Metal bioremediation by CrMTP4 over-expressing Chlamydomonas reinhardtii in comparison to natural wastewater-tolerant microalgae strains

Abstract

Metal pollution in freshwater bodies is a long-standing challenge with large expense required to clean-up pollutants such as Cd. There is widespread interest in the potentially low-cost and sustainable use of biological material to perform bioremediation, such as the use of microalgae. Efficient metal bioremediation capacity requires both the ability to tolerate metal stress and metal accumulation. Here, the role of a Chlamydomonas reinhardtii metal tolerance protein (MTP) was examined for enhanced Cd tolerance and uptake. The CrMTP4 gene is a member of the Mn-CDF clade of the cation diffusion facilitator family of metal transporters but is able to provide tolerance and sequestration for Mn and Cd, but not other metals, when expressed in yeast. Over-expression of CrMTP4 in C. reinhardtii yielded a significant increase in tolerance to Cd toxicity and increased Cd accumulation although tolerance to Mn was not increased. In comparison, the metal tolerance of three chlorophyte microalgae strains (Chlorella luteoviridis, Parachlorella hussii, and Parachlorella kessleri) that had previously been adapted to wastewater growth was examined. In comparison to wild type C. reinhardtii, all three natural strains showed significantly increased tolerance to Cd, Cu, Al and Zn, and furthermore their Cd tolerance and uptake was greater than that of the CrMTP4 over-expression strains. Despite CrMTP4 gene over-expression being a successful strategy to enhance the Cd bioremediation potential of a metal-sensitive microalga, a single gene manipulation cannot compete with naturally adapted strain mechanisms that are likely to be multigenic and due in part to oxidative stress tolerance.

Multi-genomic analysis of the Cation Diffusion Facilitator transporters from algae

“…CrMTP1 cDNA was sub-cloned into the XbaI and SacI sites of piUGpd, while CrMTP2 and CrMTP3 cDNA were sub-cloned into the BamHI and XbaI sites of piUGpd. CrMTP4 in piUGpd was generated previously (Ibuot et al 2017).…”

“…CrMTP4 has previously been confirmed to have Mn 2+ transport activity and the ability to transport Cd 2+ (Ibuot et al 2017), but the substrate specificity of the other C. reinhardtii CDF proteins is unknown. The full-length cDNA for each gene was functionally characterised by heterologous expression in yeast (Fig.…”

“…Manipulation of expression of specific transporter proteins, such as by genetic engineering, can therefore be used to enhance metal tolerance of an organism (Antosiewicz et al 2014). Moreover, manipulation of metal sequestration may also provide increased total metal content within a cell whilst still providing metal tolerance, and would be useful for metal bioremediation applications (Cheng et al 2019;Ibuot et al 2017).…”

“…For example, genomic analyses of green microalgae, such as Chlamydomonas reinhardtii, and red microalgae, such as Cyanidioschyzon merolae indicate that there are a wide variety of metal transporter pathways that are conserved between microalgae and higher plants alongside other eukaryotes such as yeast (Hanikenne et al 2005;Blaby-Haas and Merchant 2012). The identification of putative algal metal transporter genes has been complemented by other genome-scale analyses such as by proteomic and transcriptomic methods Beauvais-Flück et al 2019;Puente-Sánchez et al 2018;Khatiwada et al 2020), and subsequent functional characterisation of individual metal transporters (Ibuot et al 2017;Tsednee et al 2019;Chang et al 2020).…”

“…Within the well-studied model microalga C. reinhardtii there are five putative MTP genes (CrMTP1 to CrMTP5). Some of these genes are transcriptionally induced by Zn or Mn deficiency conditions (Allen et al 2007;Malasarn et al 2013), but to date only CrMTP4 has been functionally characterised as a Mn 2+ and Cd 2+ transporter (Ibuot et al 2017). This was determined by performing over-expression of CrMTP4 in C. reinhardtii yielding enhanced Cd tolerance, and by using yeast (S. cerevisiae) heterologous expression where CrMTP4 was able to rescue the Mn sensitive phenotype of the pmr1 yeast mutant.…”