The Microbial Palette: From Bioprospecting to Genetic Engineering of Microbial Pigments

Abstract

Microbial pigments are secondary metabolites that represent promising alternatives to synthetic colorants, offering advantages even over other natural sources. These pigments can be produced independently of seasonality and at low cost, especially when using agro-industrial residues as substrates, and their production can be optimized. Bioprospecting of microorganisms in unexplored environments offers valuable opportunities to discover safer and more efficient pigment producers. Brazil harbors vast biodiversity across multiple biomes, providing a rich reservoir for such discoveries. Biomes such as the Atlantic Forest, Pampa, Pantanal and Coastal Marine are still poorly explored with respect to the bioprospecting of pigment-producing microorganisms, representing a valuable opportunity for the discovery of novel pigments. However, several bottlenecks still hinder the regulatory approval of microbial pigments, particularly those produced by filamentous fungi, due to the frequent co-production of mycotoxins. To overcome these challenges, genetic engineering tools are crucial for eliminating mycotoxin co-production. CRISPR-Cas9, CRISPRi and CRISPR-Cpf1 have become the most widely used techniques for this purpose. Another key application of CRISPR is the enhancement of pigment yields, which can accelerate the industrial adoption of microbial pigments. Together, these two strategies, bioprospecting new environments and genetic engineering, can significantly speed up the transition from synthetic pigments to safer and more eco-friendly microbial alternatives.

1. A World of Color: From Natural to Synthetic to Microbial Pigments

From prehistoric cave walls to Petri dishes, color plays a significant role in life, as it is one of the first aspects perceived by the eye. From minerals to plants and animals, humans have extracted colors from nature for thousands of years for marking out territories, creating symbols, and personalizing things and space [1]. In the wilderness, pigments serve in the beautification or even protection of living organisms [2]. Today, pigments serve a multitude of purposes across different industrial sectors.

Diverging from dyes, pigments are mostly not soluble in water, existing as dispersed particles. Organic pigments are more stable, due to their strong intermolecular interactions, which are crucial for their existence in biological systems [2,3]. The term “biopigments”, although a redundant one, symbolizes how, in this biological context, all colorants are categorized as pigments regardless of their solubility [4].

Natural pigments can be extracted from animals, mostly mollusks and insects. One example is carmine, a red pigment containing the antipredatory kermesic acid, historically derived from dried bodies of females from the Kermes genus living on oak trees in the Ancient Mediterranean region. Similarly, carminic acid, another red pigment, was obtained from Dactylopius coccus, an insect that feeds on cacti, in the Pre-Columbian civilizations. However, due to the difficulties in producing and exploiting these animal-derived pigments, this practice has declined in popularity [1,2,5]. Pigments have also been obtained from several plants, such as archil from Rubia tinctorum and indigo dye from Indigofera tinctoria, found to be used as early as 4000 B.C. in India. Another notable example is urucum, the most important pigment used in tropical South America, extracted from the fruits of Bixa orellana. The pigment has been traditionally applied by indigenous people for dyeing cotton, painting ceramics, and body decorating [1,6,7]. On the other hand, even though they seem to be the best alternative to industries trying to sell their products as more organic and eco-friendly, pigments extracted from plants are highly sensitive to environmental changes such as heat, light, and pH, which affects reproducibility in industrial-scale production processes. Besides that, the cultivation of plants also requires large areas of land and great volumes of water dedicated to these monocultures, and the extraction of natural colorants can depend on seasonal and external factors, thereby presenting potential variations from batch to batch [2,8].

Minerals and ores were also major sources of inorganic pigments and dyes since the Paleolithic, when men would paint cave walls with charcoal and red ochre from hematite, an impure form of iron oxide, in times as far back as 30,000 B.C. [1]. Natural pigments extracted from minerals, ores, and soil that have been used for centuries, however, bring lots of ecological risks. They are prone to being contaminated by heavy metals such as lead or arsenic, which is especially problematic for the cosmetic industry. The waste discarded throughout the extraction chain could contaminate water sources used for irrigation or lead to bioaccumulation of metals in aquatic habitats, compromising agriculture and food consumption [2].

In 1856, a new era in color chemistry began when Henry Perkin synthesized Mauvein, also known as aniline, the first-ever synthetic dye [1,9,10,11]. Lots of other dyes were introduced in the following decades through artificial synthesis. These are cheaper, more stable, and more effective, and possess well-known synthesis processes, thus being very attractive and applicable to virtually every material in the industry, including food [2,9]. Synthetic dyes and pigments have since then become widely used in the textile, cosmetic, and food industries. However, prolonged exposure to artificially made colorants has been related to cytotoxic, genotoxic, allergenic, carcinogenic, and numerous other pathological effects in animals and human beings, raising controversies on whether they should still be allowed in the market [2,12]. Health problems are not the only issues; environmental concerns also arise with its indiscriminate use. The textile sector, the primary consumer of synthetic dyes, disposes of 200,000 tons of dye each cycle of production. This untreated industrial wastewater causes devastating effects on water, soil and biodiversity nearby, mainly because those dyes are nonbiodegradable, toxic and able to persist in the environment for years [13,14,15].

These different problems associated with the mainstay of pigments and dyes, whether natural or artificially synthesized, have pushed researchers and industries to search for non-toxic, safe, eco-friendly, and, nonetheless, profitable and sustainable alternatives [2,16]. This places a magnifying lens over other natural sources that were not considered before.

In Asia, for more than a thousand years, Monascus-fermented rice, known as red yeast rice or angkak, has been consumed as a functional health food [17]. Less than 30 years after Perkin’s Mauve, in 1879, a yellow pigment was obtained from milk, which was also found and fractionated from aqueous yeast extracts in 1932. In 1938, the first purification and characterization of this “beautiful yellow substance”, now called riboflavin or simply vitamin B2 (but at the time also known as lactoflavin or ovoflavin), granted the Nobel Prize in Chemistry to Richard Kuhn. Riboflavin is produced by plants and microorganisms and, nowadays, its industrial production is largely done by fermentation, using ascomycetes such as Ashbya gossypii, or genetically engineered strains of yeasts and bacteria [18]. Subsequently, several other pigment-producing microorganisms were isolated. Nowadays, the pharmaceutical, cosmetic, food, and textile industries have incorporated microbial pigments into their production lines.

A plethora of microorganisms can produce pigments through different metabolic pathways, whether as secondary metabolites or as compounds essential to their physiology and development (Figure 1). Besides being one of the most “charismatic traits” of microorganisms, as put by Ramesh et al. (2019), pigments produced by microbial communities possess a broad array of chemical compounds that sum up to a wide range of biological functions in these organisms [19].

