Investigating the dynamics of recombinant protein secretion from a microalgal host

Lauersen, Kyle J.; Huber, Isabel; Wichmann, Julian; Baier, Thomas; Leiter, Andreas; Gaukel, Volker; Kartushin, Viktor; Rattenholl, Anke; Steinweg, Christian; Riesen, Lena von; Posten, Clemens; Gudermann, Frank; Lütkemeyer, Dirk; Mussgnug, Jan H.; Kruse, Olaf

doi:10.1016/j.jbiotec.2015.05.001

Cited by 41 publications

(30 citation statements)

References 48 publications

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“…The secretion of recombinant proteins into the extracellular environment by microalgae has been described in the literature [ 8 , 9 , 21 , 25 , 29 – 32 , 42 , 43 ], and indeed, research on efficient secretion strategies is important for the development of a recombinant protein expression system. Secretion allows the use of culture manufacturing strategies, such as perfusion, which have the potential for greater productivity and the use of smaller production facilities [ 20 ].…”

Section: Discussionmentioning

confidence: 99%

Comparison of secretory signal peptides for heterologous protein expression in microalgae: Expanding the secretion portfolio for Chlamydomonas reinhardtii

2018

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Efficient protein secretion is a desirable trait for any recombinant protein expression system, together with simple, low-cost, and defined media, such as the typical media used for photosynthetic cultures of microalgae. However, low titers of secreted heterologous proteins are usually obtained, even with the most extensively studied microalga Chlamydomonas reinhardtii, preventing their industrial application. In this study, we aimed to expand and evaluate secretory signal peptides (SP) for heterologous protein secretion in C. reinhardtii by comparing previously described SP with untested sequences. We compared the SPs from arylsulfatase 1 and carbonic anhydrase 1, with those of untried SPs from binding protein 1, an ice-binding protein, and six sequences identified in silico. We identified over 2000 unique SPs using the SignalP 4.0 software. mCherry fluorescence was used to compare the protein secretion of up to 96 colonies for each construct, non-secretion construct, and parental wild-type cc1690 cells. Supernatant fluorescence varied according to the SP used, with a 10-fold difference observed between the highest and lowest secretors. Moreover, two SPs identified in silico secreted the highest amount of mCherry. Our results demonstrate that the SP should be carefully selected and that efficient sequences can be coded in the C. reinhardtii genome. The SPs described here expand the portfolio available for research on heterologous protein secretion and for biomanufacturing applications.

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Section: Discussionmentioning

confidence: 99%

Comparison of secretory signal peptides for heterologous protein expression in microalgae: Expanding the secretion portfolio for Chlamydomonas reinhardtii

2018

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“…A dedicated vector was designed in which luciferase expression in C. reinhardtii was greatly improved (up to 84%) by fusing the transgene with the previously identified secretion element of carbonic anhydrase (Lauersen et al, 2013a ). This approach was further implemented for the production of secreted ice-binding protein, a protein of considerable industrial value (Lauersen et al, 2013b , 2015 ).…”

Section: Nuclear Transformationmentioning

confidence: 99%

Transgene Expression in Microalgae—From Tools to Applications

2016

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Microalgae comprise a biodiverse group of photosynthetic organisms that reside in water sources and sediments. The green microalgae Chlamydomonas reinhardtii was adopted as a useful model organism for studying various physiological systems. Its ability to grow under both photosynthetic and heterotrophic conditions allows efficient growth of non-photosynthetic mutants, making Chlamydomonas a useful genetic tool to study photosynthesis. In addition, this green alga can grow as haploid or diploid cells, similar to yeast, providing a powerful genetic system. As a result, easy and efficient transformation systems have been developed for Chlamydomonas, targeting both the chloroplast and nuclear genomes. Since microalgae comprise a rich repertoire of species that offer variable advantages for biotech and biomed industries, gene transfer technologies were further developed for many microalgae to allow for the expression of foreign proteins of interest. Expressing foreign genes in the chloroplast enables the targeting of foreign DNA to specific sites by homologous recombination. Chloroplast transformation also allows for the introduction of genes encoding several enzymes from a complex pathway, possibly as an operon. Expressing foreign proteins in the chloroplast can also be achieved by introducing the target gene into the nuclear genome, with the protein product bearing a targeting signal that directs import of the transgene-product into the chloroplast, like other endogenous chloroplast proteins. Integration of foreign genes into the nuclear genome is mostly random, resulting in large variability between different clones, such that extensive screening is required. The use of different selection modalities is also described, with special emphasis on the use of herbicides and metabolic markers which are considered to be friendly to the environment, as compared to drug-resistance genes that are commonly used. Finally, despite the development of a wide range of transformation tools and approaches, expression of foreign genes in microalgae suffers from low efficiency. Thus, novel tools have appeared in recent years to deal with this problem. Finally, while C. reinhardtii was traditionally used as a model organism for the development of transformation systems and their subsequent improvement, similar technologies can be adapted for other microalgae that may have higher biotechnological value.

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“…The ability of P. tricornutum and C. reinhardtii to secrete antibodies and enzymes highlights these algae species to produce and secrete glycosylated antigens. For instance, C. reinhardtii was engineered to efficiently secrete the ice binding protein (LpIBP), which is a glycoprotein from Lolium perenne that is applied as food cryopreservation additive [ 215 ]; and the Venus reporter protein, which was expressed with accessory synthetic glycomodules to increase secretion and stability [ 216 ]. However, other authors working with P. tricornutum have focused on retaining antibodies at the ER to obtain simplified glycosylation patterns that favor their applicability [ 169 ].…”

Section: Perspectivesmentioning

confidence: 99%

The Potential of Algal Biotechnology to Produce Antiviral Compounds and Biopharmaceuticals

et al. 2020

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The emergence of the Coronavirus Disease 2019 (COVID-19) caused by the SARS-CoV-2 virus has led to an unprecedented pandemic, which demands urgent development of antiviral drugs and antibodies; as well as prophylactic approaches, namely vaccines. Algae biotechnology has much to offer in this scenario given the diversity of such organisms, which are a valuable source of antiviral and anti-inflammatory compounds that can also be used to produce vaccines and antibodies. Antivirals with possible activity against SARS-CoV-2 are summarized, based on previously reported activity against Coronaviruses or other enveloped or respiratory viruses. Moreover, the potential of algae-derived anti-inflammatory compounds to treat severe cases of COVID-19 is contemplated. The scenario of producing biopharmaceuticals in recombinant algae is presented and the cases of algae-made vaccines targeting viral diseases is highlighted as valuable references for the development of anti-SARS-CoV-2 vaccines. Successful cases in the production of functional antibodies are described. Perspectives on how specific algae species and genetic engineering techniques can be applied for the production of anti-viral compounds antibodies and vaccines against SARS-CoV-2 are provided.

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Investigating the dynamics of recombinant protein secretion from a microalgal host

Cited by 41 publications

References 48 publications

Comparison of secretory signal peptides for heterologous protein expression in microalgae: Expanding the secretion portfolio for Chlamydomonas reinhardtii

Comparison of secretory signal peptides for heterologous protein expression in microalgae: Expanding the secretion portfolio for Chlamydomonas reinhardtii

Transgene Expression in Microalgae—From Tools to Applications

The Potential of Algal Biotechnology to Produce Antiviral Compounds and Biopharmaceuticals

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