Pharmaceutical removal from wastewater by introducing cytochrome P450s into microalgae

Abstract

Pharmaceuticals are of increasing environmental concern as they emerge and accumulate in surface- and groundwater systems around the world, endangering the overall health of aquatic ecosystems. Municipal wastewater discharge is a significant vector for pharmaceuticals and their metabolites to enter surface waters as humans incompletely absorb prescription drugs and excrete up to 50% into wastewater, which are subsequently incompletely removed during wastewater treatment. Microalgae present a promising target for improving wastewater treatment due to their ability to remove some pollutants efficiently. However, their inherent metabolic pathways limit their capacity to degrade more recalcitrant organic compounds such as pharmaceuticals. The human liver employs enzymes to break down and absorb drugs, and these enzymes are extensively researched during drug development, meaning the cytochrome P450 enzymes responsible for metabolizing each approved drug are well studied. Thus, unlocking or increasing cytochrome P450 expression in endogenous wastewater microalgae could be a cost-effective strategy to reduce pharmaceutical loads in effluents. Here, we discuss the challenges and opportunities associated with introducing cytochrome P450 enzymes into microalgae. We anticipate that cytochrome P450-engineered microalgae can serve as a new drug removal method and a sustainable solution that can upgrade wastewater treatment facilities to function as “mega livers”.

INTRODUCTION

One of the most significant current threats to global freshwater systems and their functions for ecological and public health is the emergence of pharmaceutical contaminants (Khan et al., 2024; Luo et al., 2014; Salimi et al., 2017). Pharmaceuticals and prescription drug residues in wastewater are key contaminants of emerging concern (CEC) due to their continually increasing usage, resistance to conventional wastewater treatment processes, detection at increasing concentrations (ngL⁻¹ – μgL⁻¹) in water bodies around the world (Table 1), uncertain environmental and ecological effects, and lagging regulatory responses despite increasing awareness of the magnitude of the problem (Prada-Vásquez et al., 2021). Whereas pharmaceuticals can enter the water cycle through a variety of pathways (Figure 1), the effluents of wastewater treatment facilities (WWTF) are considered a primary source of pharmaceuticals to the environment (Wiest et al., 2018). Even in developed nations with advanced sewage treatment infrastructure, pharmaceuticals and their residues persist in effluents (Table 1) because WWTF are not able to fully remove or transform these pharmaceutical compounds, even after tertiary treatments such as chlorination, membrane filtration or UV ozonation (Luo et al., 2014) (Table 2, Table S1). Resistant to photochemical or biological breakdown in surface waters, such pharmaceutical pollutants subsequently persist in aquatic environments and pose threats to ecosystems and human health (Hejna et al., 2022). Global reviews highlight the widespread presence of pharmaceuticals and their by-products in aquatic surface environments across 71 countries (aus der Beek et al., 2016; Singer et al., 2016). Growing populations and longer human lifespans are expected to increase pharmaceutical use and exacerbate the accumulation of antibiotics, analgesics, anti-inflammatory drugs, and other drugs, in aquatic environments (Santos et al., 2007). In fact, many pharmaceutical compounds are intentionally designed to be chemically stable under ambient or physiological conditions, necessitating new environmentally friendly and cost-effective removal strategies (Samal et al., 2022).

TABLE 1 Occurrence of selected non-steroidal anti-inflammatory drugs (NSAIDs) in wastewater influents and/or effluents of different locations based on Hejna et al., 2022; Mussa et al., 2022; Oluwole et al., 2020.

Pharmaceutical	Concentration (μg/L)	Country	Source
Diclofenac	22.30	South Africa	Influent
	12.40		Effluent
	0.21–0.49	France	Effluent
	0.40	UK	Influent
	0.12		Effluent
	0.25	Japan	Influent
	0.15		Effluent
	269	Portugal	Influent
	1.30	Canada	Influent
Ibuprofen	0.29–1.38	Turkey	Influent
	0.12–1.01		Effluent
	0.15–0.18	Taiwan	Influent
	0.10–0.31		Effluent
	2.29	UK	Influent
	0.14		Effluent
	5.00–8.00	Canada	Effluent
	62.82	South Africa	Influent
	58.71		Effluent
	9.50	Korea	Influent
	0.02		Effluent
	10.60	US	Effluent
	0.23–1.05	Iran	Influent
	0.03–0.05		Effluent
	11.04–13.09	Portugal	Influent
	0.03–0.04		Effluent
	4.29–10.86	Poland	Influent
	0.02–0.64		Effluent
Naproxen	1.17	UK	Influent
	0.17		Effluent
	5.94	Korea	Influent
	0.12		Effluent
	52.30–55.00	South Africa	Influent
	13.50–20.40		Effluent
	99	Spain	Influent
	108		Effluent
	23.21	US	Influent
	0.01–1.62	Portugal	Effluent
Acetaminophen	211	UK	Influent
	11.73		Effluent
	11.30	France	Influent
	5.76	South Africa	Influent
	74.55	Korea	Influent
	0.02		Effluent
Carbamazepine	0.95	UK	Influent
	0.83		Effluent
	2.30	Canada	Influent
	0.42	Taiwan	Effluent

FIGURE 1. Routes of pharmaceutical contaminants. Pharmaceutical contaminants can enter the environment through various routes, posing potential risks to ecosystems and human health (Samal et al., 2022). One significant route is the disposal of unused or expired medications by individuals or healthcare facilities. Improper disposal methods can lead to the release of pharmaceutical compounds into water bodies, for example, through landfill leachate. Additionally, pharmaceutical manufacturing discharges and wastewater treatment plant effluents are known sources of contamination. These facilities can release trace amounts of active pharmaceutical ingredients and their by-products into surface water and groundwater through effluent discharges.