2. The Benefits of Microbial Pigments

Pigments participate in key biological processes within the cell. The chromophores in these pigment molecules carry out several functions essential to microbial survival and development. The bioactivity of these pigments makes them attractive to the industry as they serve different purposes, besides coloring. Microbial pigments can be important antioxidants, like carotenoids and xanthophylls. When absorbing Ultraviolet (UV) radiation, they protect cells against DNA damage and the formation of free radicals. Pigments such as chlorophylls and phycobilins are essential in transferring energy from light to electrons used in photosynthesis. Pigments from microorganisms have been reported to function as food conservatives and additives much less toxic than synthetically produced ones [4]. Prodiginines and tambjamines, which present a wide range of red and yellow hues, respectively, produced by both bacteria and fungi, present broad antibacterial and antifungal activities. Well-established antiviral activity is related to quinones, but phenazine compounds from Pseudomonas spp. and Streptomyces spp., and violacein isolated from Chromobacterium spp. are promising candidates against viruses. Prodigiosin from Hahella chejuensis, naphthoquinones, and anthraquinones have been reported to have algicidal, insecticidal, and herbicidal activities, respectively. Violacein is also a remarkably versatile pigment. Beyond its bright purple color, it has demonstrated antiparasitic, antiprotozoal, antileishmanial, antiulcerogenic, antilipoperoxidant, antituberculosis, and antimalarial activities. In cancer research, pigments of different classes presented antitumoral or antimetastatic activity, inducing apoptosis and cytotoxic effects against various cancer cell lines in culture or in animal models [19].

For industrial applications, microbial pigments check almost every box in the requirements list, as they come from a renewable source that lasts all year long. Not only that, they fit the new public mindset of “clean” and “green” labels, making them desirable products for the industry [20]. When producing pigments in the industry, source organisms should grow fast, easily, and have a high productivity rate in a limited space and time. They should also be nonpathogenic and not need fastidious protocols and processes for cultivation [19].

Microbial pigments have emerged as a highly promising alternative to plant-derived colorants due to their numerous biotechnological and environmental advantages. Unlike plant pigments, whose production is often dependent on seasonal variations, climate conditions, extensive agricultural land, and long cultivation periods, microbial pigments can be produced rapidly and consistently through controlled fermentation processes independent of geographical and climatic limitations. In addition, microorganisms generally exhibit higher growth rates and can be cultivated using low-cost substrates, including agro-industrial residues, contributing to waste valorization and the development of sustainable circular bioeconomy strategies. Furthermore, advances in omics technologies and genetic engineering have significantly enhanced the ability to optimize microbial strains for higher productivity and tailored pigment synthesis, making microbial platforms increasingly attractive for industrial applications. These advantages position microbial pigments as a sustainable, economically viable, and innovative alternative to conventional plant-based natural colorants [11].

When it comes to sustainable approaches, the use of natural pigments produced through biotechnological processes is associated with a lower environmental impact compared to synthetic dyes. In this context, agro-industrial residues emerge as promising substrates for pigment production, enhancing the sustainability of these processes and aligning with strategies aimed at waste valorization and circular economy practices. Brazil is one of the world’s leading producers of agricultural commodities, and this high level of production generates substantial amounts of agro-industrial residues, including cassava and sugarcane bagasse, rice husks, and coffee peels. These by-products, often underutilized, are rich in organic matter (carbon and nitrogen sources) and possess significant potential as renewable energy sources and raw materials for various industrial applications (Figure 2A,B). The valorization of these residues offers multiple advantages. From an economic perspective, their use can reduce production costs by substituting conventional raw materials and energy inputs, taking into consideration that the components of the medium can represent from 38% to 73% of the total production cost [21]. Environmentally, it helps mitigate waste disposal issues, lowering the risk of soil and water contamination. Additionally, integrating these residues into productive chains supports the principles of a circular economy, where waste is transformed into valuable resources. This approach not only addresses pollution challenges but also contributes to reducing greenhouse gas emissions and the overall carbon footprint of agro-industrial systems [21].

Aligned with pigment production in agro-industrial residues, fermentation strategies, such as submerged fermentation with optimized aeration or the use of tailored bioreactor configurations, show promising perspectives for enhancing pigment biosynthesis [11,20].

3. The Barriers Encountered When Using Microbial Pigments

In order to be used as color additives in food, drugs and cosmetics, pigments must be approved by a regulatory agency. Regulations, however, differ significantly between countries. In Brazil, the agency responsible for the approval and regulation of food additives, among other sanitary activities, is called Agência Nacional de Vigilância Sanitária (ANVISA). From a legal standpoint, Brazil’s classification of food additives originates from Resolution No. 44/1977, which groups colorants into organic (natural, artificial, synthetic, and nature-identical), inorganic, and caramel categories, and establishes general requirements for composition, labeling, and quality. However, the current legislation is mainly based on the RDC N°. 778/2023, which establishes the general principles, technological functions, and conditions of use for food additives in foods, including colorants. At the present time, 14 synthetic colorants are approved to be used as food additives (

Table 1. Artificial dyes for use in foods approved in Brazil, the United States of America (USA), and the European Union (EU).

INS	Dye	Brasil (ANVISA)	USA (FDA)	EU (EFSA)
129	Allura Red AC	✔	✔	✔
123	Amaranth/Bordeaux S	✔		✔
122	Azorubine	✔		✔
131	Blue Patent V	✔		✔
151	Brilliant Black BN	✔		✔
133	Brilliant Blue FCF	✔	✔	✔
155	Brown HT	✔		✔
121	Citrus Red n°2		✔
127	Erythrosine	✔	✔
143	Fast Green FCF	✔	✔
142	Green S			✔

✔ approved artificial dyes.

) [22].

In contrast, legislation concerning dyes in the textile industry is still relatively limited. Rather than providing a list of approved dyes, Brazilian textile regulations focus mainly on restricting or banning hazardous substances. In this context, Standard NBR 16787:2019, by the Brazilian Association of Technical Standards (ABNT), establishes analytical methods for determining certain prohibited aromatic amines derived from azo dyes in textile products. This aligns with international restrictions adopted in markets such as the European Union and aims to ensure consumer safety and reduce exposure to potentially toxic compounds in fabrics and clothing.

For the pharmaceutical industry, colorants are regulated according to standards established by ANVISA and described in the Farmacopeia Brasileira [23], which defines quality specifications, purity requirements, identification methods, and acceptable limits for substances used in medicines, including excipients such as dyes and pigments. Pharmaceutical colorants must comply with strict toxicological and quality-control criteria to ensure patient safety and product stability. Regarding cosmetics, the use of colorants is regulated by the RDC N° 628/2022 [24], which establishes the list of substances permitted as colorants in personal care products, cosmetics, and perfumes. The regulation also incorporates the MERCOSUR Resolution GMC N° 16/2012 into Brazilian legislation and defines purity criteria, restrictions, and labeling requirements for cosmetic dyes. This regulation is essential to ensure the safety of cosmetic formulations, particularly because many colorants may come into direct contact with skin, hair, nails, and mucosal surfaces.

