A novel, robust and mating-competent Chlamydomonas reinhardtii strain with an enhanced transgene expression capacity for algal biotechnology

Dementyeva, Polina; Freudenberg, Robert; Baier, Thomas; Rojek, K; Wobbe, Lutz; Weißhaar, Bernd; Kruse, Olaf

doi:10.1016/j.btre.2021.e00644

Cited by 11 publications

(12 citation statements)

References 37 publications

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“…In addition, a UVM11 strain with a cell wall and restored mating-capacity was generated, which opens the possibility of transferring transgenes by crosses and large-scale cultivation ( Neupert et al, 2020 ). To alleviate the mentioned disadvantages of the UVM4 and UVM11 strains, another walled strain with high transgene expression and mating capacity was recently isolated and awaits to be used by the Chlamydomonas community ( Dementyeva et al, 2021 ).…”

Section: Strains With Efficient and Stable Nuclear Transgene Expressionmentioning

confidence: 99%

“…The original Chlamydomonas MoClo toolkit contains 119 standardized parts including promoters, targeting signals, reporters, affinity tags, terminators, and much more ( Crozet et al, 2018 ). Many new parts were added since then ( de Carpentier et al, 2020 ; Mehrshahi et al, 2020 ; Dementyeva et al, 2021 ; Einhaus et al, 2021 ; Geisler et al, 2021 ; Kiefer et al, 2021 ; Niemeyer et al, 2021 ) and it can be anticipated that the kit will continuously grow. Very often, single target genes are tested in Chlamydomonas, for example if mutants are to be complemented ( Theis et al, 2019 ; Spaniol et al, 2022 ).…”

Section: The Chlamydomonas Modular Cloning Kit (Moclo)mentioning

confidence: 99%

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Molecular Advancements Establishing Chlamydomonas as a Host for Biotechnological Exploitation

Schroda

Remacle

2022

Front. Plant Sci.

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Chlamydomonas reinhardtii is emerging as a production platform for biotechnological purposes thanks to recent achievements, which we briefly summarize in this review. Firstly, robust nuclear transgene expression is now possible because several impressive improvements have been made in recent years. Strains allowing efficient and stable nuclear transgene expression are available and were recently made more amenable to rational biotechnological approaches by enabling genetic crosses and identifying their causative mutation. The MoClo synthetic biology strategy, based on Golden Gate cloning, was developed for Chlamydomonas and includes a growing toolkit of more than 100 genetic parts that can be robustly and rapidly assembled in a predefined order. This allows for rapid iterative cycles of transgene design, building, testing, and learning. Another major advancement came from various findings improving transgene design and expression such as the systematic addition of introns into codon-optimized coding sequences. Lastly, the CRISPR/Cas9 technology for genome editing has undergone several improvements since its first successful report in 2016, which opens the possibility of optimizing biosynthetic pathways by switching off competing ones. We provide a few examples demonstrating that all these recent developments firmly establish Chlamydomonas as a chassis for synthetic biology and allow the rewiring of its metabolism to new capabilities.

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Section: Strains With Efficient and Stable Nuclear Transgene Expressionmentioning

confidence: 99%

Section: The Chlamydomonas Modular Cloning Kit (Moclo)mentioning

confidence: 99%

Molecular Advancements Establishing Chlamydomonas as a Host for Biotechnological Exploitation

Schroda

Remacle

2022

Front. Plant Sci.

