Synthetic and Systems Biotechnology xxx (2017) 1e8

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Synthetic and Systems Biotechnology

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and-systems-biotechnology/

Production of anthocyanins in metabolically engineered

microorganisms: Current status and perspectives
Jian Zha a, Mattheos A.G. Koffas a, b, *
a
Department of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, USA
b
Department of Biological Sciences, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, USA

a r t i c l e i n f o a b s t r a c t

Article history: Microbial production of plant-derived natural products by engineered microorganisms has achieved
Received 4 August 2017 great success thanks to large extend to metabolic engineering and synthetic biology. Anthocyanins, the
Received in revised form water-soluble colored pigments found in terrestrial plants that are responsible for the red, blue and
24 September 2017
purple coloration of many flowers and fruits, are extensively used in food and cosmetics industry;
Accepted 26 October 2017
however, their current supply heavily relies on complex extraction from plant-based materials. A
promising alternative is their sustainable production in metabolically engineered microbes. Here, we
Keywords:
review the recent progress on anthocyanin biosynthesis in engineered bacteria, with a special focus on
Anthocyanin
Enzyme engineering
the systematic engineering modifications such as selection and engineering of biosynthetic enzymes,
Metabolic engineering engineering of transportation, regulation of UDP-glucose supply, as well as process optimization. These
Microbial production promising engineering strategies will facilitate successful microbial production of anthocyanins in in-
dustry in the near future.
© 2017 Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co. This is an open
access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2. Industrial applications of anthocyanins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
3. Biosynthesis of anthocyanins in plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
4. Metabolic engineering of anthocyanin production in microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
4.1. Enzyme screening and engineering for anthocyanin production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
4.2. Regulation of cofactor/cosubstrate supply for anthocyanin overproduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . 00
4.3. Engineering of specific transporters for anthocyanin production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
4.4. Optimization of culture processes for anthocyanin production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
5. Future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

Abbreviations: ANS, anthocyanidin synthase; CHI, chalcone isomerase; CHS,

chalcone synthase; 4CL, 4-coumaroyl-CoA ligase; DFR, dihydroflavonol 4-reductase; 1. Introduction
DSSC, dye-sensitized solar cell; F3H, flavanone 3-hydroxylase; F30 H, flavonoid 30 -
hydroxylase; F30 50 H, flavonoid 30 , 50 -hydroxylase; FGT, flavonoid glucosyltransfer-
As a member of the flavonoid group of polyphenols, anthocya-
ase; F3GT, flavonoid 3-O-glucosyltransferase; UV, ultraviolet.
* Corresponding author. Department of Chemical and Biological Engineering, nins are important chemicals in the plant kingdom as pigments,
Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic antioxidants, and antimicrobials (Fig. 1). With the rising interest in
Institute, 12180 Troy, NY, USA. natural nutraceuticals, there is an increasing preference for natural
E-mail address: koffam@rpi.edu (M.A.G. Koffas). food colorants such as anthocyanins, thus stimulating high demand
Peer review under responsibility of KeAi Communications Co., Ltd.

https://doi.org/10.1016/j.synbio.2017.10.005
2405-805X/© 2017 Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co. This is an open access article under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article in press as: Zha J, Koffas MAG, Production of anthocyanins in metabolically engineered microorganisms: Current status
and perspectives, Synthetic and Systems Biotechnology (2017), https://doi.org/10.1016/j.synbio.2017.10.005
2 J. Zha, M.A.G. Koffas / Synthetic and Systems Biotechnology xxx (2017) 1e8

Fig. 1. The structure of anthocyanins. Modifications (such as glycosylation, hydroxylation, methylation, and acylation) at C3’ (R1), C4’ (R2), C5’ (R3), C3 (R4), C5 (R5) and C7 (R6)
generate structural analogs. R1-R5 are functional groups derived from glycosyl, hydroxyl, methyl, and acyl units. Representative anthocyanins and their structures are listed.

