Abstract
Microalgae are naturally rich in carbohydrates, lipids, proteins, pigments, minerals, and vitamins. These metabolites make them a renewable and sustainable source of bioactive compounds in nutraceuticals, food, feed for aquaculture, and biofuels. Genetic engineering of the existing strains remains crucial in utilising and upscaling microalgae-based biorefineries. CRISPR/Cas9, TALENs, ZFNs, and genetic transformation are the established tools utilised in microalgae genetic engineering. The common genetic transformation methods include electroporation, particle bombardment (biolistics), glass-bead agitation, and Agrobacterium-mediated transformation. Expression vectors and promoters are the prerequisites in genetic engineering. The present accessibility of genome sequences and omics datasets from a diverse array of microalgae species holds promise for catalyzing strategic progress in developing a superior microalgae strain suitable for numerous applications. This paper describes the genetic engineering to enhance microalgae biomass and metabolite production, particularly lipids. The advantages and precautions to fulfil the future application of genetically engineered microalgae are also reviewed and addressed.
Article highlights
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Strategic genetic engineering optimises microalgae strains for enhanced lipid synthesis and biomolecule production.
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CRISPR/Cas9 enables precise genome modifications in microalgae, enhancing their potential for various applications.
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Establishing regulatory frameworks for genetically modified (GM) microalgae is crucial for safe deployment.
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1 Introduction
Microalgae, a diverse group of photosynthetic microorganisms, inhabit various aquatic environments, including oceans, rivers, and freshwater systems. These organisms, typically ranging from a few to several hundred micrometres, lack conventional plant structures such as roots, stems, and leaves. Instead, they possess chloroplasts and other cellular organelles that facilitate photosynthesis, converting carbon dioxide into valuable organic compounds. These compounds have potential applications in producing bioactive substances for food, feed, and biofuel industries.
Regarding taxonomy, traditionally, microalgae are broadly classified into prokaryotic and eukaryotic categories. Prokaryotic microalgae are represented by Cyanobacteria (Cyanophyceae), while eukaryotic microalgae encompass a wider diversity, classified into seven distinct phyla: Euglenozoa, Cryptista, Haptophyta, Heterokontophyta, Glaucophyta, Rhodophyta, and Chlorophyta [1, 2]. This rich taxonomic and evolutionary diversity poses unique challenges in cultivation and genetic manipulation.
Microalgae stand at the forefront of sustainable biocompound production, owing to their capability to synthesise a broad spectrum of metabolites such as proteins, carbohydrates, amino acids, lipids, polysaccharides, fatty acids, vitamins, minerals, and pigments, including carotenoids, chlorophyll, and phycocyanin. Their rapid growth rate and minimal land requirements offer a significant advantage over terrestrial plants in producing renewable, sustainable, and cost-effective bioactive compounds. This attribute positions microalgae as a promising, sustainable source of food and non-food products. Recent studies have highlighted their potential in producing nutraceuticals [3,4,5], aquaculture feeds [6,7,8], and biofuels [9,10,11], as depicted in Fig. 1. Cultivating microalgae requires sufficient light, nutrients, and carbon dioxide, but notably, they do not necessitate fertile soil, thereby mitigating land irrigation concerns.
Some microalgae species exhibit robust growth under extreme environmental conditions and limited nutrient availability. The feasibility of cultivating microalgae in wastewater and recycled water further underscores their adaptability. This cultivation approach not only treats wastewater but also generates biomass suitable for biodiesel production. Studies have indicated that using recycled water for microalgae cultivation can significantly save water usage by up to 84% and nutrient consumption by 55% [12].
Moreover, microalgae's ability to utilise carbon dioxide from industrial flue gases offers an economical approach to cultivation. [13] have demonstrated that this method can reduce cultivation costs without compromising biomass yield or bio-crude quality. This dual capacity of microalgae to serve both as an effective carbon sink and a source of valuable bioactive compounds highlights their integral role in sustainable biotechnology and environmental management. Despite the myriad benefits of microalgae in producing bioactive compounds, notable challenges impede the commercialisation of microalgal-based products. Key among these is the need for strain improvement to enhance microalgae-derived products' metabolic efficiency and economic viability. Genetic engineering emerges as a pivotal solution to these challenges, enabling the consistent production of high-yield microalgal biomass.
Microalgae produce various biomass, lipids [14], fatty acids, carotenoids, and hydrocarbon profiles [15]. Microalgae are also recognised as the next-generation sustainable feedstock in aquaculture [16] especially due to the high accumulation of lipids. To sustain aquaculture industries, lipids sources are important for the growth and development of aquatic animals. Lipid accumulation in microalgae is usually affected by growth phases [17] and cultivation conditions, including nutrients, light intensity, and temperature. In most microalgae species, lipids are highly synthesised when microalgae achieve a stationary phase of growth [18]. In addition, microalgae lipids are considered renewable energy resources with zero net carbon emissions, as microalgae can convert atmospheric carbon dioxide into biomass and other nutrition compounds through the photosynthesis process [19]. The ability to reproduce fast, higher lipids than terrestrial plants [20] and carbohydrate productivities cause microalgae to have high commercial potential [21, 22].
