Colour bio-factories: Towards scale-up production of anthocyanins in plant cell cultures

Abstract

Anthocyanins are widely distributed, glycosylated, water-soluble plant pigments, which give many fruits and flowers their red, purple or blue colouration. Their beneficial effects in a dietary context have encouraged increasing use of anthocyanins as natural colourants in the food and cosmetic industries. However, the limited availability and diversity of anthocyanins commercially have initiated searches for alternative sources of these natural colourants. In plants, high-level production of secondary metabolites, such as anthocyanins, can be achieved by engineering of regulatory genes as well as genes encoding biosynthetic enzymes. We have used tobacco lines which constitutively produce high levels of cyanidin 3-O-rutinoside, delphinidin 3-O-rutinoside or a novel anthocyanin, acylated cyanidin 3-O-(coumaroyl) rutinoside to generate cell suspension cultures. The cell lines are stable in their production rates and superior to conventional plant cell cultures. Scale-up of anthocyanin production in small scale fermenters has been demonstrated. The cell cultures have also proven to be a suitable system for production of ¹³C-labelled anthocyanins. Our method for anthocyanin production is transferable to other plant species, such as Arabidopsis thaliana, demonstrating the potential of this approach for making a wide range of highly-decorated anthocyanins. The tobacco cell cultures represent a customisable and sustainable alternative to conventional anthocyanin production platforms and have considerable potential for use in industrial and medical applications of anthocyanins.

Keywords: Anthocyanins; Blackcurrant; Natural colours; Plant cell culture.

Graphical abstract

Fig. 1

Engineered anthocyanin production in Nicotiana tabacum. (a) Anthocyanin biosynthesis pathway. Co-expression of AmDel and AmRos1 induces the expression of structural genes (dotted lines) and leads to production of high amounts of cyanidin 3-O-rutinoside (C3R) and traces of pelargonidin 3-O-rutinoside (Pel3R) in N. tabacum. Expression of a Petunia x hybrida flavonoid-3′,5′-hydroxylase (PhF3′5′H) leads to hydroxylation of the anthocyanin backbone on the B-ring and production of delphinidin 3-O-rutinoside (D3R, blue box). Expression of an anthocyanin 3-O-rutinoside-4′′′-hydroxycinnamoyl transferase from Solanum lycopersicum (Sl3AT) leads to the production of aromatically acylated cyanidin 3-O-(6′′-O-coumaroyl) rutinoside (C3couR) and cyanidin 3-O-(6′′-O-feruloyl) rutinoside (C3ferR). (b) Structures of anthocyanins produced in tobacco cultures. Abbreviations: PAL, phenylalanine ammonia-lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate-CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; F3′H, flavonoid 3′-hydroxylase; F3H, flavanone 3β-hydroxylase; DFR, dihydroflavonol 4-reductase; LDOX, leucoanthocyanidin dioxygenase; GT, glycosyltransferase; GST, glutathione S-transferase; MATE, multidrug and toxic compound extrusion transporter; ABC, ATP-binding cassette transporter.

Fig. 2

Generation of tobacco suspension cultures. (a) Stably transformed tobacco plants expressing AmDel or AmRos1 and both transcription factors together. Only AmDel/AmRos1 lines showed strong anthocyanin accumulation throughout the plant. (b) Petiole sections of the same plants. AmDel/AmRos1 plants produce anthocyanins in all tissues, whereas in AmRos1 the pigmentation is limited to the epidermis and is completely absent in AmDel plants. (c) Dedifferentiated callus from leaf tissue. (d) Suspension cultures from dedifferentiated callus cells in MS medium and microscopic images of cells of suspension cultures.

Fig. 3

Tobacco suspension cultures. (a) Shake flasks after 7 days of cultivation. (b) Microscopic images of the same suspension cultures. Scale bar 100 µm. (c) Freeze dried powder from suspension cultures. (d) Total anthocyanin amount in different suspension culture batches that have been collected over a period of six months. Error bars show standard deviation of 10 samples. (e) Total anthocyanin content in various fruits, quantified under the same conditions (n = 3).