for these compounds mainly as colorants and cosmetic additives bioinformatics tools, render microbial production of natural prod-
[1e4]. ucts facile, controllable, and cost-effective [14e16]. So far, many
The traditional means of producing anthocyanins is by extrac- plant-derived compounds have been produced, including terpe-
tion and purification from fruits, flowers, and other tissues of plants noids, alkaloids, and flavonoids, in prokaryotic and eukaryotic mi-
[5e7]. This approach has a couple of advantages such as diverse croorganisms [15,17,18]. Microbial biosynthesis of natural
sources of readily available cheap feedstocks, sophisticated flavonoids dates back to 2003 [19], and various flavonoid com-
extraction and purification techniques, and decent market recog- pounds, from flavanones to the more complicated anthocyanins,
nition (non-GMO products). And great success has been achieved in have been generated in engineered microorganisms [14]. Mean-
the development of novel extraction and purification procedures, while, microbial production of non-natural flavonoids has also been
understanding of anthocyanin biosynthesis in plants, and genetic made possible through the feeding of specific substrates [20,21]. In
engineering of plants for anthocyanin production [8e10]. However, all these cases, the producing microbes are metabolically engi-
anthocyanins isolated from plants exist as a heterogeneous mixture neered for the optimal synthesis of the target products. Coupled
of multiple types of molecules with diverse chemical structures. In with the advances in genome sequencing and DNA synthesis,
addition, anthocyanin production through plant extraction is structural biology and enzymology, and modeling of metabolic
neither stable nor sustainable, since plants produce anthocyanins networks, metabolic engineering is playing a powerful and indis-
with varied productivity, and the production relies on farmland and pensable role in microbial synthesis of on-demand compounds
irrigation, and fluctuates depending on seasonal and environ- with biochemical functions or industrial significance [22,23].
mental conditions [11e13]. An alternative is microbial production, In this review, the recent progress on anthocyanin production in
which has already demonstrated great potential in the biosynthesis genetically modified microorganisms will be described, with a
of plant-derived natural compounds. The intrinsic characteristics of main focus on metabolic engineering strategies on improving
microorganisms, such as fast growth and easy cultivation, and the in vivo production. Also, process optimization and in vitro storage of
sophisticated microbial manipulation techniques, including anthocyanins are overviewed, providing a comprehensive under-
convenient genetic modifications and readily available standing of the engineering and bioproduction processes of these

Please cite this article in press as: Zha J, Koffas MAG, Production of anthocyanins in metabolically engineered microorganisms: Current status
and perspectives, Synthetic and Systems Biotechnology (2017), https://doi.org/10.1016/j.synbio.2017.10.005
 J. Zha, M.A.G. Koffas / Synthetic and Systems Biotechnology xxx (2017) 1e8 3

molecules. complexity of anthocyanin biosynthesis and their instability,

commercial production of anthocyanins in controlled systems is
2. Industrial applications of anthocyanins still a daunting challenge.

