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

Abstract

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.

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

“…[44] The algal chloroplast, specifically that of C. reinhardtii, is well suited for genetic engineering and there is an increasing emphasis on the application of synthetic biology. [11,12,5,9] Many of these approaches are reliant on the ability to perform a series of plastome edits to the same cell line. However, conventional strategies for selection of transformants largely preclude this: methods based on photosynthetic restoration are restricted to a particular mutant host and specific locus, and can only be performed once, [41] whilst portable markers for engineering WT plastomes are currently limited to just three and these also operate on a single-use basis.…”

“…[7,8] An ever-growing 'chloroplast toolkit' for C. reinhardtii now allows routine insertion of codon-optimized transgenes into the plastome, and their high-level and regulated expression. [9] More recently, there has been a growing emphasis on the utilization of synthetic biology (SynBio) approaches to chloroplast engineering. [10][11][12]5] This has been supported by the availability of robust, well annotated genomic and transcriptomic data for the C. reinhardtii plastome [13,14] and the emergence of standardized DNA assembly methods for rapid and highthroughput design and construction of transgenes.…”

“…[17] These ambitious engineering efforts will likely require several rounds of engineering of the same strain, either to introduce multiple transgenes for metabolic engineering, or to perform plastome rearrangements and deletions. [9] However, such advanced transplastomics is currently constrained by the paucity of different selectable markers for chloroplast transformation of C. reinhardtii. [18] For example, only three bacterial genes have been developed to-date as portable markers: the aadA cassette conferring spectinomycin resistance, [19] the aphA6 cassette conferring kanamycin resistance, [20] and the ptxD cassette that allows phosphite auxotrophy.…”

A Year at the Forefront of Engineering Photosynthesis

“…Efforts to engineer photosynthesis are limited by available technologies, particularly for stable nuclear transformation of large, multigene constructs (>10 genes) into nuclear genomes ( Ort et al, 2015 ) and for the transformation of chloroplast genomes ( Bock, 2015 ; Jackson et al, 2021 ). However, introduction of point mutations to plastome-localised RbcL via a restriction enzyme-based system has been re-demonstrated with a 40% editing efficiency ( Lin et al, 2021 ).…”

A PETase enzyme synthesised in the chloroplast of the microalga Chlamydomonas reinhardtii is active against PET and polystyrene

“…To this end, microalgae represent an attractive biotechnology platform for the synthesis of recombinant proteins [24][25][26] . The advantages of using microalgae as opposed to traditional heterotrophic platforms of Escherichia coli, yeast, or CHO cells are: (i) the low-cost phototrophic cultivation of the alga in sterile, controlled photobioreactors using simple and inexpensive medium 27 ; (ii) the generally recognized as safe (GRAS) status of a number of algal species, including the chlorophyte Chlamydomonas reinhardtii 28 ; (iii) the availability of the chloroplast as a unique biosynthetic and storage compartment within the cell that contains its own minimal genetic system [29][30][31] ; and (iv) a growing interest and adoption of enabling synthetic biology principles for creating bespoke cell factories using microalgae 32 . Whilst several recent studies have reported the production of PETase in microalgal species through nuclear engineering 33,34 , the use of the chloroplast for expression of foreign genes confers several bene ts.…”