Abstract
Heavy metal polluted wastewater from industries is currently one of the major environmental concerns leading to insufficient supply of clean water. Several strategies have been implemented to overcome this challenge including the use of microalgae as heavy metal bio-removers. However, there are still limitations that prevent microalgae to function optimally. Synthetic biology is a new biological discipline developed to solve challenging problems via bioengineering approaches. To date, synthetic biology has no universally affirmed definitions; however, it is uncontroversial that synthetic biology utilizes a constructive library of genetic standardized parts to create new biological systems or to redesign existing ones with improved characteristics. In this mini-review, we present state-of-the-art synthetic biology-based approaches that can be used to enhance heavy metal bio-removal from wastewater effluents by microalgae with a narrative synthetic biology workflow (Design-Build-Test-Learn cycle) to guide future developments of more advanced systems. We also provide insights into potent genes and proteins responsible for the bio-removal processes for stepwise developments of more advanced systems. A total of 49 unique genes and proteins are listed based on their eight heavy metals (Mn, Fe, Cu, Zn, As, Cd, Hg, and Pb) bio-removal functions in transport system, cellular tolerance, synthesis of key players in heavy metal bio-removal, biotransformation of heavy metals, and gene expression regulation. Thus, with our library, genetic parts are ready to be recruited for any synthetic biology-based designs. Thereby, this mini-review identifies potential avenues of future research and maps opportunities to unleash more potential of microalgae as heavy metal bio-removers with synthetic biology.
Introduction: Overview of Synthetic Biology and Wastewater Treatment
With recent advances in biological field, it is relatively more feasible to create purposeful synthetic systems through careful designs and engineering. This rapidly emerging discipline is referred to as “synthetic biology” and, since its birth in the year 2000, has demonstrated a number of major achievements (Meng and Ellis, 2020). With its widely applicable scope, synthetic biology has been used to overcome several challenges that our world has been facing including the development of CO2 fixing heterotrophic bacteria, CRISPR-based systems including for rapid diagnostics and, particularly for algal engineering (Gleizer et al., 2019; Krishnan et al., 2020; Meng and Ellis, 2020; Kaminski et al., 2021). Synthetic biology adopts the philosophy of bioengineering and aims at creating new biological systems or redesigning existing ones for particular purposes (Deplazes, 2009). This discipline builds up on constructive libraries of genetic parts ready to be recruited for any designs.
Water pollution is one of the global concerns affecting people in certain parts of the world. To alleviate this problem, microalgae have been used and developed extensively as a biological system for wastewater treatment (Wollmann et al., 2019). Heavy metals (HMs) are often referred to metal elements that are potentially toxic to human and the environment (Duffus, 2002; Rahman and Singh, 2019). Sources of HM contamination are varied, yet mostly industrial-based (Selvi et al., 2019). Heavy metal-contaminated water is conventionally treated via several approaches including the use of microalgae as a bio-remover that takes away HMs from liquid phases (Cheng et al., 2019).
Interestingly, previous reviews on similar topics only focus on 1) Genetic engineering tools and techniques for microalgae 2) Synthetic biology approaches for HM bio-removal in all potential microorganisms 3) Synthetic biology in microalgae for biomass and chemical production and 4) The use of customized algae for HM adsorption. To give some examples, a recent review by Fajardo et al. (2020) focuses on genetic engineering techniques and tools for microalgae ranging from classical to advanced ones (Fajardo et al., 2020) without any discussions on HM bio-removal. This may indicate that genetic engineering in microalgae is still considered under development. There are a few reviews on the use of genetically modified microorganisms (GMOs) to bio-remove HMs; however, they focus on all potential microorganisms, not particularly microalgae as a host in question (Diep et al., 2018; Capeness and Horsfall, 2020). Many reviews on the development of synthetic biology toolkits for microalgae only focus on biomass and chemical production (Wang et al., 2012; Jagadevan et al., 2018; Fabris et al., 2020; Kumar et al., 2020; Naduthodi et al., 2021) but not from the HM bio-removal perspective. Although, one review aims to emphasize the use of customized algae for HM bio-removal and discusses several approaches used to enhance algal performances including biological/molecular and chemical strategies, the review only focuses on adsorption as a removal mechanism (Cheng et al., 2019). Altogether, understandings on synthetic biology-based approaches applied to all microalgal HM bio-removal mechanisms is still missing. Potential genes/proteins involved in the process and their potential are yet to be collectively reported. To fill the knowledge gap, here, synthetic biology-based strategies for microalgal HM bio-removal are comprehensively discussed based on a synthetic biology concept (Design-Build-Test-Learn cycle) and all possible mechanisms used by microalgae to bio-remove HMs 1) biosorption 2) bioaccumulation and 3) biotransformation). A collective genetic part library of genes/proteins involved in and/or have been engineered for such purposes is also constructed to enable future developments of synthetic microalgal systems for HM bio-removal. Therefore, our work fills up the missing aspect on this topic and serves as a guide for stepwise developments. For instance, if a highly effective microalga is of interest as a host to bio-remove HMs, one can utilize the synthetic biology workflow presented in this mini-review to develop such systems by which they can select a strategy and handpick the most suitable genes/proteins listed here to start off the development. Specifically, if HM transporters of a microalga are observed to be ineffective, which limits the bio-removing performances, potential HM transporters from the library provided here can be handpicked and engineered.
Microalgal Mechanisms to Remove Heavy Metals From Wastewater Effluents
There are several mechanisms that microalgae use to remove HMs from contaminated sources. Here, we classify these mechanisms into 1) biosorption, 2) bioaccumulation, and 3) biotransformation.
Microalgae can be used as biosorbents by which HMs are absorbed onto their cell surface, or to be specific, onto the functional groups (e.g., -COO−, -OH- and -PO43−) or associated proteins present on their cell walls (Monteiro et al., 2012; Kumar et al., 2015; Mantzorou et al., 2018; Salama et al., 2019). In general, these functional groups are present in negatively charged forms, thereby, attribute to the overall anionics and attract positively charged metal ions. This mechanism is called biosorption. In this regard, the components of cell wall are functionally important key players and the binding affinity of cell surface to HMs in question is a key parameter determining the effectiveness of this mechanism. Modification of cultivation conditions to alter surface properties has been demonstrated to improve Hg biosorption in Scenedesmus obtusus XJ-15 (Huang et al., 2019). Moreover, increased accumulation of extracellular polymeric substances (EPSs) such as exopolysaccharides (Xiao and Zheng, 2016; Naveed et al., 2019) have been reported as a HM stress response mechanism in Chlamydomonas reinhardtii (Li et al., 2021) presumably because HMs can immobilize onto these substances.
Some may refer to biosorption and the second mechanism presented here, bioaccumulation, as the same process since they both ultimately result in free HMs taken away from liquid phases. However, in this mini-review, we propose that they are two different mechanisms to simplify the principles and lay out plain understandings toward synthetic biology-based approaches. Bioaccumulation is defined as a process that cells uptake metal ions and accumulate them intracellularly in a so-called “import-storage system” (Diep et al., 2018). Thus, it is important to state that in biosorption, HMs can be retrieved via simple methods such as washing with chelators; however, bioaccumulation requires cell disruption (Diep et al., 2018). Bioaccumulation is divided into 2 sequential steps: 1) cellular uptake of HMs, and 2) binding of the HMs to metal-biding peptides or polyphosphates (polyP) and sequestering in inactive forms. Microalgae uptake HMs via various transporters (Blaby-Haas and Merchant, 2012). Once HMs enter the cells, they can bind to designated biomolecules. In this section, metal-binding peptides (phytochelatins and metallothioneins) and polyphosphate bodies are discussed. Phytochelatins (PCs) are one of the metal-binding peptides (Cobbett and Goldsbrough, 2002) synthesized by biosynthetic enzymes (glutamate-cysteine ligase (GCL), glutathione synthetase (GSHS), and PC-synthase (PCS)) (Balzano et al., 2020; García-García et al., 2020). Typically, HM-PC complexes are sequestered in compartmentalized vacuoles, chloroplasts, and cytosol (Nagel et al., 1996; Zitka et al., 2011). Interestingly, glutathione—the final intermediate in PC synthesis—is also involved in HM-PC interactions (Balzano et al., 2020). Thus, all PC biosynthetic enzymes play a major part in HM bio-removal as they can determine intracellular availability of PCs (see Section 3). Metallothioneins (MT) are another well-known group of metal-binding peptides allowing the same process (Balzano et al., 2020). The main difference between PCs and MTs is that MTs are proteins translated directly from encoded gene, unlike PCs, which are enzymatically synthesized from intracellularly available precursors (Cobbett and Goldsbrough, 2002). Lastly, HMs can also bind to intracellular polyphosphates (polyP) because of the anionic nature of the phosphate groups (PO43−) making up the polymer. PolyP accumulation was shown to increase upon HM stress conditions (Sanz-Luque et al., 2020) emphasizing its unrealized potential in HM-bio-removal.