Genetic engineering, as defined by [23], involves integrating foreign genetic material into the genome of a host organism, resulting in genetic modifications. This technology has proven instrumental in enhancing microalgae strains through sophisticated molecular genetics techniques, significantly boosting productivity. The evolution of genome editing technologies has now made it feasible to target and directly modify the genomic sequences of almost all eukaryotic cells, including those of microalgae.
This review delves into the molecular tools and techniques employed in microalgae to develop genetically engineered strains. It comprehensively covers the range of genetic modifications applied to microalgae, examining their efficacy and potential in strain enhancement. Furthermore, the review addresses the risks linked with genetically modified microalgae, including ecological and health concerns, and outlines strategies for effective risk management. The potential of bioengineering in microalgae, especially in the context of sustainable and efficient production of bioactive compounds, forms a core part of this discussion. This approach holds promise for industrial-scale production and contributes to the broader goal of sustainable biotechnology.
2 Molecular tools for genetic engineering in microalgae
The development of microalgae as a viable model for producing customised bioproducts necessitates a comprehensive molecular toolkit capable of precisely manipulating their genomic architecture. Such advancements are crucial for harnessing the full potential of microalgae in biotechnological applications, enabling the production of superior bioproducts. In contrast to microorganisms like yeast and bacteria, which have a wide array of engineering tools for industrial product synthesis, microalgae require a more specialised set of molecular tools. [24] highlight the disparity in the availability of genetic engineering resources between these organisms. The recent progress in identifying and refining novel genetic elements and transformation techniques positions microalgae as increasingly competitive candidates in the biotechnology sector, as illustrated in Fig. 2. This section underscores the significance of expression vectors and promoters, which are fundamental to the genetic engineering process.
2.1 Expression vectors
In the realm of synthetic biology, the construction of expression vectors is a pivotal process, enabling the manifestation of desired phenotypes and functions in model organisms. These vectors, primarily derived from plasmids, are engineered to drive the expression of target genes, thus demonstrating specific phenotypes and functionalities in model organisms. Highlighting this critical role [25] underscores the foundational impact of synthetic biology in facilitating the creation of these essential tools. The advent of the Golden Gate Modular Cloning toolkit (MoClo), as reported by [26], represents a significant advancement in this domain. This toolkit simplifies the creation of genetic vectors through a library of genetic blocks, guiding both the design and assembly processes. Moreover, it provides a robust framework for developing long-term synthetic biology systems.
The impact of such toolkits on the field of microalgae biotechnology has been profound, as indicated by [27, 28]. The MoClo toolkit, first developed for Chlamydomonas reinhardtii by [29], incorporates over 100 gene pieces, encompassing a wide range of genetic elements such as terminators, promoters, tags, untranslated regions (UTRs), antibiotic-resistance genes, introns, and reporters. This toolkit allows for the assembly of C. reinhardtii codon-optimised gene expression vectors, which are highly effective in gene expression.
In pathway engineering, where the orchestration of multi-gene transcription is essential for effective biosynthesis, the role of enzymes is critical. Recent advancements in gene stacking technologies have begun to address challenges of predictability and efficiency in metabolic engineering [28]. The integration of sequence optimisation algorithms with computational methods has shown efficacy in boosting the efficiency of gene expression vectors [30]. For instance, the modification of nuclear gene expression in C. reinhardtii to produce different synthetic enzymes resulted in expression variability up to 65-fold [28, 31], underscoring the importance of precise codon optimisation for achieving high levels of transgenic expression [31].
The development of the Chlamys Sequence Optimizer (CSO) by [32, 33] is a testament to these advancements. The CSO, which is freely available, enables users to choose from three experimentally validated codon-optimising algorithms. Integrating modular tools with expression vector creation methods significantly accelerates the engineering process, allowing researchers to concentrate on developing and scaling new microalgal products [33]. It also facilitates faster screening and enhances the expression rate of recombinant proteins and natural metabolites, thanks to a standardised set of resources and molecular toolkits [28]. As the repertoire of gene sections expands, it is anticipated that the scientific toolkits for microalgae will continue to evolve, opening new avenues in microalgal biotechnology.
2.2 Promoters
The efficacy of metabolic and recombinant product yields hinges significantly on identifying and utilising cis-regulatory elements, particularly promoters, that can drive optimal expression of transgenes [28]. A promoter, a specific DNA segment, serves as the binding site for RNA polymerase and transcription factors, thereby initiating transcription. High gene expression levels have been observed with the use of promoters from prevalent microalgae production strains [34], such as the chimeric HSP70/rbcs2 promoter (commonly known as ar1) from C. reinhardtii [35] and the fcp promoters in Phaeodactylum tricornutum.