Overall, Brazilian legislation on colorants is strongly influenced by international regulatory frameworks and scientific recommendations. ANVISA regulations are based on guidelines and safety evaluations established by organizations such as the Food and Agriculture Organization (FAO), the World Health Organization (WHO), the Codex Alimentarius Commission, MERCOSUR resolutions, the European Food Safety Authority (EFSA), and the Food and Drug Administration (FDA) [25].

In the United States, the Food and Drug Administration (FDA) classifies color additives into three categories: FD&C (Food, Drugs & Cosmetics), D&C (Drugs & Cosmetics), and Ext. D&C (External Drugs & Cosmetics), depending on their permitted applications [26]. In April 2025, the FDA and the U.S. Department of Health and Human Services announced a series of measures to phase out all petroleum-based synthetic dyes from the food supply, including the elimination of six synthetic dyes (FD&C Green N°3, FD&C Red N°.0, FD&C Yellow N°5, FD&C Yellow N°6, FD&C Blue N°1, and FD&C Blue N°2) by the end of 2026 [27]. In an effort to start the transition, in May 2025, the FDA announced the approval of Galdieria extract blue, a blue color derived from the unicellular red algae Galdieria sulphuraria, to be used as a food additive [28].

Although Monascus spp. have been traditionally used in Asia for centuries as natural bioactive colorants, their industrial use remains restricted in many countries because several species concomitantly produce citrinin, a mycotoxin associated with toxicological effects. Mycotoxins are low-molecular-weight fungal metabolites capable of causing necrosis, hepatotoxicity, bleeding, neurological disorders, carcinogenesis, and, in extreme cases, death, generally related to chronic exposure. More than 400 mycotoxins have been identified to date, produced mainly by Aspergillus, Alternaria, Claviceps, Fusarium, Penicillium, and Monascus species. This significantly limits the safe application of fungal pigments on an industrial scale. In recent years, however, advances in biotechnology have been crucial for mitigating these risks. Approaches such as the selection of non-mycotoxigenic strains, genetic improvement, modulation of cultivation parameters, gene-editing strategies targeting biosynthetic gene clusters (BGCs), and multi-omics analyses have enabled both the suppression of mycotoxin biosynthesis and the enhancement of pigment production, thereby expanding the potential of fungi as safe and sustainable sources of natural colorants (Figure 2B,C) [29,30,31,32].

Consumer perception also plays an important role in market acceptance. Increasing consumer awareness regarding the potential health risks and environmental impacts associated with synthetic dyes has driven the demand for naturally derived alternatives. In this scenario, microbial pigments present strong market potential because they align with current “green label” and clean-label trends, which prioritize natural, sustainable, and environmentally friendly products. Furthermore, many microbial pigments are associated with additional bioactive properties, such as antioxidant and antimicrobial activities, reinforcing the perception of healthier and more sustainable products. Although concerns regarding genetically engineered microorganisms may still represent a challenge in some markets, transparent labeling, safety validation, and correct advertising practices could contribute to greater public acceptance and commercialization of microbial-derived colorants [33].

We should also shed light on some of the scalability and profitability problems throughout the microbial pigment production chain. Even though there has been progress in the field, microbial pigments face several setbacks when it comes to scaling up their production to an industrial level. The extraction of microbial pigments is one of the most demanding stages of the production process, particularly when dealing with intracellular compounds. Although widely used, solvent-based methods are often laborious, require large volumes of solvent, and can provide low yields. Moreover, the use of solvents other than water or ethanol can compromise the “natural pigment” regulatory status, since most organic solvents are not recognized as natural under food and cosmetic regulations, and can present toxicity. Extraction and other downstream operations frequently account for more than half of total bioprocessing costs, mainly considering purification steps (that is the part usually more expensive in the production), underscoring the need for efficient and economically viable methodologies. Factors such as solvent selection, pigment chemistry, the complexity of fungal cell walls, and residual culture components directly influence final yield. From a biotechnological perspective, these limitations make intracellular pigments less attractive for industrial purposes compared to extracellular ones, which avoid cell-disruption steps and enable cleaner, eco-friendly and more scalable processes (Figure 2B). Consequently, developing more efficient extraction strategies compatible with large-scale production remains a central challenge for the industrial viability of microbial pigments. In addition, production in submerged tanks is the common technique used to produce pigments, because solid-state fermentation is difficult to implement on a larger scale [2,11].

The instability of some pigments can lead to degradation, color loss and reduced shelf life, which are all undesirable consequences for the industry. It is also important to acknowledge that pigments of different colors derived from the same organism may vary in their response to distinct physicochemical conditions, as observed in Monascus species. Composition of culture media, for example, can prioritize some pigments in relation to others [34]. This occurs because microbial pigments can be affected by media composition and physicochemical conditions, such as pH, temperature, oxygen availability, light exposure, carbon–nitrogen balance and agitation [35,36]. Interestingly, this responsiveness to environmental parameters represents a double-edged sword: while uncontrolled fluctuations can compromise pigment quality, deliberate manipulation of these parameters can be strategically exploited to boost production or shift the pigment profile toward more desirable hues. Thus, these microorganisms require controlled in vitro culture conditions to yield more pigments in either solid-state or submerged fermentation (Figure 2D). It is necessary to investigate the optimized culture conditions for each strain [11,37,38]. Optimization can be performed with statistical experiment designs and response surface analysis. Response surface methodology is by far the most used statistical technique to improve pigment production [39]. Despite these obstacles, the recent literature emphasizes that these challenges are not insurmountable.

4. Brazilian Biodiversity: Bioprospecting New Pigment-Producing Microorganisms

Brazilian geographical domains encompass an area of 8.51 million km² of continental area and 5.7 million km² of marine area, encompassing seven vast biomes with their own topography, pluviosity, climate, altitude and soil conditions. The Amazonia Rainforest, Atlantic Forest, Cerrado, Pampa, Caatinga, Pantanal and Coastal Marine biomes are home to the country’s living organisms (Figure 3). All biomes are populated by a great number of species, including plants, algae, animals, insects and microorganisms. Thus, Brazil is rich in unique biodiversity that is also adapted to various ecological and climate conditions in these habitats, which can sometimes be harsh.

High levels of pluviosity contrasting with drought periods, semi-arid regions with oceans, dense forests with savannas, and regions below sea level with highlands: those contrasts shape Brazilian nature and also the adaptation of organisms to these conditions. A range of molecular and evolutionary mechanisms may have played central roles in allowing microorganisms to explore new ecological niches. Molecular mechanisms include duplication events [40], intra-genomic rearrangement [40], horizontal gene transfer [41,42] and point mutations [43], the latter being considered the ultimate source of all genetic variation and fuel for evolution [44]. While the described mechanisms create genetic innovations, the fate of these characteristics depends on evolutionary forces: natural selection and genetic drift can act upon fixing novel traits [45]. This makes native biodiversity a promising source for novel metabolites for biotechnological purposes, as biosynthetic potential is highly dependent on the ecosystem [46]. Thus, having a variety of ecosystems leads to a variety of biosynthetic machineries.