TABLE 2 Various technologies for treatment of selected NSAIDs.

[Table omitted. See PDF]

Bacteria and fungi play pivotal roles in the degradation of xenobiotics, synthetic compounds foreign to the environment. Bacteria such as Pseudomonas, Bacillus, and Rhodococcus, and fungi like Aspergillus, Trichoderma, and Cunninghamella elegans (C. elegans) (Arora et al., 2016; Khan et al., 2024; Khan & Murphy, 2022a; Soares et al., 2021), have been extensively studied for their ability to metabolize and detoxify xenobiotics. Through enzymatic hydroxylation, oxidation, and conjugation pathways, these microorganisms transform xenobiotic compounds into simpler, less toxic molecules that can be assimilated into natural metabolic processes or further degraded by other organisms in the ecosystem (Miglani et al., 2022).

Like bacteria and fungi, microalgae have garnered significant attention as a promising means to perform wastewater treatment, due to their inherent ability to metabolize a wide variety of pharmaceuticals, such as diclofenac, acetaminophen, and carbamazepine (Abdelfattah et al., 2023). Photosynthetic microalgae also show rapid growth rates, high biomass productivity, and the capacity to utilize diverse carbon sources (Yin et al., 2020), even in challenging environments. These features are particularly advantageous because they allow for scalable and efficient process bioengineering. Also, microbial biofilms, including those formed by algae, play a significant role in the biodegradation of pharmaceuticals in aquatic environments. Algal biofilms, consisting of densely packed communities of algae and associated microorganisms, provide a conducive microenvironment for microbial interactions and the exchange of genetic material, further enhancing their biodegradative capabilities (Khan & Murphy, 2022b). Microalgae employ diverse mechanisms for drug removal, encompassing both adsorption onto their cell surfaces and metabolic transformations, rendering them highly effective for this purpose (Zhou et al., 2022). However, microalgae also face limitations in their ability to degrade recalcitrant pharmaceuticals because they show low expression of specific enzymes or lack certain metabolic pathways altogether. This means that a single microbial species is typically insufficient to efficiently degrade the entire complex spectrum of emerging pharmaceutical compounds in wastewater (Goswami et al., 2022).

The liver serves as the primary human organ responsible for catabolizing pharmaceuticals (Vaja & Rana, 2020). It is the site where small molecules from the digestive system first encounter the bloodstream. Many pharmaceuticals undergo enzymatic breakdown in the liver through a group of enzymes called cytochrome P450s (P450s) (Gu & Manautou, 2012). These enzymes, constituting a superfamily of haeme-containing monooxygenases, can perform selective chemical alterations to pharmaceuticals, often effectively diminishing their potency (Behrendorff, 2021). When examining the proportion of drugs metabolized by enzymes, approximately 75% can be attributed to P450s (Zhao et al., 2021). These specific P450 enzymes are closely associated with the breakdown of specific types of drugs, and their roles are extensively studied during the drug development process. Therefore, the corresponding P450 enzymes responsible for the breakdown of every approved drug are generally well understood (Zanger & Schwab, 2013).

Considering the constraints of microalgae in degrading recalcitrant pharmaceuticals, attributed to either insufficient expression of particular enzymes or the absence of necessary metabolic pathways, and the ability of the P450 enzymes in catabolizing these drugs, if wastewater microalgae could be genetically modified with P450 enzymes, they would grow their capacity to degrade wastewater pharmaceuticals. This paper aims to explore the potential of P450-engineered microalgae for sustainable and efficient removal of pharmaceutical contaminants during wastewater treatment.

MICROALGAE IN WASTEWATER TREATMENT

Oluseyi Osunmakinde et al. (2019) identified Proteobacteria, Actinobacteria, Firmicutes, and Chloroflexi as dominant prokaryotic phyla in WWTF, with functions highly associated with carbohydrate, energy, and amino acid metabolism. Numerous microalgae species, such as Anabaena cylindrica, Chlorella vulgaris, Scenedesmus obliquus, and Spirulina platensis, among others, have demonstrated their ability for cultivation in a range of environments, including domestic, municipal, and industrial wastewaters (Mennaa et al., 2015; Purba et al., 2024). Also, microalgae species like Scenedesmus, Chlorella, Botryococcus, Phormidium, and Chlamydomonas have demonstrated proficiency in removing various wastewater contaminants, including pharmaceuticals, due to their tolerance and adaptability to harsh growth conditions (Abdelfattah et al., 2023).

Phytoremediation of microalgae possesses a diverse array of metabolic pathways that enable them to transform and degrade pollutants. Through their inherent biochemical machinery, microalgae can metabolize organic compounds, breaking them down or modifying their chemical structures, thus reducing their toxicity and environmental impact (Mustafa et al., 2021). Microalgae employ various enzymatic reactions, such as oxidation, reduction, hydrolysis, and conjugation, to facilitate the transformation of pollutants into less harmful or more readily biodegradable forms (Abdelfattah et al., 2023; Mojiri et al., 2020; Wu et al., 2012). These metabolic transformations can occur through intracellular enzymatic processes or extracellular secretions, such as exopolysaccharides and enzymes (Mona & Kaushik, 2015; Mota et al., 2016; Xiao & Zheng, 2016), which contribute to the overall degradation of pollutants in the surrounding environment.