Anthocyanins have wide applications in pharmaceutical in- 4. Metabolic engineering of anthocyanin production in
dustry, food processing, cosmetic manufacturing, and solar cell microorganisms
development. Anthocyanins help to suppress neuroinflammation,
neurodegradation and brain aging by blocking interleukin-1b, tu- Engineering microorganisms for the production of natural
mor necrosis factor a, and nuclear factor-kb [24], and are promising chemicals is a promising and sustainable way of satisfying indus-
in the prevention of cancer, cardiovascular diseases, neurodegen- trial needs. As the most commonly used workhorse in metabolic
erative diseases, obesity and diabetes according to trials in animal engineering, E. coli has been extensively engineered for the
models and humans, although the underlying mechanisms are still biosynthesis of natural flavonoids such as naringenin, kaempferol,
not very clear [24e26]. and catechin [25,37e39]. Later on, the host cell range has been
Certain types of anthocyanins strongly absorb visible and ul- expanded to Saccharomyces cerevisiae and Streptomyces venezuelae
traviolet (UV) spectra owing to the specific polyphenol structure [25,40]. Recently, the amino acid-producing bacterium Corynebac-
and side groups with phenolic compounds [27], and, when applied terium glutamicum has also been reported for the production of
externally, protect human skin from aging and UV-induced damage stilbenes and flavanones [41].
[28], such as inflammation and oxidative damage in the epidermis, As important members of the flavonoid class of polyphenols,
dermis, and adnexal organs of the skin [29]. These discoveries have anthocyanins have been drawing much focus due to their
fueled the elevated application of anthocyanins in cosmetics and tremendous industrial and commercial values. In 2005, Yan et al.
skin care products [29]. cloned and expressed in E. coli the genes of flavanone 3-
In addition, anthocyanins are widely used as food colorants hydroxylase (F3H) and ANS from Malus domestica, DFR from
because of their diverse colors and nutritional properties. In the US, Anthurium andraeanum, and flavonoid 3-O-glucosyltransferase
four anthocyanin-based colorants are exempt from FDA certifica- (F3GT) from Petunia hybrida [42]. The recombinant E. coli strain
tion. The acylated anthocyanins are also commonly applied in in- produced 6.0 mg L1 of cyanidin 3-O-glucoside and 5.6 mg L1
dustry because of the improved color stability [30]. As a dye, pelargonidin 3-O-glucoside using naringenin and eriodictyol as
anthocyanins are also exploited as sensitizers in dye-sensitized precursors, respectively. Subsequent selection of plant-derived
solar cells (DSSCs), to replace the toxic, complicated, and costly enzymes, optimization of UDP-glucose pool, regulation of precur-
transition metal coordination complexes, for the conversion of sor uptake and optimization of the production process resulted in
solar energy to electricity, with slightly lower yet acceptable effi- dramatically enhanced production of pelargonidin 3-O-glucoside
ciencies compared to the traditional silicon solar cells [31e34]. and cyanidin 3-O-glucoside, with their titers reaching 113 mg L1
and 350 mg L1, using afzelechin and catechin precursors, respec-
3. Biosynthesis of anthocyanins in plants tively [43e45]. The engineered microbial production of other an-
thocyanins is summarized in Table 1 with the schematic illustration
Naturally produced in plants, anthocyanins serve to attract shown in Fig. 3. All reported recombinant producing hosts are
pollinating insects and seed dispersers, and to protect plants currently limited to E. coli derivatives.
against irradiation damages and pathogens [10,35]. Anthocyanins
in plants are synthesized via the general flavonoid pathway, 4.1. Enzyme screening and engineering for anthocyanin production
whereby three molecules of malonyl-CoA and one molecule of 4-
coumaroyl-CoA derived from phenylalanine or tyrosine are Construction and engineering of the anthocyanin pathway in
condensed by the key enzyme chalcone synthase (CHS) to form microbes involves co-expression of plant enzymes. Gene orthologs
naringenin chalcone (Fig. 2), which is further converted to its iso- encode enzymes catalyzing the same reaction in different plants
mer naringenin by chalcone isomerase (CHI). Naringenin is modi- that exhibit diverse kinetic and thermodynamic activities, and can
fied through hydroxylation by enzymes such as flavonoid 30 - thus lead to varied metabolic behaviors and distinct production
hydroxylase (F30 H) and flavonoid 30 , 50 -hydroxylase (F30 50 H), giving levels of the target compounds when they are heterologously
rise to different dihydroflavonols (dihydroquercetin, dihy- expressed in microorganisms. Hence, enzyme screening and se-
drokaempferol, and dihydromyricetin). These molecules are lection from diverse species is an important way of improving the
reduced by dihydroflavonol 4-reductase (DFR) to form leucoan- production of anthocyanins and other flavonoids [38]. Yan et al.
thocyanidins (leucocyanidin, leucopelargonidin, and leucodelphi- compared the in vivo activities of ANS from four plants, and ach-
nidin). Oxidation of leucoanthocyanidins by anthocyanidin ieved maximal cyanidin production in E. coli by P. hybrida ANS [39].
synthase (ANS) generates the unstable flavylium cation anthocya- Similarly, naringenin production in E. coli was optimized by
nidins, which are then linked to a monosaccharide residue at C3 in comparing different homologs for each of the upstream pathway
ring C or other positions through flavonoid glucosyltransferase enzymes, i.e., 4-coumaroyl-CoA ligase (4CL), CHS and CHI, and
(FGT)-catalyzed glycosylation (Fig. 1). The most common sugar unit enzyme combinations of different sources showed drastic variation
is glucose, whereas galactose and xylose are also found in natural in naringenin titer [37]. The same approach was also adopted for
anthocyanins [24]. Other modifications such as methylation on the resveratrol production in E. coli [46].
hydroxyl groups of ring B and acylation are also possible [25]. These With a suitable enzyme system containing the optimal grouping
intrinsic modifications are aimed at improving anthocyanin sta- of enzymes from proper sources, the high yield microbial produc-
bility or diversifying colors of anthocyanins [36]. tion of anthocyanins is still not always guaranteed, and the chal-
Anthocyanins in plants are transferred to and stored in vacuoles lenge lies in the heterologous expression of these plant-based
after biosynthesis. Depending on the pH in the vacuoles of different enzymes in prokaryotic cells. Typically, the genes encoding these
species, anthocyanins present vastly diverse colors with distinct enzymes need engineering modifications prior to functional
stability [2,24]. Being quite unstable at neutral and basic pH, an- expression (Fig. 3). For example, to achieve functional prokaryotic
thocyanins can be stabilized through structural decorations, low- expression of a P450 F30 50 H from Catharanthus roseus, the four
ered pH, and co-pigmentation in vacuoles [10]. Because of the codons at the 50 -end were deleted to remove the N-terminal