Biotransformation of toxic HMs into less or non-toxic forms is another strategy used by microorganisms (Chaturvedi et al., 2015). Evidently, arsenic (As) is most toxic in the forms of As (III) and As (V), respectively. These forms can be detoxified by means of oxidation [for As (III)], reduction [for As(V)], methylation and conversion to arsenosugars/arsenolipids in different diatom species (Wang et al., 2015; Papry et al., 2019). Similarly, Hg and Cr reduction was reported to be catalyzed by intracellular reductases in microalgae (Kelly et al., 2007; Lee et al., 2017; Leong and Chang, 2020), which facilitates the bio-removal of these metals. Previously with synthetic biology, Sattayawat and colleagues described a novel concept “bioderivatization” in cyanobacteria where a toxic compound produced by the cells was biotransformed into a less toxic form. This successfully alleviated the deleterious effects on microbial cells and resulted in higher production yields (Sattayawat et al., 2020).
Intriguingly, we also reported that Pediastrum duplex AARLG060, a HM bio-removing microalga, performed a combination of mechanisms to remediate Mn (Thongpitak et al., 2019). This observation suggests that several engineering strategies could be implemented concurrently to achieve more impact.
Synthetic Biology-Based Approaches as Strategies to Enhance Heavy Metal Bio-Removal From Wastewater Effluents
Although the native bio-removal mechanisms in microalgae are promising, there is still room for improvement. For instance, the use of fast-growing microalgae is preferable as this could increase total surface area for biosorption and metal uptake. However, such fast-growing microalgae are usually sensitive to high concentrations of HMs.
Several synthetic biology toolkits for microalgae have been developed over the last few years (Gimpel et al., 2016; Crozet et al., 2018; Poliner et al., 2018, 2020; de Grahl et al., 2020; Mehrshahi et al., 2020; Südfeld et al., 2021; Windhagauer et al., 2021) with a recent comprehensive review on synthetic biology-based approaches for enhancement of biomass accumulation and carbon flux partitioning (Naduthodi et al., 2021). Construction of biosynthetic pathways for attractive chemicals has also been reported (Wichmann et al., 2018, 2021; Yunus et al., 2018). Recently, a work on ground-breaking CRISPR technology in Picochlorum celeri (Krishnan et al., 2020) emphasizes the rise of toolkit development even more.
Synthetic Biology Workflow—Design
Identification of desirable characteristics is a fundamental key toward engineering success that can facilitate the first step in synthetic biology workflow—design (Figure 1). Firstly, different species or even strains of microalgae exhibit different performances as biosorbents (Kumar et al., 2015; Salama et al., 2019). This suggests that the degree of anionics plays an important role in determining microalgal performances. From this observation, modification of anionic moieties on cell walls could be the first strategy. Physically, acid treatment could alter surface functional groups and increased HM bio-removal efficiency (Almomani and Bohsale, 2021). However, this strategy also requires an extra acid treatment step. Certainly, an alternative approach is to address this through synthetic biology. The next question is, how can microalgae be genetically modified to increase the binding affinity between microalgal cell surface and HMs? A recent work by Ma and colleagues suggested that biofilm formation facilitated Scenedesmus Cd bio-removal efficiency (Ma et al., 2021). Given that this process is a stress response mechanism toward HMs (Li et al., 2021; Ma et al., 2021) and extracellular polymeric substances (EPSs) forming up the biofilm are generally negatively charged, engineering microalgae for enhanced biofilm production can be implemented to facilitate HM bio-removal. The genetic control of biofilm formation in microorganisms has been studied and several reports on manipulation of biofilm formation in bacteria have been discussed elsewhere (Perni et al., 2013; Benedetti et al., 2016). Mutagenesis studies of cyanobacteria for enhanced EPS production have also been investigated (Pereira et al., 2019; Yadav et al., 2020). However, these remain to be explored in microalgae.
FIGURE 1
Design-Build-Test-Learn (DBTL) cycle of synthetic biology for HM bio-removal. Design: (i) Biosorption (ii) Cellular uptake of HMs (iii) Bioaccumulation (iv) Native cellular responses to HM-induced stress (v) Biotransformation.
Increasing cellular uptake of HMs is another strategy employed to enhance bio-removal efficiency. Mostly, this is carried out by increasing a number or activity of HM active transporters (Zagorski and Wilson, 2004; Deng et al., 2007; Deng and Jia, 2011; Sone et al., 2013). Heterologous expression and overexpression are one of the prime synthetic biology-based approaches leading to increased protein level and activity. For example, expression of a plant transporter for Zn and Cd (AtHMA4 C-terminal domain protein) in C. reinhardtii increases cellular tolerance and bioaccumulation of these HMs compared with the wild-type (Ibuot et al., 2020).
Expression and overexpression of genes involved in metal-binding peptide synthesis have also been reported to promote HM bio-removal. As to metal-binding peptides, PCs and MTs are the prime candidates. PCs are enzymatically synthesized from naturally available precursors (Hirata et al., 2001; Bräutigam et al., 2011; Filiz et al., 2019; Balzano et al., 2020); therefore, heterologous expression and overexpression of PC biosynthetic enzymes can increase their availability and capacity (Clemens et al., 1999; Bae et al., 2001; Pal and Rai, 2010; Singh et al., 2010). The expression of MT gene from chicken in C. reinhardtii was first reported in 1999 and showed to promote Cd bioaccumulation (Cai et al., 1999). Later, other works have also shown that the expression of MT enhanced binding affinity and cellular tolerance in the same host (Sihai et al., 2008; He et al., 2011). This is also the case when expressed in E. coli (Singh et al., 2008; Jafarian and Ghaffari, 2017; Shahpiri and Mohammadzadeh, 2018). Furthermore, overexpression of metal-tolerant proteins, CrMTP4, has shown to increase Cd tolerance in C. reinhardtii by 2.29 folds and resulted in a 3.09-fold increase in Cd bioaccumulation (Ibuot et al., 2017). In a more advanced level, synthetic phytochelatins were expressed and showed to enhance the ability of HM bioaccumulation in bacteria (Bae et al., 2000, 2002). Metalloregulatory proteins also play an important role in determining the efficiency of HM bioaccumulation and homeostasis (Quinn et al., 2003; Silver and Phung, 2005; Sommer et al., 2010; Reyes-Caballero et al., 2011; Merchant et al., 2020) with examples of engineering in E. coli (Sato and Kobayashi, 1998; Yang et al., 2013).
Highly tolerant microalgae tend to remove HMs more effectively due to their ability to withstand HMs at high concentrations. Several enzymes and proteins have been reported to play a role in cellular tolerance toward HMs. For example, increased superoxide dismutase (SOD) expression was observed during Hg exposure as a defense system in Halamphora veneta (Mu et al., 2017). Glutathione peroxidase (GPX), an enzyme responsible for cellular responses to oxidative stress from HM exposure, has also been identified in C. reinhardtii (Fu et al., 2002; Dayer et al., 2008; Ma et al., 2020). Another example, thioredoxins in C. reinhardtii are also induced upon Cd exposure (Lemaire et al., 1999). On an engineering point of view, heterologous expression of C. reinhardtii Fe-assimilating protein 1 (FEA1) was shown to alleviate HM toxicity in yeast and plants (Narayanan et al., 2011). Sequestration also plays a role in HM tolerance. For instance, CrMRP2 (a member of the ATP-binding cassette (ABC) transporter subfamily) in C. reinhardtii functions in cellular sequestration of PC-Cd complex. It was shown that Cd bioaccumulation and sequestration were higher in the wild-type compared with the CrMRP2 mutant. In the same study, expression of CrMRP2 could complement Cd-sensitive yeast mutant lacking a vacuolar glutathione (GSH)-conjugate ABC transporter (Wang and Wu, 2006).
As mentioned in the previous section, reductases have been shown to facilitate HM bio-removal. Expression of mercuric reductase from Bacillus megaterium MB1 in eukaryotic microalga Chlorella sp. DT was previously studied and shown to promote Hg bio-removal (Huang et al., 2006). For more exploratory features, construction of biotransformation pathways could be an option and has previously been implemented in bacteria (Dvořák et al., 2017).
Computational-based analysis to predict the outcomes is another approach facilitating the design step. Though, computational-based approaches for HM bio-removal have not been widely used in microalgal engineering. Molecular dynamics of PCs have been studied (Pochodylo and Aristilde, 2017) to gain better understandings on the HM-PC complexes and computational approaches for redesigning metal-binding sites were reviewed (Akcapinar and Sezerman, 2017). A web tool to facilitate the design of intron-containing transgene expression in microalgae has also been established (Jaeger et al., 2019). Note that gene expression or overexpression could burden overall cellular performances; hence, in silico modelling that can predict a balance state between protein expression and energy production (or metabolic flux) could be beneficial. Recently, this principle has been applied to microalgae for bioproduction purposes (Tibocha-Bonilla et al., 2018; Zuñiga et al., 2018), but not yet for HM bio-removal.