However, transgene silencing is a major obstacle in achieving commercially viable amounts of recombinant proteins in microalgae, historically attributed to the organism's defensive mechanisms against viral invasions [36, 37]. In C. reinhardtii, for instance, transgene silencing has been linked to various factors, including the gene Mut6p, which is thought to be involved in post-transcriptional silencing through DNA degradation [38]. Additionally, certain protein factors in C. reinhardtii can induce chromatin compaction at insertion sites, further contributing to transgene silencing.
Recent advancements in microalgal transgene optimisation have concentrated on refining transformation processes to ensure the insertion of single-copy transgenes, thereby minimising silencing risks. A notable development is the use of the HSP70A promoter, which has been effective in preventing transgene silencing, as reported by [39]. Further enhancements have been achieved using high-expression promoters and flanking sequences derived from endogenous genes in Chlamydomonas, such as the Hsp70A-RbcS2 chimeric promoter and various inducible systems including copper-, nitrate-, heat-, light-, and alcohol-inducible promoters [40,41,42,43,44]. Innovative efforts have also led to the development of synthetic microalgal promoters (SAPs) in C. reinhardtii, spearheaded by [28, 45]. These SAPs, such as sap11, incorporate novel DNA motifs crucial for promoter function and display enhanced performance over the strongest known endogenous promoters. These advances not only boost the expression levels of transgenes but also offer insights into the structural dynamics of green microalgae promoters, marking significant progress in the field of microalgal biotechnology.
The approach taken by [46] involved using native cis motif elements, shape, and overall nucleotide contents from C. reinhardtii's top gene-generating genes to create these promoters. The potential for future SAPs libraries to yield further advancements in this field is substantial. These libraries have the potential to enhance the precision of gene alterations in microalgae, thereby greatly broadening the variety of promoters accessible in the genetic toolbox for primary microalgal production [47]. This area of research is poised for continued growth, augmenting the capabilities and efficiency of genetic engineering in microalgae.
2.3 Transcriptional engineering
Transcriptional engineering, a vital aspect of genetic manipulation, entails the regulation of multiple genes within a metabolic pathway through the engineering of transcriptional elements, notably transcription factors (TFs). TFs exert control over gene expression by binding to specific DNA sequences and interacting with RNA polymerase, thereby modulating transcription levels either upwards or downwards [48]. Unlike the targeted action of individual genes, TFs have the capacity to concurrently influence various segments of a metabolic pathway, thus offering a more holistic approach to genetic regulation [28].
The potential of transcriptional engineering has been exemplified in vascular plants, where it has been successfully employed to enhance anthocyanin levels [28]. In microalgae, most TFs have been identified serendipitously, with many yet to be characterised in terms of their specific functions [49]. However, insights from RNA sequencing data suggest that transcriptional regulatory networks based on TFs could effectively control multiple elements and binding sites of cellular metabolism [50]. [50] advocate applying this genetic engineering approach to augment the synthesis of various compounds in microalgae.
An example of a key transcription factor group in microalgae is the basic leucine zipper (bZIP) transcription factors, known for regulating metabolic processes like lipid production and contributing to stress responses. Modifying bZIP transcription factors presents a promising strategy for enhancing lipid production in microalgae species such as Nannochloropsis sp., as demonstrated through both endogenous and exogenous TFs [51].
In Arabidopsis thaliana, the AP2-type transcription factor WRINKLED1 (AtWRI1) is critical in regulating lipid accumulation [52]. When subjected to osmotic stresses, transformed cell lines expressing AtWRI1 exhibited a 44% increase in lipid content compared to wild types and a 36% increase under normal conditions. This finding underscores the potential of employing plant-derived TFs for heterologous gene expression in microalgae, indicating the scope for additional heterologous genetic elements [52].
The advancement of transcriptional engineering in microalgae hinges on the identification of more TFs involved in specific metabolic pathways, understanding their homologs across different microalgae species, and the establishment of standardised protocols for transcriptional engineering across species [53]. These steps are essential for harnessing the full potential of TFs in regulating gene expression, thereby enhancing the biosynthesis of desired products in microalgae.
2.4 Genome editing tools (CRISPR/Cas9, TALENs, and ZFNs)
The expansion and refinement of the biotechnology toolkit, particularly in the realm of genome engineering, represents a monumental leap in the field of microalgae research. Over the past decade, significant strides have been made in enhancing the array of tools available for the genetic engineering of microalgae, thereby bolstering their viability as a model organism for both scientific research and industrial applications.
One of the most pivotal advancements in this domain is the development of various genome editing technologies, each offering unique benefits and limitations, as summarised in Table 1. These include the Clustered Regularly Interspaced Short Palindromic Repeats with associated protein (CRISPR/Cas) systems, Transcription Activator-Like Effector Nucleases (TALENs), and Zinc-Finger Nucleases (ZFNs) [54]. These methods primarily operate by triggering DNA double-strand breaks, which then activate the cell's inherent DNA repair mechanisms. This mechanism can be utilized to either disable targeted genes or introduce donor DNA for transgene integration, as depicted in Fig. 3 [55].