Fig. 4

Phytochemical characterisation of anthocyanins produced in tobacco cultures. (a) Representative UV-chromatograms at 515 nm for lines expressing AmDel/AmRos1, AmDel/AmRos1/PhF3′5′H and AmDel/AmRos1/Sl3AT. (b) Relative amount of anthocyanins, shown as percentage of total anthocyanins calculated from integrated peak areas of respective extracted ion chromatograms. Error bars show standard deviation of 2, 9 and 5 batches. Peak annotation is given in Supplementary Fig. 3.

Fig. 5

Regioselective ¹³C labelling of anthocyanins. (a) Schematic representation of anthocyanin biosynthesis highlighting major precursors and intermediates. (b) Mass spectrum of C3R showing the isotope pattern under control conditions with unlabelled sucrose (top) and after feeding of ¹³C-sucrose (bottom). The evaluation of the carbon status is given in Supplementary Table 2. (c) Mass spectra of C3G (left) and cyanidin (right) after MS fragmentation of the C3R precursor ion showing the isotope pattern under control conditions (top) and after ¹³C-sucrose treatment (bottom).

Fig. 6

Scale-up in bioreactors. (a) Dark red leaves of an AmDel*/AmRos1 tobacco plant next to green leaves of a wildtype plant (left panel) and a suspension culture generated from AmDel*/AmRos1 leaves (right panel). (b) Cells harvested from 50 mL suspension cultures after 7 days of growth with the same amount of starting material. (c) Total amount of anthocyanins produced in 50 mL AmDel*/AmRos1 and AmDel/AmRos1 suspension cultures. Error bar shows standard deviation of anthocyanin yield for 3 batches with each construct. (d) Array of stir tank bioreactors and control units. The outer vessels allow water circulation for temperature control and were wrapped in aluminium foil to prevent light exposure. (e) Growth curves of AmDel*/AmRos1 suspensions from four different bioreactor runs, grown in LS medium at 23° after inoculation with 3% packed cell volume. Biomass is shown as average FW ± standard deviation.

Fig. 7

Anthocyanin purification from tobacco cell suspension cultures. (a) Purification scheme. (b) Representative UV-chromatograms at 515 nm and 280 nm for defatted extract and pure isolate fractions from purification of D3R. (C) Purity check by mass spectrometry analysis. MS spectra of solvent and pure isolate fractions are shown together with the MS fragmentation spectrum for confirming the identity of the isolated D3R. Abbreviations: delphinidin (D); glucoside (Glc); rutinoside (R); delphinidin 3-O-rutinoside (D3R); methanol (MeOH); acetonitrile (AcN).

Fig. 8

Anthocyanins from Arabidopsis cell cultures. (a) Callus cultures generated from Col-0 (left) and f3′h plants (right), both expressing AmDel*/AmRos1. (b) Microscopic images of cells from the same cultures. Scale bar 25 µm. (c) Anthocyanin structures detected in A. thaliana. (d) UV chromatograms at 515 nm and MS fragmentation pattern of extracts from cultures carrying the AmDel*/AmRos1 construct in Col-0 (upper panel) and in the f3′h background (lower panel). The major compounds are cyanidin 3-O-[2′′-O-(xylosyl)−6′′-O-(p-coumaroyl) glucoside] 5-O-malonylglucoside (m/z 975.24) and pelargonidin cyanidin 3-O-[2′′-O-(xylosyl)−6′′-O-(p-coumaroyl) glucoside] 5-O-malonylglucoside (m/z 959.25).

Fig. 9

Application of anthocyanins from plant cell cultures as food colourants. (a) Colours of anthocyanin extracts (200 µM C3R equivalents) from AmDel*/AmRos1 Arabidopsis cultures in 25 mM KCL pH1 and McIlvaine's citrate-phosphate buffer at various pH, as indicated, in comparison to extracts of AmDel*/AmRos1 tobacco cultures and a conventional grape culture. (b) Cupcakes decorated with icing sugar, supplemented with anthocyanins from Arabidopsis AmDel*/AmRos1 cell cultures. Preparations with acylated cyanidins had purple to dark blue colours at neutral and slightly alkaline pH (cupcakes on the left-hand side) and aubergine hues at acidic pH (lower cupcake on the right-hand side). Icing with extracts containing acylated pelargonidins had orange to red colours at acidic pH (cupcakes on the right-hand side).