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“…A feature that has enabled forward genetic mutation studies of the nuclear genome of this alga through random integration of antibiotic selection markers and knock-out of native genes (Rochaix, 1995;Zhang et al, 2014;Li et al, 2019). The over-expression of transgenes from the nuclear genome of C. reinhardtii, however, requires sophisticated transgene design (Baier et al, 2018(Baier et al, , 2020Jaeger et al, 2019;, strain domestication (Neupert et al, 2009(Neupert et al, , 2020Dementyeva et al, 2021), and high-throughput screening of transformants mediated by FP fusions to target recombinant sequences (Abdallah et al, 2022) to identify high transgene-expressing cell lines which can arise from multiple integration events (Shahar et al, 2020) and 'position effects' of NHEJ integration (Kong et al, 2014).…”

Section: Introductionmentioning

confidence: 99%

Teaching an old ‘doc’ new tricks for algal biotechnology: Strategic filter use enables multi-scale fluorescent protein signal detection

Gutiérrez

Wellman

Lauersen

2022

Front. Bioeng. Biotechnol.

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Fluorescent proteins (FPs) are powerful reporters with a broad range of applications in gene expression and subcellular localization. High-throughput screening is often required to identify individual transformed cell lines in organisms that favor non-homologous-end-joining integration of transgenes into genomes, like in the model green microalga Chlamydomonas reinhardtii. Strategic transgene design, including genetic fusion of transgenes to FPs, and strain domestication have aided engineering efforts in this host but have not removed the need for screening large numbers of transformants to identify those with robust transgene expression levels. FPs facilitate transformant screening by providing a visual signal indicating transgene expression. However, limited combinations of FPs have been described in alga and inherent background fluorescence from cell pigments can hinder FP detection efforts depending on available infrastructure. Here, an updated set of algal nuclear genome-domesticated plasmid parts for seven FPs and six epitope tags were generated and tested in C. reinhardtii. Strategic filter selection was found to enable detection of up to five independent FPs signals from cyan to far-red separately from inherent chlorophyll fluorescence in live algae at the agar plate-level and also in protein electrophoresis gels. This work presents technical advances for algal engineering that can assist reporter detection efforts in other photosynthetic host cells or organisms with inherent background fluorescence.

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“…These limitations require organism-specific part collections for a MoClo-based Synthetic Biology strategy. Accordingly, a MoClo toolkit with 119 standardized parts has been established previously for the green alga Chlamydomonas reinhardtii (Crozet et al 2018 ), and this kit is rapidly expanding (de Carpentier et al 2020 ; Dementyeva et al 2021 ; Einhaus et al 2021 ; Geisler et al 2021 ; Kiefer et al 2021 ; Mehrshahi et al 2020 ; Niemeyer et al 2021 ).…”

Section: Introductionmentioning

confidence: 99%

New destination vectors facilitate Modular Cloning for Chlamydomonas

Niemeyer

Schroda

2022

Curr Genet

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Synthetic Biology is revolutionizing biological research by introducing principles of mechanical engineering, including the standardization of genetic parts and standardized part assembly routes. Both are realized in the Modular Cloning (MoClo) strategy. MoClo allows for the rapid and robust assembly of individual genes and multigene clusters, enabling iterative cycles of gene design, construction, testing, and learning in short time. This is particularly true if generation times of target organisms are short, as is the case for the unicellular green alga Chlamydomonas reinhardtii. Testing a gene of interest in Chlamydomonas with MoClo requires two assembly steps, one for the gene of interest itself and another to combine it with a selection marker. To reduce this to a single assembly step, we constructed five new destination vectors. They contain genes conferring resistance to commonly used antibiotics in Chlamydomonas and a site for the direct assembly of basic genetic parts. The vectors employ red/white color selection and, therefore, do not require costly compounds like X-gal and IPTG. mCherry expression is used to demonstrate the functionality of these vectors.

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A novel, robust and mating-competent Chlamydomonas reinhardtii strain with an enhanced transgene expression capacity for algal biotechnology

Cited by 11 publications

References 37 publications

Molecular Advancements Establishing Chlamydomonas as a Host for Biotechnological Exploitation

Molecular Advancements Establishing Chlamydomonas as a Host for Biotechnological Exploitation

Teaching an old ‘doc’ new tricks for algal biotechnology: Strategic filter use enables multi-scale fluorescent protein signal detection

New destination vectors facilitate Modular Cloning for Chlamydomonas

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