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Abstract:

Over the last decade microalgae have gained increasing interest as a natural source of valuable compounds and as bioreactors for recombinant protein production. Currently nontransgenic microalgae have a wide range of applications in various industries, including food, feed, cosmetics and pharmaceutical.

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On the other hand, transgenic microalgae can be used as bioreactors to produce therapeutically relevant recombinant proteins. I will be discussing how this technology could simplify the production process and significantly bring down the production costs. I will also highlight which bioreactors are currently used in industry and compare their importance with microalgae bioreactors.

Introduction:

The demand for recombinant proteins is increasing globally because of their important application values in industry, diagnosis and therapy. The bioreactor systems that are currently used to produce recombinant proteins are becoming more important for producing large quantities of proteins, particularly those that have associated limitations due to cost or source availability. Based on different types of organisms, several original bioreactor systems were recently developed due to the advance in biotechniques. In general, a particular bioreactor system may be selected for a specific protein production based on costs as well as the integrity, purity and expression level of the protein. (Na Yan et al, 2016)

Algae are classified into two groups; microalgae and macroalgae. It is estimated that there are between one to ten million algae species, most of these consist of microalgae. From this wide range of species there are some species which can be cultivated and these are split into four separate groups; cyanobacteria, green algae, chrysophyte and red algae. From this group, cyanobacteria is the only photosynthetic prokaryote able to produce oxygen. (Na Yan et al, 2016)

Over the last decade or so, the importance of eukaryotic microalgae for the production of recombinant proteins  has been highlighted significantly. Research has increased in this field because microalgae have really beneficial properties compared to current bioreactors used for protein expression, these benefits include; simpler growth requirements, ease of manipulation and high growth rate, which means that they can rapidly  produce large quantities of high-value proteins at very low costs.

Importance of Algae:

Microalgae has been used as a food source for centuries but its importance in producing high value compounds commercially has recently been highlighted. Microalgae contain many natural sources including proteins, carbohydrates and lipids. They are also capable of producing metabolites such as pigments and other vitamins. One example is the green algae found in fresh water, Chlorella. It is used in human food, animal feed and aquaculture. One main property of this algae is that it has a high protein content (50/60% of dry biomass) and other nutrient values. Microalgae are able to produce carbohydrates mainly in the form of starch, glucose, sugars, and polysaccharides. Efficient polysaccharide fractions are found mainly in cyanobacteria, but are also present in green microalgae such as Chlorella or Dunaliella, and are used as dietary supplements and pharmaceuticals.

Microalgae contain chlorophyll as primary photosynthetic pigments, but they don’t fully rely on this. They are able to synthesize many other pigments that have a crucial job in increasing the efficiency of using light energy and protecting microalgae cells from photodamage effects. From an industrial perspective the carotenoid and phycobiliproteins seem to be the most valued pigments. The carotenoid β-carotene is used as a vitamin A precursor and biological antioxidant in health foods and cosmetics.

Although a number of different types of valuable compounds have been found in microalgae. From these compounds there are only a few high-value compounds commercially available today. The carotenoid β-carotene from Dunaliella, natural astaxanthin from Haematococcus, and DHA from Crypthecodinium are the three well-known compounds that are derived from microalgae. One common factor between these compounds is that they are almost all from nontransgenic microalgae. However, over the past years, transgenic microalgae have proved to be excellent bioreactors for producing many other valuable compounds, especially recombinant therapeutic proteins. (Beth A. Rasala, 2010)

 

System System characteristics
Molecular Operational
Glycosylation Gene size Sensitivity to shear stress Recombinant product yield Production time Cost of cultivation Scale-up costs Cost for storage
Bacteria None Unknown Medium Medium Short Medium High Low (−20 °C)
Yeast Incorrect Unknown Medium High Medium Medium High Low (−20 °C)
Insecta Correct, but depends on strain and product Limited High Medium to high Long High High High (liquid N2)
Mammalian cells Correct Limited High Medium to high Long High High High (liquid N2)
Plant cells Correctb Unlimited N/A High Long Low Very low Low (room temperature)
Unicellular microalgae Correctb Unlimited Low Generally low Short Very low Low Low (room temperature)

Table 1. comparison of different platforms for recombinant protein expression (Gabriel Potvin et all, 2010

 

Microalgal Transformation:

The utilization of transgenic microalgae as effective platforms for recombinant protein production depends on the establishment of stable transformation systems. Over the last 20 years, successful genetic transformation has been reported in ~22 species of microalgae, most of which are achieved by nuclear transformation. Despite these advances, up to now routine transformation is achievable only for very few species including C. reinhardtii, Volvox carteri, some species of Chlorella, and the diatom Phaeodactylum tricornutum.

