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Microalgal Genetic Engineering for Bioremediation of Heavy Metals: Comparison
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Please note this is a comparison between Version 2 by Saeed Ranjbar and Version 5 by Amina Yu.

MRecent advances encompassing molecular tools for genome editing, together with the advent of multi-omics technologies and computational approaches, have permitted the design of tailor-made microalgal cell factories with significantly improved heavy metal bioremediation capacity.

  • heavy metals
  • bioremediation
  • microalgae
  • genetic engineering
  • phycoremediation

1. Introduction

Introduction

Heavy metals (HMs) are an integral constituent of the biosphere; they are naturally recycled in the environment through various biotic and abiotic processes, as part of biogeochemical cycles [1][2][1,2]. However, the dramatic rise in urbanization and industrialization has led to the release of alarmingly toxic levels of HMs, along with many other organic and inorganic pollutants in the environment. Aside from geochemical processes beyond one’s control, as is the case of erosion, atmospheric deposition, infiltration, thermal spring activity, and volcanic eruptions, HMs are increasingly penetrating aquatic systems, as a consequence of a wide range of anthropogenic activities, for example.g., discharges of untreated effluents from mining, spontaneous leaching from intensive agriculture, petroleum refining, improved performance of petroleum-based fuels, refuse burning, electroplating, printing, power generation, and other such activities carried out by (fine) chemical and metallurgical industries in the manufacture of microelectronic devices, paints, plastics, batteries, cosmetics, and medical equipment [3][4][3,4]. Conventional methods to remove HMs from effluents are chemical precipitation, solvent extraction, ion exchange, evaporation, adsorption, nanofiltration, ultrafiltration, reverse osmosis, and electrochemical treatments; unfortunately, they often prove inefficient in terms of energy input, environmental footprint, capital investment, and operational costs. As a consequence, huge amounts of non-treated domestic and industrial waste end up in the natural environment [2][4][5][2,4,14]. Bioremediation comes as a tool to avoid such dumping (bearing several well-documented advantages [6][7][8][9][15–18]); it is defined at large as the application of biological organisms and their components to degrade, transform, sequester, mobilize, or contain environmental contaminants present in the soil, water, or air [10][19]. Various species of plants, bacteria, fungi, yeasts, and microalgae, as well as dead biomass-derived therefrom, have shown a great potential for bioremediation of HM-derived pollution in aqueous media—through metal binding and uptake [11][12][13][20–22]. Among these, phycoremediation (for i.example., use of microalgae for mitigation of organic and inorganic contamination) offers several advantages; hence, it has accordingly undergone intensive investigation for the large-scale remediation of industrial and domestic effluents, as well as HM-contaminated sites and water bodies [14][15][12,23]. Microalgae are photoautotrophic eukaryotic microorganisms, which account for most of the biologically sequestered trace metals in aquatic environments [16] [24]. Their unique metabolic plasticity, inherent capacity to grow on nonarable lands and in wastewater, using just solar light as the source of energy and atmospheric CO2 as the carbon source, and relatively high rate of cell division and growth account for such widespread occurrence; the exceptional retention capacity of HMs also contributes to make them the ideal platform to develop the next-generation technologies for wastewater treatment. Microalgae cells can accumulate HMs up to 10% of their biomass, owing to their large surface-to-volume ratio, coupled with their efficient metal binding, uptake, metabolization, and storage mechanisms [12][13][12,22]. Despite their outstanding potential, current technical and economic constraints—associated with the underlying upstream and downstream processes—have hampered the large-scale use of microalgae in HM bioremediation. Even though the integration of microalgae-mediated wastewater treatment with energy production appears logical and inevitable, the strains most commonly employed, essentially retrieved from nature in their native state, lack the robustness required by sustainable, large-scale scenarios bearing a commercial interest. Recent advances in genetic and protein engineering, complemented by an essentially unanimous orientation toward a holistic approach to the engineering of biological systems, at the expense of bioinformatic and omics tools, may soon allow for the tailor-made design of microbial cell factories for a number of end- or start-products while lowering the risks and concerns over the putative adverse effects associated to use of genetically modified organisms (GMOs). Further to knowledge on the most obvious routes to enhance energy load (stemming from hydrocarbons) and improving growth rate and photosynthetic ability in microalgae cells, effective engineering of (sustainable) cell factories, for efficient HM bioremediation, will call for work on specific genes and traits identified as relevant.