Please cite this article in press as: Zha J, Koffas MAG, Production of anthocyanins in metabolically engineered microorganisms: Current status
and perspectives, Synthetic and Systems Biotechnology (2017), https://doi.org/10.1016/j.synbio.2017.10.005
4 J. Zha, M.A.G. Koffas / Synthetic and Systems Biotechnology xxx (2017) 1e8

Fig. 2. The pathway of anthocyanin biosynthesis in plants. The general precursor phenylalanine or tyrosine derived from the shikimate pathway is converted to 4-coumaroyl-CoA
through the phenylpropanoid pathway or the combined effect of TAL and 4CL, respectively. One molecule of 4-coumaroyl-CoA is condensed with three molecules of malonyl-CoA to
form one molecule of naringenin chalcone, which is subsequently converted to naringenin by CHI. Naringenin, the major intermediate compound, undergoes various hydroxylation
to form diverse anthocyanidins. Further glycosylation and other decorations act on the anthocyanidin compounds to generate anthocyanins. Abbreviations: PAL, phenylalanine
ammonia lyase; C4H, cinnamate 4-hydroxylase; CPR, cytochrome P450 reductase; TAL, tyrosine ammonia lyase; 4CL, 4-coumaroyl-CoA ligase; CHS, chalcone synthase; CHI, chalcone
isomerase; F3H, flavanone 3-hydroxylase; F30 H, flavonoid 30 -hydroxylase; F30 50 H, flavonoid 30 , 50 -hydroxylase; DFR, dihydroflavonol reductase; ANS, anthocyanidin synthase; FGT,
flavonoid glucosyltransferase; OMT, O-methyltransferase; ACT, anthocyanin acyltransferase.

