPTG binding to the lac repressor and high CAP/cAMP complex binding upstream of the 311

promoter. High glucose concentration can result in low cAMP levels, subsequently disrupting the 312

formation of the CAP/cAMP complex and hinder downstream transcription (39). In order to 313

balance the need for good protein expression under glucose-limiting conditions while 314

maintaining cell viability, fermentation with glucose pulsing was adopted instead. Additional 1% 315

of glucose and 1 mM of catechin were supplemented by the end of first day to maintain both cell 316

viability and substrate driving force, while minimizing the detrimental effect of catabolic 317

repression. By increasing the initial substrate concentration via substrate and glucose feeding, 318

C3G production improved significantly in all three strains with different UDP-glucose pathway 319

enzymes overexpressions (Fig. 4A). The most noticeable improvement was seen in strain BL21* 320

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3AO overexpressing ycjU, cmk and ndk, which showed over 70% improvement in titer to 204.3 321

mg/L. 322

Next, induction OD600 was investigated, in which different induction ODs were tested as 323

alternatives to the manufacturer-recommended mid-log phase induction (OD600 of 0.8) for strain 324

BL21*. Our results show that as induction OD600 increases from 0.8 to 1.2, C3G production 325

remained the same. However, production increased when induction occurred later in the growth 326

phase (greater than OD600 1.4) (Fig. 4B). The highest production was attained at the highest 327

induction OD600 of 3.2 with a production of 350 mg/L. Induction OD600 was not further increased 328

as the strains had reached stationary phase at 3.2 (data not shown). The beneficial effect of 329

inducing at higher OD600 could possibly be attributed to better protein expression during a 330

nutrient-depleted state. To date, the reported titer of 350 mg/L is the highest reported 331

anthocyanin production, exhibiting a 3.5-fold increase in C3G production over the previously 332

reported highest production or 7-fold improvement if compared solely to extracellular 333

production. It should be noted that the actual amount of C3G produced could be higher than our 334

reported value as anthocyanins exist in equilibrium as a mixture of the red flavylium cation, blue 335

quinoidal base, colorless hemiketals and chalcone structures (18). Further optimization of media 336

conditions (pH, temperature, oxygen, light, metals for complex formation, copigments) to favor 337

the formation of the flavylium cation could potentially improve C3G titers using the engineered 338

E. coli strain. 339

3.6 Simple Extraction Method for C3G Purification 340

Solvent extraction has been a common method for phenolic compounds extraction. Since 341

anthocyanins are polar molecules, a simple purification process of C3G was demonstrated using 342

the organic solvent ethyl acetate. By a single-step extraction, C3G (in the polar phase) can be 343

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separated from catechin and the intermediate, cyanidin (in the organic phase) (Fig. 5, Fig. S4). 344

Remaining polar impurities including UDP-glucose, organic acids and proteins in the cell-culture 345

medium can be subsequently purified using either solid phase extraction or liquid-liquid 346

extraction (40). 347

4 Conclusions 348

Unlike most natural compounds, anthocyanins offer an easy and direct colorimetric 349

screen without the need of any coupled assay for product detection (41). We have shown for the 350

first time the development of a micro-titer plate assay capable of screening anthocyanin 351

overproducers by optimizing fermentation conditions. By optimizing the media and strain, the 352

48-hour culturing time can be significantly reduced to increase throughput. To further improve 353

the biotransformation efficiency, a more combinatorial engineering approach can be utilized, e.g. 354

direct evolution of ANS and 3GT enzymes to improve their biochemical properties and coupled 355

with synthetic biology approaches that optimize the genotype of the recombinant strain. Apart 356

from visual screening to identify improved variants, the titers can also be assessed in a more 357

quantitative manner. 358

Overall, we set out to further improve the C3G production of the original heterologous 359

two-enzyme pathway in E. coli BL21*. A more efficient system was identified utilizing an 360

expression system in which both enzymes were expressed in a bicistronic operon. Metabolic 361

engineering efforts aimed at improving the availability of known rate-limiting cofactors often led 362

to higher product formation (42, 43). In this study, we focused on augmenting the intracellular 363

pool of UDP-glucose by overexpressing another E. coli endogenous phosphoglucomutase, ycjU 364

instead of the initial pgm. The production with ycjU overexpression alone is better than the 365

coexpression of pgm and galU. Lastly, by improving culture conditions through higher initial 366

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substrate concentration, substrate and glucose pulsing and inducing at stationary phase, a C3G 367

titer of 350 mg/L was attained, representing a yield of 22.3 gr C3G per gr of catechin. This 368

optimized system, coupled with recent advances in producing high-yield, high-titers of (+)-369

catechin, offers a process that can lead to a commercially viable production of the vibrant natural 370

colorant, C3G (23, 44). In addition, C3G can be semi-purified after one-step solvent extraction 371

and its coloration is comparable to synthetic dye. 372

In conjunction with recent advances in understanding a plant’s ability to stabilize and 373

accumulate anthocyanin, novel methods to improve thermal and pH stability of anthocyanins 374

have been developed. Some notable examples include the use of the copigment quercetagetin to 375

stabilize anthocyanins derived from grape skin in a pH range of 3.0 to 5.0 (45) and the addition 376

of yeast mannoproteins that form complexes with anthocyanins to improve their half-life and 377

decrease their degradation constant at neutral pH 7.0 while maintaining their oxidant capacity 378

(46). These developments, in addition to classic metabolic engineering strategies, can guide our 379

future design in engineering an industrial E. coli strain capable of over-producing anthocyanins 380

to support the market demand for natural food colorants in a cost-effective way. 381