2. Cellular Mechanisms of HM Bioremediation in Microalgae

Cellular Mechanisms of HM Bioremediation in Microalgae

Heavy metals adversely affect the physiological health of, and may even cause severe toxicity to microalgae cells, owing to attenuation of the bioactivity of proteins, lipids, nucleic acids, pigments, and other molecules, or the generation of excessive reactive oxygen species (ROS); more specifically, they act by impairing photosynthetic machinery, inhibiting enzyme activities, and/or ceasing cell division [17][29], or else by inhibiting the normal function of the thylakoid membrane and chlorophyll biosynthesis, acidifying cytoplasm, or damaging the cell membrane [18][19][20][30–32]. Similar to other life forms, microalgae have developed, through evolution, several intracellular and extracellular adaptive mechanisms for the mitigation of HM toxicity. For instance, the physicochemical properties of the microalgal cell wall and extracellular polymeric substances (EPS) allow the binding of HM ions to functional groups on their surface, in a process generally known as biosorption [21][33]. As the interface between the intracellular compartment and external environment, the constitutive macromolecules of the cell wall possess various negatively charged functional groups, for example.g., amino, hydroxyl, carboxyl, sulfhydryl, sulfate, phosphate, carbonyl, amide, imidazole, thioether, and phenol; said moieties can bind to ions from the surrounding medium, in the absence of steric or conformational barriers [22][23][24][25][34–37]. The molecular mechanisms behind the biosorption of HMs onto the cell wall and EPS include ion exchange, chelation and complexation, hydroxide condensation, covalent binding, redox interaction, biomineralization, and precipitation of insoluble metal complexes, through electrostatic, van der Waals, or hydrophobic interactions of positively charged HM cations with negatively charged groups present on the cell surface [1][26][27][1,20,38]. The adsorption capacity of the microalgal cell wall is a metabolism-independent process; hence, it is primarily affected by such environmental factors as pH, temperature, contact time, and concentration of HM and competing ions [4][28][4,39]. Given the numerous reports on fluidity and evolution of cell wall components (for example.g., fatty acids) in response to external stimuli, a yet unknown metabolic background to this phenomenon seems to exist. On the other hand, biosorption of HMs onto EPS is regulated by the cell itself via changes in the properties of such biopolymers, as required by the nature of metabolic stress, for examplei.e., metal toxicity in the situation under scrutiny [40,41]. Both the cell wall and EPS provide, indeed, an extracellular protective layer to the cell that prevents the harmful effects of HMs, if transported into the intracellular compartment; in this fashion, cellular integrity is maintained. The distinct physiology of existing species of microalgae then accounts for the differences found in the composition and structure of such outer structures, which, in turn, drive their species- and even strain-dependent HM-biosorption capacities [1][11][1,20]. Secretion of metal-chelating proteins and specific organic acids, and subsequent endocytosis of the organometallic complexes formed, is another mechanism for extracellular HM-bioremediation, reported in microalgae cells [29][42]. Since HMs are hydrophilic in nature, a requirement exists for certain carrier molecules that facilitate their transport into the cells. Once in the cytoplasm, HM toxicity is overcome by resorting to unique metabolic mechanisms—some of which have been well-documented. Several metal efflux pumps do regulate the algal membrane permeability, by actively transporting HMs into and out of the cell [30][31][32][43–45]; the net metal flux is accordingly reduced, and may even affect the chemical speciation of the HMs, due to expulsion of trace metal complexes [33][46]. Another strategy followed by microalgae is increased expression of metal-binding amino acids, peptides, and proteins, such as metallothioneins (MTs), phytochelatins (PCs), glutathione (GSH), proline, histidine, and glutamate [34][47]. These organometallic complexes are typically transported in, but partitioned into vacuoles—so as to neutralize the otherwise toxic effects of HMs in the cytoplasm [5]. In the acidic environment of vacuoles, HMs are released from their organic carrier; while the latter may be transported back to the cytosol, HMs are most likely stabilized and chelated by sulfides or organic acids in said vacuoles [35][36][37][10,48,49]. Depending on the prevailing environmental conditions, microalgae may attain a high polyphosphate content, suitable for binding divalent HM cations, and drive them into vacuoles for further sequestration. In addition to vacuoles, excess intracellular HM loads can be transported for eventual sequestration in such other organelles as mitochondria and chloroplasts [32][45], meaning that the expression level of metal transporters in the membrane of those organelles will play an important role in determining HM-removal specificity, capacity, and rate by microalgae strains. As happens with other cell types, microalgae respond to HM-generated oxidative stress by controlling the cell redox state and overexpressing heat shock proteins (HSPs) [38][39][50,51]. MicroRNAs (miRNAs) also serve as key components of the gene regulatory network involved in cellular HM mitigation. They contribute to post-transcriptional cleavage and translational inhibition of target mRNAs, or methylation of target DNAs to regulate a particular response, aimed at maintaining cellular homeostasis, by triggering complexation of excess HMs, defense against oxidative stress, and signal transduction for biological control purposes [40][52]. Despite the scarce information available on the subject, metal-responsive transcription factors (TFs) appear to activate multiple genes responsible for HM uptake, transport, and detoxification, thus, establishing a global resistance network against HM toxicity in the cell. Therefore, identification and characterization of the set of TFs able to regulate HM stress will be of the utmost importance, in attempts to develop transgenic microalgae with improved bioremediation potential [17][41][29,53]. Sexual reproduction, expression of metal-modifying enzymes, and phenotypic plasticity are alternative mechanisms entertained by microalgae as survival tools against HM toxicity [37][49]. For instance, transcriptomic analysis of Chlorella vulgaris, following exposure to Cu cations, unfolded a marked increase in intracellular carotenoids and proline contents, and in activity of such antioxidative enzymes as catalase, peroxidase, polyphenol oxidase, and superoxide dismutase. Photosystem II (PSII) and CO2 assimilation are apparently inhibited in microalgae, as a response to metal stress; a relative reduction in growth rate and cell density has been reported for various species of microalgae upon exposure to high concentrations of Cu. A severe drop in protein levels, in parallel to an enhanced rate of carbohydrate biosynthesis, has been demonstrated in microalgae cells in the presence of HMs [42][43][54,55]. The aforementioned coordinated response of microalgae cells to metal toxicity is a result of crosstalk among different molecular networks, urged by the need for keeping cellular hemostasis. Similar to other cellular functions, the key role played by such signaling molecules as phytohormones, Ca itself, and kinases is worthy of further research [29,56].