membrane anchor, the fifth one was replaced with the start codon, oxidation of its substrates [39,42], and an equimolar amount of
and the sixth was changed from leucine to alanine to create an UDP-glucose is essential as a cosubstrate for the glycosylation of
anchor suitable for bacterial expression [47]. The new F30 50 H was cyanidin at the C3 position.
fused to a shortened P450 reductase with C. roseus origin to form a Supply of UDP-glucose is indispensable for the overproduction
chimeric hydroxylase that catalyzed the formation of quercetin. of glycosylated anthocyanins. Since UDP-glucose is also involved in
Apart from modifications of individual enzymes in the meta- many other metabolic pathways for the generation of energy, co-
bolic pathway, fused expression of multiple enzymes in successive factors, and metabolic precursors and intermediates, the global
steps is another effective method of improving anthocyanin pro- adjustment of its biosynthesis is crucial. The commonly used
duction. It has been shown that upon fusion of F3GT from Arabi- regulation schemes are overexpression of the biosynthetic genes
dopsis thaliana to the N-terminus of ANS from P. hybrida with a and partial inhibition of the degradation pathways for a higher level
pentapeptide linker, a higher titer of cyanidin 3-O-glucoside was of intracellular UDP-glucose. Attempts of such metabolic adjust-
achieved with the chimeric enzyme than with the uncoupled ANS ments in E. coli involved the upregulation of one or more genes
and F3GT. The chimeric enzyme complex was proposed to catalyze responsible for UDP-glucose biosynthesis from orotic acid (pyrE,
the successive biochemical reactions more efficiently than the in- pyrR, cmk, ndk, pgm, galU) and inhibition of the competitive UDP-
dependent enzymes, probably by creating a higher local concen- glucose consumption pathways. These modifications resulted in
tration of the unstable intermediate cyanidin, and by fueling it cyanidin 3-O-glucoside production increasing by more than 20-fold
rapidly to F3GT for glycosylation, without causing much cyanidin (from 4 mg L1 to 97 mg L1) [21,39]. In another case, the over-
degradation [39]. expression of intracellular genes pgm and galU along with the
expression of ANS and 3GT led to a 57.8% increase in cyanidin 3-O-
glucoside production [39].
4.2. Regulation of cofactor/cosubstrate supply for anthocyanin
S-Adenosyl-L-methionine is also a necessary cosubstrate for the
overproduction
production of methylated anthocyanins such as peonidin 3-O-
glucoside. By increasing the availability of S-adenosyl-L-methionine
Efficient biosynthesis of anthocyanins requires sufficient yet
through the CRISPR interference (CRISPRi)-mediated silencing of
balanced supply of cofactors and/or cosubstrates for electron
the transcriptional repressor MetJ, a twofold improvement of
transfer and enzyme activation/stabilization. For example, ferrous
peonidin 3-O-glucoside production was achieved in E. coli with a
ions and sodium ascorbate as cofactors, and 2-oxoglutarate as a
final titer of 56 mg L1 [48].
cosubstrate, are necessary for ANS to conduct a two-electron

Please cite this article in press as: Zha J, Koffas MAG, Production of anthocyanins in metabolically engineered microorganisms: Current status
and perspectives, Synthetic and Systems Biotechnology (2017), https://doi.org/10.1016/j.synbio.2017.10.005
 J. Zha, M.A.G. Koffas / Synthetic and Systems Biotechnology xxx (2017) 1e8 5

Table 1
Summary of anthocyanin production in engineered bacteria.

E. coli Genetic modifications Substrate Product Fermentation conditions Titer/mM Ref.

strain

JM109 MdF3H/AaDFR/MdANS/PhF3GT 0.25 mM Naringenin Pelargonidin 3-O-glucoside M9 minimal medium (pH 7) 0.012 [63]
0.1 mM Eriodictyol Cyanidin 3-O-glucoside plus UDP-glucose; 0.012
IPTG induction at RT
BL21* (DE3) MdF3H/AaDFR/At3GT/PhANS 0.2 mM Naringenin Pelargonidin 3-O-glucoside M9 minimal medium (pH 7) 1.34 [39]
0.2 mM Eriodictyol Cyanidin 3-O-glucoside with 2-oxoglutarate, sodium 3.88
MdF3H/AaDFR/At3GT/PhANS/DuLAR 0.2 mM Naringenin Pelargonidin 3-O-glucoside ascorbate and UDP-glucose; 2.09
0.2 mM Eriodictyol Cyanidin 3-O-glucoside IPTG induction at 30  C 4.27
MdF3H/AaDFR/At3GT/PhANS/DuLAR/ 0.2 mM Naringenin Pelargonidin 3-O-600 -O-malonylglucoside 0.18
Dv3MaT 0.2 mM Eriodictyol Cyanidin 3-O-600 -O-malonylglulcoside 0.21
BL21* (DE3) At3GT/PhANS 0.75 mM Catechin Cyanidin 3-O-glucoside 3 h IPTG induction at 30  C; 5- 5.16
fold concentration in M9
minimal medium as listed
above without UDP-glucose
BL21* (DE3) At3GT/PhANS 0.75 mM Catechin Cyanidin 3-O-glucoside Concentrated in modified M9 80
minimal medium (pH 5)
At3GT/PhANS/galU/pgm 0.75 mM Catechin Cyanidin 3-O-glucoside Same as above. 127
Fusion of At3GT and PhANS/galU/pgm 0.75 mM Catechin Cyanidin 3-O-glucoside 146
0.75 mM afzelechin Pelargonidin 3-O-glucoside 168
BL21* (DE3) Fusion of At3GT and PhANS/galU/pgm/ Catechin Cyanidin 3-O-glucoside Modified M9 medium (pH 5) 215 [21]
ndk/Dudg/galE/T(inactive) afzelechin Pelargonidin 3-O-glucoside with orotic acid (0.1 mM) 241
BL21* (DE3) At3GT/PhANS/galU/pgm/cmk/ndk/ 2.5 mM Catechin Cyanidin 3-O-glucoside Modified M9 medium (pH 5) 260 [43]
YadH/DtolC with 1% glucose, 5 mM IPTG, 2-
At3GT/PhANS/galU/pgm/cmk/ndk/ycjU 2.5 mM Catechin Cyanidin 3-O-glucoside oxoglutarate, sodium ascorbate, 252
and orotic acid.
At3GT/PhANS/cmk/ndk/ycjU 2.5 mM Catechin Cyanidin 3-O-glucoside Feed glucose and catechin 421
BL21* (DE3) At3GT/PhANS/galU/pgm/cmk/ndk/ycjU 2.5 mM Catechin Cyanidin 3-O-glucoside Induced at stationary phase; 722
modified M9 medium (pH 5)
with 1% glucose, 5 mM IPTG, 2-
oxoglutarate, sodium ascorbate,
and orotic acid; feed glucose
and catechin
BL21* (DE3) MBP-At3GT/MBP-PhANS/VvAOMT/ 3.44 mM Catechin Peonidin 3-O-glucoside Semi-rich medium AMM with 112 [48]
MetJY 2% glucose, IPTG induction
(CRISPRi) (1 mM) and production process
at 30  C