Synthetic Biology Workflow—Build
Variant libraries as a collection of genetic parts ready to be recruited are helpful in the second step of synthetic biology workflow—build (Figure 1). With Next Generation Sequencing (NGS) technology, more whole genome sequences are available for putative gene discovery. Previously, a list of metal transporters in microalgae has been reported based on a whole-genome analysis (Hanikenne et al., 2005). Similarly, metagenomic analysis allowed the discovery of a new MT family with confirmed ability to chelate Cu(I), Zn(II) and Cd(II) (Ziller et al., 2017). Moreover, putative HM tolerance genes from C. acidophila—an extremophilic green alga—are also reported based upon transcriptomic analysis (Puente-Sánchez et al., 2018).
To emphasize the potentials of employing synthetic biology in microalgal bio-removal of HMs and facilitate future designs of such systems, we accumulatively search for genes taking part in HM bio-removal and summarized in Table 1. Subsequently, we group the genes based on their functions: 1) HM transport system, 2) cellular tolerance, 3) synthesis of key players, 4) biotransformation reactions of HMs, and 5) regulation of gene expression. However, it should be noted that these genes are listed based on their reported potential in HM bio-removal mechanisms, some of them have never been confirmed to enhance the process experimentally.
TABLE 1
| Gene | HM | Original host | Heterologous expression host | Type of function | Maximum improvement (if available) | References |
|---|---|---|---|---|---|---|
| Cyanobacteria, microalgae, and related examples | ||||||
| mntA (Mn transport system ATP-binding protein) | Mn | Synechocystis sp. PCC 6803 | — | HM transport system | — | Kizawa et al. (2016) |
| mntC (Mn transporter) | Mn | Synechocystis sp. PCC 6803 | — | HM transport system | — | Kizawa et al. (2016) |
| AtHMA4-CT4 (Cd/Zn transporter) | Cd | Arabidopsis thaliana | C. reinhardtii | HM transport system | ∼2.8-folda (bioaccumulation) | Ibuot et al. (2020) |
| AtHMA4-FL2 (Cd/Zn transporter) | Zn | A. thaliana | C. reinhardtii | HM transport system | ∼3-folda (bioaccumulation) | Ibuot et al. (2020) |
| FSD (Fe superoxide dismutase (FeSOD)) | Fe | C. reinhardtii | — | Cellular tolerance (antioxidant activity) | — | Dudley Page et al. (2012) |
| MSD (Mn superoxide dismutase (MnSOD)) | Mn | C. reinhardtii | — | Cellular tolerance (antioxidant activity) | — | Dudley Page et al. (2012) |
| gpx5 (glutathione peroxidase (GPX)) | - | C. reinhardtii | — | Cellular tolerance (antioxidant activity) | — | Ma et al. (2020) |
| SOD (superoxide dismutase (SOD)) | Hg | Halamphora veneta (Kützing) Levkov | — | Cellular tolerance (antioxidant activity) | — | Mu et al. (2017) |
| CrAPX4 (ascorbate peroxidase (CrAPX)) | - | C. reinhardtii | — | Cellular tolerance (antioxidant activity) | — | Kuo et al. (2020) |
| H O -1 (Heme oxygenase-1) | Hg | C. reinhardtii | C. reinhardtii | Cellular tolerance (Suppress reactive oxygen species generation) | 48.2% increase (in cell number) | Wei et al. (2011) |
| P5CS-1 (Δ1-pyrroline-5-carboxylate synthetase) | Cd | Mothbean | C. reinhardtii | Cellular tolerance (Cd tolerance + proline accumulation) | 4.16-fold (bioaccumulation) and enhanced growth | Siripornadulsil et al. (2002) |
| H43 (Fe-deficiency- inducible gene) | Fe | Chlorococcum littorale | S. cerevisiae Fe-uptake mutant | HM uptake system or cellular tolerance | 2-fold (bioaccumulation) and partially rescued growth | Rubinelli et al. (2002) |
| CrMTP4 (Metal-tolerant protein) | Cd | C. reinhardtii | C. reinhardtii | Cellular tolerance | 2.29-fold (bioaccumulation) | Ibuot et al. (2017) |
| CrMTP4 (Metal-tolerant protein) | Mn | C. reinhardtii | C. reinhardtii | Cellular tolerance | 2.48-fold (bioaccumulation) | Ibuot et al. (2017) |
| Not specified (plasma membrane-anchored MT polymer) | Hg | Not specified | C. reinhardtii | Synthesis of key players | ∼1.45-folda (biosorption/bioaccumulation) | He et al. (2011) |
| CrPTC1 (Pi transporter) | - | C. reinhardtii | Yeast mutant YP100 | Synthesis of key players | Pi uptake | Wang et al. (2021) |
| CrVTC4 (PolyP polymerase) | - | C. reinhardtii | — | Synthesis of key players | — | Wang et al. (2021) |
| gshA (γ-glutamylcysteine synthetase) | Cd | Synthetic gene from Uniprot ID P0A6W9 | C. reinhardtii | Synthesis of key players | 32.2% increase (bioaccumulation) | Piña-Olavide et al. (2020) |
| MT gene | Cd | Chicken | C. reinhardtii | Synthesis of key players | 1.64-fold (bioaccumulation) | Cai et al. (1999) |
| PC4 (PC synthase (PCS)) | Zn | Dunaliella tertiolecta | — | Synthesis of key players | — | Hirata et al. (2001) |
| CaPCS (PC synthase (PCS)) | Cd | C. acidophila | — | Synthesis of key players | — | Nishikawa et al. (2006) |
| CaECS γ-glutamylcysteine synthetase | Cd | C. acidophila | — | Synthesis of key players | — | Nishikawa et al. (2006) |
| CrPCS (PC synthase (PCS)) | Cd | C. reinhardtii | — | Synthesis of key players | — | Bräutigam et al. (2011) |
| γ-GT (γ-glutamyl transpeptidase (γ-GT)) | Cd | C. reinhardtii | — | Synthesis of key players | — | Bräutigam et al. (2011) |
| cDNA of MT-like gene (Accession No. U96646) | Cd | Festuca rubra cv. Merlin | C. reinhardtii | Synthesis of key players | 324.83% increase (bioaccumulation) and 55.43% increase (tolerance) | Sihai et al. (2008) |
| MerA (mercuric reductase) | Hg | Bacillus megaterium MB1 | Chlorella sp. DT | Biotransformation reactions | ∼2.08-folda (bioaccumulation) and ∼1.98-folda (biotransformation) | Huang et al. (2006) |
| pcc7942_1134 (gene involved in biofilm formation) | - | Synechococcus elongatus | — | Synthesis of key players (biofilm formation) | — | Schatz et al. (2013) |
| pcc7942_1133 (gene involved in biofilm formation) | - | Synechococcus elongatus | — | Synthesis of key players (biofilm formation) | — | Schatz et al. (2013) |
| AztR (ArsR/SmtB Metalloregulator) | Zn | Anabaena PCC 7120 | E. coli | Regulation of gene expression (tolerance) | Enhanced growth | Liu et al. (2005) |
| AztR (ArsR/SmtB Metalloregulator) | Pb | Anabaena PCC 7120 | E. coli | Regulation of gene expression (tolerance) | Enhanced growth | Liu et al. (2005) |
| AztR (ArsR/SmtB Metalloregulator) | Cd | Anabaena PCC 7120 | E. coli | Regulation of gene expression (tolerance) | Enhanced growth | Liu et al. (2005) |
| Bacteria, yeasts, and related examples | ||||||
| mntA (Mn(II) and Cd importer) | Cd | Lactobacillus plantarum | E. coli | HM transport system | ∼5.3-folda (bioaccumulation) | Zagorski and Wilson (2004) |
| copA (Cu(I) importer) | Cu | Enterococcus hirae | E. coli | HM transport system | ∼2.42-folda (bioaccumulation) | Zagorski and Wilson (2004) |
| MerT and MerP (Hg2+ transport protein) | Hg | E. coli | E. coli | HM transport system | ∼2.71-folda (bioaccumulation co-express with EC20) | Bae et al. (2001) |