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Acknowledgements 383

The authors acknowledge the financial support from Chromadex. The authors appreciate C. 384

Santos, A. Qualley, M. De Mey and A. Parayil for the discussions and comments on the 385

manuscript. 386

Appendix A. Supplementary information 387

Table 1. Plasmids and strains used in the present study. 388

Plasmid or strain Relevant properties or genetic marker Source Plasmids pACYCDuet p15A ori (pACYC184), CmR Novagen pCOLADuet ColA ori, KmR Novagen pCDFDuet CloDF13 ori, StrR Novagen pETDuet ColE1 , AmpR Novagen pCDF-3A pCDF carrying A. thaliana 3GT and P. hybrida ANS fusion protein (21) pCDF-3AO pCDF carrying A. thaliana 3GT and P. hybrida ANS in an operon this study pET-pgm-galU pETDuet carrying E. coli pgm and galU (21) pCOLA-cmk-ndk pCOLADuet carrying E. coli cmk and ndk (22) pACYC-ycjU pACYCDuet carrying E. coli ycjU this study pACYC-tnaB pACYCDuet carrying E. coli tnaB this study pACYC-marA pACYCDuet carrying E. coli marA this study pACYC-tolC pACYCDuet carrying E. coli tolC this study pACYC-arcAB-tolC pACYCDuet carrying E. coli arcAB and tolC this study pACYC-yadH pACYCDuet carrying E. coli yadH this study Strains E. coli BL21* F- ompT hsdSB (rB- mB-) gal dcm rne131 (DE3) Invitrogen BL21* Δudg E. coli BL21* Δudg (22) BL21* ΔtolC E. coli BL21* ΔtolC this study BL21* 3AO E. coli BL21* pCDF-3AO this study BL21* 3A E. coli BL21* pCDF-3A (21) 389

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397 Table 2. The effect of transporter gene modifications and expression systems in the 398 production of cyanidin 3-O-glucoside. 399

400

Strain Transporter Gene Modification

Product Titera (mg/L) Fusion, pCDF-3A Operon, pCDF-3AO

BL21* b 20.7c 94.2±2.4

BL21* + pET-pgm-galU + pCOLA-cmk-ndk

b 42.2±2.2 120±12.1 ΔtolC 65.7±4.4 120±0.2 ΔtolC + pACYC-yadH 69.0±2.5 126±2.4

aAll values are the average of three trials with standard deviations. 401 bNo plasmid transformed. 402 c(21) 403 404

405 406

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Table 3. Cyanidin 3-O-glucoside production after 17hrs with UDP-glucose pathway 422 overexpressions. 423

Strain Overexpressed Gene(s) Product Titera (mg/L)

BL21* 3AO

b 84.6±1.2 ycjU 104.5±8.1 pgm, galU, cmk, ndk 107.2±0.6 ycjU, cmk, ndk 120.2±1.3 ycjU, pgm, galU 114.9±2.3 ycjU, pgm, galU, cmk, ndk 122.3±0.3

aAll values are the average of three trials with standard deviations. 424 bNo plasmid transformed. 425

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428

Figure 1. Engineered pathway for the biosynthesis of colored anthocyanin, cyanidin 3-O-429 glucoside (C3G) in E. coli. (A) Mechanism catalyzing the formation of cyanidin from (+)-catechin 430 by anthocyanidin synthase (ANS) and the transfer of a glucose group from UDP-glucose to 431 cyanidin to yield C3G by 3-O-glycosyl transferase (3GT). (B) C3G-producing strains can be 432 efficiently screened by using a colorimetric assay. 433

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436

Figure 2. Significant amount of cyanidin 3-O-glucoside (C3G) accumulated in the intracellular 437 medium. (A) Cell pellets and the extracellular medium of different C3G producing strains; (B) 438 Picture demonstration of two potential scenarios: (Left) C3G remain mostly in the cytoplasm; 439 (Middle) C3G sequestered are transported back into the cells; (Right) Amino acids and 440 antibiotics have specific transporter proteins for their uptake and efflux across cell membrane. 441

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443

444 445 Figure 3. Extracellular cyanidin 3-O-glucoside (C3G), cyanidin and (+)-catechin with the 446 overexpression of different transporter proteins. (A) C3G accumulation after 24 and 48 hrs; (B) 447 Peak area of intermediate cyanidin and substrate catechin in the extracellular medium after 24 448 hrs cultivation. Error bars represent standard deviation between biological and technical 449 triplicates. 450 451

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453

Figure 4. Cyanidin 3-O-glucoside (C3G) production as a result of optimization of culture 454 conditions including substrate concentration, carbon source concentration and induction 455 OD600 for strains harboring pCDF-3AO plasmids with the overexpression of different UDP-456 glucose pathway enzymes. (A) C3G production when cultures were pulsed with 1 mM of 457 catechin and 1% of glucose at the end of 1st day. (B) C3G production when induction OD600 was 458 increased from initial OD600 0.8 to OD600 3.2. Error bars represent standard deviation between 459 biological and technical triplicates. 460 461

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Figure 5. A titer of 350 mg/L of cyanidin 3-O-glucoside (C3G) titers was achieved using the 468 engineered E. coli. (Left) Cell culture after 2 days cultivation in clear M9 minimal medium; 469 (Middle) Partially purified anthocyanin after extraction with ethyl acetate; (Right) Impurities 470 extracted in organic phase with ethyl acetate. 471

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