Brazil holds an important part of the largest tropical forest in the world, the Amazon Rainforest. Aside from the imminent climate change, the Amazon climate is typically rainy with minimal temperature variation [47]. The Oswaldo Cruz Foundation (Fiocruz) keeps a collection of fungi isolated from the Amazon “https://collectory.sibbr.gov.br/collectory/public/show/co40” (accessed on 14 January 2026). Specimens were isolated from a wide range of samples, such as flowers, soil and water. The most abundant genera in the collection are Penicillium, Aspergillus and Trichoderma, already described in the literature as pigment producers (Figure 3) [48,49]. From native plants, endophytic fungi that produce pigments with antimicrobial and antioxidant potential have also been described [50]. The Amazonian soil has also been screened for pigment-producing fungi with interesting results and perspectives for fungi of the genera Penicillium and Aspergillus [51]. Penicillium sclerotiorum 2AV2 produces a yellow–orange sclerotiorin pigment that has a maximum absorbance in ethyl acetate at 350 nm. Rhodotorula spp. isolates from different Amazonian substrates were evaluated for their carotenoid production capacity [52].

The Brazilian territory is also composed of 15% Atlantic Forest [53]. The Atlantic Forest is the second-largest tropical forest in South America [54] and one of the most biodiverse and unique regions on Earth [55]. Barbosa et al. (2022) [56] conducted a literature survey regarding the records of fungi of the genera Aspergillus spp., Penicillium spp. and Talaromyces spp. in the Atlantic Forest area. This study led to 169 species: 68 Aspergillus spp., 79 Penicillium spp., and 22 Talaromyces spp. The latter is a genus with already described pigment-producing species [49]. Inselbergs of the Atlantic Forest zone had their surface biofilms assessed, and a cyanobacterium identified as Gloeocapsa sabulosa exhibited pigmented colonies [57]. Bioprospection of carotenoid-producing yeasts isolated from soil, flowers, tree barks, and grasses from an area close to anthropogenic activity reported 8 pigmented yeasts, being the candidate with the most potential identified as Rhodotorula mucilaginosa [58]. Other regions covered by the Atlantic Forest were explored for the prospecting of yeasts of the genus Rhodotorula, well known for carotenoid production [59]. Despite the impressive number of described species, the literature about pigment-producing microorganisms in the Atlantic Forest directly targeting this biological characteristic and its technological application is scarce.

Cerrado is a tropical savanna recognized as a global biodiversity hotspot, harboring thousands of endemic species [60]. The climate is characterized by elevated temperatures and seasonal rainfall distribution, with wet summers and dry winters [61,62]. Cerrado’s microbiome has been explored in the search for carotenoid-producing microorganisms. Screenings have shown the potential of yeasts from the genus Rhodotorula isolated from soil and plants [63], and from bacteria Occallatibacter sp. AB23 from soil (Figure 3) [64]. Occallatibacter sp. AB23 was also directly tested for resistance to oxidative stress, based on the hypothesis that carotenoids could act as cell protectors. Cells that exhibited pigmentation survived under induced oxidative stress conditions, which indicates a mechanism for adaptation to stressful conditions present in the Cerrado’s soil by the pigments.

The Caatinga is an ecological region that lies in the semiarid hinterland of northeastern Brazil, which has an extended dry period in which rainfall is scarce. Regarding Caatinga, studies have described filamentous fungi and bacterial isolates as pigment producers. Talaromyces spp. isolated from Caatinga’s soil were pointed out as producers of pigments with antimicrobial activity, and the study suggests that pigment production can be modulated in response to nutrient availability and salinity [65]. Two bacterial isolates from Caatinga’s soil identified as Kocuria palustris were described as producers of a rare carotenoid named sarcinaxanthin and exhibited photoprotective activity that can be potentially used in sunscreen development [66]. These findings corroborate studies that point out pigment synthesis as an adaptive trait for adaptation to high UV incidence levels, especially in the soil, as a consequence of the native vegetation or soil characteristics, as seen in Cerrado. Another application of pigments produced by Caatinga’s soil native microorganisms is in bioelectrodes, in which they act as redox mediators [67]. Penicillium sp. UCP 1286 and Rhizopus microsporus var. chinensis UCP 1296 lyophilized extracts were tested as natural electron shuttles for the substitution of synthetic dyes in electrodes (Figure 3).

Bioprospecting of pigment-producing microorganisms in Pampa, Pantanal and Coastal Marine is less expressive when compared to other biomes. The Pampa biome lies within the South Temperate Zone and has both subtropical and temperate climates with four well-characterized seasons [68]. The biome’s vegetation is mainly composed of grasslands with sparse shrub and tree formations. A study from Pampa described the prospection of carotenoid-producing yeasts in the Pampean region [69]. The isolation of different matrices resulted in 64 isolates exhibiting pink, yellow or orange pigmentation, and three were selected as the isolates with the highest potential for production and identified as Sporidiobolus pararoseus, Rhodotorula mucilaginosa and Pichia fermentans (Figure 3). After initial analyses, carotenoid production of these three isolates was evaluated using agro-industrial residues as substrate [70], further reaffirming that biotechnological traits can be combined with “green” practices for production optimization.

Pantanal consists of a tropical wetland in which periods of inundation and desiccation alternate annually [68], which differentiates it from other wetlands around the world. Regarding assessing the biome’s microbial communities, the literature compared to other biomes is limited. Studies described the bacterial diversity of soil islands associated with bromeliad species from an ecological perspective [71] and endophytic fungi from vegetation [72], but studies focusing on pigment-producing microorganisms were not found.

The Coastal Marine biome, often referred to as the Blue Amazon, stretches along the entire Brazilian coastline, extending from the mouth of the Oiapoque River in the far north to the Chuí River in the extreme south [73]. This vast area encompasses a wide range of ecosystems and environmental conditions, such as dunes, mangroves, coral reefs, and oceanic islands. These conditions form a reservoir of biosynthetic machinery, still in its majority underexplored especially in the context of bioprospecting for novel pigments. A study published by Pommer et al. (2026) verified the change in pigment production by Periconia belmontensis, isolated from the São Pedro and São Paulo archipelago, in different growth parameters [74]. However, the study did not directly explore the pigment’s biotechnological potential. A study on the biodiversity and anti-cancer potential of microorganisms associated with sediments from the intertidal zone of the northeastern coast of Brazil assessed 32 bacterial isolates [75]. The candidate with the most promising results in the anti-cancer assays was a red pigment-producing bacterium identified as Pseudoalteromonas rubra, and its pigment identified as prodigiosin, was suggested to be responsible for the strain’s bioactivity (Figure 1 and Figure 3).

The proportion of studies specifically targeting microbial pigments is unequal among the previously discussed biomes. The Amazon Rainforest, Cerrado and Caatinga have more studies regarding direct microbial pigments, while the Atlantic Forest, Pampa, Pantanal and the Coastal Marine have less expressive pigment bioprospection rates. And zooming in, studies focusing on microbial pigments in Pantanal were not found until now, which represents an opportunity for new bioprospecting studies with native species, and maybe native (and new) molecules. The most bioprospected pigments are carotenoids, and this may be due to their proposed photoprotection activity. Since Brazil is a country located in a global area characterized by high solar incidence, this may be an explanation for its search rates. A taxon considerably represented in bioprospection studies in Brazilian biomes is Rhodotorula, probably as a consequence of the carotenoid-targeted search bias.