| Ars (Metal-resistant gene) | As | Bacteria | — | Cellular tolerance | — | González Henao and Ghneim-Herrera (2021) |
| Cad (Metal-resistant gene) | Cd | Bacteria | — | Cellular tolerance | — | González Henao and Ghneim-Herrera (2021) |
| Pbr (Metal-resistant gene) | Pb | Bacteria | — | Cellular tolerance | — | González Henao and Ghneim-Herrera (2021) |
| CrMRP2 (ABC transporter) | Cd | C. reinhardtii | S. cerevisiae | Cellular tolerance | — | Wang and Wu (2006) |
| ArsR (Metalloregulatory protein) | As | Bacillus subtilis | E. coli | Regulation of gene expression (tolerance and transport) | 3.4 to 5.6-fold (biosorption/bioaccumulation) | Yang et al. (2013) |
| gMT (MT protein) | Pb | Corynebacterium glutamicum | E. coli | Synthesis of key players | ∼1.81-folda (bioaccumulation) | Jafarian and Ghaffari (2017) |
| gMT (MT) | Zn | Corynebacterium glutamicum | E. coli | Synthesis of key players | ∼3.06-folda (bioaccumulation) | Jafarian and Ghaffari (2017) |
| fMT (MT) | As | Fucus vesiculosus | E. coli | Synthesis of key players | 26 to 30-fold (bioaccumulation) | Singh et al. (2008) |
| OsMT1 (MT) | Hg | Rice | E. coli | Synthesis of key players | ∼10-folda (bioaccumulation) | Shahpiri and Mohammadzadeh (2018) |
| ec8 (synthetic PC (Glu-Cys)20Gly)) | Cd | Synthesized | E. coli | Synthesis of key players (Surface expression) | ∼4.75-folda (bioaccumulation) | Bae et al. (2000) |
| ec11 (synthetic PC (Glu-Cys)20Gly)) | Cd | Synthesized | E. coli | Synthesis of key players (Surface expression) | ∼7-folda (bioaccumulation) | Bae et al. (2000) |
| ec20 (synthetic PC (Glu-Cys)20Gly)) | Hg | Synthesized Bae et al. (2000) | E. coli (expression of cell surface anchored EC20) | Synthesis of key players (Surface expression) | ∼19-folda (bioaccumulation) | Bae et al. (2001) |
| ec20 (synthetic PC (Glu-Cys)20Gly)) | Hg | Synthesized Bae et al. (2000) | E. coli | Synthesis of key players (Surface expression) | ∼7-folda (bioaccumulation) | Bae et al. (2002) |
| ec20 (synthetic PC (Glu-Cys)20Gly)) | Hg | Synthesized Bae et al. (2000) | Moraxella sp | Synthesis of key players (Surface expression) | ∼6-folda (bioaccumulation) | Bae et al. (2002) |
| GFP-hMT2A (MT) | Cu | Human | S. cerevisiae | Synthesis of key players | 2.1-fold (bioaccumulation) | Geva et al. (2016) |
| Pho84p (As importer) | As | S. cerevisiae | S. cerevisiae | HM transport system | ∼1.4-folda (bioaccumulation) | Shen et al. (2012) |
| PpMT1.1a (Metallothionein-like genes) | Cu | Physcomitrella patens (Moss) | S. cerevisiae (∆cup1) | Synthesis of key players | ∼1.78-folda (bioaccumulation) | Pakdee et al. (2019) |
| MT4b (MT) | Cu | Arabidopsis thaliana | S. cerevisiae (∆cup1) | Synthesis of key players | ∼2-folda (bioaccumulation) | Guo et al. (2008) |
| MT4a (MT) | Zn | Arabidopsis thaliana | S. cerevisiae (Δzrc1, Δcot1) | Synthesis of key players | ∼4.88-folda (bioaccumulation) | Guo et al. (2008) |
| Combination of strategies | ||||||
| GlpF (As(III)-specific transporter) and fMT | As | E. coli and Fucus vesiculosus | E. coli | HM transport system and synthesis of key players | 43.05-fold (bioaccumulation) | Singh et al. (2008) |
| MerP and MerT with synthetic ec20 | Hg | E. coli and synthesized | E. coli | HM transport system and synthesis of key players | ∼19-folda (bioaccumulation) | Bae et al. (2001) |
List of genes involved or reported on heavy metal (HM) bio-removal.
Data obtained by estimation from reported graphs.
In synthetic biology, genetic circuits can be designed and constructed in a manner that would maximize system performances. In this sense, refactoring native genetic parts—promoters, terminators—is a useful approach to enhance protein expression. Promoter and terminator engineering in C. reinhardtii have been studied and reported (Einhaus et al., 2020). To boost up the engineered constructs even further, fine-tuning of regulatory systems could be implemented. This strategy has been illustrated in microalgae, though in the area not related to HM bio-removal (Sun et al., 2018). Additionally, protein redesign based on computational predictions could be implemented experimentally in this step.
Synthetic Biology Workflow—Test and Learn
The final two steps in synthetic biology workflow are to test and to learn from the newly designed system (Figure 1). In these steps, system performances are assessed and factors affecting the system are taken into consideration for the second round of the Design-Build-Test-Learn (DBTL) cycle. The goal of each cycle is to achieve a more optimal system, thus observations made from the previous cycle essentially put each piece of jigsaws together toward better performances. For instance, a bacterial system expressing synthetic phytochelatins showed enhanced HM bioaccumulation (Bae et al., 2000); however, it was learnt that HM uptake was a rate-limiting step of the process. Thus, the same system was improved in the second round of DBLT cycle by expressing anchored synthetic phytochelatins on the cell surface or expressing the synthetic phytochelatins together with HM transporters to overcome the limitation (Bae et al., 2001).
Prospects of Synthetic Biology-Based Approaches for Microalgal Heavy Metal Bio-Removal
Synthetic biology builds solely on genetic manipulations. Thus, legislations that are in effect to control the use of genetically modified organisms (GMOs) in each country are the major restrictions. Moreover, relatively few numbers of genetic parts and tools compared with that of other microorganisms also limit the development of microalgal chassis for synthetic biology, though the knowledge is building up (Tran and Kaldenhoff, 2020; Sproles et al., 2021). Omics information is also required for accurate computational predictions. This allows us to foresee cellular states after modifications and facilitates the system design. Altogether, despite such limitations, we expected that synthetic biology will gain more attention as a tool to improve microalgal HM bio-removal from wastewater effluents in the near future.
Conclusion
In microalgae, synthetic biology is often investigated as a tool to improve biomass and chemical production. Here, we discuss synthetic biology from another point of view—for heavy metal bio-removal from wastewater. A descriptive Design-Build-Test-Learn synthetic biology cycle is also presented for the first time to serve as a direction for straightforward implementations of synthetic biology for microalgal HM bio-removal. To facilitate this even further, we construct a genetic part library of genes and proteins involved in the process and/or reported to be engineered in a synthetic biology manner that in future, each part can be handpicked directly from our library.
Statements
Author contributions
PS conceptualized and drafted the manuscript. IY, NN, NM, WP-A, JP ,and CP revised the manuscript critically and approved the final draft.
Funding
This research work was partially supported by Chiang Mai University.