Whereas microalgae possess inherent pollutant removal capabilities, their natural metabolic pathways often face limitations in degrading recalcitrant organic compounds commonly found in wastewater. This is because microalgae have primarily evolved to thrive in aquatic environments and perform functions such as photosynthesis and nutrient uptake (Aron et al., 2021). Hence, their enzymatic machinery is tailored to support these metabolic processes rather than the degradation of complex organic pollutants like pharmaceuticals (Almaguer et al., 2021). Also, microalgae typically inhabit environments with fluctuating nutrient concentrations, where they compete with other microorganisms for resources. Consequently, they may allocate resources towards strategies that optimize their growth and survival under prevailing conditions, rather than investing in the production of specialized enzymes for pollutant degradation. The metabolic limitations of microalgae impede their effectiveness in removing and degrading specific organic contaminants like pharmaceuticals such as naproxen, sulfamethoxazole, and erythromycin in wastewater treatment systems (Ghasemi et al., 2011; Patel et al., 2019; Vingiani et al., 2019).

To overcome these limitations and enhance the pharmaceutical degradation capabilities, genetic engineering can be applied to introduce specific enzymes and metabolic pathways into microalgae, thereby expanding their metabolic repertoire (Chowdhury et al., 2023). For example, by introducing enzymes with broad substrate specificity, such as P450s, microalgae can acquire the necessary catalytic machinery to tackle recalcitrant pharmaceuticals effectively thereby improving the overall efficiency of wastewater treatment.

Benefits of using microalgae for heterologous expression

The utilization of microalgae for heterologous expression offers a range of benefits that make them an attractive platform for various applications. Microalgae are amenable to environmental conditioning to enhance their growth rates which allows for efficient production of desired proteins or compounds within a relatively short timeframe (Grama et al., 2022). Additionally, microalgae offer a scalable and cost-effective option for heterologous expression. The cultivation of microalgae typically requires less resources compared to traditional expression systems. They can thrive in simple media with sunlight as an energy source, reducing the need for complex growth conditions and expensive supplements (Jareonsin & Pumas, 2021). Also, by incorporating the biorefinery concept, the biomass obtained from harvested microalgae can be utilized to produce carbon-neutral fuels, valuable pigments, feeds for fish and animals, biofertilizers, bioplastics, and carbon dioxide emission mitigation (Okeke et al., 2022). This integrated approach promotes a circular economy, emphasizing minimal waste to the environment and fostering sustainability.

Microalgae can undergo genetic engineering or manipulation through various techniques to introduce desired traits or characteristics (Gutiérrez & Lauersen, 2021; Kumar et al., 2020). One common method is transformation, where foreign DNA is introduced into microalgae cells through techniques like glass bead transformation (Economou et al., 2014; Neupert et al., 2012), electroporation (Chen & Hu, 2019), or biolistic transformation (Remacle et al., 2006). Once integrated into the microalgae's genome, this foreign DNA can lead to the expression of desired traits, such as increased biomass productivity (Zhao et al., 2023) or improved stress tolerance (Siripornadulsil et al., 2002). Gene editing techniques, such as CRISPR-Cas9, allow for precise modifications to specific genes within the microalgae genome (Song et al., 2022). This enables researchers to create microalgae with enhanced traits tailored to specific applications (Dhokane et al., 2023; Görlich et al., 2019). RNA interference (RNAi) is another technique used to suppress the expression of specific genes in microalgae by introducing short RNA molecules that bind to target mRNA, preventing their translation into protein (Molnar et al., 2009). These genetic engineering and manipulation techniques offer powerful tools for improving microalgae for various purposes, including biofuel production, bioremediation, and the production of high-value compounds compared to the bacterial and fungal hosts (Table 3).

TABLE 3 An overview of the heterologous expression of human cytochrome P450s in bacterial and fungal surrogate cell factories.

Aspect	Bacteria	Fungi
Genetic tractability	High, well-established genetic manipulation tools	Moderate, only some fungal species amenable to genetic engineering
Protein folding	May require codon optimization and chaperone co-expression for correct folding	Generally competent in proper folding and post-translational modifications
Post-translational modifications	Limited, often lack eukaryotic post-translational machinery	Extensive, capable of diverse post-translational modifications
Cofactor availability	May lack eukaryotic cofactor synthesis pathways	Typically possess eukaryotic cofactor synthesis pathways
Metabolic burden	Low, efficient metabolic pathways and expression systems	Moderate, may experience metabolic burden with high-level expression
P450 expression	High	Low compared to bacteria
Scale-up potential	Highly scalable with established fermentation processes	Moderate scalability, may require optimization for large-scale production
Examples of commonly used hosts	Escherichia coli	Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, Aspergillus sp.

P450 ENZYMES Overview and role in xenobiotic metabolism

P450s are a class of haeme–thiolate proteins containing iron, specifically belonging to the cytochrome b type (Miles et al., 2000). The name P450 is derived from the wavelength (450 nm) at which the carbon monoxide derivative of the reduced cytochrome exhibits maximum absorption (Werck-Reichhart & Feyereisen, 2000). While the P450 enzymes are proficient in catalysing a myriad of other chemical reactions such as epoxidation, dealkylations, deaminations, and desulfurations, the prototypical biotransformation facilitated by P450s is the monooxygenase reactions (Esteves et al., 2021). Akin to other monooxygenases, one atom of molecular oxygen is reduced to water, and another is incorporated into the substrate, leading to various metabolic transformations (Rudolf et al., 2017). The transfer of reducing equivalents from NADPH to P450 is orchestrated by a flavoprotein enzyme known as NADPH–cytochrome P450 reductase, often abbreviated as P450 reductase (Hamdane et al., 2009). This enzymatic process plays a pivotal role in the overall functionality of P450s, enabling them to catalyse diverse oxidation reactions essential in the metabolism of various substrates (Guengerich, 2018) (Table 4).

