Accelerating Chloroplast Engineering: A New System for Rapid Generation of Marker-Free Transplastomic Lines of Chlamydomonas reinhardtii

‘Marker-free’ strategies for creating transgenic microorganisms avoid the issue of potential transmission of antibiotic resistance genes to other microorganisms. An already-established strategy for engineering the chloroplast genome (=plastome) of the green microalga Chlamydomonas reinhardtii involves the restoration of photosynthetic function using a recipient strain carrying a plastome mutation in a key photosynthesis gene. Selection for transformant colonies is carried out on minimal media, such that only those cells in which the mutated gene has been replaced with a wild-type copy carried on the transgenic DNA are capable of phototrophic growth. However, this approach can suffer from issues of efficiency due to the slow growth of C. reinhardtii on minimal media and the slow die-back of the untransformed lawn of cells when using mutant strains with a limited photosensitivity phenotype. Furthermore, such phototrophic rescue has tended to rely on existing mutants that are not necessarily ideal for transformation and targeted transgene insertion: Mutants carrying point mutations can easily revert, and those with deletions that do not extend to the intended transgene insertion site can give rise to a sub-population of rescued lines that lack the transgene. In order to improve and accelerate the transformation pipeline for C. reinhardtii, we have created a novel recipient line, HNT6, carrying an engineered deletion in exon 3 of psaA, which encodes one of the core subunits of photosystem I (PSI). Such PSI mutants are highly light-sensitive allowing faster recovery of transformant colonies by selecting for light-tolerance on acetate-containing media, rather than phototrophic growth on minimal media. The deletion extends to a site upstream of psaA-3 that serves as a neutral locus for transgene insertion, thereby ensuring that all of the recovered colonies are transformants containing the transgene. We demonstrate the application of HNT6 using a luciferase reporter

ADA: an open-source software platform for plotting and analysis of data from laboratory photobioreactors

Algal biotechnology has received significant attention over the past two decades in fields ranging from biofuels to cosmeceuticals. However, the development of domesticated or genetically engineered microalgal strains for commercial applications depends on accurate and reliable growth data. To this end, several companies have developed lab-scale photobioreactors (PBRs) that enable precision control of conditions and automated growth recording. Whilst the transition from manual control of conditions and measurements to automated systems has allowed researchers to greatly improve the accuracy and scope of cultivation experiments, it has also presented novel challenges. The most pertinent of these being the analysis of the copious quantities of data produced. A standard PBR experiment can contain tens or even hundreds of thousands of data points, and often features outliers, noise, and a requirement for datasets to be calibrated with a standard curve or merged with replicates. Furthermore, complex analysis of multiple curves may be required in order to extract information such as the gradient or fit to a growth model. This can be laborious, time consuming and is not standardized between research groups. Proprietary software provided with most PBRs tends to lack these more advanced features and is typically unable to process data from other PBR manufacturers. To address these issues, we have developed the Algal Data Analyser (ADA), an open-source software platform providing the tools to rapidly plot and analyse microalgal data. ADA can simultaneously interpret datasets from three major PBR suppliers (Algenuity, Industrial Plankton, Photon Systems Instruments), and can also incorporate data from manual readings. Users can rapidly produce standardized, publication ready plots, and analyse multiple growth curves in parallel. Future iterations of ADA will include compatibility with datasets from other PBR suppliers as they become available, with the aim of making it a universal platform for all PBR data

A PETase enzyme synthesised in the chloroplast of the microalga Chlamydomonas reinhardtii is active against post-consumer plastics

Polyethylene terephthalate hydrolases (PETases) are a newly discovered and industrially important class of enzymes that catalyze the enzymatic degradation of polyethylene terephatalate (PET), one of the most abundant plastics in the world. The greater enzymatic efficiencies of PETases compared to close relatives from the cutinase and lipase families have resulted in increasing research interest. Despite this, further characterization of PETases is essential, particularly regarding their possible activity against other kinds of plastic. In this study, we exploited for the first time the use of the microalgal chloroplast for more sustainable synthesis of a PETase enzyme. A photosynthetic-restoration strategy was used to generate a marker-free transformant line of the green microalga Chlamydomonas reinhardtii in which the PETase from Ideonella sakaiensis was constitutively expressed in the chloroplast. Subsequently, the activity of the PETase against both PET and post-consumer plastics was investigated via atomic force microscopy, revealing evidence of degradation of the plastics

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.Chloroplast SynBio research in the authors’ groups is funded by grants BB/R016534/1 and BB/R01860X/1 from the U.K.’s Biotechnology and Biological Sciences Research Council

CpPosNeg: A positive-negative selection strategy allowing multiple cycles of marker-free engineering of the Chlamydomonas plastome.

The chloroplast represents an attractive compartment for light-driven biosynthesis of recombinant products, and advanced synthetic biology tools are available for engineering the chloroplast genome ( = plastome) of several algal and plant species. However, producing commercial lines will likely require several plastome manipulations, and this will present issues with respect to selectable markers: there are a limited number of markers available, these can be used only once in a serial engineering strategy, and it is undesirable to retain marker genes for antibiotic resistance in the final transplastome. To address these problems, we have designed a rapid iterative marker system for the green microalga Chlamydomonas reinhardtii that allows creation of marker-free transformants starting from wild-type strains. The system employs a dual marker encoding a fusion protein of E. coli aminoglycoside adenyltransferase (conferring spectinomycin resistance) and a variant of E. coli cytosine deaminase (conferring sensitivity to 5-fluorocytosine). Initial selection on spectinomycin allows stable transformants to be established and driven to homoplasmy. Subsequent selection on 5-fluorocytosine results in rapid loss of the dual marker through intramolecular recombination between the markers's 3'UTR and the 3'UTR of the introduced transgene(s). We demonstrate the versatility of the CpPosNeg system by serial introduction of reporter genes into the plastome. This article is protected by copyright. All rights reserved

A PETase enzyme synthesised in the chloroplast of the microalga Chlamydomonas reinhardtii is active against post-consumer plastics

Abstract Polyethylene terephthalate hydrolases (PETases) are a newly discovered and industrially important class of enzymes that catalyze the enzymatic degradation of polyethylene terephatalate (PET), one of the most abundant plastics in the world. The greater enzymatic efficiencies of PETases compared to close relatives from the cutinase and lipase families have resulted in increasing research interest. Despite this, further characterization of PETases is essential, particularly regarding their possible activity against other kinds of plastic. In this study, we exploited for the first time the use of the microalgal chloroplast for more sustainable synthesis of a PETase enzyme. A photosynthetic-restoration strategy was used to generate a marker-free transformant line of the green microalga Chlamydomonas reinhardtii in which the PETase from Ideonella sakaiensis was constitutively expressed in the chloroplast. Subsequently, the activity of the PETase against both PET and post-consumer plastics was investigated via atomic force microscopy, revealing evidence of degradation of the plastics