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Publisher’s note
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
References
1
AkcapinarG. B.SezermanO. U. (2017). Computational Approaches for De Novo Design and Redesign of Metal-Binding Sites on Proteins. Biosci. Rep.37, 1–12. 10.1042/BSR20160179
2
AlmomaniF.BhosaleR. R. (2021). Bio-sorption of Toxic Metals from Industrial Wastewater by Algae Strains Spirulina Platensis and Chlorella Vulgaris: Application of Isotherm, Kinetic Models and Process Optimization. Sci. Total Environ.755, 142654. 10.1016/j.scitotenv.2020.142654
3
BaeW.MehraR. K.MulchandaniA.ChenW. (2001). Genetic Engineering of Escherichia coli for Enhanced Uptake and Bioaccumulation of Mercury. Appl. Environ. Microbiol.67, 5335–5338. 10.1128/aem.67.11.5335-5338.2001
4
BaeW.MulchandaniA.ChenW. (2002). Cell Surface Display of Synthetic Phytochelatins Using Ice Nucleation Protein for Enhanced Heavy Metal Bioaccumulation. J. Inorg. Biochem.88, 223–227. 10.1016/S0162-0134(01)00392-0
5
BalzanoS.SardoA.BlasioM.ChahineT. B.Dell’AnnoF.SansoneC.et al (2020). Microalgal Metallothioneins and Phytochelatins and Their Potential Use in Bioremediation. Front. Microbiol.11, 1–16. 10.3389/fmicb.2020.00517
6
BenedettiI.de LorenzoV.NikelP. I. (2016). Genetic Programming of Catalytic Pseudomonas Putida Biofilms for Boosting Biodegradation of Haloalkanes. Metab. Eng.33, 109–118. 10.1016/j.ymben.2015.11.004
7
Blaby-HaasC. E.MerchantS. S. (2012). The Ins and Outs of Algal Metal Transport. Biochim. Biophys. Acta (Bba) - Mol. Cell Res.1823, 1531–1552. 10.1016/j.bbamcr.2012.04.010
8
BräutigamA.SchaumlöffelD.Preud'hommeH.ThondorfI.WesenbergD. (2011). Physiological Characterization of Cadmium-Exposed Chlamydomonas Reinhardtii. Plant Cell Environ.34, 2071–2082. 10.1111/j.1365-3040.2011.02404.x
9
CaiX.-H.BrownC.AdhiyaJ.TrainaS. J.SayreR. T.BrownC.et al (1999). Growth and Heavy Metal Binding Properties of Transgenic Chlamydomonas Expressing a Foreign Metallothionein Gene. Int. J. Phytoremediation1, 53–65. 10.1080/15226519908500004
10
CapenessM. J.HorsfallL. E. (2020). Synthetic Biology Approaches Towards The Recycling of Metals From the Environment. Biochem. Soc. Trans.48, 1367–1378. 10.1042/BST20190837
11
ChaturvediA. D.PalD.PentaS.KumarA. (2015). Ecotoxic Heavy Metals Transformation by Bacteria and Fungi in Aquatic Ecosystem. World J. Microbiol. Biotechnol.31, 1595–1603. 10.1007/s11274-015-1911-5
12
ChenW.BaeW.MehraR.MulchandaniA. (2002). Enhanced Bioaccumulation of Heavy Metals by Bacterial Cells with Surface-Displayed Synthetic Phytochelatins. ACS Symp. Ser.806, 411–418. 10.1021/bk-2002-0806.ch024
13
ChengS. Y.ShowP.-L.LauB. F.ChangJ.-S.LingT. C. (2019). New Prospects for Modified Algae in Heavy Metal Adsorption. Trends Biotechnol.37, 1255–1268. 10.1016/j.tibtech.2019.04.007
14
ClemensS.KimE. J.NeumannD.SchroederJ. I. (1999). Tolerance to Toxic Metals by a Gene Family of Phytochelatin Synthases from Plants and Yeast. EMBO18, 3325–3333. 10.1093/emboj/18.12.3325
15
CobbettC.GoldsbroughP. (2002). Phytochelatins and Metallothioneins: Roles in Heavy Metal Detoxification and Homeostasis. Annu. Rev. Plant Biol.53, 159–182. 10.1146/annurev.arplant.53.100301.135154
16
CrozetP.NavarroF. J.WillmundF.MehrshahiP.BakowskiK.LauersenK. J.et al (2018). Birth of a Photosynthetic Chassis: A MoClo Toolkit Enabling Synthetic Biology in the Microalga Chlamydomonas Reinhardtii. ACS Synth. Biol.7, 2074–2086. 10.1021/acssynbio.8b00251
17
DayerR.FischerB. B.EggenR. I. L.LemaireS. D. (2008). The Peroxiredoxin and Glutathione Peroxidase Families in Chlamydomonas Reinhardtii. Genetics179, 41–57. 10.1534/genetics.107.086041
18
de GrahlI.RoutS. S.Maple-GrødemJ.ReumannS. (2020). Development of a Constitutive and an Auto-Inducible High-Yield Expression System for Recombinant Protein Production in the Microalga Nannochloropsis Oceanica. Appl. Microbiol. Biotechnol.104, 8747–8760. 10.1007/s00253-020-10789-4
19
DengX.JiaP. (2011). Construction and Characterization of a Photosynthetic Bacterium Genetically Engineered for Hg2+ Uptake. Bioresour. Tech.102, 3083–3088. 10.1016/j.biortech.2010.10.051
20
DengX.YiX. E.LiuG. (2007). Cadmium Removal from Aqueous Solution by Gene-Modified Escherichia coli JM109. J. Hazard. Mater.139, 340–344. 10.1016/j.jhazmat.2006.06.043
21
DeplazesA. (2009). Piecing Together a Puzzle. EMBO Rep.10, 428–432. 10.1038/embor.2009.76
22
DiepP.MahadevanR.YakuninA. F. (2018). Heavy Metal Removal by Bioaccumulation Using Genetically Engineered Microorganisms. Front. Bioeng. Biotechnol.6, 1–20. 10.3389/fbioe.2018.00157
23
DuffusJ. H. (2002). "Heavy Metals" a Meaningless Term? (IUPAC Technical Report). Pure Appl. Chem.74, 793–807. 10.1351/pac200274050793
24
DvořákP.NikelP. I.DamborskýJ.de LorenzoV. (2017). Bioremediation 3.0 : Engineering Pollutant-Removing Bacteria in the Times of Systemic Biology. Biotechnol. Adv.35, 845–866. 10.1016/j.biotechadv.2017.08.001
25
EinhausA.BaierT.RosenstengelM.FreudenbergR. A.KruseO. (2021). Rational Promoter Engineering Enables Robust Terpene Production in Microalgae. ACS Synth. Biol.10, 847–856. 10.1021/acssynbio.0c00632
26
FabrisM.AbbrianoR. M.PerniceM.SutherlandD. L.CommaultA. S.HallC. C.et al (2020). Emerging Technologies in Algal Biotechnology: Toward the Establishment of a Sustainable, Algae-Based Bioeconomy. Front. Plant Sci.11, 1–22. 10.3389/fpls.2020.00279
27
FajardoC.DonatoM.CarrascoR.Martínez‐RodríguezG.ManceraJ. M.Fernández‐AceroF. J. (2020). Advances and Challenges in Genetic Engineering of Microalgae. Rev. Aquacult12, 365–381. 10.1111/raq.12322
28
FilizE.SaracogluI. A.OzyigitI. I.YalcinB. (2019). Comparative Analyses of Phytochelatin Synthase (PCS) Genes in Higher Plants. Biotechnol. Biotechnological Equipment33, 178–194. 10.1080/13102818.2018.1559096
29
FuL.-H.WangX.-F.EyalY.SheY.-M.DonaldL. J.StandingK. G.et al (2002). A Selenoprotein in the Plant Kingdom.Mass Spectrometry Confirms that an Opal Codon(UGA) Encodes Selenocysteine in Chlamydomonas Reinhardtii Glutathione Peroxidase. J. Biol. Chem.277, 25983–25991. 10.1074/jbc.M202912200
30
García-GarcíaJ. D.Sánchez-ThomasR.SaavedraE.Fernández-VelascoD. A.Romero-RomeroS.Casanova-FigueroaK. I.et al (2020). Mapping the Metal-Catalytic Site of a Zinc-Activated Phytochelatin Synthase. Algal Res.47, 101890. 10.1016/j.algal.2020.101890
31
GevaP.KahtaR.NakonechnyF.AronovS.NisnevitchM. (2016). Increased Copper Bioremediation Ability of New Transgenic and Adapted Saccharomyces cerevisiae Strains. Environ. Sci. Pollut. Res.23, 19613–19625. 10.1007/s11356-016-7157-4
32
GimpelJ. A.Nour-EldinH. H.ScrantonM. A.LiD.MayfieldS. P. (2016). Refactoring the Six-Gene Photosystem II Core in the Chloroplast of the Green Algae Chlamydomonas Reinhardtii. ACS Synth. Biol.5, 589–596. 10.1021/acssynbio.5b00076
33
GleizerS.Ben-NissanR.Bar-OnY. M.AntonovskyN.NoorE.ZoharY.et al (2019). Conversion of Escherichia coli to Generate All Biomass Carbon from CO2. Cell179, 1255–1263.e12. 10.1016/j.cell.2019.11.009