TABLE 4 An overview of the commonly produced metabolites by the P450 enzymes.

Drug component	Metabolites produced by P450s
Diclofenac	P450 enzyme	Metabolites
	CYP2C9, CYP5208A3, CYP5313D1	4′-hydroxy diclofenac
	CYP3A4	5′-hydroxy diclofenac
Carbamazepine	CYP2B6, CYP3A4, CYP3A7	3-hydroxycarbamazepine
	CYP2C8, CYP2C19, CYP3A4, CYP3A5, CYP3A7	10,11-epoxycarbamazepine
Naproxen	CYP1A2, CYP2C8, CYP2C9	O-desmethyl naproxen
Acetaminophen	CYP2E1	N-acetyl-p-benzoquinone imine
Ibuprofen	CYP2C9, CYP3A4/5, CYP5208A3, CYP5313D1	Hydroxy ibuprofen or carboxy ibuprofen

P450 enzymes play a crucial role in the degradation of xenobiotics, including drugs, pollutants, dyes, agrochemicals, and per- and poly-fluorinated alkyl substances (PFAS) (Khan et al., 2023; Khan & Murphy, 2022b). These enzymes facilitate the oxidative metabolism of these foreign compounds, transforming them into more water-soluble and excretable forms, thereby aiding in detoxification and reducing their potential toxicity in biological systems. P450 enzymes facilitate the phase I metabolism of xenobiotics (Table 4) by introducing diverse chemical modifications, primarily through the oxidation reactions (Esteves et al., 2021). Oxidation of the pharmaceutical compounds increases their water solubility, making it easier for the body to eliminate them through urine or bile (Li et al., 2019). Studies have revealed that the human genome includes 57 potentially functional, protein-coding P450 genes (Nebert et al., 2013). These genes are categorized based on sequence homology into 18 families and 44 subfamilies (Zhao et al., 2021). Notably, within families 1, 2, 3, and 4, there are more than a dozen P450 isozymes primarily accountable for the metabolism of drugs and various xenobiotics (Esteves et al., 2021).

Structural arrangement of cytochrome P450 systems

There are two main classes of cytochromes P450 distinguished by their electron-supporting systems: (a) the bacterial type and (b) the microsomal type (Figure 2) (Bernhardt, 2006). As shown in Figure 2, the bacterial-type P450 systems consist of the respective P450, a ferredoxin reductase, and a ferredoxin. The ferredoxin reductase receives electrons from NADPH, which are then transferred to the P450 by the soluble ferredoxin (2a). Conversely, the microsomal P450 systems, characterized by a two-component structure, comprise the membrane-bound P450 reductase and the corresponding microsomal P450. The P450 reductase obtains electrons for redox reactions from NADPH and subsequently interacts with the P450 (2b). An exceptional instance of this protein complex paradigm is the bacterial P450 enzyme CYP102 (BM3) isolated from Bacillus megaterium, where both the P450 and the reductase are housed within a single enzyme (2c) (Bernhardt, 2006; Hannemann et al., 2007; Omura, 2010).

FIGURE 2. Overview of the P450 systems. (A) Bacterial-type P450 systems include the P450, ferredoxin reductase, and ferredoxin, with electron transfer from NADPH to the P450 mediated by the soluble ferredoxin. (B) Microsomal P450 systems, where electron transfer for redox reactions occurs from NADPH to the P450 through the P450 reductase. (C) bacterial P450 enzyme CYP102 (BM3) from Bacillus megaterium, where both the P450 and the reductase are fused within a single enzyme. “Created with BioRender.com”.

CHALLENGES OF INTRODUCING P450S INTO MICROALGAE Choice of P450 enzymes

Commonly expressed P450 enzymes in model microorganisms include fungal CYP5208A3 and CYP5313D1 from Cunninghamella elegans for degrading pyrethroids, non-steroidal anti-inflammatory drugs (NSAIDs), per- and poly-fluorinated alkyl substances (PFAS) (Khan & Murphy, 2022a). CYP51 for sterol biosynthesis and drug metabolism (Zhang et al., 2019). Additionally, CYP1A1/CYP1A2 and CYP2E1 play essential roles in xenobiotic detoxification, while CYPBM3 finds applications in biotechnology. Human isoforms like CYP3A, CYP2D6, CYP2C, CYP1A2, and CYP2E1 are desirable due to their efficiency in metabolizing approximately 70%–80% of clinically used drugs (Zanger & Schwab, 2013).

However, challenges arise with mammalian P450s, predominantly due to their membrane-bound nature (Šrejber et al., 2018), resulting in relatively low activities and inherent instability (Guengerich et al., 1996). Attempts at genetic engineering to enhance the specific activity of human P450s towards drugs have shown limited success, yielding modest increases, usually below 10% (Kumar & Halpert, 2005). In contrast, microbial P450s demonstrate greater stability (Munro et al., 2002; Narhi & Fulco, 1986), with specific activity towards natural substrates ranging from 100- to 1000-fold higher compared to the highest activities observed with human P450s (Bernhardt, 2006). Moreover, genetic engineering efforts on BM3 from Bacillus megaterium, as reported by Reinen et al. (2011), have identified four mutants (M02, MT35, MT38, and MT43) capable of producing P450-mediated metabolites for 41 of the 43 drugs tested, metabolizing 77% of the drug library by more than 20%. These BM3 mutants address limitations commonly associated with human P450 isoforms mentioned above and present themselves as ideal candidates for diverse biotechnological applications, demonstrating their potential for overcoming challenges associated with drug metabolism in wastewater treatment processes (Reinen et al., 2011).

