The Algal Chloroplast as a Testbed for Synthetic Biology Designs Aimed at Radically Rewiring Plant Metabolism

Sustainable and economically viable support for an ever-increasing global population requires a paradigm shift in agricultural productivity, including the application of biotechnology to generate future crop plants. Current genetic engineering approaches aimed at enhancing the photosynthetic efficiency or composition of the harvested tissues involve relatively simple manipulations of endogenous metabolism. However, radical rewiring of central metabolism using new-to-nature pathways, so-called “synthetic metabolism”, may be needed to really bring about significant step changes. In many cases, this will require re-programming the metabolism of the chloroplast, or other plastids in non-green tissues, through a combination of chloroplast and nuclear engineering. However, current technologies for sophisticated chloroplast engineering (“transplastomics”) of plants are limited to just a handful of species. Moreover, the testing of metabolic rewiring in the chloroplast of plant models is often impractical given their obligate phototrophy, the extended time needed to create stable non-chimeric transplastomic lines, and the technical challenges associated with regeneration of whole plants. In contrast, the unicellular green alga, Chlamydomonas reinhardtii is a facultative heterotroph that allows for extensive modification of chloroplast function, including non-photosynthetic designs. Moreover, chloroplast engineering in C. reinhardtii is facile, with the ability to generate novel lines in a matter of weeks, and a well-defined molecular toolbox allows for rapid iterations of the “Design-Build-Test-Learn” (DBTL) cycle of modern synthetic biology approaches. The recent development of combinatorial DNA assembly pipelines for designing and building transgene clusters, simple methods for marker-free delivery of these clusters into the chloroplast genome, and the pre-existing wealth of knowledge regarding chloroplast gene expression and regulation in C. reinhardtii further adds to the versatility of transplastomics using this organism. Herein, we review the inherent advantages of the algal chloroplast as a simple and tractable testbed for metabolic engineering designs, which could then be implemented in higher plants.

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Recombinant Protein Production in Microalgae: Emerging Trends

Protein and Peptide Letters ◽

10.2174/0929866526666191014124855 ◽

2020 ◽

Vol 27 (2) ◽

pp. 105-110 ◽

Cited By ~ 4

Author(s):

Niaz Ahmad ◽

Muhammad Aamer Mehmood ◽

Sana Malik

Keyword(s):

Chloroplast Genome ◽

Recombinant Proteins ◽

Large Scale ◽

Higher Plants ◽

Scale Up ◽

Chloroplast Transformation ◽

Point Of View ◽

Nuclear Transformation ◽

Scale Production ◽

Algal Chloroplast

: In recent years, microalgae have emerged as an alternative platform for large-scale production of recombinant proteins for different commercial applications. As a production platform, it has several advantages, including rapid growth, easily scale up and ability to grow with or without the external carbon source. Genetic transformation of several species has been established. Of these, Chlamydomonas reinhardtii has become significantly attractive for its potential to express foreign proteins inexpensively. All its three genomes – nuclear, mitochondrial and chloroplastic – have been sequenced. As a result, a wealth of information about its genetic machinery, protein expression mechanism (transcription, translation and post-translational modifications) is available. Over the years, various molecular tools have been developed for the manipulation of all these genomes. Various studies show that the transformation of the chloroplast genome has several advantages over nuclear transformation from the biopharming point of view. According to a recent survey, over 100 recombinant proteins have been expressed in algal chloroplasts. However, the expression levels achieved in the algal chloroplast genome are generally lower compared to the chloroplasts of higher plants. Work is therefore needed to make the algal chloroplast transformation commercially competitive. In this review, we discuss some examples from the algal research, which could play their role in making algal chloroplast commercially successful.

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CRIMoClo plasmids for modular assembly and orthogonal chromosomal integration of synthetic circuits in Escherichia coli

Journal of Biological Engineering ◽

10.1186/s13036-019-0218-8 ◽

2019 ◽

Vol 13 (1) ◽

Cited By ~ 1

Author(s):

Stefano Vecchione ◽

Georg Fritz

Keyword(s):