TALENs are engineered tools that combine the FokI cleavage domain with DNA-binding domains from TALE proteins. These transcription activator-like effectors (TALE) proteins have repeat domains, each recognising a single base pair. TALENs create targeted double-strand breaks (DSBs) in DNA, activating repair pathways that allow precise genome editing [54, 56]. Essentially, the TALENs system acts as a custom-made restriction enzyme that can target specific sites on the genome. In a recent study, researchers used both meganucleases and TALE nucleases to transform the diatom P. tricornutum. They successfully edited seven genes important for lipid synthesis [57], leading to improved oil production in the modified strain. This demonstrates the potential of TALENs for precise genetic modifications and enhancing desired traits in microalgae.
ZFNs are engineered tools that combine the nonspecific DNA cleavage domain from the FokI restriction enzyme with zinc-finger proteins. Similar to TALENs, ZFNs create targeted DSBs in DNA, activating DNA damage response pathways. The zinc-finger domains are designed to guide ZFNs to specific genomic sites, allowing precise genome editing [54, 56]. By engineering these domains to target precise DNA sequences, researchers successfully applied ZFNs to modify the gene encoding channelrhodopsin-1 in C. reinhardtii [58].
Among these, CRISPR/Cas9 has emerged as a particularly transformative tool. It functions as a site-directed gene editor, enabling precise genome modifications in microalgae. This technology is derived from a bacterial immune response, where a single guide RNA (sgRNA) directs the Cas9 protein to target and cleave exogenous DNA sequences. This method has been effectively adapted for genetic engineering in microalgae [23, 59], offering a high degree of control over gene expression and addressing issues such as transgene silencing.
Implementing CRISPR/Cas9 and related technologies in microalgae genetic engineering has led to significant breakthroughs, extending the scope of possible modifications. This technique is applicable to microalgae and a wide range of microorganisms, revolutionising the field of genetic modification [23]. The advancements in CRISPR/Cas9 technology have pushed the boundaries of gene modification and device development, allowing for altering a diverse array of eukaryotic genomes [62].
The application of CRISPR/Cas9 technology in microalgae has marked a significant advancement in genetic engineering, particularly in diatoms like P. tricornutum. [63] highlights its critical role in both gene function analysis and the enhancement of industrial traits such as increased lipid content and biomass production. [64] demonstrated how CRISPR/Cas9 can be used to achieve stable genotype alterations in P. tricornutum, maintaining the modified genotype through successive generations. Further, the CRISPR-based approach has been employed to target the CpSRP54 gene, which is instrumental in indirect carbon fixation processes in P. tricornutum. This modification, as explored in studies including [65], underscores the potential of CRISPR/Cas9 to fine-tune metabolic pathways that are crucial for improving carbon utilisation and storage, thereby enhancing the organism's utility in industrial applications.
However, challenges have arisen in applying CRISPR/Cas9 in microalgae. The initial attempts at employing CRISPR/Cas9 in microalgae in 2014 encountered issues, such as Cas9 toxicity in C. reinhardtii when the CRISPR/Cas9 gene was constitutively expressed [66, 67]. Despite these hurdles, [28] reported successful CRISPR-mediated nucleotide replacement in C. reinhardtii, albeit with the persistent issue of Cas9 toxicity. Using oligonucleotide-directed mutagenesis (ODM) techniques by [66] also achieved genome modification in C. reinhardtii, although the mutagenic rates were low.
CRISPR-mediated gene modification in microalgae has primarily focused on producing gene knockout mutants instead of inserting protein-encoding genes [28]. While it has been a valuable tool in metabolic engineering to boost the production of natural microalgal chemicals, its use in generating high-value recombinant proteins has been limited. This limitation stems from C. reinhardtii's (a commonly used strain for producing recombinant proteins) insufficient homologous recombination (HR) pathways for homology-mediated editing [28].
Genome editing in microalgae typically involves recombinant nuclear nucleases that recognise specific sequences and create double-strand breaks (DSBs) in the DNA [68]. Non-homologous end joining (NHEJ) repairs these breaks, often resulting in indel mutants within the target genes [69]. On the other hand, HR is a more precise mechanism involving the exchange of information between two identical sequences and is essential for inserting foreign DNA into specific genome locations. Due to the lack of HR in C. reinhardtii, NHEJ-mediated gene modification has been more prevalent [70].
Future research is expected to advance HR-mediated gene editing in C. reinhardtii [71]. Such developments would significantly enhance the prospects of protein engineering in microalgae, opening new avenues for producing complex and high-value recombinant proteins, thereby expanding the scope and impact of microalgal biotechnology.
2.5 Genetic transformation techniques
In the field of microalgal genetic engineering, the introduction of optimised expression vectors into the microalgal genome is a critical step. This process requires techniques that ensure high efficiency and minimise the time spent on post-transformational screening [28]. Several genetic transformation techniques have been developed and successfully applied to microalgae, including electroporation, biolistic particle bombardment (biolistics), glass bead agitation, and Agrobacterium-mediated transformations [72]. Each of these methods has its own set of advantages and disadvantages, as detailed in Table 2.