Beyond UDP-glucose and S-adenosyl-L-methionine, sodium host microorganisms, introduction of transporters from plants is
ascorbate is another necessary ingredient to sustain the over- also a possible route to enhancing the transportation of substrates
production of anthocyanins. It has been found that the addition of and products for improved anthocyanin production in engineered
sodium ascorbate significantly increases the consumption of the microbes. Anthocyanins in their natural plant hosts are transported
substrate catechin and the production of anthocyanin 3-O-gluco- to and accumulate in vacuoles by specific transporters [40,45]. In
side [39]. But for the cosubstrate 2-oxoglutarate, extra addition is maize, anthocyanin transport is largely dependent on an ATP-
generally unnecessary, which may be due to 2-oxoglutarate being binding cassette transporter ZmMRP3 present in the tonoplast
an intermediate compound in the Krebs cycle and its supply being [49], and the deposition of anthocyanins in vacuoles is achieved by
commonly abundant [39]. glutathione S-transferase encoded by Bronze-2 [41]. In other plants,
Hþ-gradient-dependent transporters are also related to anthocy-
anin transportation. The gene Transparent Testa12 in Arabidopsis
4.3. Engineering of specific transporters for anthocyanin production encodes a secondary transporter-like protein belonging to the
MATE family, which is predicted to participate in the vacuolar
Many target products in engineered microorganisms are toxic to transport of anthocyanins via an Hþ-antiport mechanism. Indeed,
the host strains, thereby inhibiting their high-titer production. A the mutant lacking this gene exhibits greatly reduced proantho-
feasible strategy to continuously biosynthesize these compounds at cyanidin accumulation in vacuoles [50].
acceptable levels is to transfer the products from cytoplasm to These transporters have not been tested in bacterial strains for
extracellular environments, where the harmful products are their functions in the efflux of anthocyanins or in the improvement
attenuated, through specific efflux pumps. First reported by Dunlop of anthocyanin production. Given that transfer of anthocyanins into
et al. for improved production of biofuels in E. coli upon introduc- plant vacuoles and outside microbial hosts both involve delivery
tion of an array of efflux pumps responsible for the biofuel export across membrane bilayers that are composed of ordered lipids with
from the producing cells [44], this approach has been extended to anchored integral proteins, it would be highly interesting to
many systems, including the biosynthesis of anthocyanins for investigate the potential potency of plant-derived transporters and
higher titers. A cyanidin 3-O-glucoside-associated efflux pump combine them with bacterial transporters in microbial hosts.
YadH has been identified and its overexpression led to 15% more
production of anthocyanins [43]. Moreover, deletion of another
efflux pump TolC, probably responsible for the secretion of the 4.4. Optimization of culture processes for anthocyanin production
substrate catechin, further promoted the titer of cyanidin 3-O-
glucoside. The highly unstable nature of anthocyanins is a problematic
Apart from engineering of the transporters naturally present in issue for their microbial production. Unlike in plants where