Membrane-bound nature of P450 enzymes

The membrane-bound nature of P450 enzymes poses significant challenges for their heterologous expression in other hosts. This membrane association, primarily within the endoplasmic reticulum of eukaryotic cells and the inner mitochondrial membrane, is essential for their functional regulation but complicates their expression in non-native systems. Achieving proper integration into the host cell membrane is difficult, as it requires maintaining the correct orientation to lipophilic substrates that are typically embedded in the lipid bilayer. The stability and catalytic efficiency of CYP enzymes rely on their membrane environment, which must be replicated in the host system to ensure functional enzyme expression (Kaluzhskiy et al., 2024). Additionally, CYP enzymes need to interact with other proteins, such as cytochrome P450 reductase, for electron transfer during catalysis, necessitating the co-expression of these accessory proteins. The specific lipid composition of the native membrane also modulates enzyme activity, and discrepancies in the lipid environment of the host can impair enzyme functionality. Therefore, replicating the spatial and biochemical context of CYP enzymes in heterologous systems is challenging but crucial for their effective expression and activity (Guengerich, 2008; McDonnell & Dang, 2013; Omura, 2010).

P450/reductase interaction

One hurdle in heterologous expression of human P450 is the necessity for P450 to interact with an active electron donor, whether bound to a membrane or fused together (Šrejber et al., 2018) as highlighted in Section 3.3; hence, overcoming this challenge becomes crucial for successful expression (Durairaj et al., 2016; Durairaj & Li, 2022). One approach to address this is through the co-expression of P450 and P450 reductase (Quehl et al., 2016). The co-expression strategy becomes a viable solution if the microalgae's cellular context permits the necessary interplay between P450 and the electron donor (Durairaj & Li, 2022). Achieving optimal co-expression necessitates careful consideration of the specificity of CYPs and their varying affinities for redox partners (Esteves et al., 2020, 2023; Pikuleva et al., 1999). In general, the recombinant eukaryotic P450s are reconstituted with the P450 reductases belonging to three major sources of origin: (i) endogenous P450 reductases, (ii) homologous P450 reductases, and (iii) heterologous P450 reductases (Durairaj & Li, 2022). Ensuring this compatibility is crucial for efficient electron transfer, enabling the activation of the P450 for substrate oxidation (Agrawal et al., 2008). In the case of endogenous P450 reductases, eukaryotic hosts naturally pair with native or endogenous redox partners to supply reducing equivalents. For instance, in yeast systems, recombinant eukaryotic P450s typically pair with the native S. cerevisiae NADPH reductase (Ide et al., 2012). This native reductase autonomously satisfies the P450 electron transfer chain, eliminating the need for exogenous reductases (Nazir et al., 2011). Research consistently shows that coupling P450s with cognate or homologous redox partners leads to enhanced monooxygenation activities (Durairaj et al., 2015; Neunzig et al., 2013). For example, whereas the endogenous P. pastoris P450 reductase was incompatible with heterologously expressed human CYP2D6, co-expressing the homologous human P450 reductase favoured activity in P. pastoris (Dietrich et al., 2005). In the case of heterologous P450 reductases, P450s can be reconstituted with heterologous P450 reductases from various origins, spanning different species and kingdoms (Theron et al., 2019). For instance, prokaryotic systems often employ surrogate or heterologous redox partners to reconstitute bacterial P450 activities (Liu et al., 2022).

Also, to streamline the heterologous expression process further, the exploration of naturally fused proteins such as P450 BM3 (Girvan & Munro, 2016) or engineered constructs becomes particularly beneficial (Ugalde et al., 2018). Through genetic engineering, artificial constructs can be designed to achieve the same fusion (Zuo et al., 2017), creating a catalytically self-sufficient system within the microalgae. Another potential approach to address the challenges associated with P450/reductase interactions would be to selectively express the P450s in the algae chloroplast (Mellor et al., 2023; Nielsen et al., 2013). Photosystem II demonstrates efficient absorption of solar energy, facilitating the photolysis of water molecules and generating electrons for downstream metabolic pathways during photosynthesis (Barber, 2016). In microalgae, the total cellular biosynthetic electron demand is significantly lower than the produced electrons (Santos-Merino et al., 2021), suggesting that the surplus solar energy could theoretically be harnessed further by introducing heterogeneous catalytic elements (Zheng et al., 2022). These elements, provided they can interact with the photosystem proteins to acquire electrons, may leverage the excessively absorbed solar energy. Therefore, if the P450s can be expressed in the algae chloroplasts, P450s can utilize the excess reducing power associated with photosynthesis to drive the P450 catalytic cycle (Gangl et al., 2015; Lassen et al., 2014; Xue et al., 2014).

Furthermore, P450 reductases, such as NADPH: P450 reductase, play a crucial role in the biotransformation of drugs, for example, the anticancer drug flutamide. In the case of flutamide, NADPH: P450 reductase assists in converting the drug into its metabolites through a series of oxidative reactions mediated by specific P450 enzymes. Therefore, P450 reductases serve as key components in drug metabolism pathways, contributing to the efficacy and safety of therapeutic interventions (Khan & Murphy, 2022b).

