Natural pigments derived from plants and microorganisms: classification, biosynthesis, and applications

Summary

Pigments, as coloured secondary metabolites, endow the world with a rich palette of colours. They primarily originate from plants and microorganisms and play crucial roles in their survival and adaptation processes. In this article, we categorize pigments based on their chemical structure into flavonoids, carotenoids, pyrroles, quinones, azaphilones, melanins, betalains, flavins, and others. We further meticulously describe the colours, sources, and biosynthetic pathways, including key enzymatic steps and regulatory networks that control pigment production, in both plants and microorganisms. In particular, we highlight the role of transport proteins and transcription factors in fine‐tuning these pathways. Finally, we introduce the use of pigments in practical production and research, aiming to provide new insights and directions for the application of coloured compounds in diverse fields, such as agriculture, industry, and medicine.

Introduction

There are over 100,000 substances in nature capable of producing colours, with the majority relying on chemical colours displayed by pigments, excluding a portion that exhibits bioluminescence and structural colours. As secondary metabolites, pigments are a broad class of coloured compounds that serve as a significant source of a vast array of colours, endowing the world with their vibrant splendour. They mainly come from plants and microorganisms and play crucial roles in their survival and adaptation mechanisms. For example, chlorophylls, the most abundant pigments in nature, are primarily found in green plants. They play a crucial role in providing power for plant growth and development by absorbing light energy (Lee et al., 2024). Some pigments, like melanins, protect plants and fungi from damage caused by excessive light. They act as sunscreen, absorbing harmful UV radiation and preventing oxidative stress in plants and microorganisms (Gessler et al., 2014). Brightly coloured pigments such as flavonoids and carotenoids in flowers help attract pollinators, like butterflies and bees, aiding in the process of pollination (Raguso, 2004).

Pigments showcase an expansive spectrum of hues, ranging from deep blues to dazzling reds, vibrant oranges to rejuvenating greens (Figure 1). Due to their diverse range of colours, pigments are widely used in the food, beverage, textile, and cosmetics industries (Lu et al., 2023). Many pigment biosynthetic pathways have been elucidated through hundreds of years of research (Figure 2). Currently, an increasing number of researchers are focusing on the application of pigments, aiming to create an expanded range of natural and visually appealing products by leveraging plants or micros as bases and employing synthetic biology techniques.

Figure 1.

Representative pigments that produce different colours and their corresponding natural sources are as follows: The red hue in red rice is attributed to monascorubrin, while shrimp's red colour originates from astaxanthin. The orange colouration in carrots and oranges stems from α‐carotene. Maize and yellow leaves derive their yellow colour from zeaxanthin and quercetin, respectively. Chlorophyll a is responsible for the green colour of green algae and green plants. Blueberries and flowers owe their blue colour to malvidin and indigo blue pigments. The indigo shade in mulberries and eggplants is due to various types of anthocyanins. Similarly, the violet colour in flowers and onions is also a result of anthocyanins.

Figure 2.

A simplified flowchart illustrating the biosynthetic pathway of pigments. In this diagram, purple boxes highlight essential precursor compounds, whereas blue boxes represent various types of pigments. Ru5P, ribulose 5‐phosphate; 3PG, 3‐phosphoglycerate; PEP, phosphoenolpyruvate; L‐DOPA, 3,4‐dihydroxy‐L‐phenylalanine; OSB, o‐succinylbenzoate; DMAPP, dimethylallyl diphosphate; IPP, isopentenyl diphosphate; Acetyl‐CoA, Acetyl coenzyme A; ALA, 5‐aminolevulinic acid.

In this review, we classify pigments into the following categories based on their chemical structures, including flavonoids, carotenoids, pyrroles, quinones, azaphilones, melanins, betalains, flavins, and others. Moreover, we have extensively examined the pigments discovered in plants and microorganisms, categorizing them and detailing their properties according to their varied chemical structures (Table 1). Furthermore, we have devoted significant attention to the biosynthetic pathways of diverse pigments and the latest research advancements achieved in the targeted synthesis of specific pigments within plants and microorganisms. Additionally, we have incorporated insights from synthetic biology, exploring how these principles can be applied to enhance or manipulate pigment production in both natural and heterologous systems. We hope that these research findings can provide a valuable reference for future studies and offer more insights into cultivating multi‐coloured plants and the heterologous expression of pigments.

Table 1.

Representative molecules of natural pigments

Classification	Pigment	Colour	Source	Reference
Flavonols	Quercetin	Citron yellow	Plants	Li et al. (2016)
	Rutin	Pale yellow	Plants	Goyal and Verma (2023)
	Chlorflavanin	Yellow	Plants	Zhang et al. (2023)
	Digicitrin	Yellow	Plants	Meierw and Fuerst (1962)
	Myricetin	Light yellow	Plants	Song et al. (2021)
Flavones	Apigenin	Yellow	Plants	Lee et al. (2015)
	Chrysin	Pale yellow	Plants	Mani and Natesan (2018)
	Luteolin	Pale yellow	Plants	Imran et al. (2019)
Flavanones	Naringenin	Beige brow	Plants	Zang et al. (2019)
	Hesperidin	Pale yellow	Plants	Pandey and Khan (2021)
Flavanols	Epicatechin	White	Plants	Zhao and Dixon (2009)
	Gallocatechin	Light brown	Plants	Hammerbacher et al. (2018)
Isoflavones	Genistein	Pale yellow	Plants	Sharifi‐Rad et al. (2021)
	Daidzein	White	Plants	Shimada et al. (2012)
Chalcones	Licochalcone A	Yellow to orange	Plants	Li et al. (2022)
Anthocyanins	Pelargonidin	Orange, blue purple	Plants	Xu et al. (2021)
	Cyanidin	Red, blue purple	Plants	Sharma et al. (2020)
	Delphinidin	Blue purple	Plants	Lozoya‐Gloria et al. (2023)
	Peonidin	Red, blue purple	Plants	Fang (2015)
	Petunidin	Orange, reddish brown	Plants	Zimman and Waterhouse (2002)
	Malvidin	Purplish red	Plants	Merecz‐Sadowska et al. (2023)
Carotenes	ζ‐Carotene	Pale yellow	Plants, Rhodotorula spp. (fungi)	Liu et al. (2020c)
	α‐Carotene	Red	Plants, Rhodotorula spp. (fungi)	Zhao et al. (2022)
	β‐Carotene	Yellow	Plants, Blakeslea trispora (fungi)	Almagro et al. (2022)
	Lycopene	Red, pink	Plants, Rhodospirilum rubrum (bacteria)	Wei et al. (2023)
Xanthophylls	Neoxanthin	Yellow	Plants, Algae	Giossi et al. (2020)
	Fucoxanthin	Yellow	Plants, Algae	Muradian et al. (2015)
	Violaxanthin	Yellow	Plants	Takemura et al. (2021)
	Antheraxanthin	Yellow	Plants, Photosynthetic bacteria	Zhao et al. (2024)
	Lutein	Yellow	Plants, Scenedesmus spp., Chlorella spp., Rhodophyta spp.	Sun et al. (2016)
	Cathaxanthin	Red	Crustaceans, Corynebacterium	Rebelo et al. (2020)
	Zeaxanthin	Orange	Plants, Scenedesmus spp., Chlorella spp., Rhodophyta spp.	Giordano and Quadro (2018)
	Astaxanthin	Red	Shrimp, Haematococcus pluvialis	Zhang et al. (2020)
Tetrapyrrole	Chlorophyll	Green	Plants, bacteria	Bollivar (2006)
	Heme	Red	Escherichia coli, yeasts, and plants	Koreny et al. (2022)
	Siroheme	Brown	Plants, Micro	Tripathy et al. (2010)
	Bilin	Yellow, green, red	all kingdoms of life except the Archaea	Dammeyer and Frankenberg (2008)
	Cyanocobalamin	Red	Propionobacterium shermanii (bacteria)	Balabanova et al. (2021)
	Phycocyanobilin	Blue	Algae	Rockwell et al. (2023)
	Phycoerythrobilin	Red	Algae	Dammeyer and Frankenberg (2006)
	Bacteriochlorophyll	Green	Rhodopseudomonas sp. (bacteria)	Canniffe and Hunter (2014)
Tripyrrole	Prodigiosin	Red	Serratia sp. (bacteria)	Han et al. (2021)
Bipyrrole	Tambjamine	Yellow	Pseudoalteromonas tunicate (bacteria)	Picott et al. (2020)
Naphthoquinones	Shikonin/Alkannin	Purple, red, yellow	Boraginaceae family plants	Yadav et al. (2022a)
	Bikaverin	Red	Fusarium oxysporum (fungi)	Santos and Bicas (2021)
	Juglone	Yellow‐brown	Juglandaceae family plants	McCoy et al. (2018)
	Chimaphilin	Yellow	Chimaphila and Pyrola	Ageenko et al. (2022)
	Xylindein	Cyan	Chlorociboria spp. (bacteria)	Court et al. (2020)
	Actinorhodin	Blue	Streptomyces coelicolor (bacteria)	Bartel et al. (1990)
Anthraquinones	Alizarin	Red	Rubia tinctorum (plant)	Leistner (1973)
	Purpurin	Red	R. tinctorum (plant)	Leistner (1973)
	Emodin	Yellow	Rheum emodi (plant), Polygonum multiflorum (plant), Aspergillus sp. (fungi)	Semwal et al. (2021)
Others quinones	Ubiquinone (coenzyme Q)	Orange‐red	Prokaryotes and Eukaryotes	Fernández‐Del‐Río and Clarke (2021)
	Phylloquinone (vitamin K1)	Yellow	Plants	Basset et al. (2017)
	Menaquinone (vitamin K2)	Yellow	Bacteria	Meganathan and Kwon (2009)
Azaphilones	Citrinin	Yellow	Penicillium, Monascus, and Aspergillus	He and Cox (2016)
	Asperfuranone	Not documented	Aspergillus nidulans	Chiang et al. (2009b)
	Monascin	Yellow	Monascus spp.	Chen et al. (2017b)
	Ankaflavin	Yellow	Monascus spp.	Chen et al. (2017b)
	rubropunctatin	Orange	Monascus spp.	Chen et al. (2017b)
	Monascorubrin	Orange	Monascus spp.	Chen et al. (2017b)
	Monascorubramine	Red	Monascus spp.	Chen et al. (2017b)
	Rubropunctamine	Red	Monascus spp.	Chen et al. (2017b)
Melanins	Eumelanin	Dark brown‐black	Bacteria, fungi	Lee et al. (2022)
	Pheomelanin	Yellow‐red	Bacteria, fungi	Lee et al. (2022)
	Pyomelanin	Brown‐black	Bacteria, fungi	Perez‐Cuesta et al. (2020a)
	Allomelanin	Orange	Bacteria, fungi	Perez‐Cuesta et al. (2020a); Sone et al. (2018a)
	GHB melanin		Fungi	Weijn et al. (2013a)
	Plant melanin	Dark brown‐black	Plants	Glagoleva et al. (2020)
Betacyanins	Betanin	Bluish‐red	Beta vulgaris	Sepulveda‐Jimenez et al. (2005)
	Amaranthin	Reddish‐rose	Celosia cristata	Sasaki et al. (2005)
	Gomphrenin I		Dorotheanthus bellidiformis	Heuer et al. (1996)
	Bougainvillein V	Magenta	Bougainvillea peruviana	Ohno et al. (2023)
Betaxanthins	Miraxanthin V	Yellow	B. vulgaris	Kobayashi et al. (2001)
	Portulacaxanthin II	Yellow	Portulaca grandiflora	Gandia‐Herrero et al. (2005)
	Indicaxanthin	Yellow	Opuntia ficus‐indica	Allegra et al. (2019)
Flavins	Riboflavin (vitamin B2)	Yellow	All kingdoms of live	Averianova et al. (2020)
	Flavin mononucleotide (FMN)	Yellow	All kingdoms of live	Liu et al. (2019b)
	Flavin adenine dinucleotide (FAD)	Yellow	All kingdoms of live	Liu et al. (2019b)
	Roseoflavin	Yellow	Streptomyces davawensis, Streptomyces cinnabarinus	Schwarz et al. (2016)
	Deazaflavins	Yellow	Methanogens, Actinobacteria	Philmus et al. (2015)
Styrylpyrones	Hispidin	Yellow	Hymenochaetaceae family (fungi)	Lee and Yun (2011)
	Hypholomine	Yellow	Hymenochaetaceae family (fungi)	Lee and Yun (2011)
	Phelligridin	Yellow	Hymenochaetaceae family (fungi)	Lee and Yun (2011)
	Dihydrokavain	Yellow	Angiosperm plants	Zhang et al. (2018)
Phenazines	Pyocyanin	Green	Bacteria	Cátia et al. (2024)
	2‐hydroxy‐phenazine	Yellow‐orange	Bacteria	Cátia et al. (2024)
Indigoidine	Indigoidine	Blue	Bacteria	Brachmann et al. (2012)