3. Genetic Engineering Targets to Improve Microalgal HM Bioremediation Capacity

Genetic Engineering Targets to Improve Microalgal HM Bioremediation Capacity

3. 1. Metal Transportation

In C. reinhardtii, metal transporters have been classified into two main groups. Group A transporters, including natural resistance-associated macrophage proteins (NRAMP), zinc-regulated transporters (ZRT), iron-regulated transporters (IRT), Zrt-Irt-like proteins (ZIP), and Fe- (FTR) and Cu-transporter (CTR) families, ensure metal trafficking from the extracellular environment to the cytosol (as HM uptake), and from the cytosol into the vacuoles (as HM storage) [44][87]. Conversely, group B transporters, including members of the families of cation diffusion facilitators (CDF), P1B-type ATPases, FerroPortiN (FPN), and the Ca2+-sensitive cross-complementer1/Vacuolar iron transporter1 (Ccc1/VIT1), reduce cytosolic metal concentration via active efflux of metal ions and organometallic complexes into the extracellular surroundings, should metal concentrations exceed cellular requirements [45][12]. NRAMP transporters utilize the transmembrane proton gradient to mediate the transport of divalent cations to the cytoplasm [46][88]. Overexpression of NRAMP1 was reported in Auxenochlorella protothecoides under a high concentration of Cd in the medium [47][89]. Similarly, the upregulation of gene encoding NRAMP1, ZIP, and CTR transporters in Dunaliella acidophila was reported to improve Cd uptake [48][90]. The role of ZIP transporter genes in uptaking and sequestering Cd and Hg has also been demonstrated in C. reinhardtii [47][49][89,92]. Furthermore, genes that code for phosphate transporters (PTA) and aquaglycoporin (AQP) have been observed to increase As uptake in Chlamydomonas eustigma and Microcystis aeruginosa [50][16][93,94]. Ibuot et al. [51][95] overexpressed CrMTP4, a metal-tolerant protein (MTP) from the Mn-CDF clade of cation diffusion facilitator family of metal transporters, in C. reinhardtii; marked increases in resistance to Cd toxicity, and in bioaccumulation efficiency due to increased transfer to and storage of Cd in acidic vacuoles were found. P1B-type ATPases—also known as heavy metal ATPases (HMAs)—are a class of metal transporters present across all taxa, including higher plants and macroalgae; HMAs play a critical role in metal trafficking across cell membranes [52][53][97,98]. Ibuot et al. [51][95] heterologously overexpressed AtHMA4—a plant Cd and Zn transporter from Arabidopsis thaliana—in C. reinhardtii, and recorded increases in uptake and bioaccumulation of Cd and Zn by the transformed microalga. Ramírez-Rodríguez et al. overexpressed an arsenic hyperaccumulator, Acr3, which was localized in the vacuolar membrane; it acts as an efflux pump, and leads to a 1.5- to 3-fold increase in As removal capacity, as compared to wild type [54][104]. Furthermore, two highly conserved vacuolar proton pumps—vacuolar proton-ATPase (V-ATPase) and vacuolar proton pyrophosphatase (V-PPase)—generate the energy necessary to transport most solutes into the vacuoles [55][10]. Moreover, expression of a universally expressible plasma membrane H+-ATPase (PMA) in C. reinhardtii led to a 3.2-fold increase in photoautotrophic production, under the high CO2 concentrations of (toxic) flue gas; this piece of evidence further highlighted the great potential of efflux pumps in microalgal bioengineering [56][106]. Given the requirement for such thiols and thiol-containing compounds as GSH and PCs, posed by the cellular defense against HM toxicity, sulfur metabolism plays an imperative role in microalgal HM mitigation. The upregulation of biotin biosynthesis genes, which are involved in sulfur metabolism, and S-transporter genes was reported in C. reinhardtii when exposed to Hg2+ [49][92]. Genes involved in S-assimilation pathways, including those encoding methionine synthase (mete) and sulfite reductase (sir1), were also found to undergo upregulation in C. reinhardtii under HM stress. Additionally, overexpression of amino acid transporter genes in C. reinhardtii has been linked to an increase in Cd detoxification [57][107]. Other known HM transporters, well-characterized in plants but less so in microalgae, include multidrug resistance-associated proteins (MRP), ABC transporters of the mitochondrion (ATM), pleiotropic drug resistance (PDR) transporters, yellow-stripe-like (YSL) transporters, and Ca2+ cation antiporters (CAX) [58][53]. Proteomic analysis of high HM tolerance and accumulation in Euglena gracilis unfolded a significant increase in expression of the major facilitator superfamily (MFS) transporters, cadmium/zinc-transporting ATPase, and HM transporting P1B-ATPase, as well as metal-binding, thiol-rich proteins, following HM exposure. A major MFS transporter involved in HM compartmentalization in cellular organelles experienced a 5.5-fold increase in expression level upon the presence of HMs in the medium. Two P1B-ATPases, HMA2 and HMA3, known for their role in HM efflux, in and out of the cell and vacuoles, respectively, were also upregulated by ca. 3.6-fold, and further claimed to be key mediators of metal homeostasis in HM-exposed microalgae. In addition, a transmembrane TrkA transporter, involved in potassium transport and sodium/sulfate symport, showed a 6.5-fold increased expression once exposed to HM [59][108]. The feasibility of expressing bacterial metal transporters in plants has been widely demonstrated [60][61][109,110]; this approach also appears to be feasible in attempts to increase phycoremediation capacity in microalgae. Heterologous overexpression of metal ion/H+ antiporters CAX2 and CAX4, from A. thaliana in Nicotiana tabacum, increased uptake and sequestration of Cd, Zn, and Mn by 70–80% in transgenic plants, as compared to wild type [62][63][111,112]. Similar results were reported when a vacuolar ZAT Zn transporter from animal origin was overexpressed in A. thaliana [64][113]. Such ABC transporters as MRP2 were shown to be significantly upregulated; they increased HM uptake and sequestration in the vacuoles of C. reinhardtii and D. acidophila cells [49][65][66][92,114,115]. An ABC-type YCF1 transporter has been identified in yeasts and plants as a vector for the transport of cytosolic GSH-complexed Cd into vacuoles [67][116]. Heterologous expression of the YCF1 gene in A. thaliana yielded transgenic plants with increased Cd and Pb tolerance [68][117]. In another study, the human multidrug resistance-associated protein (hMRP1) gene—encoding an ABC-type multidrug resistance-associated transporter—was overexpressed in N. tabacum; the transformants exhibited higher tolerance against Cd when compared to wild type. Notably, hMRP1 is a well-known protein involved in the multidrug resistance of cancer cells, where it performs an efficient efflux of a wide range of cytotoxic compounds, including HMs [69][118]. Endogenous ABC transporters from the MRP subclass have also been characterized in microalgae as key mediators of metal homeostasis. Among seven MRPs identified in the genome of C. reinhardtii, four are glutathione S-conjugate pumps present in the vacuolar membrane, and able to transport metal-GSH complexes into the vacuoles [70][85].