Availability of the substrates

The challenge in expressing P450 lies in the localization within the cell and its impact on substrate availability. When targeting the nucleus for expression, the substrate is typically readily available, as it can freely diffuse in the cytoplasm. However, if the expression is directed to the chloroplast, a more intricate scenario unfolds as proteins expressed in the chloroplast are contained within its boundaries, requiring the substrate to traverse the chloroplast membrane for metabolic processing. Overcoming this challenge involves considerations not only about expressing the enzyme within the chloroplast but also ensuring that the substrate can efficiently enter this compartment for effective metabolic transformations. This intricate interplay between P450 expression localization and substrate availability adds a layer of complexity to the design and optimization of heterologous expression systems, especially in organisms like microalgae.

Enzyme stability and activity

Maintaining the stability and activity of P450 enzymes in the microalgal cellular environment poses several challenges. One major challenge is the oxygen sensitivity of these enzymes. P450 enzymes require oxygen for their catalytic activity, but excessive oxygen levels can lead to the production of reactive oxygen species (ROS) and damage the enzyme (Veith & Moorthy, 2018). To address this, microalgae cultivation systems can be optimized to control oxygen levels and minimize oxidative stress. Strategies such as bubble column reactors, photobioreactors with efficient gas exchange, and oxygen-scavenging systems can help maintain the stability and activity of P450 enzymes.

Protein folding is another critical factor affecting enzyme stability and activity. P450 enzymes often require specific chaperones and cofactors for proper folding and functional assembly (Graham-Lorence et al., 1995; Peterson & Graham, 1998; Werck-Reichhart et al., 2000). Genetic engineering approaches can be employed to co-express chaperones and cofactors alongside the P450 genes to enhance enzyme stability and activity (Schlapschy & Skerra, 2011; Wu et al., 2009). Additionally, protein engineering techniques, such as directed evolution which involves iterative rounds of mutagenesis and screening to select variants with improved stability, activity, or substrate specificity (Cobb et al., 2013) and rational design, can be utilized to optimize the folding properties of the enzymes. Hence, implementing these approaches in the selected P450 enzymes can enhance their catalytic efficiency, expand substrate specificity, and improve the overall efficacy of pharmaceutical drug degradation processes during wastewater treatment.

Genetic transformation and expression efficiency

Genetic transformation of microalgae and achieving high expression efficiency of P450 genes present certain limitations. Microalgae species have varying transformation efficiencies and may require specific protocols for successful gene delivery. Optimizing gene delivery methods, such as developing more efficient transformation techniques like nanoparticle-mediated delivery or gene gun systems, can help improve transformation efficiency (Gutiérrez & Lauersen, 2021). Furthermore, the integration of foreign genes into the microalgal genome needs to be stable and predictable to ensure long-term expression. Promoter choice, regulatory elements, and selection markers can influence gene expression levels and stability, requiring careful consideration and optimization (Doron et al., 2016; Rasala & Mayfield, 2011).

Choosing between nucleus and chloroplast transformation for P450 expression involves a comprehensive evaluation of various factors, each presenting distinct advantages and challenges. Nuclear transformation serves as a conventional and easily understandable avenue for genetic engineering. The nuclear genome is a well-established target for integrating foreign genes, utilizing techniques familiar to molecular biologists. Integration into the nucleus is relatively straightforward compared to the chloroplast genome. However, nuclear transformation is a random process, lacking precise control over the location or copy number of foreign gene integration (Doron et al., 2016; Leon & Fernandez, 2007; Zhang et al., 2014).

Conversely, chloroplast transformation offers unique advantages. With approximately 80 copies of the chloroplast genome, achieving high expression levels becomes more feasible. This is particularly significant for P450, where robust expression is pivotal for substantial enzymatic activity. Precise transgene integration can be accomplished through homologous recombination, providing an advantage over the random integration seen in nuclear transformation. Additionally, chloroplast transgenes are less susceptible to RNAi-mediated silencing, and the chloroplast's lower protease content enhances the conducive environment for protein expression (Cutolo et al., 2022; Purton et al., 2013; Wannathong et al., 2016). Furthermore, chloroplasts provide an enriched environment in terms of NADPH, a critical cofactor for cytochrome P450 enzymatic activity as discussed in Section 3.3. This advantage can notably impact the efficiency of specific enzymatic reactions, making chloroplast transformation an attractive option for certain applications. However, challenges arise in terms of substrate availability due to the compartmentalized nature of chloroplasts, as mentioned earlier in P450/reductase interaction.

INTRODUCING P450S INTO MICROALGAE: OPPORTUNITIES Cases for the successful expression of P450s using the reducing power from thylakoid membranes

Forskolin, derived from Coleus forskohlii, has medicinal uses like treating hypertension and asthma (Pateraki et al., 2017). Despite its importance, its synthesis is challenging due to its specific structure (Ye et al., 2009). 13R(+) manoyl oxide, found in C. forskohlii's cork cells, serves as forskolin's precursor (Pateraki et al., 2014). Lauersen et al. (2018) showed that co-expressing a truncated C. forskohlii microsomal P450 (CfCYP76AH16) in the microalgae Chlamydomonas reinhardtii chloroplast catalyse the hydroxylation of heterologous 13R(+) manoyl oxide to 9-OH 13R(+) manoyl oxide. Remarkably, this CfCYP76AH16 used native chloroplast electrons, eliminating the need for a dedicated P450 reductase. This represents the first controlled functionalization of a non-native metabolite in a green algal cell through metabolic engineering, suggesting green algae's potential for heterologous P450 production and light-driven bioproduction concepts.