The small number of studies targeting microbial diversity in the Atlantic Forest, Pampa, Pantanal and Coastal Marine does not reflect their relevance, since those are unique biomes in terms of biotic and abiotic factors, and the first three are also classified as endangered due to anthropogenic activity. However famous or neglected in terms of social popularity, the microbiological potential of all of these biomes, in terms of the number of species and biotechnology, is still underestimated compared to their projections of biodiversity magnitude. The studies previously discussed represent a modest highlight of the unique findings and characteristics of these biomes, which may drive the emergence of distinctive evolutionary traits and complex ecological interactions, often associated with biotechnologically relevant functions, including pigment biosynthesis.

Genome mining of native microbes in search of BGCs can also be an alternative for bioprospecting novel genetic resources in these highly selective environments. Native genes encoding the previously described metabolites can be expressed in more efficient metabolic machinery, such as well-known synthetic biology chassis, to allow production on higher scales and the development of products based on Brazil’s native microorganisms.

These findings indicate that Brazilian microbial biodiversity is an unexplored potential source of novel biotechnological resources and is especially overlooked in terms of pigment-producing microorganisms. Much more than a source of biotechnological products, the bioprospection of microorganisms is also a way to demonstrate the importance of biomes as a reservoir of novel species, applications and products arising from the heterogeneity of ecosystems found in the Brazilian territory from a non-extractive perspective. The search for microorganisms capable of producing biotechnological commodities and generating economic interest is also important for conservation, since habitat protection stimuli are sometimes based on the utilitarian value of what is found in native environments.

5. Omics Approaches for Pigment Discovery

Interest in secondary metabolites has been growing significantly, both from a scientific and economic standpoint. Currently, there are approximately 500,000 described secondary metabolites of different sources. Of those, approximately 70,000 compounds are obtained from microorganisms [76]. These numbers highlight the vast chemical diversity of microbial metabolites, emphasizing their significant potential for further exploration. Advances in omics technologies have facilitated the search for new microbial metabolites, including pigments.

The progress of bioinformatics and the development of whole-genome sequencing technologies have substantially expanded the discovery of microbial pigments through genome mining approaches. By analyzing biosynthetic gene clusters, researchers can predict the capacity of an organism to produce pigments, even when these compounds are not expressed under standard laboratory conditions. Tools such as antiSMASH enable the identification of genes associated with carotenoids, melanins and polyketide pigments in both fungi and bacteria. In that sense, genome mining can reveal silent pathways encoding potentially novel metabolites, greatly increasing the diversity of candidate pigments for industrial applications [77,78]. An example of genomic application in the identification of BGCs is the research conducted by Stannius et al. (2025) [79]. Using a comparative genomic approach based on a collection of pigment-producing isolates, the authors identified the biosynthetic gene cluster responsible for the production of a melanin-like pigment in Bacillus subtilis and characterized the cultivation parameters associated with its synthesis. The study also discusses how sequence-based genome mining approaches enable the identification of potential BGCs using similarity to already known clusters. This homology-based approach can, however, halt the identification of truly novel BGCs.

Transcriptomic approaches aim to understand gene expression patterns associated with microbial pigment biosynthesis under different environmental and physiological conditions. Through RNA sequencing (RNA-seq) or RT-qPCR, researchers can identify differentially expressed genes involved in pigment production, associating it to variations in pH, temperature, salinity, nutrient availability and stress. These can help elucidate the regulatory networks controlling secondary metabolism and pigment biosynthesis [77,78].

Metabolomic approaches play a crucial role in elucidating the chemical profile of microorganisms, representing a powerful strategy for the characterization and identification of secondary metabolites, including bioactive compounds and microbial pigments. By employing advanced analytical techniques such as Liquid Chromatography–Mass Spectrometry (LC–MS), Gas Chromatography–Mass Spectrometry (GC–MS) and Nuclear Magnetic Resonance (NMR), metabolomics enables the comprehensive analysis of metabolite profiles under specific conditions. These approaches provide detailed information regarding the diversity of metabolites produced over time, allowing a better understanding of microbial metabolic behavior. Furthermore, metabolomics provides direct evidence of the compounds effectively synthesized by the organism, reducing the gap between genetic potential and metabolic expression [77,78].

Environmental and cultivation variables can play a fundamental role in modulating both gene expression and metabolite production in microorganisms. Factors such as nutrient composition, carbon and nitrogen sources, pH, temperature, salinity, aeration, and oxidative stress can activate or repress biosynthetic pathways associated with pigment production. At the transcriptomic level, these variations may alter the expression of genes involved in precursor biosynthesis, regulatory proteins, and secondary metabolism pathways. Simultaneously, metabolomic analyses can reveal changes in the abundance and diversity of intracellular and extracellular metabolites, reflecting metabolic adaptations. In this context, omics analyses are essential for identifying how different parameters modulate metabolic pathways and trigger the production of specific pigments or bioactive molecules [77,78].

The integration of omics approaches, such as genomics, transcriptomics, and metabolomics, offers a more comprehensive understanding of pigment biosynthesis at multiple biological levels. While genomics reveals the biosynthetic potential encoded within the genome and transcriptomics identifies actively expressed genes, metabolomics confirms the final metabolic products generated by the cell.

In an integrative approach of transcriptomics and metabolomics, Huang et al. (2021) [80] observed that ammonium-based nitrogen sources promoted higher pigment production. Metabolomic analyses identified significant alterations in metabolites, revealing correlations between red pigment production and intracellular amino acid metabolism, while yellow and orange pigments were associated with nucleotides. Simultaneously, transcriptomic analyses demonstrated that red pigment production was strongly associated with primary metabolic pathways, whereas orange pigment biosynthesis was linked to secondary metabolic pathways, and distinct regulatory pathways were associated with yellow pigment production. By integrating both datasets, the study showed that pigment diversity in Monascus is not controlled solely by the pigment biosynthetic gene cluster, but also by broader metabolic and regulatory networks influenced by nitrogen availability. These findings highlight the importance of combining metabolomic and transcriptomic approaches to better understand and optimize microbial pigment production.

A multi-analysis approach was proposed by Ma et al. (2023) [81] to study the pigment synthesis pathway in Auricularia cornea. Genomic analysis revealed that the A. cornea genome contains 32 secondary metabolite biosynthetic gene clusters, including 20 terpene synthases, one type I polyketide synthase (PKS), two indole synthases, one nonribosomal peptide synthase (NRPS), and eight NRPS-like clusters. Transcriptomic analyses, including KEGG enrichment and Gene Set Enrichment Analysis (GSEA), demonstrated that pigment biosynthesis was strongly associated with the shikimate pathway, which is linked to glycolysis and the pentose phosphate pathway. Several core genes enriched in these pathways were differentially expressed between pigmentation phenotypes, suggesting that pigment formation in A. cornea is closely regulated by central carbon metabolism. Metabolomic profiling further identified differential accumulation of metabolites associated with shikimate and aromatic compound metabolism, supporting the hypothesis.