3. 2. Metal Chelation

MTs are a group of genetically encoded (class I and II), or enzymatically-synthesized (class III), polypeptides, ubiquitously found in living organisms and playing an important role in metal homeostasis and trafficking [71][72][121,122]. They contain a few aromatic residues (<10%), but a high proportion (15–35%) of cysteine and, to a lesser extent, histidine residues; such structure accounts for the high metal-binding capacity of MTs [73][74][123,124]. The high variability observed in the amino acid sequence of MTs—even among closely related organisms—implies a previous active evolutionary change, responsive to environmental conditions and cellular signals [75][5]. Class I MTs consist of two smaller Cys-rich domains, and a large spacer region in between. Class II MTs are low-molecular-weight (6–7 kDa) proteins, possessing three Cys-rich domains, separated by 10–15 residues; they are located in the cytosol, and mainly involved in the control of intracellular concentrations of metals at regular levels. Class III MTs—also known as phytochelatins (PCs)—are enzymatically synthesized thiol-containing oligopeptides; they are typically composed of three amino acid residues, viz. γ-Glu, Cys, and Gly [76][125]. Enhanced activity of the enzymes involved in phytochelatin biosynthesis, along with a marked increase in the supply of GSH and PCs upon exposure to HMs, have been well-documented for several microalgae species [77][78][79][80][81][82][83][84][85][86][128,129,130,131,132,133,134,135,136,137]. Recently, overexpression of a synthetic gene (gshA) encoding for γ-glutamylcysteine synthetase was shown to significantly increase Cd tolerance of C. reinhardtii [87][88][138,139]. Furthermore, the CrGNAT gene, encoding an acetyltransferase involved in histone methylation and chromatin remodeling, was overexpressed in C. reinhardtii; under toxic concentrations of Cu, a marked increase in the cell population, chlorophyll accumulation, and photosynthesis efficiency were observed compared to wild type, while CrGNAT knockdown lines with antisense exhibited sensitivity to Cu stress [89][140]. In one study, phytochelatin synthetase from wheat was overexpressed in tobacco and the transgenic plants were able to produce 100-fold biomass in HM-contaminated soils when compared to hyperaccumulator Thlaspi caerulescens [90][141]. Cai et al. [91][149] authored the first report on the heterologous expression of MTs in microalgae; overexpression of a chicken MT-II in cell wall-deficient mutants of C. reinhardtii increased tolerance to Cd and enhanced sequestration thereof by about two-fold in the transgenic microalgae. In another study, a fusion protein composed of low CO2-induced plasma membrane protein and MT-II polymer was expressed in Chlamydomonas sp., which led to a five-fold increase in Cd uptake by transformants relative to wild type [92][24]. These Itauthors also evaluated the metal recovery capacity of transgenic microalgae, retrieved from contaminated sediments, using in situ sonications, and found that it was twice that of its wild counterpart [93][150].Among 98 different types of metal-binding proteins with increased expression, when exposed to HM, MTs were surprisingly not detected in E. gracilis cells [59][108]. The heterologous expression of phytochelatin synthase (OAS-TL or PCS) from A. thaliana in Mesorhizobium huakuii, increased Cd accumulation in this transgenic bacteria by 25-fold [60][109]. MT genes have also been reported in Synechococcus, as well as in seven other blue-green microalga strains [94][30]. It has been suggested that oligopeptide chain length and cysteine residue distribution determine the capacity of MTs and their host cells for HM binding and remediation. Some microalgae strains, bearing higher HM tolerance, appear indeed to synthesize MTs with longer chain length and more frequent cysteine residues. This realization may be taken advantage of to engineer microalgae with improved HM removal capacity, via heterologous expression of enzymes for synthesis of long cysteine-rich MTs, from hyper-tolerant species, in some transgenic strains [95][96][155,156]. Orthophosphate