Escherichia Coli ◽

Synthetic Biology ◽

Integrated Circuits ◽

High Efficiency ◽

Genetic Circuit ◽

Dna Assembly ◽

Chromosomal Integration ◽

Golden Gate ◽

Genetic Circuits ◽

E Coli

Abstract Background Synthetic biology heavily depends on rapid and simple techniques for DNA engineering, such as Ligase Cycling Reaction (LCR), Gibson assembly and Golden Gate assembly, all of which allow for fast, multi-fragment DNA assembly. A major enhancement of Golden Gate assembly is represented by the Modular Cloning (MoClo) system that allows for simple library propagation and combinatorial construction of genetic circuits from reusable parts. Yet, one limitation of the MoClo system is that all circuits are assembled in low- and medium copy plasmids, while a rapid route to chromosomal integration is lacking. To overcome this bottleneck, here we took advantage of the conditional-replication, integration, and modular (CRIM) plasmids, which can be integrated in single copies into the chromosome of Escherichia coli and related bacteria by site-specific recombination at different phage attachment (att) sites. Results By combining the modularity of the MoClo system with the CRIM plasmids features we created a set of 32 novel CRIMoClo plasmids and benchmarked their suitability for synthetic biology applications. Using CRIMoClo plasmids we assembled and integrated a given genetic circuit into four selected phage attachment sites. Analyzing the behavior of these circuits we found essentially identical expression levels, indicating orthogonality of the loci. Using CRIMoClo plasmids and four different reporter systems, we illustrated a framework that allows for a fast and reliable sequential integration at the four selected att sites. Taking advantage of four resistance cassettes the procedure did not require recombination events between each round of integration. Finally, we assembled and genomically integrated synthetic ECF σ factor/anti-σ switches with high efficiency, showing that the growth defects observed for circuits encoded on medium-copy plasmids were alleviated. Conclusions The CRIMoClo system enables the generation of genetic circuits from reusable, MoClo-compatible parts and their integration into 4 orthogonal att sites into the genome of E. coli. Utilizing four different resistance modules the CRIMoClo system allows for easy, fast, and reliable multiple integrations. Moreover, utilizing CRIMoClo plasmids and MoClo reusable parts, we efficiently integrated and alleviated the toxicity of plasmid-borne circuits. Finally, since CRIMoClo framework allows for high flexibility, it is possible to utilize plasmid-borne and chromosomally integrated circuits simultaneously. This increases our ability to permute multiple genetic modules and allows for an easier design of complex synthetic metabolic pathways in E. coli.

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Endogenous Fluctuations of DNA Topology in the Chloroplast of Chlamydomonas reinhardtii

Molecular and Cellular Biology ◽

10.1128/mcb.18.12.7235 ◽

1998 ◽

Vol 18 (12) ◽

pp. 7235-7242 ◽

Cited By ~ 42

Author(s):

Maria L. Salvador ◽

Uwe Klein ◽

Lawrence Bogorad

Keyword(s):

Chlamydomonas Reinhardtii ◽

Chloroplast Genome ◽

Continuous Light ◽

Dna Supercoiling ◽

Dna Topology ◽

Chloroplast Gene ◽

Chloroplast Gene Expression ◽

Endogenous Fluctuations ◽

Unicellular Green Alga ◽

In Cells

ABSTRACT DNA supercoiling in the chloroplast of the unicellular green algaChlamydomonas reinhardtii was found to change with a diurnal rhythm in cells growing in alternating 12-h dark–12-h light periods. Highest and lowest DNA superhelicities occurred at the beginning and towards the end of the 12-h light periods, respectively. The fluctuations in DNA supercoiling occurred concurrently and in the same direction in two separate parts of the chloroplast genome, one containing the genes psaB, rbcL, andatpA and the other containing the atpB gene. Fluctuations were not confined to transcribed DNA regions, indicating simultaneous changes in DNA conformation all over the chloroplast genome. Because the diurnal fluctuations persisted in cells kept in continuous light, DNA supercoiling is judged to be under endogenous control. The endogenous fluctuations in chloroplast DNA topology correlated tightly with the endogenous fluctuations of overall chloroplast gene transcription and with those of the pool sizes of most chloroplast transcripts analyzed. This result suggests that DNA superhelical changes have a role in the regulation of chloroplast gene expression in Chlamydomonas.

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Erratum to: No training required: experimental tests support homology-based DNA assembly as a best practice in synthetic biology

Journal of Biological Engineering ◽

10.1186/s13036-015-0013-0 ◽

2016 ◽

Vol 10 (1) ◽

Cited By ~ 1

Author(s):

Afnan Azizi ◽

Wilson Lam ◽

Hilary Phenix ◽

Lioudmila Tepliakova ◽

Ian J. Roney ◽

...

Keyword(s):

Synthetic Biology ◽

Best Practice ◽

Experimental Tests ◽

Dna Assembly

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DNA assembly for synthetic biology: from parts to pathways and beyond

Integrative Biology ◽

10.1039/c0ib00070a ◽

2011 ◽

Vol 3 (2) ◽

pp. 109-118 ◽

Cited By ~ 209

Author(s):

Tom Ellis ◽

Tom Adie ◽

Geoff S. Baldwin

Keyword(s):

Synthetic Biology ◽

Dna Assembly

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Modular and Integrative Vectors for Synthetic Biology Applications in Streptomyces spp.