Electroporation is the most efficient and commonly used method for nuclear transformations in microalgae, particularly C. reinhardtii [73]. This technique is favoured for its relative ease of use and high efficiency, capable of producing a significantly higher number of successful transformants with fewer false positives than other methods [74]. Electroporation involves the application of an electric pulse to temporarily permeabilise the cell membrane, allowing foreign DNA to enter the cells. To optimise the electroporation process, researchers have developed tools and methods for fine-tuning the parameters involved. [75] highlighted the importance of optimising this procedure for maximum efficiency. Additionally, [76] identified optimal conditions for electroporation, including light intensities, voltage settings, and cell concentrations. These findings provide valuable insights for determining the most effective experiment conditions.
The integration of the plastid genome via electroporation, coupled with a nuclear-targeted construction, has marked a milestone in the field of microalgal biotechnology, notably with the first successful chloroplast transformation of Nannochloropsis sp. [28]. However, the transformation of chloroplasts in Nannochloropsis sp. poses challenges due to the organism's small size and the yet-to-be-characterised components of its cell wall, suggesting avenues for further methodological improvements [77]. Despite these challenges, electroporation remains the predominant method for transforming various microalgal species [78]. Nonetheless, alternative transformation methods may be more effective for certain taxa. For instance, [79] reported that haptophytes had been successfully transformed using both biolistic and Agrobacterium-mediated approaches, with biolistic yielding the most favourable results.
In the diatom P. tricornutum, the biolistic delivery of CRISPR/Cas9 ribonucleoproteins facilitated the creation of double-gene knockouts of a photoreceptor-encoding gene with an efficiency of 65–100%, establishing a DNA-free editing system for the species [82]. Additionally, the glass bead agitation method, which involves vortexing microalgae cells with glass beads, has been employed to transform artificial microRNA into C. reinhardtii, aiming to regulate gene expression and enhance fatty acid content. This method is noted for its simplicity and the absence of specialised equipment requirements, although it only applies to cells with compromised cell walls or strains pretreated with autolysin [28].
Agrobacterium-mediated transformation has demonstrated higher success rates in generating transformants compared to the glass bead method, as indicated by [83]. This method, however, requires the co-culture of transgenic bacteria with microalgae. In response, more streamlined techniques, such as electroporation, have been developed to offer simpler, cost-effective alternatives [84]. Although Agrobacterium-mediated transformation generally results in fewer transgene-silenced transformants, it does not significantly outperform electroporation in Chlamydomonas, where it is often more labour-intensive and prone to generating false positives [85].
The variety of available transformation techniques underscores the necessity of selecting an appropriate method tailored to the unique requirements and characteristics of the target microalgal species. Ongoing refinement and adaptation of these methods are critical for the progressive capabilities of microalgal genetic engineering, opening new avenues in biotechnological applications.
3 Genetic engineering of microalgae for enhanced biorefinery capabilities
The genetic engineering of microalgae to enhance biorefinery capabilities represents a transformative approach to sustainable biotechnology. Microalgae can be optimised through metabolic engineering to achieve higher productivity and efficiency, improving their economic viability and sustainability. Given the critical role of microalgae in the nutraceutical and food industries, aquaculture feed, and biofuel production, considerable attention has been focused on elucidating and manipulating their biosynthetic pathways [86].
The strategic application of genetic engineering in microalgae targets optimising lipid synthesis, biomolecule production, and photosynthetic efficiency, among other traits. This optimisation is achieved using various genetic engineering techniques tailored to the specific metabolic profiles and biorefinery potentials of different microalgal species. These approaches are crucial for enhancing the efficiency and economic viability of microalgae in biotechnological applications. The following are key examples demonstrating how genetic engineering has effectively improved microalgae for diverse biorefinery purposes:
3.1 Enhancement of lipid production
The advancement of lipid synthesis in microalgae via genetic engineering marks a crucial advancement for multiple sectors, especially in the production of third-generation biofuels and dietary supplements. The ability to boost triacylglycerol (TAG) and polyunsaturated fatty acid (PUFA) production, including omega-3 fatty acids, through metabolic engineering not only promises cost reduction but also presents a sustainable substitute for petroleum-derived goods and fish oils. These traditional sources are restricted by environmental and resource constraints [87,88,89].
Key enzymes in the lipid biosynthesis pathway, such as malonyl-CoA: ACP transacylase (MAT) and acetyl-CoA synthetase (ACS), have been identified as targets for metabolic engineering to increase the production of microalgal PUFAs to commercially viable levels [28, 90]. ACS plays a crucial role in lipid biosynthesis by converting acetate into acetyl-CoA, a fundamental molecule in fatty acid production [91]. Meanwhile, MAT is involved in forming malonyl-ACP intermediates, essential for both polyketide and fatty acid synthesis in species like Schizochytrium sp. [90].