polymers—also known as polyphosphates (polyP)—have been implicated with the accumulation of HMs in both prokaryotic and eukaryotic organisms [97][98][157,158]. Biosynthesis of polyP in microalgae is regulated by the activity of exopolyphosphatase, or else through compartmentalization mechanisms, mainly with the contribution of acidocalcisome membrane transporters [99][159]. The functions performed by polyP in microalgae include cycling phosphorus in the ocean, acting as a phosphorus reservoir in the cell, and providing cellular defense against nutrient, osmotic, thermal, and HM stresses [99][100][159,160]. Consequently, polyP formation facilitates HM sequestration and storage and may regulate the chelation and compartmentalization of such toxic ions [101][102][44,161]. PolyP synthesis in prokaryotes is catalyzed chiefly by (reversible) ATP-specific kinases, PPK1, and PPK2, using GTP as a substrate [103][162]. Overexpression of PPK1 in cyanobacterium Synechococcus doubled its polyP content [104][163]. Most genes and proteins associated with polyP biosynthesis in eukaryotes remain unknown, while overexpression of prokaryotic PPK1 was found toxic for yeast and plant cells [105][106][164,165]. Instead of PPK, the vacuolar transporter chaperone (VTC) complex appears to be responsible for polyP synthesis in Chlamydomonas, whereas exopolyphosphatases (PPX) are the key enzymes responsible for its degradation [107][166]. The important role played by polyP in microalgal HM sequestration justifies examining how genetic engineering will affect the (characterized) genes; identification of other genes and enzymes associated with polyP metabolism might allow effective manipulation of said key cellular component for bioremediation purposes. It must be noted, however, that changes in phosphorous level—as one of the most critical nutrients in microalga cultivation—may have unpredictable and disturbing effects upon P homeostasis in the cell. Amino acids are known to function as major players in cellular defense against metal and oxidative stress. Under toxic levels of Cd and limitation of N, C. vulgaris increased its ability to accumulate ketogenic and glucogenic amino acids, as well as such metal-binding amino acids as proline, histidine, and glutamine [108][167]. Proline behaves as a signaling molecule; its metabolic roles include regulation of intracellular osmotic pressure, prevention of protein denaturation, maintenance of membrane integrity, stabilization of enzymes, and quenching of toxic ROS, under various biotic and abiotic stress conditions, in both plants and microalgae. Furthermore, Pro accumulated in the cytosol contributes to alleviating metal stress via chelation of HMs and regulation of water potential [109][110][111][168,169,170]. A mothbean pyrroline-5-carboxylate synthase (P5CS), and a fusion protein composed of chicken MT-II and a plasma membrane protein were separately overexpressed in C. reinhardtii. The MT-expressing microalgae showed significantly enhanced tolerance to toxic concentrations of Cd, and its Cd-binding capacity increased by 2- to 5-fold compared to wild type. Furthermore, the P5CS-expressing microalgae produced 80% more free Pro and showed a 4-fold increase in its Cd-binding capacity. Proline was accordingly claimed to contribute to HM tolerance by enhancing GSH and PC biosynthesis, as well as reducing free radical damage, via physical quenching of oxygen singlets and chemical reaction with hydroxyl radicals [112][171]. Overexpression of the HISN3 gene—encoding phosphoribosylformimino-5-aminoimidazole carboxamide ribonucleotide isomerase—induced a moderate increase in His accumulation, and significantly enhanced Ni tolerance in transgenic C. reinhardtii compared to wild type [113][173]. Similarly, C. reinhardtii cells were transformed with the HAL2 gene, which regulates the synthesis of Cys, leading to a five-fold increase in metal binding capacity of the transgenic microalgae [92][24]. Another amino acid osmolyte, glycine-betaine, was also shown to be overproduced by upregulation of serine decarboxylase (SDC1) in C. reinhardtii cells, under Cd stress [57][107].

