Achievements and challenges of genetic engineering of the model green alga Chlamydomonas reinhardtii

Tran, Nam Trung; Kaldenhoff, Ralf

doi:10.1016/j.algal.2020.101986

Cited by 38 publications

(20 citation statements)

References 138 publications

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“…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.…”

Section: Prospects Of Synthetic Biology-based Approaches For Microalgal Heavy Metal Bio-removalmentioning

confidence: 99%

Synthetic Biology-Based Approaches for Microalgal Bio-Removal of Heavy Metals From Wastewater Effluents

Sattayawat

Yunus

Noirungsee

et al. 2021

Front. Environ. Sci.

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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.

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Section: Prospects Of Synthetic Biology-based Approaches For Microalgal Heavy Metal Bio-removalmentioning

confidence: 99%

Synthetic Biology-Based Approaches for Microalgal Bio-Removal of Heavy Metals From Wastewater Effluents

Sattayawat

Yunus

Noirungsee

et al. 2021

Front. Environ. Sci.

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“…The green model microalga Chlamydomonas reinhardtii [1] has become a powerful biotechnological production host for a wide range of recombinant proteins [2][3][4] and metabolites [5][6][7]. Innovative tools for metabolic engineering, gene editing and synthetic biology are developing rapidly [8][9][10][11][12][13][14]. However, heterologous gene expression requires selection systems to establish sufficient expression levels and to prevent transgene silencing.…”

Section: Introductionmentioning

confidence: 99%

The Spermidine Synthase Gene SPD1: A Novel Auxotrophic Marker for Chlamydomonas Reinhardtii Designed by Enhanced CRISPR/Cas9 Gene Editing

Freudenberg¹,

Wittemeier²,

Einhaus³

et al. 2022

Preprint

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Biotechnological application of the green microalga Chlamydomonas reinhardtii hinges on the availability of selectable markers for effective expression of multiple transgenes. However, biological safety concerns limit the establishment of new antibiotic resistance genes and until today, only few auxotrophic markers exist for C. reinhardtii. The recent improvements in gene editing via CRISPR/Cas9 allows directed exploration of new endogenous selectable markers. Since editing frequencies with CRISPR/Cas9 techniques are often low, the Cas9-sgRNA ribonucleoprotein (RNP) delivery protocol was strategically optimized by applying nitrogen starvation to the pre-culture, increasing editing frequencies from 10% to 66% after pre-selection. Probing the essential polyamine biosynthesis pathway, the spermidine synthase gene (SPD1) is shown to be a potent selectable marker with versatile biotechnological applicability. Very low levels of spermidine (0.75 mg/L) were required to maintain normal mixotrophic and phototrophic growth in newly designed spermidine auxotrophic strains. Complementation of these strains with a synthetic SPD1 gene was achieved when the mature protein was targeted to either the cytosol or the chloroplast. This work highlights the potential of new selectable markers for biotechnology as well as basic research and proposes an effective pipeline for the identification of new auxotrophies in C. reinhardtii.

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“…Among several eukaryotic algae, the unicellular green alga Chlamydomonas reinhardtii has been the most extensively studied because sexual reproduction in this alga has facilitated genetic studies—especially those on the cilium and photosynthesis, which are absent in yeast. As a result, genetic modification procedures were established in this alga >30 years ago ( Harris et al 2009 , Tran and Kaldenhoff 2020 ). Recently, procedures for efficient genetic modification have also been developed in some other unicellular eukaryotic algae, such as the red alga C. merolae (described below), the green alga Ostreococcus tauri ( Corellou et al 2009 , Sanchez et al 2019 ), the diatom Phaeodactylum tricornutum ( Butler et al 2020 ) and the eustigmatophytes Nannochloropsis spp.…”

Section: Introductionmentioning

confidence: 99%

The Unicellular Red AlgaCyanidioschyzon merolae—The Simplest Model of a Photosynthetic Eukaryote

Miyagishima

Tanaka

2021

Plant and Cell Physiology

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Several species of unicellular eukaryotic algae exhibit relatively simple genomic and cellular architecture. Laboratory cultures of these algae grow faster than plants and often provide homogeneous cellular populations exposed to an almost equal environment. These characteristics are ideal for conducting experiments at the cellular and subcellular levels. Many microalgal lineages have recently become genetically tractable, which have started to evoke new streams of studies. Among such algae, the unicellular red alga Cyanidioschyzon merolae is the simplest organism; it possessses the minimum number of membranous organelles, only 4,775 protein-coding genes in the nucleus, and its cell cycle progression can be highly synchronized with the diel cycle. These properties facilitate diverse omics analyses of cellular proliferation and structural analyses of the intracellular relationship among organelles. C. merolae cells lack a rigid cell wall and are thus relatively easily disrupted, facilitating biochemical analyses. Multiple chromosomal loci can be edited by highly efficient homologous recombination. The procedures for the inducible/repressive expression of a transgene or an endogenous gene in the nucleus and for chloroplast genome modification have also been developed. Here we summarize the features and experimental techniques of C. merolae and provide examples of studies using this alga. From these studies, it is clear that C. merolae – either alone or in comparative and combinatory studies with other photosynthetic organisms – can provide significant insights into the biology of photosynthetic eukaryotes.

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Achievements and challenges of genetic engineering of the model green alga Chlamydomonas reinhardtii

Cited by 38 publications

References 138 publications

Synthetic Biology-Based Approaches for Microalgal Bio-Removal of Heavy Metals From Wastewater Effluents

Synthetic Biology-Based Approaches for Microalgal Bio-Removal of Heavy Metals From Wastewater Effluents

The Spermidine Synthase Gene SPD1: A Novel Auxotrophic Marker for Chlamydomonas Reinhardtii Designed by Enhanced CRISPR/Cas9 Gene Editing

The Unicellular Red AlgaCyanidioschyzon merolae—The Simplest Model of a Photosynthetic Eukaryote

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