Flavonoids

Flavonoids are a class of water‐soluble pigments containing a C6–C3–C6 skeleton structure, consisting of two benzene rings connected by a three‐carbon bridge, which are exclusively synthesized in higher plants (Saito et al., 2013). Flavonoid compounds play various significant roles in plants, such as serving as signal molecules, phytoalexins, detoxifying agents, and stimulants for the germination of spores. Furthermore, they also play a crucial role in seed germination, protecting against ultraviolet radiation, and enhancing cold and drought resistance (Samanta et al., 2011). As one of the largest groups of plant secondary metabolites, flavonoids have amassed over 10,000 distinct varieties since the term was coined in 1949 (Tursunov et al., 2023; Williams and Grayer, 2004). According to the chemical structures and modifications, flavonoids are usually divided into several families, including chalcones, flavonols, flavones, flavanones, flavanols, isoflavones, and anthocyanins (Zhang et al., 2021). Notably, flavonols, flavones, chalcones, and anthocyanins primarily contribute to the pigmentation of plants, with flavonols and flavones exhibiting yellow and chalcones appearing orange. Anthocyanins are extremely unstable, and they need to be transformed into stable anthocyanins in plants through glycosylation and other pathways. The colour of anthocyanins in plants is affected by pH. As the pH value changes, anthocyanins show different colours: red when pH < 3, colourless when 3 < pH < 7, purple when pH = 7, and blue when pH > 7 (Ying et al., 2018).

The biosynthesis of flavonoids begins with the amino acid phenylalanine, which is produced via the shikimate pathway (Figure 3). This biosynthetic journey continues through the general phenylpropane pathway (GPP), which is primarily driven by enzymes such as phenylalanine ammonia lyase (PAL), cinnamate 4‐hydroxylase (C4H), 4‐coumarate coenzyme A ligase (4CL), and chalcone synthase (CHS) to synthesize chalcone. Subsequently, chalcone isomerase (CHI) catalyses the conversion of chalcone into naringenin, a crucial precursor for flavonoid biosynthesis. Naringenin serves as the starting point for the production of specific flavonoid components, which serves as the central branch point in the flavonoid biosynthesis pathway. Firstly, naringenin can be converted to other flavanones under the action of flavanone 3′‐hydroxylase (F3'H) or flavanone 3′,5′‐hydroxylase (F3'5'H). Secondly, flavone synthase (FNS) converts flavanones into flavones. It is noteworthy that there is another pathway, a root‐specific flavone pathway for flavone synthesis in Scutellaria baicalensis (Zhao et al., 2016a). Thirdly, isoflavone synthase (IFS) guides a fraction of flavanones into the isoflavone pathway. The others are produced under the action of hydroxyisoflavanone dehydratase (HID). Finally, dihydroflavonols (DHF) are formed from flavanone through the catalytic action of flavanone 3‐hydroxylase (F3H), serving as a common precursor for the biosynthesis of flavonols, anthocyanidins, and flavanols. Flavonol synthase (FLS) converts DHF to flavonols, like quercetins. Dihydroflavonol 4‐reductase (DFR) transformed DHF into colourless leucoanthocyanidins, which are then further converted into brightly coloured anthocyanins under the catalysis of anthocyanidin synthase (ANS). Flavanols are partly derived from the conversion of leucoanthocyanidins catalysed by leucoanthocyanidin reductase (LAR) and partly from the conversion of anthocyanidins catalysed by anthocyanidin reductase (ANR) (Dixon and Pasinetti, 2010; Koes et al., 2005; Liu et al., 2021).

Figure 3.

Biosynthetic network of the main flavonoids in plants. The illuminated spots exhibit the hues of various substances, while the dashed arrow indicates multiple reactions occurring during the step. The display of different colours signifies the synthetic routes of various flavonoid types. The core intermediate substances and precursors are highlighted with black boxes. PAL, phenylalanine ammonia lyase; FNS, flavone synthase; F6H, flavonoid 6‐hydroxylase; C4H, cinnamic acid 4‐hydroxylase; 4CL, 4‐coumarate: CoA ligase; CHS, chalcone synthase; CHR, chalcone reductase; CHI, chalcone isomerase; IFS, isoflavone synthase; HID, 2‐hydroxyisoflavanone dehydratase; F3' H, flavanone 3'‐hydroxylase; F3H, flavanone 3‐hydroxylase; DFR, dihydroflavonol 4‐reductase; ANS, anthocyanidin synthase; FLS, flavonol synthase; LAR, leucoanthocyanidin reductase; ANR, anthocyanidin reductase (Liu et al., 2021).