Moreover, understanding the genetic and metabolic mechanisms underlying pigment biosynthesis creates new opportunities for genetic engineering and synthetic biology applications. The identification of key genes, regulatory pathways, and metabolic bottlenecks can support the rational engineering of microbial strains with enhanced pigment yields, reduction in the production of mycotoxins, and optimized industrial performance. Such approaches contribute not only to the development of more efficient bioprocesses but also to the sustainable production of natural pigments for applications in the food, pharmaceutical, cosmetic, and textile industries.

6. Genetic Engineering Applications in Microbial Pigment Production

Microorganisms can act as biofactories when heterologous BGCs are introduced into their cells. This approach represents a promising alternative for scaling up the production of natural pigments. It is particularly relevant because natural producers often present limitations such as low yields and challenges in pigment extraction and purification. Well-known pigments such as violacein and lycopene are naturally produced by bacteria and fungi, respectively. However, these native organisms typically generate these compounds in low quantities, which limits their industrial applicability [82]. Advances in genomic engineering technologies, particularly tools such as CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and its variants, offer significant advantages by enabling more efficient production of a wide range of high-value pigments. Through the construction, reconstruction, optimization, and regulation of metabolic pathways, these tools allow for the use of diverse host organisms under controlled conditions to enhance metabolite production [83]. In this section, we will focus on how to enhance pigment and decrease mycotoxin production using the CRISPR method and its variants.

The application of the CRISPR technology in bacteria is already well established. In contrast, its use in yeasts and, particularly, in filamentous fungi, is still challenging. These organisms possess cell walls primarily composed of chitin, which can hinder the efficient delivery of CRISPR components. Moreover, their genomes are generally more complex than those of prokaryotes, adding further challenges to genetic manipulation. In the case of filamentous fungi, their multicellular organization introduces additional layers of complexity for gene editing. Despite these limitations, recent advances have demonstrated promising results, expanding the possibilities for genetic engineering in these microorganisms [84]. Recent studies employ CRISPR-based approaches to enhance pigment production in algae, bacteria, yeasts, and filamentous fungi (and, in the case of fungi, to also reduce or eliminate mycotoxin biosynthesis) (

Table 2. Genetic engineering of algae, bacteria, yeast, and filamentous fungi through CRISPR-based methods.

Organism	Family	Pigment or Compound	Color	Host Organism	Metabolic Engineering Strategies Used	Modified Genes	Outcomes	References
Algae	Carotenoids	Zeaxanthin	Yellow	Chlamydomonas reinhardtii	CRISPR-Cas9 ribonucleoprotein-mediated knock-in	Double knockout of LCYE and ZEP	The double knockout mutant had a 60% higher zeaxanthin yield and content after 3 days of cultivation	Song et al., 2020 [85]
Bacteria	Lycopene	Lycopene	Bright red	Pantoea dispersa MSC14	CRISPR-Cas9	Knockout of crtY, overexpression of ctrI	crtY knockout and crtI overexpression enabled lycopene production in P. dispersa MSC14	(Lai et al. 2024) [86]
	Indol pigments	Violacein	Purple	Escherichia coli	CRISPRi uses a single-molecular guide RNA (sgRNA)	Repression of tyrR, pykF, cra, ptsG, pykA, sdaA, and tnaA	A 5′-shortened sgRNA CRISPRi library enabled low-cost phenotype-associated gene screening and improved violacein production in E. coli	(Jeong et al. 2023) [87]
Yeasts	Carotenoids	β-carotene	Orange	Saccharomyces cerevisiae CEN.PK2-1c	CRISPR-Cas9	Integration of crtE, crtYB and crtI	Metabolic engineering increased β-carotene and β-ionone production in S. cerevisiae	(López et al. 2020) [88]
	Apocarotenoid dicarboxylic acid	Crocetin	Red	Saccharomyces cerevisiae BY4741	CRISPR-Cas9 based multiplex genome integration	Integration of CCD2 and ALDH genes	CRISPR-Cas9-mediated multi-copy integration and temperature-regulated expression of CCD2 and ALDH optimized crocetin biosynthesis in S. cerevisiae	(Liu et al. 2020) [89]
		β-carotene and Zeaxanthin	Orange and Yellow	Yarrowia lipolytica Po1f	CRISPR-iCas9	Integration of carRA/carB/GGS1/tHMG	CRISPR-iCas9 enabled efficient multi-gene editing and carotenoid pathway engineering in Y. lipolytica, accelerating carotenoid pathway engineering through one-step integration of several genes	(Chen et al. 2023) [90]
	Lycopene	Lycopene	Red	Pichia pastoris GS115	CRISPR-Cpf1a	Deletion of DPP1 and LPP1. downregulation of Erg9	CRISPR/Cpf1-mediated metabolic and lipid engineering enabled record-high lycopene production by optimizing terpene pathway flux and lipid synthesis in P. pastoris	(Zhang et al. 2023) [91]
Filamentous Fungi	Mycotoxin	Citrinin	–	Monascus purpureus RP2	CRISPR-Cas9	Knockout of ctnA and overexpression of ctnA	ctnA gene interference altered morphology and secondary metabolism in M. purpureus, reducing citrinin production while enhancing pigment biosynthesis for potential industrial optimization	(Zhang et al. 2025) [92]
			–	Monascus purpureus KL-001	CRISPR-Cas9 mediated marker-based deletion (CMBD) and CRISPR-Cas9 mediated marker-free deletion (CMFD)	Deletion of citrinin BGC	A dual-plasmid CRISPR/Cas system enabled efficient large-gene-fragment deletions and stable elimination of citrinin biosynthesis while improving Monascus Red production in Monascus	(Liu et al. 2020) [93]
	Monascus pigments	–	Red, orange and yellow pigments	Monascus ruber KACC46666	CRISPR-Cas9	Inactivation of MpigI and MpigI’ (mutation insertion)	CRISPR/Cas9 engineering of the negative regulators MpigI and MpigI′ enhanced Monascus pigment biosynthesis without inducing citrinin production in Monascus	(Ree Yoon et al. 2023) [94]
	Phenylpropanoid-class of polyketides	α-pyrones homopyrones A and B	Yellow	Aspergillus homomorphus IBT21893	CRISPR-Cas9	Deletion of ahpA and truncated mutant of ahpB	CRISPR/Cas9-mediated deletion of ahpA and ahpB identified the biosynthetic gene cluster responsible for phenylpropanoid-type yellow pigment production in A. homomorphus	(Futyma et al. 2021) [95]

).