Caffeic acid, a phenolic natural compound found in all plant species, is recognized for its antioxidant, anti-inflammatory, and anticancer properties, making it a widely utilized substance (Chao et al., 2009; Gülçin, 2006; Rajendra Prasad et al., 2011). However, the current commercial production of caffeic acid relies on chemical synthesis, a method marred by low yield and significant environmental concerns. A pilot study by Xue et al. (2014) explored the potential use of cyanobacteria as a “photo-bioreactor” to produce caffeic acid, by introducing the plant caffeic acid synthase P450 CYP98A3 (C3H) from Arabidopsis thaliana into Synechocystis sp. PCC 6803. The introduction of a codon-optimized C3H-encoding gene into Synechocystis enabled the successful expression of the plant P450, with localization onto the thylakoid membrane. Operating under photosynthetic growth conditions, the cyanobacterium, along with other biosynthetic enzymes, successfully generated the anticipated product, caffeic acid showcasing the feasibility of utilizing algal thylakoids as a suitable platform for studying membrane-bound P450s (Kim et al., 2011).

Dhurrin, a cyanogenic glycoside found in various plants, can constitute up to 30% of the dry mass of etiolated sorghum seedlings (Sibbesen et al., 1994). With potent antimicrobial properties and cytotoxic effects that deter herbivory in certain insects and animals, the biosynthetic pathway of dhurrin in Sorghum bicolor involves the crucial enzyme CYP79A1. By fusing the soluble catalytic domain of CYP79A1 with a cyanobacterial photosystem I-derived subunit for thylakoid localization, successful expression of the eukaryotic P450 occurred in Synechococcus sp. PCC 7002. This engineered enzyme exhibited light-dependent activity both in vitro and in vivo (Lassen et al., 2014). Additionally, a separate study by Gangl et al., 2015 demonstrated the successful expression of CYP79A1 in the eukaryotic green algae C. reinhardtii. Upon incorporation of the CYP79A1-encoding gene into the chloroplast genome of C. reinhardtii, the P450 enzyme was consistently expressed and positioned within the chloroplast membrane (Gangl et al., 2015).

Atrazine, a widely employed pesticide globally, has gained recognition as a substantial environmental pollutant (Hayes et al., 2010). Mammalian P450 CYP1A1 has been shown to effectively convert atrazine into deisopropylated and de-ethylated products, both in vitro and in vivo (Kawahigashi et al., 2005; Zheng et al., 2022). The expression of CYP1A1 from Rattus norvegicus (brown rat) in the cyanobacteria Synechococcus sp. PCC 7002 revealed that the protein could be localized to the thylakoid membrane through its native N-terminal membrane-bound domain. Notably, the Synechococcus sp. PCC 7002 strain expressing CYP1A1 demonstrated the ability to degrade atrazine in a light-dependent manner, showcasing its potential in environmentally friendly atrazine remediation (Berepiki et al., 2016).

Although references on heterologous P450 expression in microalgae are limited, the findings not only on cyanobacteria but also on microalgae underscore the significant promise of utilizing the reducing power of the thylakoid membranes of microalgae as cost-effective and environmentally friendly light-driven P450 bioreactors and biosynthetic platforms.

Exploring novel P450s

The exploration of novel P450 enzymes from diverse sources is crucial to expand the repertoire of pollutant degradation capabilities in microalgae. The vast diversity of microorganisms in various ecological niches provides a rich resource for discovering new enzymes with unique catalytic activities (Tian et al., 2021). By screening microbial communities and metagenomic libraries, it is possible to identify novel P450 enzymes with desired properties, such as the ability to degrade specific pollutants (Mishra et al., 2021). Functional screening assays can further evaluate the catalytic activities of these enzymes, enabling the identification of promising candidates for engineering microalgae with enhanced pollutant degradation capabilities (Bouhajja et al., 2017).

Scale-up and field application

Scaling up P450-engineered microalgae for field application in wastewater treatment systems presents both challenges and opportunities. Challenges include the need to achieve large-scale cultivation of microalgae and ensure stable expression of engineered enzymes over extended periods (da Silva & Reis, 2015). Considerations such as reactor design, culture medium optimization, and the integration of engineered microalgae into existing treatment infrastructure need to be addressed. Efforts should focus on developing cost-effective and efficient cultivation strategies, selecting suitable reactor configurations, and optimizing culture conditions to maximize pollutant removal efficiency at the large-scale level (Borowitzka & Vonshak, 2017; Novoveská et al., 2023).

Integration into existing wastewater treatment technologies

Pseudomonas fluorescens HK44 stands as the first recombinant microorganism to undergo a complete U.S. EPA biotechnology risk assessment review, securing environmental release approval for bioremediation applications (Ripp et al., 2000). Despite conducting environmental research tests under controlled, replicated conditions, the scalability of these tests to simulate open environmental release to subsurface vadose conditions remains a key consideration. The study explicitly demonstrated the viability and maintenance of recombinant microorganisms designed for in situ bioremediation upon environmental introduction. While extensive laboratory studies on genetically modified organisms in bioremediation exist (Ranjbar & Malcata, 2022), as of the present, there is a dearth of reported outdoor cultivation of genetically modified microalgae (Sayler & Ripp, 2000). Nevertheless, the strategic combination of P450-engineered microalgae with other wastewater treatment technologies such as activated sludge systems (Chen et al., 2019; Nguyen et al., 2020), biofilters (Chaudhary et al., 2003; Fu et al., 2019; Sauter et al., 2023), high-rated algal ponds (Mehrabadi et al., 2015), and constructed wetlands (Wang et al., 2022; Zhao et al., 2022) holds promise for synergistic effects providing oxygen and nutrients to the attached microbial communities, potentially elevating overall treatment efficiency and pharmaceutical contaminant removal (Arias et al., 2018; Guo et al., 2023).