Several innovative genetic strategies have been employed to boost lipid production in microalgae. In C. reinhardtii, the overexpression of endogenous chloroplast acyl-CoA synthetase (ACS) genes using the HSP70A and RBCS2 promoters has led to significant increases in ACS transcripts and triacylglycerol (TAG) accumulation, particularly under nitrogen-depleted conditions with acetate supplementation [91, 92]. Additionally, employing the TEF promoter to overexpress malonyl-CoA transacylase (MAT) has notably enhanced docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) yields [28]. Other species have also shown promising results. For instance, targeting ACCase in C. cryptica and N. saprophila increased enzyme activity, though it did not affect lipid content [93]. Conversely, targeting ACS genes in Schizochytrium sp. led to an 11.3% increase in fatty acids and a 29.9% increase in biomass [94]. In N. oceanica, manipulation of NoMCAT genes resulted in increases of 36% in total fatty acids and 31% in neutral lipids, alongside improved growth and photosynthetic efficiency [95]. Furthermore, targeting the PtTE gene in P. tricornutum enhanced the accumulation of shorter chain-length fatty acids, incorporating 75–90% into TAGs without significant fatty acid secretion [96]. Lastly, in C. reinhardtii, targeting DtTE genes led to a substantial increase in neutral lipids and total fatty acids without affecting growth [97].
These breakthroughs in genetic engineering underscore the potential for microalgae to serve as a scalable and sustainable source of essential omega-3 fatty acids like DHA and EPA, which are crucial for human health due to their roles in embryonic development and their anti-inflammatory properties [98]. The advancements in microalgal lipid production through genetic engineering promise to enhance microalgae's economic viability as a source of biofuels and nutraceuticals and contribute to sustainable practices in these industries. Further exploration and optimisation of genetic engineering techniques in microalgae are expected to continue advancing their role in biorefineries, making them an even more valuable resource for producing a wide range of lipid-based products. Examples of genetic engineering for enhanced lipid content in microalgae are shown in Table 3.
3.2 Enhancement of other biomolecules
The enhancement of carotenoid production in microalgae through genetic engineering holds significant promise for various applications, particularly in human health [123]. Carotenoids, a diverse group of pigments found abundantly in microalgae, are not only crucial for efficient photosynthesis by absorbing light energy and protecting chlorophyll but also offer potential health benefits, including the prevention of diseases such as lung cancer [124,125,126]. Given the well-characterised carotenoid biosynthesis pathway and the identification of its key genes, metabolic engineering techniques have been increasingly applied to boost carotenoid production in microalgae [127].
Phytoene synthase (PSY), which catalyses the initial step in carotenoid biosynthesis, is considered a rate-determining enzyme in the pathway, leading to the production of phytoene [128]. This is followed by the action of enzymes such as ζ-carotene desaturase (ZDS), phytoene desaturase (PDS), and carotene cis–trans isomerase (CRISCO), which convert phytoene into lycopene, a key intermediate for the synthesis of high-value carotenoids [73, 129]. By increasing the expression of genes upstream from lycopene synthesis, such as PSY and PDS, the production of carotenoids can be significantly enhanced. Studies have shown that regulating the PSY gene in microalgae species like P. tricornutum, Haematococcus pluvialis, and C. reinhardtii can increase carotenoid synthesis [130]. Similarly, manipulating PDS gene expression has been demonstrated to boost carotenoid production in various microalgae [131].
The process of converting lycopene into α- or β-type carotene and subsequently into lutein and zeaxanthin involves specific lycopene cyclases and carotene cyclases/hydrolases [132, 133]. While there is a scarcity of publications on the genetic engineering of these enzymes in microalgae, their regulation could potentially increase carotenoid production under certain conditions, such as nitrogen depletion.
Furthermore, the oxidation of β-carotene or zeaxanthin in species like H. pluvialis and C. zofingiensis can lead to the synthesis of higher-value biocompounds such as canthaxanthin, astaxanthin, or violaxanthin [134]. Enhancing or introducing the β-carotene oxygenase (BKT) enzyme, crucial for astaxanthin production, into model organisms has been a focus of research due to astaxanthin's applications in anti-inflammatory and skincare products [135, 136]. The successful introduction of BKT in C. reinhardtii, leading to astaxanthin production, exemplifies the potential of genetic engineering in this area [137].
While traditional approaches to increase carotenoid production have often relied on modifying culture conditions or inducing stress, the exploration of genetic engineering offers a more targeted and efficient strategy to enhance carotenoid biosynthesis in microalgae. As research continues to evolve, genetic engineering techniques are expected to play a pivotal role in maximising the production of carotenoids and other valuable biomolecules in microalgae, expanding their applications in health, nutrition, and beyond. Other examples of microalgae genetic engineering for enhanced carotenoids are given in Table 4.