7. CRISPR-Mediated Gene Editing

7.1. CRISPR-Cas9

The genome-editing technology CRISPR-Cas9 employs the Cas9 endonuclease and a guide RNA molecule (gRNA) to induce specific double-strand DNA breaks. After cleavage, cellular DNA repair mechanisms can generate targeted or off-target modifications, such as mutations, gene knockouts, or insertions following the cleavage [96]. This approach enables the modification of genes involved in pigment biosynthesis. For instance, the system was used for a dual purpose: deletion of the lycopene cyclase gene (crtY) and overexpression of the octahydrolycopene dehydrogenase gene (crtI) in Pantoea dispersa MSC14, increasing lycopene production associated with Plackett–Burman design and response surface methodology optimization [86]. In another study, a Chlamydomonas reinhardtii double deletion mutant was generated by CRISPR-Cas9, knocking out genes encoding for LCYE (lycopene epsilon cyclase) and ZEP (zeaxanthin epoxidase), which increased by 60% the production of zeaxanthin and facilitated the downstream steps [85]. These demonstrate the effectiveness of CRISPR-Cas9 for redirecting metabolism toward carotenoid biosynthesis. However, under conditions of large-scale fermentation, increasing the production of pigment can favor its accumulation and affect cellular homeostasis, leading to metabolic burden and reduced growth of the microorganism [97]. CRISPR-Cas9 was also employed to integrate the heterologous carotenogenic genes crtE, crtYB, and crtI from Xanthophyllomyces dendrorhous into Saccharomyces cerevisiae CEN.PK2-1c. This strategy enabled the accumulation of β-carotene, followed by its conversion into β-ionone, an aroma-relevant compound [88]. Another example is the production of crocetin, a carotenoid derived from plants, using S. cerevisiae BY4741. Using a similar approach, production efficiency was enhanced by incorporating multiple copies of the carotenoid genes 9,10-dioxygenase (CCD2) and aldehyde dehydrogenase (ALDH), both essential for crocetin biosynthesis [89]. Similarly, strategies involving the integration of multiple copies of genes from one organism into another can boost pigment production. However, excessive overexpression of heterologous pathways can compromise strain stability and intensify competition for vital metabolites, such as acetyl-CoA and nicotinamide adenine dinucleotide phosphate (NADPH), for example. Striking a balance between precursor supply and pathway flux remains a major challenge in carotenoid metabolic engineering [98].

In Monascus purpureus, researchers manipulated the regulator gene involved in the biosynthesis of the mycotoxin citrinin (ctnA). The deleted mutant exhibited a reduction in citrinin content to 22% of the wild-type (WT) level, alongside the formation of smaller, more compact pellets and increased pigment production. In contrast, ctnA overexpression led to a 120% increase in citrinin, with minimal impact on pellet morphology and pigment synthesis [92]. In another study, two CRISPR-Cas9 systems were developed in order to eliminate large genomic fragments, allowing the removal of a 15 kb BCG associated with citrinin biosynthesis in the industrial strain M. purpureus KL-001. Researchers successfully eliminated citrinin production in mutant strains and achieved a 2–5% increase in Monascus red pigment yields [93]. These results highlight the potential of CRISPR-Cas9 not only to remove harmful mycotoxins but also to enhance pigment production. To optimize pigment production in important fungi that have applications in the food and pharmaceutical industries, CRISPR-Cas9 was employed to modify specific negative regulatory genes, thereby enhancing pigment biosynthesis, MpigI and MpigI′ in Monascus ruber KACC46666. The MpigI16-7 mutant produced 2.5, 12.4 and 18.5 times more yellow, orange and red pigments, respectively, than the WT strain [94]. Finally, using genomic analyses and gene deletions in Aspergillus homomorphus, which produces natural yellow pigments, homopirones A and B, phenoylpropanoid derivatives and polyketides, the gene cluster was identified. This cluster includes the gene polyketide synthase ahpAcinnamoyl-CoA (ahpA) that helps the production of these pigments [95]. Despite these advances, CRISPR-mediated engineering in filamentous fungi remains constrained by several challenges, including low transformation efficiency, the structural complexity of fungal cells, and organism-specific DNA repair mechanisms. These factors can negatively affect the reproducibility and scalability of genome editing approaches. In addition, most studies have been conducted at a laboratory scale, and there is still limited knowledge regarding long-term genetic stability and fermentation performance under industrial conditions. Consequently, these limitations continue to represent major barriers to large-scale pigment production.

7.2. CRISPRi Gene Repression

The CRISPR interference system (CRISPRi) uses a deactivated protein complex, guided by single guide RNA (sgRNA), to target specific DNA sequences. By binding to these sequences, the system blocks gene expression without altering the DNA itself. This approach facilitates gene function analysis and improves the efficiency of metabolic pathway engineering. Researchers developed a CRISPRi system in Methylorubrum extorquens AM1, a methylotrophic bacterium. The system was able to reduce the expression of a central metabolism gene encoding for the serine hydroxymethyl transferase enzyme (glyA), by up to 96.6%. It was also capable of reducing crtI expression by 97.7%. Additionally, repression of the squalene-hopene cyclase gene (shc), involved in hopanoid biosynthesis, led to a 1.9-fold increase in carotenoid production. Furthermore, a new functional gene (designated META1_3670) was identified among thousands of mutants [99]. In another study, violacein production in Escherichia coli was enhanced through the suppression or deletion of 17 genes. This was achieved by constructing a library of truncated and randomized sgRNAs within a CRISPRi approach, which significantly increased violacein yields. Notably, seven of the identified genes (tyrR, pykF, cra, ptsG, pykA, sdaA, and tnaA) had previously been associated with improving intracellular levels of L-tryptophan, the precursor of violacein [87]. In contrast to strategies based on permanent gene inactivation, CRISPRi enables reversible and tunable repression of metabolic pathways, offering greater flexibility for metabolic engineering applications. This precise modulation of gene expression has been associated with reduced cellular burden and enhanced robustness during pigment biosynthesis.