In addition to being directly influenced by the aforementioned structural genes, the biosynthesis of flavonoids is also regulated by various transcription factors, the most important of which is the MBW (MYB–bHLH–WD40) protein complexes (Xu et al., 2015). Furthermore, with the deepening of research on flavonoid biosynthetic pathways, transcription factors from families such as B‐box, WRKY, and bZIP have also been proven to participate in flavonoid synthesis (Bai et al., 2019; Li et al., 2021; Wang et al., 2022). Since the biosynthesis of flavonoids occurs on the cytosolic side of the endoplasmic reticulum but ultimately accumulates in the vacuoles, an efficient flavonoid transport and accumulation system is required to transfer them from the endoplasmic reticulum to the vacuole (Zhao and Dixon, 2010). Currently, there are three transport modes for flavonoids to enter the vacuole from the cytoplasm, namely glutathione S‐transferase‐mediated transport, membrane transporter‐mediated transport, and vesicle‐mediated transport (Kaur et al., 2021). Although the regulatory factors involved in flavonoid biosynthesis have been widely studied, the research on the transcriptional regulation of transporter genes by transcription factors is still not thorough enough. Further in‐depth studies are needed to determine whether different flavonoid transcription factors can selectively activate specific transport mechanisms.

Carotenoids

Carotenoids, also known as isoprene derivatives, constitute a vast lipid‐soluble isoprenoid family. Predominantly, they are C40 tetraterpenoids arising from the sequential condensation of eight C5 isoprene units. Occasionally, among nonphotosynthetic bacteria, novel carotenoids with elongated C45 or C50, or shortened C30 backbones are observed. Since the beginning of the 19th century, scientists have uncovered over a thousand varieties of natural carotenoids, which can be synthesized de novo in photosynthetic bacteria, fungi, algae, and plants (DellaPenna and Pogson, 2006; Yabuzaki, 2017). Carotenoids play crucial roles in photosynthesis and photoprotection in photosynthetic bacteria, algae, and plants, and they also act as antioxidants, colour attractants, and precursors of plant hormones (Maoka, 2019). Carotenoids are classified into two main groups: carotenes and xanthophylls, distinguished by their elemental composition. The diverse colours of carotenoids are attributed to the length of their carbon skeletons and the number of double bonds present. Compounds like canthaxanthin, α‐carotene, lycopene, and astaxanthin possess a significant number of conjugated double bonds, rendering them red in colour. Except for α‐crustacyanin, which exhibits blue, most carotenoids tend to display a yellow colour (Ashokkumar et al., 2023; Maoka, 2019; Meléndez‐Martínez et al., 2015).

Photosynthetic cyanobacteria and plants can synthesize C5 isoprenoid precursors through the 2‐C‐methyl‐derythritol‐4‐phosphate pathway (MEP), while plants can also produce these precursors via the mevalonate pathways (MVA). However, bacteria and fungi primarily derive C5 isoprenoid precursors through the MVA pathway. However, there are a few bacterial species of Streptomyces that utilize both the MVA and the MEP pathway for the generation of isoprenoid precursors (Liang et al., 2018). As is shown in Figure 4, two molecules of GGPP are condensed into phytoene by phytoene synthase (PSY, CrtB in bacteria). PSY serves as a pivotal rate‐limiting enzyme in carotenoid biosynthesis, marking the initial decisive step and constituting a critical bottleneck. Overexpression of PSY in plants or microorganisms can significantly enhance the rate and total amount of carotenoid biosynthesis (Fraser et al., 2002; Rao et al., 2024). Subsequently, phytoene is desaturated into all‐trans‐lycopene by phytoene desaturase (PDS), ζ‐carotene desaturase (ZDS), and carotenoid isomerase (CRTISO). Unlike plants, which require four enzymes to desaturate and isomerize phytoene into all‐trans‐lycopene, bacteria and fungi utilize a single enzyme, CrtI, to catalyse all the desaturation and isomerization steps. All‐trans‐lycopene is cyclized into α‐carotene (α pathway) and β‐carotene (β pathway) in reactions catalysed by lycopene ε (LYCE) and β‐cyclase (LYCB, CrtY in bacteria), respectively. Lutein is formed by the action of α‐carotene ring‐ε hydroxylase (CHYE) via the α pathway; β‐carotene is converted into violaxanthin via the β pathway in a reaction catalysed by β‐carotene hydroxylase (CHYB, CrtZ in bacteria). It is noteworthy that while only plants and certain fungi possess the ability to synthesize lutein, the range of carotenoids produced by bacteria is extensive. Shortened C30 and elongated C45 or C50 carotenoids, like diapolycopen and decaprenoxanthin, are synthesized only in microorganisms (DellaPenna and Pogson, 2006; González‐Peña et al., 2023; Sandmann, 2022; Wang et al., 2007).

Figure 4.

Biosynthetic pathway of natural carotenoids. In this figure, the illuminated spots display the colours of various substances. Specifically, the carotenoids belonging to C30 and C50 categories are denoted in blue. The main biosynthetic pathways are divided into α and β pathways, which are distinctly colour‐coded as green and orange, respectively. The dashed arrow indicates that multiple reactions occur within that particular step. Crucial precursors in the biosynthesis process are emphasized with black boxes. MEP, 2‐C‐methyl‐derythritol‐4‐phosphate pathway; MVA, mevalonate pathways; FPP, farnesyl pyrophosphate; GGPP, geranylgeranyl diphosphate; GGPPS, GGPP synthase; PSY, phytoene synthase; PDS, phytoene desaturase; ZDS, ξ‐carotene desaturase; CRTISO, carotenoid isomerase; LCYB, lycopene β‐cyclase; LYCE, lycopene ε‐cyclase; CHYB, β‐carotene hydroxylase; CHYE, ε‐carotene hydroxylase; ZEP, zeaxanthin epoxidase; VDE, violaxanthin de‐epoxidase (DellaPenna and Pogson, 2006).

It is currently known that the transcription factors regulating the synthesis of plant carotenoids mainly control the metabolic flux of the pathway by regulating the expression of enzyme genes in the carotenoid synthesis pathway. Among them, the gene PSY is positively regulated by transcription factors MAD‐RIN, SlCMB1, TAGL1, SlMBP15, FUL1, FUL2, and SlNAC4, and negatively regulated by transcription factors MADS1, Sl‐MBP8, SlFYFL, RAP2.2, SlAP2a, PIF, SlAN2, and SlNAC1 (Liu et al., 2015; Llorente et al., 2017; Yin et al., 2017); the gene PDS is positively regulated by transcription factors SlCMB1 and SlMBP15, and negatively regulated by transcription factors SlMBP8, SlFYFL, RAP2.2, and SlAN2 (Xie et al., 2014); the gene ZDS is positively regulated by transcription factors SlMBP15, FUL1, and FUL2, and negatively regulated by transcription factors SlMBP8, SlFYFL, and SlAN2 (Meng et al., 2015); the gene LYCB is positively regulated by the transcription factor SlNAC4 and negatively regulated by transcription factors SlAP2a and SlNAC1; the gene LYCE is positively regulated by the transcription factor SlNAC4 (Mingku et al., 2014). In addition, the molecular chaperone OR, as a major post‐transcriptional regulator of PSY, also participates in the regulation of carotenoid synthesis (Sun et al., 2023).

Pyrroles

Pyrroles refer to a class of heterocyclic compounds with a five‐membered ring structure, featuring one or more nitrogen heteroatoms. Based on the number of pyrrole rings present, they are categorized primarily as tetrapyrroles, tripyrroles, and dipyrroles. Among these, tetrapyrroles predominantly encompass chlorophylls, phycobilins, cobalamin, heme, and siroheme. Prodigiosin serves as a primary representative of tripyrroles, while dipyrroles are primarily composed of benjaminin. Among them, the functions of chlorophylls and phycobilins are to act as light‐harvesting pigment complexes involved in the energy supply of plants, cyanobacteria, and algae during photosynthesis (Reinbothe et al., 2010). Heme and siroheme participate in redox reactions, electron transfer, oxygen transport, and other processes within organisms (Tripathy et al., 2010). Cobalamin, prodigiosin, and benjaminin are synthesized by microorganisms. Cobalamin serves as a cofactor in numerous metabolic pathways, whereas psilocybin and benomycen may be involved in competition among microorganisms (Balabanova et al., 2021).

