A chimeric hydrolase-PTXD transgene enables chloroplast-based heterologous protein expression and non-sterile cultivation of Chlamydomonas reinhardtii

Cutolo, Edoardo Andrea; Tosoni, Matteo; Barera, Simone; Herrera‐Estrella, Luis; Dall’Osto, Luca; Bassi, Roberto

doi:10.1016/j.algal.2021.102429

Cited by 7 publications

(7 citation statements)

References 53 publications

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“…For immunodecoration experiments, total cellular extracts were obtained using a previously described protocol starting from approximately 3 × 10 7 pelleted cells [ 134 ]. Approximately 5 µg of proteins were separated via SDS-PAGE and transferred to a nitrocellulose membrane, and subsequently probed using the following primary antibodies α-LHCSR1 (AS142819, Agrisera, Vännäs, Sweden) α-LHCSR3 (AS142766), and α-ATPase β-subunit (AS05085), as well as the previously described [ 55 ] primary α-PSBS antibody, which was kindly provided by Prof. Stefano Caffarri (Aix-Marseille Universitè, France).…”

Section: Methodsmentioning

confidence: 99%

Proteomic characterization of a lutein-hyperaccumulating Chlamydomonas reinhardtii mutant reveals photoprotection-related factors as targets for increasing cellular carotenoid content

McQuillan,

Cutolo,

Evans

et al. 2023

Biotechnol Biofuels

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Background Microalgae are emerging hosts for the sustainable production of lutein, a high-value carotenoid; however, to be commercially competitive with existing systems, their capacity for lutein sequestration must be augmented. Previous attempts to boost microalgal lutein production have focussed on upregulating carotenoid biosynthetic enzymes, in part due to a lack of metabolic engineering targets for expanding lutein storage. Results Here, we isolated a lutein hyper-producing mutant of the model green microalga Chlamydomonas reinhardtii and characterized the metabolic mechanisms driving its enhanced lutein accumulation using label-free quantitative proteomics. Norflurazon- and high light-resistant C. reinhardtii mutants were screened to yield four mutant lines that produced significantly more lutein per cell compared to the CC-125 parental strain. Mutant 5 (Mut-5) exhibited a 5.4-fold increase in lutein content per cell, which to our knowledge is the highest fold increase of lutein in C. reinhardtii resulting from mutagenesis or metabolic engineering so far. Comparative proteomics of Mut-5 against its parental strain CC-125 revealed an increased abundance of light-harvesting complex-like proteins involved in photoprotection, among differences in pigment biosynthesis, central carbon metabolism, and translation. Further characterization of Mut-5 under varying light conditions revealed constitutive overexpression of the photoprotective proteins light-harvesting complex stress-related 1 (LHCSR1) and LHCSR3 and PSII subunit S regardless of light intensity, and increased accrual of total chlorophyll and carotenoids as light intensity increased. Although the photosynthetic efficiency of Mut-5 was comparatively lower than CC-125, the amplitude of non-photochemical quenching responses of Mut-5 was 4.5-fold higher than in CC-125 at low irradiance. Conclusions We used C. reinhardtii as a model green alga and identified light-harvesting complex-like proteins (among others) as potential metabolic engineering targets to enhance lutein accumulation in microalgae. These have the added value of imparting resistance to high light, although partially compromising photosynthetic efficiency. Further genetic characterization and engineering of Mut-5 could lead to the discovery of unknown players in photoprotective mechanisms and the development of a potent microalgal lutein production system.

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Section: Methodsmentioning

confidence: 99%

Proteomic characterization of a lutein-hyperaccumulating Chlamydomonas reinhardtii mutant reveals photoprotection-related factors as targets for increasing cellular carotenoid content

McQuillan,

Cutolo,

Evans

et al. 2023

Biotechnol Biofuels

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“…This metabolic enhancement has dual functions as a transformation marker and in reducing contamination by other organisms that cannot metabolize phosphite. 68 , 69 Numerous recombinant proteins have been produced in this organelle, and iterative transformations have been made possible by the development of chloroplast selection marker recycling methods. 70 …”

Section: Key Developments In Engineering Algal Genomesmentioning

confidence: 99%

The synthetic future of algal genomes

Goold,

Moseley,

Lauersen

2024

Cell Genomics

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“…A rich genetic toolkit allows plastome manipulation in non-model species [273], including marker-free and metabolism-dependent selection strategies [274][275][276]. Among these, the type D phosphite dehydrogenase (PtxD) gene, which enables the oxidization of non-assimilable phosphite ions in phosphate, is a biosafe solution to maintain axenic cultures [277,278], especially in mixotrophic conditions where pest contamination risk is high [279].…”

Section: Production Of Heterologous Immunotherapeutics In Microalgaementioning

confidence: 99%

The Clinical Promise of Microalgae in Rheumatoid Arthritis: From Natural Compounds to Recombinant Therapeutics

Cutolo,

Caferri,

Campitiello

et al. 2023

Marine Drugs

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Rheumatoid arthritis (RA) is an invalidating chronic autoimmune disorder characterized by joint inflammation and progressive bone damage. Dietary intervention is an important component in the treatment of RA to mitigate oxidative stress, a major pathogenic driver of the disease. Alongside traditional sources of antioxidants, microalgae—a diverse group of photosynthetic prokaryotes and eukaryotes—are emerging as anti-inflammatory and immunomodulatory food supplements. Several species accumulate therapeutic metabolites—mainly lipids and pigments—which interfere in the pro-inflammatory pathways involved in RA and other chronic inflammatory conditions. The advancement of the clinical uses of microalgae requires the continuous exploration of phytoplankton biodiversity and chemodiversity, followed by the domestication of wild strains into reliable producers of said metabolites. In addition, the tractability of microalgal genomes offers unprecedented possibilities to establish photosynthetic microbes as light-driven biofactories of heterologous immunotherapeutics. Here, we review the evidence-based anti-inflammatory mechanisms of microalgal metabolites and provide a detailed coverage of the genetic engineering strategies to enhance the yields of endogenous compounds and to develop innovative bioproducts.

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A chimeric hydrolase-PTXD transgene enables chloroplast-based heterologous protein expression and non-sterile cultivation of Chlamydomonas reinhardtii

Cited by 7 publications

References 53 publications

Proteomic characterization of a lutein-hyperaccumulating Chlamydomonas reinhardtii mutant reveals photoprotection-related factors as targets for increasing cellular carotenoid content

Proteomic characterization of a lutein-hyperaccumulating Chlamydomonas reinhardtii mutant reveals photoprotection-related factors as targets for increasing cellular carotenoid content

The synthetic future of algal genomes

The Clinical Promise of Microalgae in Rheumatoid Arthritis: From Natural Compounds to Recombinant Therapeutics

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