3.3. Metal Biotransformation

Xenobiotic or endobiotic chemicals in a cell are metabolized to fewer toxic products by resorting to biotransformation. The detoxification pathways of HMs in microalgae consist of several enzymatic reactions, developed ab initio by the cell to reduce their toxic nature, by confining the metal ions to organic structures, where their oxidative potential is severely constrained [114][35]. In C. vulgaris, a chromate reductase (ChrR) has been characterized that converts Cr(VI) to Cr(III), via an enzymatic reaction, involving oxidation of GSH, which confers a high tolerance against Cr toxicity [115][116][178,179]. Two bacterial genes, gsh1 and arsC (arsenate reductase) were overexpressed in A. thaliana, thus, resulting in transgenes bearing significantly higher tolerance to As(V) than wild type [117][180]. Moreover, an arsenate reductase (CrACR2s), found in C. reinhardtii, was shown to reduce arsenate to (less toxic) arsenite, with the extra electrons transferred to glutaredoxin [118][181]. In several microalgal strains, arsenic is biotransformed through various mechanisms—the most common being reduction of As(V) to As(III)—complemented by methylation of As(III) to monomethylarsonate (MMA), brought about by oxidase and S-adenosylmethionine (SAM), followed by conversion of MMA(V) to dimethylarsinate (DMA(V)), which is further reduced to DMA(III). Finally, DMA(III) is converted to a range of organoarsenicals, for example.g., arsenolipids, arsenosugars, arsenobetaine, and arsenoribosides [119][120][121][122][123][182,183,184,185,186]. By the same token, the expression of mercuric reductase permits biotransformation of Hg2+ to elemental Hg and metacinnabar (β-HgS), in strains of microalgae Selenastrum minutum, Chlorella fusca, and Galdiera sulphuraria [124][187]. In attempts to improve the ability of Chlorella spp. DT to detoxify mercury, a bacterial mercuric reductase (merA) gene from Bacillus megaterium was overexpressed in this microalga; the resulting transformants exhibited a two-fold increase in Hg2+ bioremoval capacity compared to wild type [125][188]. The applicability of this method had been previously demonstrated in various plants when enhancing their tolerance against mercury. Organomercurial lyase mediates the protonolysis of organic mercury to Hg2+, while mercuric reductase reduces Hg2+ to Hg0; hence, overexpression of cytosolic MerA and ER-located MerB, using (microalgae-specific) codon-optimized genes might prove an effective approach to improve the HM-biotransformation capacity of microalgae [126][201]. Remember that the Mer operon also possesses the coding genes for three inner membrane transporters (MerC, MerT, and MerF), involved in the transport of mercury into the cytosol; periplasmic MerP is responsible for funneling metal ions (preferably ionic mercury) to those inner membrane metal transporters [127][202]. Therefore, expression of MerP or a fusion of MerC/T/F to a metal chelator (as membrane-bound Hg2+ trap) will putatively improve the ability of the microalgae to bioremove mercury [126][201]. Given the environmental concerns over the excessive release of Hg0, as volatile metabolites, into the atmosphere by such engineered species, it might be safer to sequester ionic mercury inside the cell via binding to a chelating molecule. Cytochromes-P450 (CYPs) are as well recognized for their role in the enzymatic biotransformation of toxic molecules. Other HM volatilization mechanisms described in microalgae include a photoreduction pathway, found in C. vulgaris, for the biotransformation of Cr [128][208], intracellular and extracellular biosyntheses of metal nanoparticles, and reductive interactions with functional groups of biomolecules in- and outside the cell [129][130][131][132][40,209,210,211]. Despite the improved efficiency and wide range of applications that HM-volatilizing genes and proteins might offer to the phycoremediation process, the underlying pathways for biotransformation of HMs in microalgae (and plants) remain mostly unknown and, thus, still require extensive in-depth research.

