The application of genetic and metabolic engineering can be used to enhance the production of desired metabolites, which has ceased due to the inherent limitation of metabolic capacity. As microalgae are a very diverse group and source of numerous metabolites, the latest biotechnological approach can be used as an opportunity to enhance the production [48]. Previously, with the help of mutagenesis and genetic engineering production of different microbial carotenoids has been enhanced [48,134]. Conventionally to improve the strains or to achieve a high yield of carotenoids, microalgal strains are subjected to mutagenesis [135]. Although different mutagens are used to develop the desired strains, mutagenesis via radiation is found to be a suitable approach to develop high-yield strains [136]. In a study, Dunaliella bardawil, after exposure to UV radiation induced the production of β-carotene in the mutant strain; similar results were reported with the strain of Scendesmus and Chlorella [137,138]. In another study, mutagen N-nitro-N-nitrosoguanidine has been reported to enhance astaxanthin production in Haematoccus pluvialis [139]. In a study, Yi et al. [140] use UV radiation to induce mutation in the Phaeodactylum tricornutum to enhance the production of carotenoids. Similarly, Sivaramakrishnan and Incharoensakdi [141] also used UV radiation to induce mutagenesis in Scenedesmus sp. to enhance the production of lipids. Further, Trovão et al. [142] briefly discussed random mutagenesis to improve the microalgal strain. 5.1. Metabolic and Genetic EngineeringMetabolic engineering refers to deliberate modulation in the metabolic pathway of an organism with the objective of producing desired molecules important as medicine, fuel, and pharmaceuticals for commercial application [143,144,145]. Metabolites are a diverse array of intermediate and final products in biosynthetic pathways. Few metabolites are known to regulate enzymatic activity [146]. Therefore, understanding the synthesis of cellular metabolites under given environmental conditions could help in manipulating the desired pathway in selected microalgae. The important information regarding the metabolites in terms of quality and quantity has been made quite possible by different forms of advanced mass spectroscopy in spite of considerable variations in chemical attributes, including molecular weight, polarity, volatile nature, and miscibility. So far, most of the microalgal metabolic engineering research have focused on lipid profiling under changing environmental parameters. Very limited information on carotenoid synthesis is available. The in situ investigation of single isolated cells using single-probe mass spectrometry could unravel the diversity of hidden metabolites more effectively under a given set of conditions [147]. This technique holds promise in future research rendered by minimized experimental errors and cellular complexities. Contrary to genomic and transcriptomic databases, the microalgal metabolite database is largely lacking, suggesting extensive research. Although metabolic models relying on the genome are available, however, experimental investigation-based metabolite information is limited. Some of the existing databases such as KEGG (https://www.genome.jp/kegg/pathway.html (accessed on 12 October 2022)), Reactome (https://reactome.org (accessed on 12 October 2022)), and Metacyc (https://metacyc.org (accessed on 12 October 2022)) containing useful metabolites information based on experiments as well as predictions may be considered for further exploration.The process followed to enhance the microalgal carotenoid production by metabolic engineering is similar to the strategies followed in higher plants. This includes the modulation and regulation of enzyme biosynthetic pathways and formation of the metabolic sink, enhancing particular metabolite flux by interfering with their cellular metabolic process. However, this strategy needs extensive knowledge of biosynthetic enzymes and necessary information on how to modulate the cellular metabolism to enhance the flux [148].The overexpression of particular enzymes, which can control the flux of particular products, or the enzyme present in the rate determination stage of the enzymatic reaction, can help achieve the enhanced desired metabolites without interfering with other metabolites. Thus, it is not mandatory to overexpress all the enzymes of the particular biosynthetic pathways to the same extent [149,150]. Although, it is not necessary that a single enzyme can control the flux of the particular desired metabolite. It is regulated through various enzymes by coordinated expression. Thus, during carotenoid synthesis, multiple enzymes must be overexpressed for enhanced production [151].Manipulated expression of genes, including PSY, PDS, BKY, OR, BCH, and LCY under the control of different promoters, responsible for carotenoid synthesis, has been demonstrated in different algal species mostly belonging to Chlamydomonas, Chlorella, Haematococcus, and Dunaliella [152]. Studies have reported various microalgal strains such as Chlorella zofingiensis and Haematococcus pluvialis, and Chlamydomonas reinhardtii, whose gene has been modified for regulating carotenoid production [153]. Recent investigations have reported some genes and their metabolic pathways required to regulate carotenoid synthesis. For example, strains Chlorella vulgaris, Chlamydomonas reinhardtii, and Volvox carteri have regulatory enzymes such as phytoene synthase, encoded by the PSY gene responsible for the synthesis and regulation [154,155]. Similarly, strain D. salina contains two classes of PSY gene families’ upregulated during stress conditions, resulting in enhanced production of carotenoids [156]. Similarly, Couso et al. [157] reported upregulation of carotenoid synthesis in the Chlamydomonas reinhardtii under stress. Vidhyavathi et al. [158] reported altered expression of the carotenoids synthesis gene in the Haematococcus pluvialis strain under nutrient-stress conditions.The principle behind any cell’s metabolic engineering is to understand the biochemical pathway of the cell in order to manipulate biochemical steps through modifying enzymatic activity, which leads to the creation of a sink, enhancing the flux