Additionally, algal biofilms could provide a targeted system for in situ bioremediation (Khan & Murphy, 2022a). Genetically modified algae with enhanced metabolic activities may uptake and degrade various pharmaceutical compounds at rates/efficiencies beyond those achieved by native/autochthonous communities. The biofilm structure may enhance the retention time of wastewater, allowing for more effective interaction between the algae and contaminants (Fanesi et al., 2019). Additionally, biofilms protect algae from environmental stressors and increase their stability and resilience, leading to sustained bioremediation performance.

Techno-economic analysis and cost-effectiveness

Techno-economic aspects are pivotal considerations in the implementation of P450-engineered microalgae in wastewater treatment. While this approach shows promise as an alternative to traditional tertiary treatment methods like UV irradiation, ozonation, and membrane filtration, its viability hinges on various factors (Pei et al., 2019). One significant advantage lies in its potential to leverage existing infrastructure within conventional treatment systems, thereby minimizing the need for extensive capital investments in infrastructure upgrades (Amenorfenyo et al., 2019). Additionally, P450-engineered microalgae offer the prospect of a more sustainable and cost-effective solution for pharmaceutical degradation in wastewater, as they harness natural biological processes rather than relying on energy-intensive or chemical-based treatments (Ebah et al., 2022). However, the economic feasibility of this approach depends on factors such as the scalability of P450-engineered microalgae cultivation, the efficiency of pharmaceutical degradation achieved, and the comparative costs of implementation and operation against conventional treatment methods (Borowitzka & Vonshak, 2017). Comprehensive techno-economic assessments are essential to evaluate the long-term viability and competitiveness of P450-engineered microalgae in wastewater treatment scenarios, guiding decision-making towards sustainable and economically sound solutions.

CONCLUSIONS

Utilizing P450-engineered microalgae for wastewater treatment holds significant potential as a sustainable and effective approach to help tackle the emerging pharmaceutical contamination problem (Figure 3). By genetically engineering microalgae to express P450 enzymes, we may enhance their inherent pharmaceutical degrading capabilities (Table 5), making them efficient and versatile tools for wastewater treatment. The rapid growth rates, photosynthetic efficiency, and capacity to utilize carbon dioxide from wastewater, converting it into biomass, are advantageous characteristics that make microalgae an appealing choice for pharmaceutical removal over bacteria or alternative technologies.

FIGURE 3. An overview of P450 expression in microalgae. “Created with BioRender.com”.

TABLE 5 List of metabolites produced by algae when exposed to selected pharmaceuticals, with mechanisms likely mediated by inherent P450 enzymes present in each alga.

[Table omitted. See PDF]

However, there are several challenges associated with implementing genetically modified microalgae in wastewater treatment on a large scale. One challenge is the optimization of genetic engineering techniques to ensure stable and efficient expression of P450 enzymes in microalgae. This requires identification of the most suitable enzyme variants and optimizes their expression levels. However, once overcome, this challenge presents an opportunity to tailor or condition microorganism species or communities to selectively address pharmaceuticals that pose greater difficulties at specific sites than others.

Another challenge is the scalability of microalgae cultivation and wastewater treatment systems. Designing and operating large-scale bioreactors or open pond systems that can support the growth of genetically engineered microalgae requires careful engineering and consideration of factors such as light availability, nutrient supply, and contamination control (Borowitzka & Vonshak, 2017). Furthermore, the potential release of genetically modified microalgae into the environment raises concerns about ecological impacts. Adequate risk assessments and containment strategies must be implemented to prevent unintended consequences, such as the spread of genetically modified organisms to natural ecosystems (Sayler & Ripp, 2000). Potential alternatives for the use of genetically modified microalgae include the use of specific enzymes ex vivo (enzymatic bioremediation) and enzyme immobilization (Al-Maqdi et al., 2021).

Despite these challenges, the future prospects for utilizing P450-engineered microalgae in wastewater treatment are promising. To address limitations and optimize the efficiency and cost-effectiveness of the technique, different strains of microalgae may be explored, novel genetic engineering techniques employed, and the understanding of the metabolic pathways involved in pollutant degradation leveraged. Moreover, the development of integrated systems that combine microalgae-based wastewater treatment with other complementary technologies, such as anaerobic digestion or membrane filtration, could enhance the overall effectiveness and economic viability of the process and release the burden on these other tertiary treatment processes. In conclusion, investing in this area of study is crucial to address the increasing global water pollution problem and move towards more environmentally friendly and efficient wastewater treatment processes.

AUTHOR CONTRIBUTIONS

Thamali Kariyawasam: Writing – original draft; writing – review and editing; investigation; data curation. Christian Helvig: Conceptualization. Martin Petkovich: Conceptualization; supervision; funding acquisition; project administration. Bas Vriens: Conceptualization; funding acquisition; writing – review and editing; project administration; supervision.

FUNDING INFORMATION

This research was supported by a New Frontiers in Research Fund – Exploration 2020 (NFRFE-2020-00832), Canada.

CONFLICT OF INTEREST STATEMENT

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.