Based on their colouration, pyrrole compounds can be categorized into several distinct groups. Green is the most prevalent colour among these, primarily represented by various types of chlorophylls. Following green, we find red pyrroles such as heme, cobalamin, phycoerythrobilin, and prodigiosin. Blue pyrroles are represented by phycocyanobilin, while yellow pyrroles are exemplified by tambjamine. Except that cobalamin is water‐soluble, almost all pyrroles are lipid‐soluble. The diverse range of colours reflects the varying structural and functional properties that pyrrole compounds possess in nature.

Chlorophyll is the most ubiquitous pyrrole pigment in nature, encompassing over 100 species discovered to date (Shen et al., 2023). Despite the intricate nature of their synthesis pathways, a predominant and unifying mechanism has been identified as the primary route for chlorophyll production. Initially, glutamate serves as the fundamental starting material for synthesizing 5‐aminolevulinic acid (ALA), a crucial precursor in biological systems. ALA can also be produced by the enzymatic reaction of glycine and succinyl‐CoA in bacteria. Two ALA molecules condense under the catalysis of ALA dehydratase (ALAD, Pbgs in bacteria) to form porphobilinogen (PBG), a precursor that contains a single pyrrole ring. Then, four PBG molecules undergo condensation, catalysed by porphobilinogen deaminase (PBGD, HmbS in bacteria), to form hydroxymethylbilane, a linear tetra‐pyrrole compound. Hydroxymethylbilane undergoes a cyclization reaction catalysed by uroporphyrinogen III synthase (UROS) to form uroporphyrinogen III, a cyclic tetra‐pyrrole compound. In different biosynthetic pathways, metal ions are inserted into the tetra‐pyrrole ring, determining the function of the final product. In porphyrins, magnesium is inserted to form chlorophyll, cobalt is inserted to form cobalamin, and iron is inserted to form heme. The transformation of DV‐PChlide a into 3,8‐divinyl‐chlorophyllide a (DV Chlide a) is catalysed by protochlorophyllide oxidoreductases (PORs), promoting its efficient conversion. PORs are key enzymes in chlorophyll synthesis, and so far, their crucial roles in chlorophyll synthesis, chloroplast development, and photomorphogenesis in plants have been confirmed in multiple species such as arabidopsis, tobacco, barley, and rice (Frick et al., 2003; Talaat, 2013). After the final two steps of the reaction, phytyl combines with chlorophyllide a to form chlorophyll a, and chlorophyll a can also undergo subsequent reactions to produce other chlorophylls (Figure 5) (Tanaka et al., 2011; Yang et al., 2024).

Figure 5.

Biosynthetic pathway of three representative pyrroles. The synthesis pathways of chlorophyll a (a), prodigiosin and tambjamines (b) are denoted by orange, green, and pink colours, respectively. The rendered light spots show the colours of different substances. The hollow arrow suggests multiple reactions in the step. ALAD, 5‐aminoleculinic acid dehydratase; PBGD, porphobilinogen deaminase; UROS, uroporphyrinogen III synthase; UROD, uroporphyrinogen III decarboxylase; CPO, coproporphyrinogen III oxidase; PPO, protoporphyrinogen IX oxidase; MgCh, Mg‐chelatase; MgMT, Mg‐protoporphyrin IX methyltransferase; MgCY, Mg‐protoporphyrin IX monomethylester cyclase; POR, protochlorophyllide oxidoreductase; DVR, 3,8‐divinyl protochlorophyllide a 8‐vinyl reductase; CHLG, chlorophyll synthase; MAP, 2‐methyl‐3amyl‐pyrrole; MBC, 4‐methoxy‐2,2′‐bipyrrole‐5‐carbaldehyde; DDEA, cis‐dodec‐3en‐1‐amine (Paul et al., 2022; Picott et al., 2020; Tanaka et al., 2011).

The biosynthesis of chlorophyll is a complex and exquisite process, involving fine‐tuning by multiple factors (such as light, hormones, circadian rhythms, etc.) and multi‐level regulatory factors (including transcription factors, post‐transcriptional regulatory factors, post‐translational modification factors, etc.) (Brzezowski et al., 2015). Among these regulatory mechanisms, transcriptional regulation is a widely studied type across different species. Currently, multiple transcription factor families have been reported to participate in the regulation of chlorophyll synthesis, including the bHLH, bZIP, MYB, MADS, and TCP families (Koichi and Tatsuru, 2016; Liu et al., 2020a; Toledo‐Ortiz et al., 2014; Wu et al., 2020; Zheng et al., 2022).

Prodigiosin, a microbial secondary metabolite red pigment, possesses a distinctive methoxypyrrole skeleton. As is shown in Figure 5, with the catalytic action of pigC, two key intermediates, 2‐methyl‐3‐n‐amylpyrrole (MAP) and 4‐methoxy‐2,2′‐bipyrrole‐5‐carbaldehyde (MBC), synthesize prodigiosin (Han et al., 2021; Paul et al., 2022). Tambjamines, similar to prodiginines, originate from microorganisms and possess a series of clinically relevant biological activities. TamQ has recently been shown to form tambjamines by fusing MBC and cis‐dodec‐3en‐1‐amine (DDEA) (Picott et al., 2020).

Quinones

Quinones, characterized by six‐membered unsaturated rings harboring two carbonyl moieties and a vinylene group, represent a prominent class of pigmented compounds. Typically, these compounds exhibit colours ranging from yellow to red, with occasional variations observed in shades of green, brown, or violet (Dulo et al., 2021). Naturally, quinones are distributed across various biological domains, including animals, plants, and microorganisms. Notable examples of quinones include the dietary supplement ubiquinone (coenzyme Q10), the ancient dye alizarin extracted from the madder plant, and vitamins K1 (phylloquinone) and K2 (menaquinone).

Bacterial quinones are synthesized via the polyketide synthase (PKS) pathway and the o‐succinylbenzoate (OSB) pathway. The PKS pathway involves the utilization of acetyl‐CoA (coenzyme A) and malonyl‐CoA as substrate precursors, leading to the formation of a polyketide intermediate tethered to an acyl carrier protein (ACP). Subsequent cyclization events at distinct molecular sites yield either naphthoquinone or anthraquinone ring scaffolds, followed by further structural modifications (Mund and Čellárová, 2023). The OSB pathway utilizes chorismate as a substrate, undergoing enzymatic conversion to isochorismate catalysed by an isochorismate synthase. Subsequent enzymatic reactions yield OSB, which is then conjugated with CoA to form OSB‐CoA. Further transformations lead to the formation of DHNA‐CoA, subsequently liberated to yield free DHNA (Widhalm and Rhodes, 2016), which will contribute to menaquinone, naphthoquinone, and anthraquinone biosynthesis.

Fungal quinones are synthesized by various species, particularly those belonging to the genera Aspergillus, Penicillium, Talaromyces, Fusarium, and Arthrinium. The core structure of these quinones is primarily assembled through the PKS pathway. Unlike bacteria, which rely on type II PKS for quinone synthesis, fungi utilize type I PKS. Various modifications, such as methylation, prenylation, halogenation, amination, and acetylation, are often applied to the core structure, resulting in a wide diversity of fungal quinone compounds (Christiansen et al., 2021).

Plant quinones are synthesized through various pathways. In addition to the PKS pathway and the OSB pathway found in bacteria, plants also utilize the 4‐hydroxybenzoic acid (HBA) and the homogentisic acid (HGA) pathways for quinone biosynthesis. The HBA pathway is involved in the production of naphthoquinones. Starting with phenylalanine, this pathway initiates the synthesis of 4HBA, which undergoes a series of enzymatic transformations to form ubiquinones. Additionally, 4HBA can be converted into 3‐geranyl‐4HBA, which, in combination with GPP, leads to the formation of geranylhydroquinones. Further modifications in certain plant species may introduce additional ring structures to geranylhydroquinones (Yadav et al., 2022b). The HGA pathway, primarily identified in plants of the Pyroloideae subfamily within the Ericaceae family, is crucial for naphthoquinone synthesis. This pathway begins with the conversion of tyrosine to HGA, followed by enzymatic conversion to toluquinol. Toluquinol then reacts with dimethylallyl diphosphate (DMAPP) to produce dimethylallyl‐toluquinol, which serves as the precursor for assembling the second ring structure of the quinone molecule (Ageenko et al., 2022).

In the case of quinones synthesized via PKS, the primary precursors are acetyl‐CoA and malonyl‐CoA (Figure 6). Enhancing the availability of these precursors by fortifying acetyl‐CoA carboxylase can significantly increase the titres of endocrocin and emodin (Sun et al., 2019). In contrast, quinones produced through the shikimate pathway utilize chorismate as a key intermediate (Figure 6). This pathway is subject to feedback inhibition. Overexpression of aroADE leads to inhibition of menaquinone biosynthesis (Yang et al., 2019), while introducing aroG and aroF mutants can alleviate this feedback, thereby enhancing quinone biosynthesis (Liu et al., 2023). Furthermore, the quorum‐sensing regulator SinR, which is essential for biofilm formation, is also implicated in the regulation of menaquinone biosynthesis. This regulator likely balances cellular growth with product synthesis (Wu et al., 2021). Additionally, maintaining balanced levels of cofactors such as NADPH is vital for the production of isoprene (Liu et al., 2019a) and polyketides (Wang et al., 2024), both of which are integral components of numerous quinones.