to produce required metabolites [159,160,161]. The overexpression of the gene directing the synthesis of a particular enzyme leads to the production of the desired metabolite, not interfering with other metabolite production. Therefore, only the enzyme of the final step of any biochemical pathway required for the formation of the desired product may be taken into account for overexpression. For example, overexpression of the gene leading to the synthesis of the PDS enzyme, a rate-limiting step, is crucial for ζ-carotene production [162]. The role of different genes involved in carotenoid production is presented in Table 3.Nuclear overexpression of endogenously mutated PDS gene in the chloroplast may lead to enhanced astaxanthin production. A study conducted on Chlorella zofingiensis and Haematococcus pluvialis by performing overexpression of nuclear mutated endogenous gene PDS resulted in enhanced astaxanthin content by 26% and 32%, respectively [148,149]. A similar study was conducted in the chloroplast of Haematococcus pluvialis, which showed a 90% enhancement in astaxanthin content [150]. Moreover, it is a coordinated action of multiple enzymes whose overexpression results in the production of specific carotenoids. Even the downregulation of specific enzymes has helped to overexpress specific enzymes [151]. For example, in a study, the downregulation of the LCYE gene led to the overproduction of β-carotene. A study also reported that the ZEP gene knockout mutant, achieved through CRISPER-CAS9 in Chlamydomonas reinhardtii, resulted in higher zeaxanthin content than the wild type [178].Further study showed the integration of the bkt gene of Haematococcus pluvialis by using metabolic engineering in the chloroplast of Dunaliella salina, which leads to the production of astaxanthin [179]. However, the overproduction of several carotenoids is obstructed through feedback inhibition and encountered overexpression of feedback-resistant enzymes. The metabolic sink is considered a reservoir of specific metabolites, which are transported to the sites, and the overproduction of specific metabolites is inhibited through feedback inhibition. However, it has been observed that in microalgae, Haematococcus lipid act as a metabolic sink that regulates carotenoid production [180,181]. Similarly, Rabbani et al. [182] reported that the triacylglycerol deposition induced the β-carotene synthesis in Dunaliella bardawil. 5.2. Selection of Hosts and Transformation MethodsThe strategies of transformation play a significant role in the genetic manipulation of microalgae to achieve the desired products. Successful transformation based on electroporation, biolistic approach, polyethylene glycol, silicon carbide whiskers, glass bead, and conjugation been reported in the numbers of microalgae [183,184]. In addition, the selection of transformed cells involves the application of selectable markers, including resistance to antibiotics, herbicides, and auxotrophic markers. The selection of specific transformation techniques and markers, however, may vary from species to species. The considerable diversity in the cellular system of microalgae, nevertheless, limits the usefulness of microbe and plant-derived markers for selection [185]. However, such as other mechanisms, transformation techniques also have some merits and demerits. Such as rapid growth, reduced risk of contamination, uniformity (single cell microalgae proteins) in the products of microalgae has advantages over the plant system [186]. But the lesser efficacy of carotenoids productions in microalgal cells has been considered a a major disadvantage in comparison to plant systems. During the transformation, selection and identification of the host cell that will have incorporated with foreign genes and their ability of expression have been considered as a prime factor for successful transformation. The host structure and method of transformation also have a prominent role in transformation [187]. A general screening of transformants has been carried out with the addition of antibiotics or herbicides in the media, so optimization of culture media and sensitivity against the antibiotics or their concentration must be predefined [188,189]. The first successful nuclear transformation has been reported in the Chlamydomonas reinhardtii, a single-cell green alga and also considered a model organism because of its well-studied genomes [190]. Initially, the transformation has been executed using the biolistic approach, glass bead agitation, and electroporation [191,192,193,194]. In the recent past, various authors used transformation techniques to overexpress particular genes in microalgae. For example, Steinbrenner and Sandmann [150] improve the biosynthesis of astaxanthin by the transformation of phytoene desaturase in Haematococcus pluvialis. Kumari et al. [195] overexpressed the plant’s Brassica oleracea “OR” gene in C. reinhardtii. Simon et al. [163] reported enhanced production of violaxanthin and zeaxanthin after transforming the BCH gene of Chlamydomonas reinhardtii in Dunaliella salina.In spite of substantial achievement in transformation, low efficiency has been recorded in most algae except Chlamydomonas in comparison to plants. The direct transfer of bacterial vectors into diatom through conjugation has emerged as an efficient approach to transformation [184]. The technique bears multiple benefits over traditional methods, including the transfer of large-sized genomes, natural replication of episomal vectors, deletion of the transferred gene in the absence of selection pressure, and minimal chances of epigenetic influences [196]. Studies have presented the generation of trans-gene-free mutants in P. tricornutum and Nannochloropsis oceanica using conjugation-based genome editing mediated by CRISPR/cas9 [197,198]. The transformation efficiency was twenty to a hundred times greater. However, mutant visibility in transformants was considerably delayed owing to reduced Cas9 expression [197]. Therefore, successful transformation in suitable microalgal species relying on available methods is an important strategy for the enhanced production of desired carotenoids. Further improvements in this direction would raise the productivity of carotenoids for commercial application.