Applied and Environmental Microbiology ◽

10.1128/aem.00485-19 ◽

2019 ◽

Vol 85 (16) ◽

Cited By ~ 1

Author(s):

Céline Aubry ◽

Jean-Luc Pernodet ◽

Sylvie Lautru

Keyword(s):

Synthetic Biology ◽

Anticancer Agents ◽

Gene Clusters ◽

Bioactive Molecules ◽

Dna Assembly ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Dna Cloning ◽

Specialized Metabolism ◽

Assembly Method

ABSTRACT With the development of synthetic biology in the field of (actinobacterial) specialized metabolism, new tools are needed for the design or refactoring of biosynthetic gene clusters. If libraries of synthetic parts (such as promoters or ribosome binding sites) and DNA cloning methods have been developed, to our knowledge, not many vectors designed for the flexible cloning of biosynthetic gene clusters have been constructed. We report here the construction of a set of 12 standardized and modular vectors designed to afford the construction or the refactoring of biosynthetic gene clusters in Streptomyces species, using a large panel of cloning methods. Three different resistance cassettes and four orthogonal integration systems are proposed. In addition, FLP recombination target sites were incorporated to allow the recycling of antibiotic markers and to limit the risks of unwanted homologous recombination in Streptomyces strains when several vectors are used. The functionality and proper integration of the vectors in three commonly used Streptomyces strains, as well as the functionality of the Flp-catalyzed excision, were all confirmed. To illustrate some possible uses of our vectors, we refactored the albonoursin gene cluster from Streptomyces noursei using the BioBrick assembly method. We also used the seamless ligase chain reaction cloning method to assemble a transcription unit in one of the vectors and genetically complement a mutant strain. IMPORTANCE One of the strategies employed today to obtain new bioactive molecules with potential applications for human health (for example, antimicrobial or anticancer agents) is synthetic biology. Synthetic biology is used to biosynthesize new unnatural specialized metabolites or to force the expression of otherwise silent natural biosynthetic gene clusters. To assist the development of synthetic biology in the field of specialized metabolism, we constructed and are offering to the community a set of vectors that were intended to facilitate DNA assembly and integration in actinobacterial chromosomes. These vectors are compatible with various DNA cloning and assembling methods. They are standardized and modular, allowing the easy exchange of a module by another one of the same nature. Although designed for the assembly or the refactoring of specialized metabolite gene clusters, they have a broader potential utility, for example, for protein production or genetic complementation.

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No training required: experimental tests support homology-based DNA assembly as a best practice in synthetic biology

Journal of Biological Engineering ◽

10.1186/s13036-015-0006-z ◽

2015 ◽

Vol 9 (1) ◽

Cited By ~ 6

Author(s):

Afnan Azizi ◽

Wilson Lam ◽

Hilary Phenix ◽

Lioudmila Tepliakova ◽

Ian J Roney ◽

...

Keyword(s):

Synthetic Biology ◽

Best Practice ◽

Experimental Tests ◽

Dna Assembly

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A reversible memory switch for plant synthetic biology based on the phage PhiC31 integration system

10.1101/656223 ◽

2019 ◽

Cited By ~ 1

Author(s):

Bernabé-Orts Joan Miquel ◽

Quijano-Rubio Alfredo ◽

Mancheño-Bonillo Javier ◽

Moles-Casas Victor ◽

Selma Sara ◽

...

Keyword(s):

Synthetic Biology ◽

Cell Fate ◽

Regulatory Element ◽

Living Organism ◽

Inducible Promoter ◽

Chemical Stimulus ◽

Toggle Switch ◽

Genetic Switch ◽

Whole Plants ◽

Plant Synthetic Biology

ABSTRACTPlant synthetic biology aims to contribute to global food security by engineering plants with new or improved functionalities. In recent years, synthetic biology has rapidly advanced from the setup of basic genetic devices to the design of increasingly complex gene circuits able to provide organisms with novel functions. While many bacterial, fungal and mammalian unicellular chassis have been extensively engineered, this progress has been delayed in plants due to their complex multicellular nature and the lack of reliable DNA devices that allow an accurate design of more sophisticated biological circuits. Among these basic devices, gene switches are crucial to deploying new layers of regulation into the engineered organisms. Of special interest are bistable genetic toggle switches, which allow a living organism to exist in two alternative states and switch between them with a minimal metabolic burden. Naturally occurring toggle switches control important decision-making processes such as cell fate and developmental events. We sought to engineer whole plants with an orthogonal genetic toggle switch to be able to regulate artificial functions with minimal interference with their natural pathways. Here we report a bistable toggle memory switch for whole plants based on the phage PhiC31 serine integrase and its cognate recombination directionality factor (RDF). This genetic device was designed to control the transcription of two genes of interest by inversion of a central DNA regulatory element. Each state of the device is defined by one transcriptionally active gene of interest, while the other one remains inactive. The state of the switch can be reversibly modified by the action of the recombination actuators, which were administered externally (e.g. via agroinfiltration), or produced internally in response to an inducible chemical stimulus. We extensively characterized the kinetics, memory, and reversibility of this genetic switch in Nicotiana benthamiana through transient and stable transformation experiments using transgenic plants and hairy roots. Finally, we coupled the integrase expression to an estradiol-inducible promoter as a proof of principle of inducible activation of the switch.

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