3.3 Improvement of photosynthetic efficiency
The transformation of atmospheric carbon dioxide into energy-rich organic compounds is fundamental for sustaining life on Earth, with carbon fixation playing a pivotal role in this process. Carbon fixation involves the conversion of inorganic carbon from the atmosphere into organic compounds by autotrophic organisms, including photoautotrophic organisms like cyanobacteria, plants, and microalgae. These organisms utilise the Calvin cycle, among other mechanisms, to convert carbon dioxide and water into organic carbon, facilitated by chlorophyll [148,149,150].
Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo) is the primary enzyme responsible for carbon fixation, acting at the initial stage of the Calvin cycle. RuBisCo, consisting of large and small subunits encoded by chloroplast and nuclear DNA, catalyses carbon dioxide and ribulose-1,5-bisphosphate conversion into 3-phosphoglycerate. It also catalyses a side reaction converting oxygen and ribulose-1,5-bisphosphate into 2-phosphoglycolate, a metabolite detrimental to cell growth [151,152,153,154].
Efforts to genetically modify RuBisCo aim to enhance its selectivity and velocity, reduce unproductive side reactions, and improve overall activity. However, challenges remain in enhancing both selectivity and velocity simultaneously due to the inherent limitations of RuBisCo [155, 156]. The exploration of heterologously expressed RuBisCo variations, particularly in red microalgae, shows promise for boosting carbon fixation efficiency beyond the capabilities of native RuBisCo [157, 158]. In addition to RuBisCo optimisation, controlling metabolic flux and regulating the Calvin cycle pathway is crucial for enhancing carbon fixation. Strategies such as the bypassing of the photorespiratory pathway via the introduction of phosphoglycolate rerouting enzymes can lead to increased carbon fixation rates and biomass formation, highlighting the significance of metabolic flux regulation in optimising photosynthetic efficiency [73, 159, 160].
Furthermore, the overexpression of key Calvin cycle enzymes, such as sedoheptulose-1,7-bisphosphatase, has been identified as a method to boost photosynthetic efficiency and carbon fixation, underscoring the potential for targeted genetic interventions to improve carbon fixation efficiency [161, 162]. However, the enhancement of carbon fixation does not solely rely on modifying individual enzymes within the Calvin Cycle, as the overall balance of flux through the cycle is essential for achieving high microalgae biomass, as shown in Fig. 4. These advances in genetic engineering and metabolic pathway optimisation offer promising avenues for increasing the efficiency of carbon fixation in microalgae, contributing to the development of more sustainable and productive systems for converting atmospheric carbon dioxide into valuable organic compounds.
4 Management of genetic engineering in microalgae
Managing genetic engineering in microalgae needs to be considered to ensure safety, efficacy, and ethical practices. Even though genetic engineering is one of the most powerful technologies in improving the production value of microalgae metabolites, it leads to divergent opinions on the safety of genetically modified microalgae for human health and the environment [163].
Despite the potential of genetic engineering to enhance microalgal performance, there are significant environmental and ecological concerns associated with these technologies that are yet to be fully debated. Globally, the deployment of transgenic and recombinant microalgae is governed by stringent regulations and policies, ensuring careful assessment and management of any associated risks [164, 165]. This regulatory framework is crucial as it not only safeguards biodiversity but also ensures that technological advancements in microalgal research are responsibly aligned with policy developments. Moreover, while international collaborations continue to drive forward the genetic enhancement of microalgae, the translation of these advancements into commercial applications remains restricted. [155] note that, despite extensive research efforts, commercial exploitation of genetically modified microalgae is still largely prohibited, underscoring the need for a balanced approach that weighs innovation against potential environmental impacts.
Navigating the realm of genetic engineering in microalgae requires a careful balance of its potential advantages against possible risks, with a strong emphasis on ethical and regulatory considerations. Adopting a cautious and responsible approach towards genetic modifications in microalgae is crucial. The application of genetically modified microalgae in fields like agriculture and biotechnology draws significant attention, yet it simultaneously raises concerns regarding safety for the environment, animals, and humans—all primary consumers of microalgae products [60]. Therefore, establishing rules and regulations concerning genetically modified microalgae is vital for setting clear guidelines to safeguard the environment, animal welfare, and public health. In various countries, these rules and regulations are critical in overseeing genetically modified microalgae research, development, and commercialisation [163]. These regulations typically address risk assessment, labelling, containment, and monitoring strategies, ensuring that genetically modified microalgae are managed carefully to prevent adverse effects.
Regulatory frameworks overseeing genetically modified microalgae vary significantly among different nations, particularly concerning their use in food or feed and their release into the environment. In the United States, the oversight of genetically modified microalgae is overseen by three primary agencies: the United States Department of Agriculture (USDA), the Food and Drug Administration (FDA), and the Environmental Protection Agency (EPA). These agencies are responsible for ensuring public health and environmental safety in relation to genetically modified organisms (GMOs). Conversely, in the European Union (EU), regulation is managed by the European Commission, the European Food Safety Authority (EFSA), and individual EU Member States. India's regulatory approach to GMOs is led by the Genetic Engineering Approval Committee (GEAC), situated within the Ministry of Environment, Forest and Climate Change (MoEFCC) [166, 167].