Figure 6.

Biosynthetic pathways of natural quinones. The rendered light spots show the colours of different substances. The hollow arrow suggests multiple reactions in the step. The HBA, HGA, OSB, and polyketide pathways are indicated in blue, orange, green, and magenta, respectively. The core intermediates are highlighted with black boxes. PAL, phenylalanine ammonia‐lyase; CA4H, cinnamate 4‐hydroxylase; 4CL, 4‐coumarate‐CoA ligase; 4HCT, 4‐hydroxybenzoyl‐CoA thioesterase; LEPGT, 4‐hydroxybenzoate geranyltransferase; CYP, cytochrome P450; TAT, tyrosine aminotransferase; HPD, 4‐hydroxyphenylpyruvate dioxygenase; ICS, isochorismate synthase; α‐KG, α‐Ketoglutarate; SES, 2‐succinyl‐5‐enolpyruvyl‐6‐hydroxy‐3‐cyclohexene‐1‐carboxylate synthase; SHS, 2‐succinyl‐6‐hydroxy‐2,4‐cyclohexadiene‐1‐carboxylate synthase; OSS, o‐succinylbenzoate synthase; OCL, o‐succinylbenzoate‐CoA ligase; DCS, 1,4‐dihydroxy‐2‐naphthoyl‐CoA synthase; DCT, 1,4‐dihydroxy‐2‐naphthoyl‐CoA hydrolase; ACS, acetate‐CoA ligase; ACC, acetyl‐CoA carboxylase; SEPHCHC, 2‐Succinyl‐5‐enolpyruvyl‐6‐hydroxy‐3‐cyclohexene‐1‐carboxylate; SHCHC, (1R,6R)‐6‐Hydroxy‐2‐succinylcyclohexa‐2,4‐diene‐1‐carboxylate; OSB, o‐succinylbenzoate; DHNA, 1,4‐dihydroxy‐2‐naphthoic acid; PEP, phosphoenolpyruvate; PKS, polyketide synthase (Ageenko et al., 2022).

Membrane engineering and transport mechanisms are critical for the effective biosynthesis of quinones, particularly those with isoprene side chains, which are lipid‐soluble. Transcriptomic analyses have demonstrated that the expression levels of signal receptors, transmembrane transporters, and components of signal transduction correlate with menaquinone biosynthesis (Cui et al., 2020). Furthermore, modifications to the composition of the cell membrane may enhance menaquinone production (Wang et al., 2019). The transport of menaquinone within cells is mediated by ATP‐binding cassette (ABC) transporters (Matsuo et al., 2023), although the mechanisms of secretion in microorganisms remain to be elucidated.

Azaphilones

Azaphilones constitute a group of fungal metabolites characterized by a highly oxygenated pyrano‐quinone bicyclic chromophore. This bicyclic chromophore confers this class of compound colours ranging from yellow to red. Their designation as “azaphilones” stems from their pronounced affinity for nitrogen nucleophiles. Azaphilones are biosynthesized by various fungi, including species of Aspergillus, Chaetomium, and Monascus, belonging to the Ascomycota and Basidiomycota phyla (Pavesi et al., 2021).

Azaphilones are produced through a polyketide pathway using acetyl‐CoA and malonyl‐CoA as primary substrates for chain formation, with occasional involvement of S‐adenosyl methionine (SAM). The biosynthesis begins with the product template (PT) domain of PKS forming the first ring in the polyketide chain. Subsequent modifications after release lead to the formation of the second ring, a key step contributing to the structural diversity of azaphilones, which follow three distinct biosynthetic pathways (Figure 7).

Figure 7.

Biosynthetic pathways of natural azaphilones. The rendered light spots show the colours of different substances. The hollow arrow suggests multiple reactions in the step. The citrinin biosynthetic pathway in Monascus, the asperfuranone biosynthetic pathway in Aspergillus nidulans, and the general pathway of azaphilone synthesis are depicted in blue, orange, and green, respectively. The core substrate is highlighted with a black box. SAM, S‐adenosyl methionine; PT, product template; R, reductase; ACC, acetyl‐CoA carboxylase (Chen et al., 2017a).

The first pathway, commonly referred to as the citrinin pathway, is utilized by Monascus species for citrinin synthesis. Following release from ACP, the molecule undergoes reduction by the CitS reductase (R) domain in conjunction with the hydrolase CitA. The second ring formation between the aldehyde and methyl groups, catalysed by oxidoreductase CitE, yields the final product, citrinin (He and Cox, 2016). The second pathway is utilized by Aspergillus nidulans for asperfuranone synthesis. After release from ACP, the molecule undergoes hydroxylation at C‐3 by the hydroxylase AfoD, followed by C‐7 hydroxylation by hydroxylase AfoF. Spontaneous condensation of the resulting 7‐OH group with the aldehyde group forms the second ring (Chiang et al., 2009a). The third pathway, the most prevalent for azaphilone biosynthesis, yields molecules such as monascin and ankaflavin. The characteristic of this pathway is the formation of the second ring between two carbonyl groups catalysed by a monooxygenase (Chen et al., 2017a). Various modifications of the hydroxyl and R₁ groups may occur, potentially preceding second ring formation (Figure 7).

In Monascus, azaphilone biosynthesis is intricately connected to the glycolytic pathway, with the biosynthesis of pyruvate and fructose‐1,6‐bisphosphate serving as rate‐limiting steps (Duan et al., 2022). Redirecting the metabolic flux of pyruvate toward malonyl‐CoA, a key precursor for PKS, significantly enhances azaphilone production. Concurrently, suppression of fatty acid biosynthesis diverts additional malonyl‐CoA toward azaphilone biosynthesis. Moreover, maintaining an optimal balance between cell growth and azaphilone production is critical. A two‐stage fermentation process, employing glucose followed by sucrose as carbon sources, effectively increases both cell density and azaphilone yield (Duan et al., 2022). In addition, the cyclic adenosine monophosphate (cAMP) signalling pathway plays a pivotal role in regulating azaphilone biosynthesis. Deletion of the mrPDE gene, which elevates intracellular cAMP levels, results in a marked increase in azaphilone production (Liu et al., 2020b).

Melanin

Melanin, a group of pigmented compounds found in a wide range of organisms from bacteria and fungi to plants and animals, is composed of polymeric structures made from different monomeric units. This discussion focuses on microbial and plant melanins. Microbial melanins include five main types: eumelanin, pheomelanin, pyomelanin, allomelanin, and GHB (γ‐L‐glutaminyl‐4‐hydroxybenzene) melanin. Plant melanins, derived from monophenolic precursors, share some characteristics with microbial melanins but exhibit greater diversity and complexity.

Eumelanin, pheomelanin, pyomelanin, and allomelanin are produced by both bacteria and fungi, each through distinct biosynthetic pathways. The synthesis of eumelanin and pheomelanin is catalysed by either tyrosinase (Tyr1) or laccase (Lac1) (Singh et al., 2021), using tyrosine as the starting material and 3,4‐dihydroxy‐L‐phenylalanine (L‐DOPA) as a crucial intermediate. The key difference is that pheomelanin synthesis requires the presence of cysteine (see Figure 8). Pyomelanin biosynthesis begins with tyrosine and involves a tyrosine aminotransferase (TAT) and a dioxygenase (HppD) (Schmaler‐Ripcke et al., 2009; Singh et al., 2018). Allomelanin, on the other hand, is produced via a PKS pathway (Perez‐Cuesta et al., 2020b; Sone et al., 2018b; Tsai et al., 1999). This pathway utilizes acetyl‐CoA and malonyl‐CoA as substrates, which undergo a series of condensation reactions to form trihydroxynaphthalene (THN). THN is subsequently converted to dihydroxynaphthalene (DHN), the monomeric unit, through a series of reductase, dehydratase, and oxidase reactions.

Figure 8.