4. Oxidative Stress Response Regulation

The physiological, biochemical, and gene expression characteristics of C. reinhardtii were evaluated under toxic concentrations of Cu; inhibition of cell growth and photosynthesis, variation of total chlorophyll content, and marked increase in lipid peroxidation were accordingly observed [133][212]. Similarly, transcriptional analysis of D. salina and C. reinhardtii, exposed to Cd and Pb, respectively, revealed that the encoding genes of several antioxidant enzymes were upregulated in microalga cells subjected to HM stress [65][134][114,213]. Along with the upregulation of antioxidant enzymes, the overexpression of thioredoxin (Trx), heat shock proteins (HSPs), and carotenoids were documented in A. protothecoides, C. vulgaris, and C. reinhardtii cells, in response to toxic levels of HMs [135][136][57][54,80,107]. A similar analysis on Amphora coffeaeformis, Navicula salinicola, and D. salina under HM stress unfolded a marked increase in antioxidant defense-related genes, a response shared by all three species of microalgae [137][214].

5. Metal Stress Response Regulation

Cellular response to HM stress, and consequent detoxification mechanisms, are mainly regulated by key components in the metal regulatory network held by microalgae. Several regulatory molecules contribute to the control of the HM-detoxifying factors, via genome-wide changes in gene expression; this leads the cell, in turn, toward a particular biochemical state that minimizes the adverse effects of HMs. The role of TFs, phytohormones, and miRNAs will be discussed below, for being the master regulators of metal stress response in microalgae. TFs are DNA-binding proteins, which interact with enhancer or promoter sequences of a cluster of genes, to regulate their transcript levels in the cell [138][221]. Metal response element (MRE)-binding transcription factor-1 (MTF-1) is the main metal-sensing TF found in eukaryotes. Zn binding to its zinc finger domain reversibly and directly activates the DNA-binding activity of MTF-1. The activated MTF-1 is then transported to the nucleus, and assists histone acetyltransferase p300 in binding specific promoters, so as to induce or repress transcription [139][222]. Aside from high intracellular concentrations of Zn, MTF-1 can be indirectly activated by Cd or Cu, as a result of the oxidative stress triggered by HMs. The genes upregulated by MTF-1, including Znt1 and Znt2 (zinc efflux transporters) [140][223], Zip10 (zinc influx transporter), Gclc (glutamate-cysteine ligase catalytic subunit), Ndrg1 (N-myc downstream regulated 1), Sepw1 (GSH-binding selenoprotein), TXNRD2 (thioredoxin reductase 2), FPN1 (FerroPortiN 1), and Csrp1 (cysteine- and glycine-rich protein 1), have all been reported to contain multiple copies of MRE motif 5′-TGCRCNC-3′ in their UTR; hence, the latter seems to be an MTF-1-binding cis-regulatory element [141][224]. Furthermore, it was demonstrated that MTF-1-dependent activation of MT gene promoters requires the presence of zinc-saturated MTs in a cell-free transcription system, whereas thionein (the metal-free form of MT) inhibits activation of MTF-1 [142][225]. WRKY13 was claimed as another metal stress-related TF in A. thaliana, where it activates transcription of PDR8, an ABC transporter involved in Cd extrusion. Overexpression of WRKY13 led to a decrease in Cd accumulation and enhancement in Cd tolerance of transgenic plants, whereas WRKY13 loss-of-function mutants exhibited increased accumulation of Cd and sensitivity thereto [143][228]. Hence, such putative TFs as WRKY13, or such transporters as PDR8, involved in the regulation of HM extrusion, could allow for the design of transgenic microalga cells with lower metal extrusion ability and enhanced HM accumulation. Note, however, that the cellular stress conferred by the resulting hyperaccumulation of HMs must be balanced, in parallel with overproduction of components of metal detoxification mechanisms (e.g., metal chelators and vacuolar metal transporters) in transgenic microalgae. Phytohormones are signaling molecules, bearing a wide array of cellular functions in higher plants and microalgae, and aimed at retaining growth plasticity during development. The role of phytohormones in harmonizing the cellular response to HM toxicity (and other abiotic and biotic stresses) has been well-documented [144][145][146][147][56,234,235,236]. Cytokinins (CKs), gibberellic acid (GA), auxins, abscisic acid (ABA), brassinosteroids (BRs), jasmonic acid (JA), ethylene (ET), and salicylic acid (SA) are the main classes of phytohormones. Although their exact mechanisms of action are mostly unknown, they have been claimed to prevent the degradation of photosynthetic pigments, monosaccharides, and proteins and activate antioxidant defense responses required to sustain the growth of microalgae under stress conditions [144][145][148][56,234,237]. Interestingly, the exogenous application of phytohormones has proven effective to improve HM tolerance in microalgae [149][150][235,238]. In view of this fact, attempts to improve the bioremediation capacity of microalgae should rationally resort to optimization of their phytohormone profiles via genetic engineering approaches, as previously demonstrated in plants[151][152] [245,246]. However, with few exceptions, the coding genes for phytohormone biosynthesis remain essentially uncharacterized in microalgae [153][258]; hence, further omics analyses are warranted to characterize algal genes associated with phytohormone biosynthesis and map endogenous hormone signaling networks in microalgae. Several decades of extensive research on the molecular configuration of biological systems has indicated that a large network of microRNAs (miRNAs) regulate the expression pattern of genes in the cell, in parallel to TFs and hormones. These small, noncoding RNAs form specific secondary structures that enable them to bind target mRNAs and this may lead to cleavage or repression of the translation of said mRNAs [154][259]. As in many other cellular processes, miRNAs play a key role in controlling HM stress response, mostly by regulating the expression of the corresponding TFs [155][260]. Nonetheless, miRNAs involved in metal uptake and transport, sulfate allocation and assimilation, protein folding and assembly, metal chelation, antioxidant system, phytohormone signaling, growth/reproduction regulation, and miRNA biogenesis and action themselves, are also of importance for affecting HM stress response in microalgae [156][157][158][261,262,263].