Further illustrating the global landscape (Table 5), countries like Australia, India, Brazil, and Argentina have established dedicated authorities for managing GMOs, including genetically modified microalgae. Meanwhile, nations like Malaysia, while not having specific legislation for genetically modified microalgae, manage GMOs within the broader context of the National Biosafety Board's biosafety and environmental protection frameworks [168]. These regulatory measures are foundational to ensuring the responsible development of genetically modified microalgae and their integration into biotechnological and sustainable development initiatives.
As the field of genetic engineering continues to evolve, so will international regulatory frameworks. These adjustments are essential for addressing both the emerging challenges and opportunities presented by GM microalgae, ensuring their ethical advancement and application in a manner that aligns with global standards for safety and sustainability.
5 Risk and disadvantages
The exploration of genetically modified microalgae as a solution for bioindustrial challenges must be approached with caution, given the substantial risks associated with their proliferation and interaction with natural ecosystems. The absence of widespread outdoor cultivation of genetically modified microalgae likely stems from concerns over the unpredictability of their effects in open cultivation systems, a caution underscored by research from [155, 169]. The primary apprehensions involve the potential for genetically modified microalgae to outcompete native species due to their enhanced adaptability, leading to possible ecological disruptions [170]. This competitive edge necessitates implementing stringent biosecurity measures to mitigate the risks of introducing genetically modified strains into local environments [169].
Moreover, the risk of gene flow between genetically modified microalgae and sexually compatible wild species introduces additional complexities, potentially resulting in unforeseen environmental impacts [171]. Therefore, legislation related to genetically modified microalgae must prioritise the protection of local ecosystems through comprehensive biosecurity protocols that guard against the release of foreign species. The ecological considerations extend beyond the mere presence of genetically modified strains, demanding an analysis of their interaction with and impact on the stability of native microalgal communities [172].
A cost–benefit analysis is needed to understand better the use of genetically modified microalgae [173]. Biosafety regulations for genetically modified microalgae must include strict monitoring and assessment of cultivation, handling, and health risks [174]. Genetically modified microalgae can be seen as a solution for the techno-economic problems of the microalgal industry. Therefore, it is essential that all parties, including legislators and business advocates, work out a plan for future genetically modified microalgae use. According to [175], various federal governments worldwide must establish laws and regulations that govern the safe use of genetically modified microalgae for human and ecological benefit. In summary, while genetic engineering in microalgae holds great promise, it is crucial to proceed cautiously and carefully consider the potential risks and disadvantages before deploying this technology in real-world applications.
6 Conclusion and future prospects
Microalgae represent a promising, sustainable biomanufacturing platform for producing economically valuable compounds with potential for long-term viability. The business model surrounding microalgae necessitates the development of processes that are not only financially appealing but also environmentally sustainable, alongside achieving higher levels of technological readiness. The genetic engineering aimed at producing robust microalgal strains is at the heart of enhancing the economic viability of microalgal processes.
Advances in high-throughput technology and molecular biology tools have aided the biotechnological approach to improve the performance of microalgae strains. New advancements have significantly expanded the molecular toolkit and simplified the engineering process, enabling more rapid study and development in this sector. Improved strains of eukaryotic microalgae are being developed for the cost-effective generation of biofuels and bioproducts. Combining microalgal multi-omics datasets and modern molecular techniques provides a strategic roadmap for strain enhancement. Progress in this research will inevitably result in strains that can be used to make beneficial industrial products. However, the economic feasibility of genetically altered strains will be decided by their safety for human health and the environment once they are produced. As a result, strict controls and monitoring are advised to evaluate the environmental and human health concerns of using GM microalgae.
Data availability
The authors confirm that the data supporting the findings of this study are available in the article.
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Acknowledgements
Ahmad Hanafi Ahmad Kamal received the Sultan Ismail Nasiruddin Shah Terengganu Scholarship, and Muhammad Faiz Zakaria received the Universiti Malaysia Terengganu Scholarship (BUMT) award. The graphical figures were created with https://www.biorender.com.
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This research was funded by TAPE-RG (Grant no. TAPERG/2022/UMT/1441).
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Conceptualisation: Malinna Jusoh and Aziz Ahmad; writing original draft: Ahmad Hanafi Ahmad Kamal, Nor Farahdevah Mohd Hamidi and Muhammad Faiz Zakaria; preparing figures and tables: Ahmad Hanafi Ahmad Kamal; review and editing: Malinna Jusoh, Aziz Ahmad, Mohd Razif Harun and Thirukanthan Chandra Segaran; funding acquisition: Malinna Jusoh.
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Ahmad Kamal, A.H., Mohd Hamidi, N.F., Zakaria, M.F. et al. Genetically engineered microalgae for enhanced bioactive compounds. Discov Appl Sci 6, 482 (2024). https://doi.org/10.1007/s42452-024-06116-5
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DOI: https://doi.org/10.1007/s42452-024-06116-5