Biosynthetic pathways of natural melanins. The rendered light spots show the colours of different substances. The hollow arrow suggests multiple reactions in the step. Biosynthetic pathways of plant melanin, pyomelanin, pheomelanin, eumelanin, GHB melanin, and allomelanin are indicated in blue, orange, brown, green, magenta, and grass green, respectively. Alb, Ayg, Arp, and Abr enzymes are derived from Aspergillus fumigatus . The core intermediates are highlighted with black boxes. PPO, polyphenoloxidase; TAT, tyrosine aminotransferase; HppD, 4‐hydroxyphenylpyruvate dioxygenase; Tyr1, tyrosinase; Lac1, laccase; 4ABS, 4‐aminobenzoate synthase; 4ABH, 4‐aminobenzoate hydroxylase; GGT, γ‐Glutaminyltransferase; ACS, acetate‐CoA ligase; ACC, acetyl‐CoA carboxylase; PEP, phosphoenolpyruvate; GHB; γ‐L‐glutaminyl‐4‐hydroxybenzene; GBQ, γ‐L‐glutaminyl‐3,4‐benzoquinone; THN, trihydroxynaphthalene; T3HN, 1,3,8‐tetrahydroxynaphthalene; DHN, dihydroxynaphthalene (Glagoleva et al., 2020).

GHB melanin, a brown biopolymer found exclusively in fungal organisms, follows a unique biosynthetic pathway (Weijn et al., 2013b). Initially, chorismate is converted to 4‐aminobenzenol by the enzymes 4‐aminobenzoate synthase (4ABS) and 4‐aminobenzoate hydroxylase (4ABH). Next, 4‐aminobenzenol is converted to GHB with the assistance of γ‐glutaminyltransferase (GGT) and glutamic acid. Downstream, GHB undergoes transformations mediated by polyphenoloxidase (PPO) or tyrosinase enzymes, resulting in the formation of γ‐L‐glutaminyl‐3,4‐dihydroxybenzene (GDHB) and γ‐L‐glutaminyl‐3,4‐benzoquinone (GBQ) from GHB. GBQ will serve as the basic building block of GHB melanin.

Plant melanins, which range in colour from brown to black, are mainly found in the seed envelope, although their exact biological role is not fully understood. These melanins are nitrogen‐free and are produced from monophenols derived from chorismate. PPO catalyses the conversion of monophenols into o‐diphenol and then into o‐quinone. This o‐quinone readily participates in nonenzymatic reactions, such as polymerization with other phenol molecules, thiol groups, amino groups, and similar compounds. Despite the diversity of plant melanins, their enzymatic conversion relies solely on PPO (Glagoleva et al., 2020).

With the exception of allomelanin, most melanins utilize chorismate as a common substrate (Figure 8). As such, enhancing the shikimate pathway by alleviating feedback inhibition can significantly improve their biosynthesis (Chávez et al., 2013). Furthermore, the key enzymes involved in melanin production, such as tyrosinases and laccases, require copper (Cu) for proper functionality. Therefore, the addition of Cu at an appropriate stage of fermentation or the modification of Cu transporters can help balance cell growth with product synthesis (Chávez et al., 2013). In some Streptomyces species, the expression of tyrosinase is regulated by the transcriptional regulator AdpA, which is also implicated in morphological development (Zhu et al., 2005). Melanin biosynthesis is frequently induced by a range of stress factors, such as nutrient deprivation and increased temperatures, indicating its potential role in cellular adaptation to environmental fluctuations (Pavan et al., 2020).

Betalains

Betalains constitute a group of pigmented compounds primarily encountered in plants, notably prevalent within the order Caryophyllales. However, their presence extends beyond botanical realms to certain basidiomycete fungi, including species within the Amanita and Hygrocybe genera (Stintzing and Schliemann, 2007), as well as the bacterial species Gluconacetobacter diazotrophicus (Contreras‐Llano et al., 2019). In most plant species, betalains serve as complete substitutes for anthocyanins, typically exhibiting a mutually exclusive distribution within a given plant. These compounds play a pivotal role in conferring vivid chromatic displays observed in the fruits, flowers, and stems of diverse plant species.

Betalains are categorized into two distinct classes: betacyanins and betaxanthins. Betacyanins, ranging in colour from red to violet, arise from the glycosylation of hydroxy groups on betanidin, a product derived from betalamic acid and 2‐carboxy‐2,3‐dihydro‐5,6‐dihydroxyindole (cyclo‐DOPA). In contrast, betaxanthins, spanning hues from yellow to orange, originate from the combination of betalamic acid with amino acids or amides.

The biosynthesis of betalamic acid stands as a pivotal process in betalain biosynthesis (Figure 9). This pathway involves two enzymatic reactions leading to the formation of betalamic acid from tyrosine. Initially, tyrosine undergoes hydroxylation catalysed by a prevalent hydroxylase, resulting in the formation of L‐DOPA (Halaouli et al., 2006; Zaidi et al., 2014). Subsequently, a dioxygenase facilitates the opening of the L‐DOPA ring, yielding 4,5‐seco‐DOPA, which exhibits limited stability and spontaneously transforms into betalamic acid. Concurrently, L‐DOPA can undergo oxygenation to generate cyclo‐DOPA via an oxygenase (Christinet et al., 2004; Halaouli et al., 2006; Hatlestad et al., 2012; Zaidi et al., 2014). The combination of cyclo‐DOPA with betalamic acid results in the spontaneous formation of betanidin. Betacyanins are then synthesized through the addition of glucosides under the catalysis of glycosyltransferase. Hydroxylase is prevalent in plants and microorganisms, while dioxygenase is mainly found in plant species such as Portulaca grandiflora and Beta vulgaris (Christinet et al., 2004; Hatlestad et al., 2012) and microbial species Amanita muscaria (Girod and Zryd, 1991) and G. diazotrophicus (Contreras‐Llano et al., 2019).

Figure 9.

Biosynthetic pathways of natural betalains. The rendered light spots show the colours of different substances. The biosynthetic pathway of betacyanins is depicted in green, while the pathway for betaxanthins is shown in orange. The core intermediates are highlighted with black boxes. L‐DOPA, 3,4‐Dihydroxy‐L‐phenylalanine; DODA, DOPA dioxygenase. CYP76AD1 is a specific hydroxylase from Beta vulgaris (Zaidi et al., 2014).

Tyrosine serves as the precursor for betalains, making the enhancement of tyrosine biosynthesis advantageous for boosting betalain production. A common strategy to achieve this involves alleviating feedback inhibition in the shikimate pathway by introducing feedback‐insensitive enzymes, such as AroG in bacteria and arogenate dehydrogenase (ADH) in plants (Polturak and Aharoni, 2019). Additionally, betalain biosynthesis is regulated by MYB transcription factors in plants, which bind to the promoters of betalain biosynthetic genes (Xi et al., 2019; Xie et al., 2023). Betalains primarily accumulate within plant cells (Polturak et al., 2017), although they have been successfully exported in yeast (Grewal et al., 2018). This transport is likely mediated by the QDR2 and APL1 transporters (Babaei et al., 2023). Introducing betalain transporters into heterologous expression hosts may enhance both secretion and overall productivity.

Flavins

Flavins represent a group of yellow organic compounds characterized by the presence of an isoalloxazine ring structure. They are widely distributed across diverse organisms, including plants, animals, fungi, and bacteria. Riboflavin, also known as vitamin B2, serves as the archetypal flavin compound, with other biologically significant flavins derived from it. Flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) are notable derivatives of riboflavin, playing pivotal roles in various biological processes.

In plants and bacteria, flavins are synthesized from guanosine 5′‐triphosphate (GTP) derived from purine metabolism, as well as ribulose 5‐phosphate (Ru5P) obtained from the pentose phosphate pathway (PPP). The conversion of Ru5P to L‐3,4‐dihydroxybutan‐2‐one 4‐phosphate (DHPB) is facilitated by DHPB synthase, followed by the catalytic action of 6,7‐dimethyl‐8‐(D‐ribityl)lumazine (DrL) synthase to form DrL in the presence of 5‐amino‐6‐(1‐D‐ribitylamino)uracil (ArP), which originates from GTP through a series of enzymatic reactions involving GTP cyclohydrolase, deaminase, reductase, and phosphatase. Eventually, DrL is converted to riboflavin by a kinase enzyme (Figure 10).

Figure 10.

Biosynthetic pathways of natural flavins. The rendered light spots show the colours of different substances. The hollow arrow suggests multiple reactions in the step. The biosynthetic pathway of ArP is depicted in orange, while the pathway for DrL is shown in green. The core intermediates are highlighted with black boxes.GTP, Guanosine 5′‐triphosphate; DARPP, 2,5‐diamino‐6‐(1‐D‐ribosylamino)pyrimidin‐4(3H)‐one 5′‐phosphate; ARPP, 5‐amino‐6‐(ribosylamino)‐2,4‐(1H,3H)‐pyrimidinedione 5′‐phosphate; DArPP, 2,5‐diamino‐6‐(1‐D‐ribitylamino)pyrimidin‐4(3H)‐one 5′‐phosphate; ArPP, 5‐amino‐2,6‐dioxy‐4‐(5′‐phospho‐D‐ribitylamino)pyrimidine; ArP, 5‐amino‐6‐(1‐D‐ribitylamino)uracil; Ru5P, ribulose 5‐phosphate; DHPB, L‐3,4‐dihydroxybutan‐2‐one 4‐phosphate; DrL, 6,7‐dimethyl‐8‐(D‐ribityl)lumazine; FMN, Flavin mononucleotide; FAD, Flavin adenine dinucleotide (Haase et al., 2013).