34
GonzálezHenaoS.Ghneim-HerreraT. (2021). Heavy Metals in Soils and the Remediation Potential of Bacteria Associated with the Plant Microbiome. Front. Environ. Sci.9, 1–17. 10.3389/fenvs.2021.604216
35
GuoW.-J.MeetamM.GoldsbroughP. B. (2008). Examining the Specific Contributions of Individual Arabidopsis Metallothioneins to Copper Distribution and Metal Tolerance. Plant Physiol.146, 1697–1706. 10.1104/pp.108.115782
36
HanS.HuZ.LeiA. (2008). Expression and Function Analysis of the Metallothionein-like (MT-like) Gene from Festuca Rubra in Chlamydomonas Reinhardtii Chloroplast. Sci. China Ser. C51, 1076–1081. 10.1007/s11427-008-0136-3
37
HanikenneM.KrämerU.DemoulinV.BaurainD. (2005). A Comparative Inventory of Metal Transporters in the Green Alga Chlamydomonas Reinhardtii and the Red Alga Cyanidioschizon Merolae. Plant Phys.137, 428–446. 10.1104/pp.104.054189.428
38
HeZ.SiripornadulsilS.SayreR. T.TrainaS. J.WeaversL. K. (2011). Removal of Mercury from Sediment by Ultrasound Combined with Biomass (Transgenic Chlamydomonas Reinhardtii). Chemosphere83, 1249–1254. 10.1016/j.chemosphere.2011.03.004
39
HirataK.TsujimotoY.NambaT.OhtaT.HirayanagiN.MiyasakaH.et al (2001). Strong Induction of Phytochelatin Synthesis by Zinc in marine green Alga, Dunaliella Tertiolecta. J. Biosci. Bioeng.92, 24–29. 10.1016/s1389-1723(01)80193-6
40
HuangC.-C.ChenM.-W.HsiehJ.-L.LinW.-H.ChenP.-C.ChienL.-F. (2006). Expression of Mercuric Reductase from Bacillus Megaterium MB1 in Eukaryotic Microalga Chlorella Sp. DT: An Approach for Mercury Phytoremediation. Appl. Microbiol. Biotechnol.72, 197–205. 10.1007/s00253-005-0250-0
41
HuangR.HuoG.SongS.LiY.XiaL.GaillardJ.-F. (2019). Immobilization of Mercury Using High-Phosphate Culture-Modified Microalgae. Environ. Pollut.254, 112966. 10.1016/j.envpol.2019.112966
42
IbuotA.DeanA. P.McIntoshO. A.PittmanJ. K. (2017). Metal Bioremediation by CrMTP4 Over-expressing Chlamydomonas Reinhardtii in Comparison to Natural Wastewater-Tolerant Microalgae Strains. Algal Res.24, 89–96. 10.1016/j.algal.2017.03.002
43
IbuotA.WebsterR. E.WilliamsL. E.PittmanJ. K. (2020). Increased Metal Tolerance and Bioaccumulation of Zinc and Cadmium inChlamydomonas Reinhardtiiexpressing a AtHMA4 C‐terminal Domain Protein. Biotechnol. Bioeng.117, 2996–3005. 10.1002/bit.27476
44
JaegerD.BaierT.LauersenK. J. (2019). Intronserter, an Advanced Online Tool for Design of Intron Containing Transgenes. Algal Res.42, 101588. 10.1016/j.algal.2019.101588
45
JafarianV.GhaffariF. (2017). A Unique Metallothionein-Engineered in Escherichia coli for Biosorption of Lead, Zinc, and Cadmium; Absorption or Adsorption?Microbiology86, 73–81. 10.1134/S0026261717010064
46
JagadevanS.BanerjeeA.BanerjeeC.GuriaC.TiwariR.BawejaM.et al (2018). Recent Developments in Synthetic Biology and Metabolic Engineering in Microalgae towards Biofuel Production. Biotechnol. Biofuels11, 1–21. 10.1186/s13068-018-1181-1
47
KaminskiM. M.AbudayyehO. O.GootenbergJ. S.ZhangF.CollinsJ. J. (2021). CRISPR-based Diagnostics. Nat. Biomed. Eng.5, 643–656. 10.1038/s41551-021-00760-7
48
KellyD. J. A.BuddK.LefebvreD. D. (2006). Biotransformation of Mercury in pH-Stat Cultures of Eukaryotic Freshwater Algae. Arch. Microbiol.187, 45–53. 10.1007/s00203-006-0170-0
49
KizawaA.KawaharaA.TakimuraY.NishiyamaY.HiharaY. (2016). RNA-seq Profiling Reveals Novel Target Genes of LexA in the Cyanobacterium Synechocystis Sp. PCC 6803. Front. Microbiol.7, 1–14. 10.3389/fmicb.2016.00193
50
KrishnanA.CanoM.BurchT. A.WeissmanJ. C.PosewitzM. C. (2020). Genome Editing Using Cas9-RNA Ribonucleoprotein Complexes in the High-Productivity marine Alga Picochlorum Celeri. Algal Res.49, 101944. 10.1016/j.algal.2020.101944
51
KumarG.ShekhA.JakhuS.SharmaY.KapoorR.SharmaT. R. (2020). Bioengineering of Microalgae: Recent Advances, Perspectives, and Regulatory Challenges for Industrial Application. Front. Bioeng. Biotechnol.8, 914. 10.3389/fbioe.2020.00914
52
KumarK. S.DahmsH.-U.WonE.-J.LeeJ.-S.ShinK.-H. (2015). Microalgae - A Promising Tool for Heavy Metal Remediation. Ecotoxicology Environ. Saf.113, 329–352. 10.1016/j.ecoenv.2014.12.019
53
KuoE. Y.CaiM.-S.LeeT.-M. (2020). Ascorbate Peroxidase 4 Plays a Role in the Tolerance of Chlamydomonas Reinhardtii to Photo-Oxidative Stress. Sci. Rep.10, 1–12. 10.1038/s41598-020-70247-z
54
LeeL.HsuC.-Y.YenH.-W. (2017). The Effects of Hydraulic Retention Time (HRT) on Chromium(VI) Reduction Using Autotrophic Cultivation of Chlorella Vulgaris. Bioproc. Biosyst. Eng.40, 1725–1731. 10.1007/s00449-017-1827-6
55
LemaireS.KeryerE.SteinM.SchepensI.Issakidis-BourguetE.Gérard-HirneC.et al (1999). Heavy-Metal Regulation of Thioredoxin Gene Expression inChlamydomonas Reinhardtii. Plant Physiol.120, 773–778. 10.1104/pp.120.3.773
56
LeongY. K.ChangJ.-S. (2020). Bioremediation of Heavy Metals Using Microalgae: Recent Advances and Mechanisms. Bioresour. Tech.303, 122886. 10.1016/j.biortech.2020.122886
57
LiC.ZhengC.FuH.ZhaiS.HuF.NaveedS.et al (2021). Contrasting Detoxification Mechanisms of Chlamydomonas Reinhardtii under Cd and Pb Stress. Chemosphere274, 129771. 10.1016/j.chemosphere.2021.129771
58
LiuT.GoldenJ. W.GiedrocD. P. (2005). A Zinc(II)/Lead(II)/Cadmium(II)-Inducible Operon from the CyanobacteriumAnabaenaIs Regulated by AztR, an α3N ArsR/SmtB Metalloregulator. Biochemistry44, 8673–8683. 10.1021/bi050450+
59
MaX.ChenY.LiuF.ZhangS.WeiQ. (2021). Enhanced Tolerance and Resistance Characteristics of Scenedesmus Obliquus FACHB-12 with K3 Carrier in Cadmium Polluted Water. Algal Res.55, 102267. 10.1016/j.algal.2021.102267
60
MaX.ZhangB.MiaoR.DengX.DuanY.ChengY.et al (2020). Transcriptomic and Physiological Responses to Oxidative Stress in a Chlamydomonas Reinhardtii Glutathione Peroxidase Mutant. Genes11, 1–26. 10.3390/genes11040463
61
MantzorouA.NavakoudisE.PaschalidisK.VerveridisF. (2018). Microalgae: a Potential Tool for Remediating Aquatic Environments from Toxic Metals. Int. J. Environ. Sci. Technol.15, 1815–1830. 10.1007/s13762-018-1783-y
62
MehrshahiP.NguyenG. T. D. T.Gorchs RoviraA.SayerA.Llavero-PasquinaM.Lim Huei SinM.et al (2020). Development of Novel Riboswitches for Synthetic Biology in the Green Alga Chlamydomonas. ACS Synth. Biol.9, 1406–1417. 10.1021/acssynbio.0c00082
63
MengF.EllisT. (2020). The Second Decade of Synthetic Biology: 2010-2020. Nat. Commun.11, 1–4. 10.1038/s41467-020-19092-2
64
MerchantS. S.SchmollingerS.StrenkertD.MoseleyJ. L.Blaby-HaasC. E. (2020). From Economy to Luxury: Copper Homeostasis in Chlamydomonas and Other Algae. Biochim. Biophys. Acta (Bba) - Mol. Cell Res.1867, 118822. 10.1016/j.bbamcr.2020.118822
65
MonteiroC. M.CastroP. M. L.MalcataF. X. (2012). Metal Uptake by Microalgae: Underlying Mechanisms and Practical Applications. Biotechnol. Prog.28, 299–311. 10.1002/btpr.1504
66