Please cite this article in press as: Zha J, Koffas MAG, Production of anthocyanins in metabolically engineered microorganisms: Current status
and perspectives, Synthetic and Systems Biotechnology (2017), https://doi.org/10.1016/j.synbio.2017.10.005
6 J. Zha, M.A.G. Koffas / Synthetic and Systems Biotechnology xxx (2017) 1e8

Fig. 3. The strategies applied in metabolic engineering of E. coli for the biosynthesis of anthocyanins. The modifications of the anthocyanin-producing strains focus on the
enzymes in the metabolic pathways, the transport of substrates and products, and the supply of cosubstrate UDP-glucose. The current production process is based on dividing the
whole biocatalysis into two stages, i.e., cell growth and enzyme production at normal pH in the first stage, and anthocyanin production and accumulation at a lower pH in the
second stage.

naturally synthesized anthocyanins are stored and stabilized in effect of oxygen on anthocyanin production.
vacuoles [10,51], there is a lack of anthocyanin stabilization Temperature is a global and key factor affecting cell viability and
mechanisms in bacterial cells that serve as artificial producing expression of heterologous proteins. Fluctuations in temperature
hosts. And since the intracellular pH is around 7 for most bacteria often lead to highly variable folding behaviors and production
commonly used for metabolic engineering under their normal yields of certain proteins, and thus imposing indirect influence on
growth conditions, the synthesized anthocyanins are extremely microbial production of useful compounds. In a co-culture system
labile. To stabilize anthocyanins in microbial hosts, a two-step producing afzelechin, the induction temperature of 20  C gave rise
biocatalysis has been proposed [39]. In the first step, cells are to the highest titer of 22.9 mg L1 among different co-cultures,
cultured in medium at pH 7, where normal cell growth and whereas the titer was only 6.1 mg L1 at 10  C [37].
expression of heterologous enzymes are maintained. In the second
step, cells at a certain growth stage are transferred to fresh medium 5. Future perspectives
with pH adjusted to 5 to minimize anthocyanin degradation (Fig. 3).
Protective agents such as glutamate can be added to protect cells At present, the cost of anthocyanins from recombinant bacteria
under low pH. Such a strategy helped to elevate the titer of cyanidin is still much higher than that from plant extraction, and one of the
3-O-glucoside in E. coli by ~15-folde38.9 mg L1 compared to the most important causes is the relatively low titer, which is attributed
traditional single-step production (2.5 mg L1) [39]. to low efficiencies and poor capabilities of the introduced path-
Apart from pH, several other factors also play important roles in ways. Thus, to maximize the potential of the biosynthetic pathways
anthocyanin production, such as induction time-point, substrate while minimizing the side effects of anthocyanin overproduction
feeding, amount of dissolved oxygen, and temperature. Lim and on microbial hosts through a series of regulation and optimization
coworkers found that induction at the stationary phase was will be the key task for microbial supply of anthocyanins. The rapid
optimal for cyanidin 3-O-glucoside production in engineered E. coli, expansion of genome sequencing and DNA synthesis capabilities,
and that pulsing of glucose and catechin improved anthocyanin leading to the discoveries of new enzymes and pathways, as well as
production [43]. The effect of induction point could be a conse- redesign of key enzymes based on present knowledge, has already
quence of differential protein expression at different growth stages. resulted in the construction of new biosynthetic pathways that can
The improvement from glucose feeding may lie in the increased be expressed in microorganisms for the production of known an-
supply of UDP-glucose, whereas the additional supplementation of thocyanins. Meanwhile, further optimization of anthocyanin pro-
catechin forms a driving force for the bioconversion. duction in microorganisms will continue to also focus on flexible
Dissolved oxygen has a dual effect on anthocyanin production. regulation of UDP-glucose supply, as well as the identification of
On the one hand, oxygen is key to the function of ANS; on the other efficient and specific transporters for anthocyanins and relative
hand, oxygen may oxidize anthocyanins. Thus, an optimal supply of substrates. Systematic engineering of UDP-glucose supply is a great
oxygen is critical. In a study of microbial eriodictyol conversion, challenge for further optimization given that UDP-glucose is also a
improved catechin synthesis was achieved by increasing the con- precursor for the biosynthesis of other bioactive compounds such
centration of dissolved oxygen, which might be related to elevated as trehalose and glycogen, and is involved in the biosynthesis of
NADPH supply [38]. However, currently there are no reports on the glycopolymers that are vital for cell metabolism, cell signaling, and

