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MoCloro : an extension of the Chlamydomonas reinhardtii modular cloning toolkit for microalgal chloroplast engineering

Authors: Xavier Melero‐Cobo; Marçal Gallemí; Marc Carnicer; Elena Monte; Antoni Planas; Pablo Leivar;

MoCloro : an extension of the Chlamydomonas reinhardtii modular cloning toolkit for microalgal chloroplast engineering

Abstract

Abstract Photosynthetic microalgae are promising green cell factories for the sustainable production of high‐value chemicals and biopharmaceuticals. The chloroplast organelle is being developed as a chassis for synthetic biology as it contains its own genome (the plastome) and some interesting advantages, such as high recombinant protein titers and a diverse and dynamic metabolism. However, chloroplast engineering is currently hampered by the lack of standardized cloning tools and Design‐Build‐Test‐Learn workflows to ease genomic and metabolic engineering. The MoClo (Modular Cloning) toolkit based on Golden Gate assembly was recently developed in the model eukaryotic green microalgae Chlamydomonas reinhardtii to facilitate nuclear transformation and engineering. Here, we present MoCloro as an extension of the MoClo that allows chloroplast genome engineering. Briefly, a Golden Gate‐compatible chloroplast transformation vector (pWF.K.4) was constructed, which contains homologous arms for integration at the petA site in the plastome. A collection of standardized parts (promoters, terminators, reporter and selection marker genes) was created following the MoClo syntax to enable easy combinatorial assembly of multi‐cassettes in the destination pWF.K.4 vector. The functionality of the biobricks was assayed by constructing and assessing the expression of several multigenic constructs. Finally, a generic vector pK4 was constructed for easy Golden Gate cloning of 5′ and 3′ homologous arms, allowing targeting at alternative plastome integration sites. This work represents a significant advancement in technology aimed at facilitating more efficient and rapid chloroplast transformation and engineering of green microalgae.

Country
Spain
Keywords

Chloroplasts, Clonatge, Genetic Vectors, 58, Genomics, 575, Genòmica, Transformation, Genetic, Technical Advance, Cloroplasts, Microalgae, Microalgues, Cloning, Molecular, Genetic Engineering, Genome, Chloroplast, Chlamydomonas reinhardtii, Cloning

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    popularity
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    Top 10%
    influence
    This indicator reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).
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    impulse
    This indicator reflects the initial momentum of an article directly after its publication, based on the underlying citation network.
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selected citations
These citations are derived from selected sources.
This is an alternative to the "Influence" indicator, which also reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).
BIP!Citations provided by BIP!
popularity
This indicator reflects the "current" impact/attention (the "hype") of an article in the research community at large, based on the underlying citation network.
BIP!Popularity provided by BIP!
influence
This indicator reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).
BIP!Influence provided by BIP!
impulse
This indicator reflects the initial momentum of an article directly after its publication, based on the underlying citation network.
BIP!Impulse provided by BIP!
8
Top 10%
Average
Top 10%
Green
hybrid