MuW.JiaK.LiuY.PanX.FanY. (2017). Response of the Freshwater Diatom Halamphora veneta (Kützing) Levkov to Copper and Mercury and its Potential for Bioassessment of Heavy Metal Toxicity in Aquatic Habitats. Environ. Sci. Pollut. Res.24, 26375–26386. 10.1007/s11356-017-0225-6
67
NaduthodiM. I. S.ClaassensN. J.D’AdamoS.van der OostJ.BarbosaM. J. (2021). Synthetic Biology Approaches to Enhance Microalgal Productivity. Trends Biotechnol.39, 1019–1036. 10.1016/j.tibtech.2020.12.010
68
NagelK.AdelmeierU.VoigtJ. (1996). Subcellular Distribution of Cadmium in the Unicellular green Alga Chlamydomonas Reinhardtii. J. Plant Physiol.149, 86–90. 10.1016/S0176-1617(96)80178-7
69
NarayananN. N.IhemereU.ChiuW. T.SiritungaD.RajamaniS.SinghS.et al (2011). The Iron Assimilatory Protein, FEA1, from Chlamydomonas Reinhardtii Facilitates Iron-specific Metal Uptake in Yeast and Plants. Front. Plant Sci.2, 1–13. 10.3389/fpls.2011.00067
70
NaveedS.LiC.LuX.ChenS.YinB.ZhangC.et al (2019). Microalgal Extracellular Polymeric Substances and Their Interactions with Metal(loid)s: A Review. Crit. Rev. Environ. Sci. Tech.49, 1769–1802. 10.1080/10643389.2019.1583052
71
NishikawaK.OnoderaA.TominagaN. (2006). Phytochelatins Do Not Correlate with the Level of Cd Accumulation in Chlamydomonas Spp. Chemosphere63, 1553–1559. 10.1016/j.chemosphere.2005.09.056
72
PageM. D.AllenM. D.KropatJ.UrzicaE. I.KarpowiczS. J.HsiehS. I.et al (2012). Fe Sparing and Fe Recycling Contribute to Increased Superoxide Dismutase Capacity in Iron-Starved Chlamydomonas Reinhardtii. Plant Cell24, 2649–2665. 10.1105/tpc.112.098962
73
PakdeeO.SongnuanW.PanvisavasN.PokethitiyookP.YokthongwattanaK.MeetamM. (2019). Functional Characterization of Metallothionein-like Genes from Physcomitrella Patens: Expression Profiling, Yeast Heterologous Expression, and Disruption of PpMT1.2a Gene. Planta250, 427–443. 10.1007/s00425-019-03173-8
74
PalR.RaiJ. P. N. (2010). Phytochelatins: Peptides Involved in Heavy Metal Detoxification. Appl. Biochem. Biotechnol.160, 945–963. 10.1007/s12010-009-8565-4
75
PapryR. I.IshiiK.MamunM. A. A.MiahS.NaitoK.MashioA. S.et al (2019). Arsenic Biotransformation Potential of Six marine Diatom Species: Effect of Temperature and Salinity. Sci. Rep.9, 1–16. 10.1038/s41598-019-46551-8
76
PereiraS. B.SousaA.SantosM.AraújoM.SerôdioF.GranjaP.et al (2019). Strategies to Obtain Designer Polymers Based on Cyanobacterial Extracellular Polymeric Substances (EPS). Ijms20, 1–18. 10.3390/ijms20225693
77
PerniS.HackettL.GossR. J.SimmonsM. J.OvertonT. W. (2013). Optimisation of Engineered Escherichia coli Biofilms for Enzymatic Biosynthesis of L-Halotryptophans. AMB Expr.3, 1–10. 10.1186/2191-0855-3-66
78
Piña-OlavideR.Paz-MaldonadoL. M. T.Alfaro-De La TorreM. C.García-SotoM. J.Ramírez-RodríguezA. E.Rosales-MendozaS.et al (2020). Increased Removal of Cadmium by Chlamydomonas Reinhardtii Modified with a Synthetic Gene for γ-glutamylcysteine Synthetase. Int. J. Phytoremediation22, 1269–1277. 10.1080/15226514.2020.1765138
79
PochodyloA. L.AristildeL. (2017). Molecular Dynamics of Stability and Structures in Phytochelatin Complexes with Zn, Cu, Fe, Mg, and Ca: Implications for Metal Detoxification. Environ. Chem. Lett.15, 495–500. 10.1007/s10311-017-0609-3
80
PolinerE.ClarkE.CummingsC.BenningC.FarreE. M. (2020). A High-Capacity Gene Stacking Toolkit for the Oleaginous Microalga, Nannochloropsis Oceanica CCMP1779. Algal Res.45, 101664. 10.1016/j.algal.2019.101664
81
PolinerE.FarréE. M.BenningC. (2018). Advanced Genetic Tools Enable Synthetic Biology in the Oleaginous Microalgae Nannochloropsis Sp. Plant Cell Rep37, 1383–1399. 10.1007/s00299-018-2270-0
82
Puente-SánchezF.DíazS.PenachoV.AguileraA.OlssonS. (2018). Basis of Genetic Adaptation to Heavy Metal Stress in the Acidophilic green Alga Chlamydomonas Acidophila. Aquat. Toxicol.200, 62–72. 10.1016/j.aquatox.2018.04.020
83
QuinnJ. M.KropatJ.MerchantS. (2003). Copper Response Element and Crr1-dependent Ni 2+ -Responsive Promoter for Induced, Reversible Gene Expression in Chlamydomonas Reinhardtii. Eukaryot. Cell2, 995–1002. 10.1128/EC.2.5.995-1002.2003
84
RahmanZ.SinghV. P. (2019). The Relative Impact of Toxic Heavy Metals (THMs) (Arsenic (As), Cadmium (Cd), Chromium (Cr)(VI), Mercury (Hg), and lead (Pb)) on the Total Environment: an Overview. Environ. Monit. Assess.191, 419. 10.1007/s10661-019-7528-7
85
Reyes-CaballeroH.CampanelloG. C.GiedrocD. P. (2011). Metalloregulatory Proteins: Metal Selectivity and Allosteric Switching. Biophysical Chem.156, 103–114. 10.1016/j.bpc.2011.03.010
86
RubinelliP.SiripornadulsilS.Gao-RubinelliF.SayreR. (2002). Cadmium- and Iron-Stress-Inducible Gene Expression in the green Alga Chlamydomonas Reinhardtii : Evidence for H43 Protein Function in Iron Assimilation. Planta215, 1–13. 10.1007/s00425-001-0711-3
87
SalamaE.-S.RohH.-S.DevS.KhanM. A.Abou-ShanabR. A. I.ChangS. W.et al (2019). Algae as a green Technology for Heavy Metals Removal from Various Wastewater. World J. Microbiol. Biotechnol.35, 1–19. 10.1007/s11274-019-2648-3
88
Sanz-LuqueE.BhayaD.GrossmanA. R. (2020). Polyphosphate: A Multifunctional Metabolite in Cyanobacteria and Algae. Front. Plant Sci.11, 1–21. 10.3389/fpls.2020.00938
89
SatoT.KobayashiY. (1998). The Ars Operon in the Skin Element of Bacillus Subtilis Confers Resistance to Arsenate and Arsenite. J. Bacteriol.180, 1655–1661. 10.1128/jb.180.7.1655-1661.1998
90
SattayawatP.YunusI. S.JonesP. R. (2020). Bioderivatization as a Concept for Renewable Production of Chemicals that Are Toxic or Poorly Soluble in the Liquid Phase. Proc. Natl. Acad. Sci. USA117, 1404–1413. 10.1073/pnas.1914069117
91
SchatzD.NagarE.SenderskyE.ParnasaR.ZilbermanS.CarmeliS.et al (2013). Self-suppression of Biofilm Formation in the cyanobacteriumSynechococcus Elongatus. Environ. Microbiol.15, 1786–1794. 10.1111/1462-2920.12070
92
SelviA.RajasekarA.TheerthagiriJ.AnanthaselvamA.SathishkumarK.MadhavanJ.et al (2019). Integrated Remediation Processes toward Heavy Metal Removal/recovery from Various Environments-A Review. Front. Environ. Sci.7, 66. 10.3389/fenvs.2019.00066
93
ShahpiriA.MohammadzadehA. (2018). Mercury Removal by Engineered Escherichia coli Cells Expressing Different rice Metallothionein Isoforms. Ann. Microbiol.68, 145–152. 10.1007/s13213-018-1326-2
94
ShenM. W. Y.ShahD.ChenW.Da SilvaN. (2012). Enhanced Arsenate Uptake in Saccharomyces cerevisiae Overexpressing the Pho84 Phosphate Transporter. Biotechnol. Prog.28, 654–661. 10.1002/btpr.1531
95
SilverS.PhungL. T. (2005). A Bacterial View of the Periodic Table: Genes and Proteins for Toxic Inorganic Ions. J. Ind. Microbiol. Biotechnol.32, 587–605. 10.1007/s10295-005-0019-6
96
SinghS.KangS. H.LeeW.MulchandaniA.ChenW. (2009). Systematic Engineering of Phytochelatin Synthesis and Arsenic Transport for Enhanced Arsenic Accumulation in E. Coli. Biotechnol. Bioeng.105, 780–785. 10.1002/bit.22585
97