7.3. CRISPRi-Cas9/Cpf1/Cas12a

The CRISPRi-Cas9 method uses the deactivated Cas9 protein complex, in which two mutations preclude its endonuclease activity [100]. CRISPRi-Cas9 technology has been successfully applied to Yarrowia lipolytica, which has low efficiency and limited yield in genetic modification. This method, based on D147Y and P411T mutations, achieved over 98% efficiency in gene disruption via non-homologous end-joining repair and up to 100% for single genes. It also enabled easier and rapid integration of large genetic fragments. As a result, optimized strains produced more β-carotene and zeaxanthin by dry weight [90]. The combination of control offered by CRISPRi and the knowledge of CRISPR-Cas9 systems provides greater flexibility for dynamic metabolic regulation, making the system highly useful. Nevertheless, the effectiveness of transcriptional repression can fluctuate considerably, contingent on the promoter architecture and the positioning of the sgRNA. The CRISPRI-Cpf1 system replaces dCas9 with dCpf1. The Cpf1, also known as Cas12a, is an endonuclease with this activity depleted in CRISPRi systems and uses a CRISPR RNA (crRNA) with a component [101]. Cas12a overcomes a few limitations encountered when using Cas9, and it can also target multiple genes at once [102], making it more efficient for pigment production. In addition, Cpf1 recognizes a T-rich PAM sequence (5′-TTTV-3′ or 5′-TTTN-3′), which is located on the 5′ side of the target sequence. On the contrary, Cas9 typically requires a G-rich PAM sequence (5′-NGG-3′), located on the 3′ side. These differences significantly impact genome editing in microorganisms with varying GC content and need to be considered during experiment planning. Researchers have deleted the DPP1 and LPP1 genes in Pichia pastoris, as well as negatively regulated Erg9 using the CRISPR-Cas12a system, which improved lycopene production [91]. These systems offer distinct advantages for multiplex genomic engineering because they have the functionality of processing multiple crRNAs concurrently. This characteristic is of particular value in the context of engineered synthetic pathways of a complex nature. But, the efficiency of editing non-model microorganisms remains inconsistent, necessitating further optimization of delivery systems and genome editing protocols.

While CRISPR and its variants show a lot of biotechnological potential in enhancing pigment production and reducing mycotoxins, there are still challenges. Getting CRISPR systems in non-standard microorganisms, mainly filamentous fungi, and biosafety regulation is tough. However, advances in the development of more specific and versatile CRISPR systems, allied to approaches such as machine learning for predicting more effective edits, open up many new possibilities for improving the production of microbial pigments in a sustainable, economical and cost-effective way [82].

7.4. Challenges and Future Perspectives of CRISPR-Based Pigment Engineering

Despite the considerable advancement that CRISPR-based technologies have brought to the engineering of microbial pigments, their widespread industrial application remains constrained by technical, metabolic and commercial limitations. A pivotal challenge resides in achieving equilibrium among metabolic fluxes within the modified pathways. In many cases, excessive expression of heterologous biosynthetic genes has been shown to increase the metabolic load, disrupt the intracellular balance of precursors and cofactors, and promote the accumulation of toxic intermediates, ultimately reducing cell growth and productivity. Consequently, success in pigment overproduction depends not only on pathway activation but also on precise control of precursor availability, cofactor regeneration, and competing metabolic pathways [103].

In order to circumvent these issues, a number of different CRISPR technologies have been developed. Each of these approaches has its own unique set of advantages and disadvantages. The CRISPR-Cas9 system has proven to be highly efficient for the permanent disruption of genes and the introduction of target genomic modifications. This property renders it suitable for the elimination of competing pathways and the integration of heterologous biosynthetic genes. However, Cas9-mediated double-strand breaks have been demonstrated to induce toxicity, off-target mutations and genomic instability, especially in non-model microorganisms with poorly characterized genomes. In contrast, CRISPRi systems permit the precise calibration of transcriptional repression, thereby ensuring the potential for reversibility without the introduction of DNA cleavage. This feature renders them advantageous for the regulation of essential pathways and the minimization of cellular stress. Nevertheless, incomplete repression and variability dependent on the generated sgRNA can limit the reproducibility and precision of the engineering process. In the context of extensive gene editing, the use of Cas12a/Cpf1 technology confers substantial benefits, due to its capacity to process multiple crRNAs concurrently [104]. This functionality enables the orchestrated regulation of intricate biosynthetic networks. However, its application remains less optimized in various industrial microorganisms.

Another critical issue related to the engineering of non-model microorganisms, including filamentous fungi, cyanobacteria and microalgae. Despite the fact that these organisms present potential metabolic capabilities that make them attractive for pigment biosynthesis, genetic manipulation of them remains problematic due to low transformation rates, complex cellular structures, limited molecular toolkits, and the diversity of DNA repair mechanisms [105]. In filamentous fungi, multinucleated hyphae and heterogeneous genome editing results have the potential to further complicate strain stabilization and industrial reproducibility [105,106]. Notably, pigments derived from these fungi are frequently linked to mycotoxins, which can result in the suppression of pigment production for reasons related to human safety [107]. In addition, the question of industrial scalability remains insufficiently explored in most CRISPR-based pigment engineering studies. These limitations underscore the necessity of integrating genomic engineering with systems biology, adaptive evolution in the laboratory, and strategies for optimizing upstream and downstream bioprocesses.

The future advancement of microbial pigment production will depend on the development of more genomic engineering platforms, the optimization of approaches based on CRISPR systems and the development of new techniques. In this context, artificial intelligence (AI) and machine learning have the potential to make a significant contribution to research endeavors, particularly accelerating pathway discovery, aiding genome assembly and annotation, predicting metabolic flux and development and optimizing industrial scaling. The combination of AI-assistance approaches with systems-level metabolic modeling, synthetic biology and systems metabolic engineering may enable the development of more robust, stable and economically viable microbial cell factories for the sustainable production of pigments [82,108].

8. Future Perspectives

The integration of bioprospecting new under-exploited environments, commonly found in Brazilian biomes, with advanced genetic engineering techniques will increase the possibilities of new microbial pigments that can be used in several industries. Strategic bioprospecting initiatives, combined with CRISPR variants, may reveal new species capable of producing high-value pigments without the co-production of toxic metabolites. Another important direction involves microbial pigment production using sustainable bioprocesses. The use of agro-industrial residues as nutrient sources, combined with optimized fermentation strategies and bioreactor design, can further reduce production costs and environmental impact. It is important to highlight that many pigments remain undiscovered due to the difficulty of accessing their native producers, which are often found in extreme or poorly explored environments such as deep-sea ecosystems (Coastal Marine biome) and dense tropical jungles (Amazon forest), regions where it is estimated that up to 80% of organisms are still unknown. These habitats harbor an immense and largely untapped microbial diversity, representing a significant reservoir of novel metabolic capabilities, including the production of unique pigments with potential biotechnological applications. Furthermore, a major limitation in studying these microorganisms lies in the fact that approximately 90% of bacteria and archaea cannot be cultured under standard laboratory conditions; in other words, the vast majority are considered nonculturable [109]. In this context, alternative strategies have gained increasing attention, particularly the application of “omics” tools, such as genomics, metagenomics, transcriptomics, and metabolomics, which enable the investigation of genetic potential and metabolic profiles directly from environmental samples, bypassing the need for cultivation [78]. These approaches allow researchers to identify biosynthetic gene clusters, predict pigment production pathways, and even guide heterologous expression of target compounds in model organisms. Despite their great potential, omics-based methodologies are still underexplored in the field of microbial pigment discovery [39]. Another emerging strategy is transport engineering to enhance the secretion of intracellular pigments (apolar pigments that generally need more toxic solvents to extract). This approach involves the heterologous expression or overexpression of endogenous ATP-binding cassette (ABC) transporters and Major Facilitator Superfamily (MFS) transporters to promote pigment efflux from microbial cell factories. This way, we can begin to investigate what wonders are at the end of this microbial rainbow.