Fungi exhibit disparate pathways in the biosynthesis of 5‐amino‐2,6‐dioxy‐4‐(5′‐phospho‐D‐ribitylamino)‐pyrimidine (ArPP) from 2,5‐diamino‐6‐(1‐D‐ribosylamino)‐pyrimidin‐4(3H)‐one 5′‐phosphate (DARPP) compared to bacteria and plants. In bacteria and plants, deamination precedes reduction, whereas fungi exhibit the reverse. Furthermore, the identification of phosphatase catalysing the dephosphorylation of DARPP remains elusive in fungi, while counterparts in bacteria and plants have been characterized (Haase et al., 2013).

Riboflavin biosynthesis is regulated by an FMN riboswitch. When FMN is present at high concentrations, it binds to the riboswitch, thereby repressing transcription of the riboflavin biosynthetic operon (You et al., 2021). A promising approach to overcome this feedback inhibition is to mutate the FMN riboswitch, located upstream of the operon (Lins et al., 2021; Pedrolli et al., 2015a; Wang et al., 2018). Additionally, the RibR protein can bind to the secondary structure of the FMN riboswitch, thereby inhibiting its function, and its activity is closely linked to intracellular sulphur metabolism (Pedrolli et al., 2015b). Similarly, the purine biosynthesis pathway is also subject to feedback inhibition. Strategies such as disrupting the inhibitor PurP (Shi et al., 2014) and mutating the FMN riboswitch in the pur operon (Yang et al., 2021) have proven effective in alleviating this inhibition. Furthermore, riboflavin production and secretion are enhanced under stress conditions, such as UV radiation and iron deficiency, possibly as a protective mechanism for the cells (Jones et al., 2015). Riboflavin transport is potentially mediated by the permease RibM in Streptomyces (Hemberger et al., 2011), the membrane protein YeeO in E. coli (McAnulty and Wood, 2014), and a specific transporter in B. subtilis (Pedrolli et al., 2015b).

Others

Styrylpyrones

Styrylpyrones constitute a class of yellow compounds distinguished by a styryl group (a phenyl group linked to an ethylene bridge) conjugated to a pyrone ring. These compounds are predominantly produced by fungi of the Hymenochaetaceae family, such as Phellinus and Inonotus (Lee and Yun, 2011), and angiosperm plants such as Piperaceae and Lauracea (Zhang et al., 2018).

The styryl group of styrylpyrones typically originates from phenylpropanoid pathways, with phenylalanine or tyrosine serving as the precursor. The pyrone ring, an essential structural element of styrylpyrones, is frequently derived from polyketide biosynthesis pathways. In this process, PKSs catalyse the condensation of simple acyl‐CoA units, usually malonyl‐CoA, to form a polyketide chain. This polyketide chain subsequently undergoes cyclization and reduction to form the pyrone ring structure. The terminal step in the biosynthesis of styrylpyrones involves the coupling of the pyrone and styryl moieties, a process generally catalysed by specific enzymes, such as ketosynthases within the PKS complex (Figure 11) (Lee and Yun, 2011).

Figure 11.

Biosynthetic pathway of styrylpyrones, phenazines, and indigoidine. The rendered light spots show the colours of different substances. The hollow arrow suggests multiple reactions in the step. The biosynthetic pathways of styrylpyrones, phenazines, and indigoidine are shown in blue, orange, and green, respectively. The core intermediates are highlighted with black boxes. Phz enzymes are derived from Pseudomonas. PAL, phenylalanine ammonia‐lyase; C4H, cinnamate 4‐hydroxylase; C3H, coumarate 3‐hydroxylase; PKS, polyketide synthetase; NRPS, nonribosomal peptide synthetase; PEP, phosphoenolpyruvate; ADIC, 2‐amino‐2‐desoxyisochorismic acid; DHHA, trans‐2,3‐dihydro‐3‐hydroxyanthranilic acid; AOCHC, 6‐amino‐5‐oxocyclohex‐2‐ene‐1‐carboxylic acid; HHPDC, hexahydrophenazine‐1,6‐dicarboxylic acid; THPCA, tetrahydrophenazine‐1,6‐carboxylic acid; DHPCA, 5,10‐dihydrophenazine‐1‐carboxylic acid; PCA, phenazine‐1‐carboxylic acid (Cátia et al., 2024; Lee and Yun, 2011; Pang et al., 2020).

In Inonotus obliquus, styrylpyrone biosynthesis is regulated through the enzyme S‐nitrosylation. During a nitrosative burst, nitric oxide (NO) accumulates, leading to the S‐nitrosylation of PAL, 4CL, and styrylpyrone synthase (SPS), which inhibits their enzymatic activities. Conversely, the denitrosylation of these enzymes by thioredoxin proteins, in coordination with thioredoxin reductase, plays a crucial role in maintaining redox balance and modulating the catalytic functions of the styrylpyrone biosynthetic enzymes (Zhao et al., 2016b).

Phenazines

Phenazines are characterized by a tricyclic structure incorporating two nitrogen atoms within the aromatic ring system. These compounds exhibit a broad spectrum of colours, including purple, blue, green, yellow, red, and brown. Several bacterial species, particularly those belonging to the genera Pseudomonas, Streptomyces, and Burkholderia, are known to synthesize phenazines.

The biosynthesis of phenazines originates from the shikimate pathway. The genes responsible for phenazine biosynthesis are typically organized in a clustered operon, comprising a series of five enzymes that catalyse the conversion of chorismate into phenazine‐1‐carboxylic acid (PCA), the core structure of phenazines (Figure 11) (Cátia et al., 2024). Phenazine biosynthesis in Pseudomonas is regulated by a quorum sensing system, the GacA/GacS two‐component signal transduction system, sigma factors, as well as environmental factors like nutrient availability and temperature. The regulatory mechanisms have been thoroughly documented in a previous review (Biessy and Filion, 2018).

Indigoidine

Indigoidine is a blue pigment synthesized by various bacterial species, including Dickeya dadantii, Streptomyces lavendulae, Streptomyces aureofaciens, and Photorhabdus luminescens (Brachmann et al., 2012; Chu et al., 2010; Novakova et al., 2010; Takahashi et al., 2007). This natural colourant exhibits considerable stability across a wide range of pH conditions and temperatures and is more sustainable and environmentally friendly compared to synthetic dyes, rendering it highly suitable for applications in cosmetics and textiles (Mohammad et al., 2021).

The biosynthesis of indigoidine is mediated by a single‐modular nonribosomal peptide synthetase (NRPS), which catalyses the formation of the final product through the condensation of two moles of L‐glutamine (Figure 11). In addition to essential domains such as the adenylation domain, thiolation domain, and thioesterase domain, indigoidine synthetase also encompasses an oxidoreductase domain, which is crucial for the formation of the double bond within the ring structure (Pang et al., 2020).

In S. lavendulae, the biosynthesis of indigoidine is regulated by the γ‐butyrolactone autoregulator IM‐2. In this regulatory network, the IM‐2‐specific receptor FarA acts as a negative regulator by binding to the autoregulatory elements of FarR2 and FarR3, which both serve as positive regulators of indigoidine biosynthesis (Kurniawan et al., 2014, 2016). In Erwinia chrysanthemi, the biosynthesis of indigoidine is repressed by PecS, which binds to the promoter regions of indA and indC (Reverchon et al., 2002). Additionally, PecM is involved in the efflux of indigoidine (Rouanet and Nasser, 2001). The industrial‐scale production of indigoidine in Corynebacterium glutamicum has been successfully achieved (Ghiffary et al., 2021). This success is largely due to the bacterium's ability to produce large quantities of glutamic acid, which can be readily converted to the substrate indigoidine.