SinghS.MulchandaniA.ChenW. (2008). Highly Selective and Rapid Arsenic Removal by Metabolically Engineered Escherichia coli Cells Expressing Fucus Vesiculosus Metallothionein. Appl. Environ. Microbiol.74, 2924–2927. 10.1128/AEM.02871-07
98
SiripornadulsilS.TrainaS.VermaD. P. S.SayreR. T. (2002). Molecular Mechanisms of Proline-Mediated Tolerance to Toxic Heavy Metals in Transgenic Microalgae. Plant Cell14, 2837–2847. 10.1105/tpc.004853
99
SommerF.KropatJ.MalasarnD.GrossoehmeN. E.ChenX.GiedrocD. P.et al (2010). The CRR1 Nutritional Copper Sensor in Chlamydomonas Contains Two Distinct Metal-Responsive Domains. Plant Cell22, 4098–4113. 10.1105/tpc.110.080069
100
SoneY.NakamuraR.Pan-HouH.ItohT.KiyonoM. (2013). Role of MerC, MerE, MerF, MerT, And/or MerP in Resistance to Mercurials and the Transport of Mercurials in Escherichia coli. Biol. Pharm. Bull.36, 1835–1841. 10.1248/bpb.b13-00554
101
SprolesA. E.FieldsF. J.SmalleyT. N.LeC. H.BadaryA.MayfieldS. P. (2021). Recent Advancements in the Genetic Engineering of Microalgae. Algal Res.53, 102158. 10.1016/j.algal.2020.102158
102
SüdfeldC.HubáčekM.FigueiredoD.NaduthodiM. I. S.van der OostJ.WijffelsR. H.et al (2021). High-throughput Insertional Mutagenesis Reveals Novel Targets for Enhancing Lipid Accumulation in Nannochloropsis Oceanica. Metab. Eng.66, 239–258. 10.1016/j.ymben.2021.04.012
103
SunX.-M.RenL.-J.ZhaoQ.-Y.JiX.-J.HuangH. (2018). Microalgae for the Production of Lipid and Carotenoids: A Review with Focus on Stress Regulation and Adaptation. Biotechnol. Biofuels11, 1–16. 10.1186/s13068-018-1275-9
104
ThongpitakJ.PekkohJ.PumasC. (2019). Remediation of Manganese-Contaminated Coal-Mine Water Using Bio-Sorption and Bio-Oxidation by the Microalga Pediastrum Duplex (AARLG060): A Laboratory-Scale Feasibility Study. Front. Microbiol.10, 1–14. 10.3389/fmicb.2019.02605
105
Tibocha-BonillaJ. D.ZuñigaC.Godoy-SilvaR. D.ZenglerK. (2018). Advances in Metabolic Modeling of Oleaginous Microalgae. Biotechnol. Biofuels11, 1–15. 10.1186/s13068-018-1244-3
106
TranN. T.KaldenhoffR. (2020). Achievements and Challenges of Genetic Engineering of the Model green Alga Chlamydomonas Reinhardtii. Algal Res.50, 101986. 10.1016/j.algal.2020.101986
107
WangB.WangJ.ZhangW.MeldrumD. R. (2012). Application of Synthetic Biology in Cyanobacteria and Algae. Front. Microbio.3, 1–15. 10.3389/fmicb.2012.00344
108
WangL.JiaX.ZhangY.XuL.MenandB.ZhaoH.et al (2021). Loss of Two Families of SPX Domain-Containing Proteins Required for Vacuolar Polyphosphate Accumulation Coincides with the Transition to Phosphate Storage in green Plants. Mol. Plant14, 838–846. 10.1016/j.molp.2021.01.015
109
WangT.WuM. (2006). An ATP-Binding Cassette Transporter Related to Yeast Vacuolar ScYCF1 Is Important for Cd Sequestration in Chlamydomonas Reinhardtii. Plant Cell Environ29, 1901–1912. 10.1111/j.1365-3040.2006.01566.x
110
WangY.WangS.XuP.LiuC.LiuM.WangY.et al (2015). Review of Arsenic Speciation, Toxicity and Metabolism in Microalgae. Rev. Environ. Sci. Biotechnol.14, 427–451. 10.1007/s11157-015-9371-9
111
WeiY. Y.ZhengQ.LiuZ. P.YangZ. M. (2011). Regulation of Tolerance of Chlamydomonas Reinhardtii to Heavy Metal Toxicity by Heme Oxygenase-1 and Carbon Monoxide. Plant Cell Physiol.52, 1665–1675. 10.1093/pcp/pcr102
112
WichmannJ.BaierT.WentnagelE.LauersenK. J.KruseO. (2018). Tailored Carbon Partitioning for Phototrophic Production of (E)-α-bisabolene from the green Microalga Chlamydomonas Reinhardtii. Metab. Eng.45, 211–222. 10.1016/j.ymben.2017.12.010
113
WichmannJ.LauersenK. J.BiondiN.ChristensenM.GuerraT.HellgardtK.et al (2021). Engineering Biocatalytic Solar Fuel Production: The PHOTOFUEL Consortium. Trends Biotechnol.39, 323–327. 10.1016/j.tibtech.2021.01.003
114
WindhagauerM.AbbrianoR. M.AshworthJ.BaroloL.Jaramillo-MadridA. C.PerniceM.et al (2021). Characterisation of Novel Regulatory Sequences Compatible with Modular Assembly in the Diatom Phaeodactylum Tricornutum. Algal Res.53, 102159. 10.1016/j.algal.2020.102159
115
WollmannF.DietzeS.AckermannJ. U.BleyT.WaltherT.SteingroewerJ.et al (2019). Microalgae Wastewater Treatment: Biological and Technological Approaches. Eng. Life Sci.19, 860–871. 10.1002/elsc.201900071
116
XiaoR.ZhengY. (2016). Overview of Microalgal Extracellular Polymeric Substances (EPS) and Their Applications. Biotechnol. Adv.34, 1225–1244. 10.1016/j.biotechadv.2016.08.004
117
YadavA. P. S.DwivediV.KumarS.KushwahaA.GoswamiL.ReddyB. S. (2020). Cyanobacterial Extracellular Polymeric Substances for Heavy Metal Removal: A Mini Review. J. Compos. Sci.5, 1. 10.3390/jcs5010001
118
YangT.LiuJ.-W.GuC.ChenM.-L.WangJ.-H. (2013). Expression of Arsenic Regulatory Protein in escherichia Coli for Selective Accumulation of Methylated Arsenic Species. ACS Appl. Mater. Inter.5, 2767–2772. 10.1021/am400578y
119
YunusI. S.WichmannJ.WördenweberR.LauersenK. J.KruseO.JonesP. R. (2018). Synthetic Metabolic Pathways for Photobiological Conversion of CO2 into Hydrocarbon Fuel. Metab. Eng.49, 201–211. 10.1016/j.ymben.2018.08.008
120
ZagorskiN.WilsonD. B. (2004). Characterization and Comparison of Metal Accumulation in Two Escherichia coli Strains Expressing Either CopA or MntA, Heavy Metal-Transporting Bacterial P-type Adenosine Triphosphatases. Abab117, 33–48. 10.1385/abab:117:1:33
121
ZillerA.YadavR. K.CapdevilaM.ReddyM. S.VallonL.MarmeisseR.et al (2017). Metagenomics Analysis Reveals a New Metallothionein Family: Sequence and Metal-Binding Features of New Environmental Cysteine-Rich Proteins. J. Inorg. Biochem.167, 1–11. 10.1016/j.jinorgbio.2016.11.017
122
ZitkaO.KrystofovaO.SobrovaP.AdamV.ZehnalekJ.BeklovaM.et al (2011). Phytochelatin Synthase Activity as a Marker of Metal Pollution. J. Hazard. Mater.192, 794–800. 10.1016/j.jhazmat.2011.05.088
123
ZuñigaC.LeveringJ.AntoniewiczM. R.GuarnieriM. T.BetenbaughM. J.ZenglerK. (2018). Predicting Dynamic Metabolic Demands in the Photosynthetic Eukaryote Chlorella Vulgaris. Plant Physiol.176, 450–462. 10.1104/pp.17.00605
Summary
Keywords
synthetic biology, heavy metal, microalgae, bio-removal, wastewater treament
Citation
Sattayawat P, Yunus IS, Noirungsee N, Mukjang N, Pathom-Aree W, Pekkoh J and Pumas C (2021) Synthetic Biology-Based Approaches for Microalgal Bio-Removal of Heavy Metals From Wastewater Effluents. Front. Environ. Sci. 9:778260. doi: 10.3389/fenvs.2021.778260
Received
16 September 2021
Accepted
08 November 2021
Published
24 November 2021
Volume
9 - 2021
Edited by
Yongjun Wei, Zhengzhou University, China
Reviewed by
Adel Al-Gheethi, Universiti Tun Hussein Onn Malaysia, Malaysia
Dillirani Nagarajan, National Cheng Kung University, Taiwan
Updates
Copyright
© 2021 Sattayawat, Yunus, Noirungsee, Mukjang, Pathom-Aree, Pekkoh and Pumas.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Pachara Sattayawat, pachara.sattayawat@cmu.ac.th
This article was submitted to Water and Wastewater Management, a section of the journal Frontiers in Environmental Science
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