Please cite this article in press as: Zha J, Koffas MAG, Production of anthocyanins in metabolically engineered microorganisms: Current status
and perspectives, Synthetic and Systems Biotechnology (2017), https://doi.org/10.1016/j.synbio.2017.10.005
 J. Zha, M.A.G. Koffas / Synthetic and Systems Biotechnology xxx (2017) 1e8 7

defense systems. Recently, a dCas9-based toolbox has been devel- industrial applications. In this review, we summarized recent
oped to regulate the expression of multiple genes simultaneously in progress on metabolic engineering of anthocyanin production in
E. coli [52,53]. This approach can be exploited to fine-tune the levels microorganisms, with particular emphasis on E. coli, the only
of a variety of enzymes, transporters and cofactors associated microorganism that has been used for the recombinant production
directly or indirectly with anthocyanin production, for the identi- of these molecules. We presented the strategies that have been
fication of commitment steps, rate-limiting factors, and potential applied in engineering the biosynthetic pathways, the host strains,
regulation points. and the bioreaction processes. By introducing these modifications,
Pathway balancing is another important factor that should be the production of cyanidin 3-O-glucoside reached over 300 mg L1,
taken into consideration for process optimization of anthocyanin which is promising for industrial applications. However, many
production. A balanced pathway reduces metabolic burden difficulties still remain to be overcome to further improve the
imposed on host cells during the overproduction of anthocyanins production and to increase the commercial competitiveness of the
while maintaining normal cell growth and metabolism to the most microbial production, such as poor expression and improper
extent. Many techniques and tools are currently available to bal- balancing of genes involved in the microbial biosynthetic pathways
ance metabolic pathways, such as ePathBrick vectors, the ePa- of anthocyanins, and engineering of transporters for the efficient
thOptimize scheme, combinatorial promoter engineering, secretion of these molecules into the extracellular medium. There
synthetic RNA switches, and biosensor-based dynamic regulation is, however, little doubt, given the great strides that have been
of key intracellular metabolites [54e60]. made in optimizing recombinant microorganisms in the past 15
Besides pathways-associated factors, inefficient transportation years, that engineered microorganisms will become one of the
and lack of storage organelles of anthocyanins can also lead to most competitive sources of anthocyanin molecules in the near
compromised production. Microbial hosts lack the machinery of future.
anthocyanin transport and stabilization inherent in plant hosts,
where anthocyanins are moved from the cytoplasm to the vacuole Conflict of interest
and stored stably [61]. Currently, the path of anthocyanin move-
ment inside microbial cells and across cell membranes is poorly The authors declare no commercial or financial conflict of
understood, and there is no research on the interactions between interest.
anthocyanins and structural components of the producing mi-
crobes. The elucidation of these mechanisms, together with the Acknowledgement
introduction or engineering of specific transporters, will benefit the
optimal production of anthocyanins to a great extent. Funding for some of the work reviewed in this manuscript was
In addition, the feeding of expensive flavonoid precursors in- provided by the National Science Foundation grant number IIP-
creases the overall cost of anthocyanin production. The de novo 1549767.
production of pelargonidin 3-O-glucoside (10 mg L1) from glucose
has been accomplished with a poly-culture system [62]. Leveraging
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Please cite this article in press as: Zha J, Koffas MAG, Production of anthocyanins in metabolically engineered microorganisms: Current status
and perspectives, Synthetic and Systems Biotechnology (2017), https://doi.org/10.1016/j.synbio.2017.10.005