Application and future perspectives

Colours play an extremely important role in our daily lives, having a significant impact on our emotions, psychology, culture, aesthetics, and physical health. Therefore, researchers are aiming to achieve the goals of enhancing ornamental value, reducing chemical pollution, and improving people's lives by expressing certain natural pigments through heterologous expression (Table 2). One of the most widely applied scenarios is the alteration of flower colours (Sasaki and Nakayama, 2015). In 1987, a groundbreaking accomplishment in the modification of flower colour through genetic engineering was announced, involving the manipulation of the anthocyanin biosynthetic pathway in petunias by utilizing a DFR gene from maize (Meyer et al., 1987). Subsequently, Tanaka successfully cultivated novel flower colour varieties, including brick‐red petunias and violet carnations, through the expression of heterologous flavonoid genes (Tanaka et al., 1998). Later, a bluish rose cultivar was genetically engineered by downregulating the endogenous DFR gene and overexpressing the Iris × hollandica DFR gene, in addition to the viola F3'5'H gene (Katsumoto et al., 2007). In addition to affecting the synthesis of flavonoids, researchers have also tried to change the colour of flowers by regulating the synthesis of carotenoids. In 2000, researchers constructed the Old Gold tomato mutant, which produces flowers with an orange hue due to its ability to synthesize more lycopenes (Ronen et al., 2000). Recently, researchers have once again obtained blue roses through a new approach. To obtain blue roses, white roses were injected with a dual‐expression plasmid containing bacterial idgS and sfp genes, which convert L‐glutamine into a blue pigment indigoidine (Nanjaraj Urs et al., 2019). Compared with hybrid breeding and mutation breeding for improving flower colour, plant genetic engineering offers a novel approach. However, species specificity has led to slow progress in genetic engineering for breeding, accompanied by instability in the results. Significant differences exist among different plants, and even within the same species, the outcomes of genetic modification can vary.

Table 2.

Application of nature pigments

Host species	Colour change	Pigment	Reference
Petunia	Pale pink to brick red	Anthocyanins	Meyer et al. (1987)
Carnation	White to violet	Anthocyanins	Tanaka et al. (1998)
Rose	Pink to violet	Anthocyanins	Katsumoto et al. (2007)
Tomato	Yellow to pale yellow	Carotenoids	Bird et al. (1991)
Tomato	Yellow to old golden	Carotenoids	Ronen et al. (2000)
Rose	White to blue	Indigoidine	Nanjaraj Urs et al. (2019)
Rice	White to pale yellow	Carotenoids	Ye et al. (2000)
Rice	White to orange yellow	Carotenoids	Paine et al. (2005)
Rice	White to red	Carotenoids	Zhu et al. (2018)
Rice	White to orange red	Carotenoids	Ha et al. (2019)
Tomato	Red to green	Carotenoids and chlorophyll	Yang et al. (2023)
Cotton	White to pink	Betalain	Ge et al. (2023)
Maize	Yellow to ruby	Betalain	Wang et al. (2023)

Researchers mainly alter the content of flavonoids in flowers to change their colours, while another popular research focuses on regulating the colour of rice by using carotenoids (Welsch and Li, 2022). In 2000, Golden Rice 1 was obtained by introducing the entire beta‐carotene biosynthesis pathway into rice endosperm in one go (Ye et al., 2000). Five years later, researchers obtained Golden Rice 2 with a more vivid colour by transferring carotenoid synthesis genes from different species into rice (Paine et al., 2005). In recent years, more and more researchers have participated in the study of Golden Rice and obtained rice with more diverse colours. Zhu et al. successfully gained yellow, orange, and red rice (Zhu et al., 2018), and Ha et al. created rice with deep yellow, pink, and orange‐red colours containing zeaxanthin, ketocarotenoids, and ketolutein, respectively (Ha et al., 2019). The same principle has also been applied to tomatoes. Researchers proposed a rapid breeding strategy using CRISPR/Cas9‐mediated multiplex gene editing of three fruit colour‐related genes (PSY1, MYB12, and SGR1) to generate tomato lines with diverse fruit colours from red‐fruited cultivars. They achieved colourful tomatoes by either decreasing the amounts of carotenoids or enhancing the level of chlorophyll (Yang et al., 2023). However, these research subjects, being food rather than ornamental flowers, are difficult for the public to accept.

Furthermore, researchers also aim to utilize natural pigments to create more environmentally friendly and colourful clothing. Currently, natural‐coloured cotton includes brown cotton, whose pigment is tannin. Additionally, pink cotton has been artificially cultivated by introducing the pathway for betanin synthesis (Chen et al., 2023; Ge et al., 2023). However, the pigment metabolites in both examples have high water solubility, which is not ideal for practical applications in cotton. The reason is that the pigments in cotton should not be easily washed away. Moreover, the thermal stability of many pigment metabolites is also insufficient, which further limits the potential applications of coloured cotton. Therefore, when modifying colour, we cannot simply express pigment metabolite synthesis pathways in cotton fibres; we must also consider reducing water solubility. For instance, it might be feasible to introduce pathways for lipid‐soluble metabolite synthesis. Alternatively, we could explore the chemical coupling of metabolites with cotton cellulose, which would likely enhance stability.

Compared with pigment synthesis in plant systems, which is mainly in the laboratory stage, pigment synthesis in microbial systems has entered the industrial stage. Microbial pigments are now utilized in various industrial sectors, including cosmetics, textiles, and food. Here, we aim to examine the current state of microbial pigment applications, identify the challenges encountered in this domain, and provide insights from the perspective of synthetic biology.

Many microorganisms produce pigments to absorb UV radiation, providing them with protection. This characteristic makes these pigments ideal candidates for sunscreen formulations. Research on prodigiosin and violacein has demonstrated that they can enhance the sun protection factor of products by more than 10% (Suryawanshi et al., 2015). Additionally, various microbial pigments, such as lycopene and astaxanthin, possess strong antioxidant properties, helping to shield human skin from oxidative stress (Davinelli et al., 2018). These pigments also serve as natural colourants in cosmetics like lipsticks and nail polish; for instance, phycocyanin, extracted from Cyanobacteria, is used as a colouring agent in eye makeup (Roy et al., 2024). However, the use of microbial pigments in cosmetics remains limited due to a lack of information on their effectiveness and safety. Furthermore, their higher cost makes them less competitive compared to synthetic colourants. Synthetic biology offers significant potential to address these challenges. By developing automated screening platforms, we can efficiently gather valuable data on these colourants. Additionally, creating a database that links molecular structure with safety information will aid in constructing AI models capable of providing accurate predictions through in silico analysis. Moreover, using synthetic biology methods to develop cell factories could help reduce production costs.

The ideal microbial pigments for dyeing textiles should be water‐soluble and secreted outside the cells, enabling direct use of cell cultures for dyeing without the need for chemical solvent extraction. Indigo, a blue dye widely used for jeans, is water‐insoluble and cannot be secreted by cells, leading to toxicity when it accumulates in large amounts. Currently, indigo must be extracted from cells before it can be used (Linke et al., 2023). Synthetic biology approaches, such as introducing exporter enzymes, developing whole‐cell reaction systems, or constructing surface display production systems, may help enable the secretion of pigments outside the cells. Many pigments also face challenges in terms of washability after being applied to textiles (Hernández et al., 2021). While chemical modification of pigment molecules could address this issue, the complexity of such modifications and the abundance of natural pigments suggest that setting up automated, high‐throughput screening facilities to identify suitable pigments may be a more efficient solution. Additionally, many pigments serve as protective agents for microorganisms by defending against UV radiation, which enhances microbial survival. However, UV exposure often causes pigments to fade, which is undesirable for textile applications. Microbial pigments such as violacein and prodigiosin are known to be sensitive to UV light, limiting their use in the textile industry (Kramar and Kostic, 2022). With advances in AI, constructing models to analyse the UV stability of pigments based on their chemical structures could provide an effective solution to this problem.

Microbial pigments have been widely used as colourants in the food industry. Common examples include canthaxanthin, astaxanthin, phycocyanin, riboflavin, β‐carotene, and lycopene. These pigments not only add colour to food and beverages but also offer additional benefits such as antioxidant and antibacterial properties. However, producing food‐grade pigments requires highly controlled fermentation conditions, which leads to high production costs. Another challenge is that some pigment‐producing microbes produce toxins, raising safety concerns (Sen et al., 2019). Using synthetic biology, we can address these issues by engineering GRAS (Generally Recognized as Safe) microbial hosts to serve as cell factories. Production can be further enhanced by optimizing the microbial chassis and fermentation conditions using machine learning combined with multi‐omics approaches. This would effectively tackle both safety and cost concerns. Another limitation is that many microbial pigments are unstable when exposed to light or oxygen, restricting their use in the food industry. Techniques like microencapsulation and nanoformulation offer promising solutions to improve pigment stability under these conditions (Sen et al., 2019).

Currently, plant and microbial pigments are typically engineered within their own systems. The main challenge is the significant differences in gene expression and regulatory mechanisms between plant and microbial systems. However, with advancements in bioinformatics and omics technologies, more information about the biosynthetic machinery and regulatory pathways of natural products, including pigments, is being uncovered. This growing knowledge will help bridge the gap between plant and microbial systems, enabling the possibility of cross‐system production of pigments and expanding the potential for more efficient and diverse pigment production.