6. Cell-Surface Bioengineering

It was recently suggested that the metallosorption properties of the cell surface can be effectively modified to improve HM phycoremediation capacity and specificity of microalgae, in what has been termed cell surface engineering, or cell surface display [159][160][1,279]. This is possible by expressing metal-binding proteins (for example.g., MTs and PCs) fused with an anchoring motif on the cell surface [161][162][280,281]. He et al. [93][150] expressed a membrane-anchored MT polymer in C. reinhardtii, using the said approach, and reported a marked increase in Hg removal capacity of the transgenic microalga versus its wild type. Given their relatively easy manipulation and rapid growth, yeasts and bacteria offer a rich source of experimental data on cell-surface engineering applications, which may then be used as a road map to conduct similar experiments in microalgae. For example, a 5-fold increase in the Pb2+ biosorption capacity of S. cerevisiae was observed upon anchoring short metal-binding NP peptides (harboring the CXXEE metal fixation motif of bacterial Pb2+-transporting P1-type ATPases) to AGα1Cp on the yeast cell wall [163][286]. Moreover, a surface-exposed MerR (a metalloregulatory protein bearing high affinity and selectivity toward Hg) enhanced the Hg2+ adsorption capacity of E. coli by 6-fold versus wild type [164][287]. The Hg removal capacity of transgenic C. reinhardtii was also expanded by expressing a membrane-anchored MT polymer [93][150]. Similarly, recombinant E. coli overexpressing MT fused to the outer membrane domain of a maltose transporter (LamB) and exhibited a 15-20-fold increase in Cd binding capacity compared to wild type [165][288]. Cell surface engineering technology has also been suggested for the recovery of precious metals from wastewater, forbearing lower costs, and improved selectivity for the target metal compared to conventional methods [166][167][290,291]. Given the wide range of ligands susceptible of display on the cell surface—including metal-binding moieties for bioremediation of HMs—cell surface engineering will likely play an important role as a biotechnological tool in the near future [168][294].

7. Conclusion

Conclusion

Despite being one of the most sophisticated biological systems regarding resistance to and transformation of heavy metals, the evidence shows that further improvements are possible in such phycoremediation ability—namely through specifically designed genetic modifications. Acceleration, yet under tight control, of the underlying forces of natural evolution will likely play an important role in theour quest toward restoration of the lost ecological balance while furthering knowledge of the processes supporting life. Genetic engineering holds immense potential, yet careful experimentation and implementation are vital. Harnessing this potential and converting it into a useful technology for a better future, demands rational approaches, rather than prohibitive regulations tout court and strict banning; carefully designed bench- and industrial-scale efforts, complemented by open and transparent communication between science and technology stakeholders, and to the society at large, constitute the only reasonable and effective path thereto.

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