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The most common method for microalgal transformation are based on the fact that microalgal cells can endure temporary permeability of the cell membrane which enables plasmid DNA to pass through the membrane and enter the cell. There are many methods which can be used to make the cell membrane permeable. microparticle bombardment is the preferred method of transformation for previously untransformed species of microalgae. This method simply uses DNA-coated gold or tungsten microprojectiles which are delivered into cells by a particle gun. One main advantage of this method is that it can used on any type of cell, despite the thickness of cell wall or its rigidity.

Electroporation is another method used for transforming special cells of microalgae, such as naked cells, protoplasts, cell-wall-reduced mutants, and other thin-walled cells. This method works by temporarily disturbing the lipid bilayer on the membrane by an electric pulse which allows the DNA molecules to enter. This technique has been used previously has been successfully achieved in C. reinhardtii, Dunaliella salina, Chlorella vulgaris, Ostreococcus tauri, and red algae Cyanidioschyzon merolae. (Yangmin Gong, 2011)

Current bioreactors:

Currently the industry uses bacteria and yeast based bioreactors for the production of recombinant proteins, because their genome is very easily manipulated and they can be cultivated with ease at low costs. However, it is not as easy to use these systems because they have their own down sides. Firstly, bacteria are unable to perform the post-transcriptional, control of gene expression at the RNA level, and post-translation modifications, including glycosylation, phosphorylation and disulphide bond formation, which is vital for the correct folding and assembly of complex proteins. On the other hand, eukaryotic yeasts are able to perform these modifications, but their profile are unstable for therapeutic proteins which are to be used for animal or human consumption. Furthermore, the recombinant proteins in yeasts are at times hyperglycosylated, which changes immunogenic epitopes ( the part of an antigen molecule to which an antibody attaches to), and the high-mannose glycosylation performed in these systems causes the half-life of proteins to decrease in a living organism. (Franziska Hempel, 2011)

However, the industry doesn’t totally rely on bacteria or yeast based bioreactors. To overcome their complications, mammalian, insect or plant cell bioreactors are used. Many recombinant eukaryotic proteins have been correctly produced, processed and gathered in these cell-based reactors. First of all, mammalian cell-based bioreactors are very expensive to develop and maintain, and have complex nutrient requirements, poor oxygen and nutrient distribution, waste accumulation, contamination by pathogens, and high sensitivity of cells to shear.

Secondly, in comparison to mammalian bioreactors insect cells are easier to culture because they are more tolerant to changes in osmolarity and by-product accumulation, and generally lead to higher recombinant protein expression levels. Similar to mammalian cells insect cells also have a complex nutrient requirements.

Finally, plant-based bioreactors are much less expensive than mammalian and insect bioreactors. They are also resistant to most animal-infecting pathogens. They do however have slow growth cycles and are linked with problems relating to environmental contamination by genetically modified plants. Although, the glycosylation profiles between animal and plant cells is different, protein folding remains stable.(Gabriel Potvin, 2011). Another disadvantage that hinders the use of plants as successful recombinant protein-producing system is that they have different types of glycosylation patterns compared with animal cells, which may alter the function of the recombinant protein or even decrease immunogenicity. In addition another barrier includes the controversial issues of regulations and safety, especially with regard to the risk of gene flow via transgenic pollen. Studies on gene flow between transgenic plants and native races have been reported, such as genes that encode Bacillus thuringiensisproteins in corn and herbicide resistance genes in canola. Finally, much time is required from the transformation step to the acquisition of a purified protein. Moreover, the purification of proteins from plants is inconvenient because they cannot be secreted.  (Na Yan et al, 2016)

Advantages of microalgae as bioreactors:

There are many advantages of using microalgae as bioreactors for protein expression, compared with other bioreactors. First of all, microalgae double their biomass within 24 hours, and there is a relatively short period from the generation of primary microalgae transformants to sufficient numbers for large scale production.

Secondly, recombinant proteins are expressed from the nuclear, chloroplast and mitochondrial genomes of a few microalgae species. In contrast to bacteria, eukaryotic microalgae posses complex post- traditional modification pathways.

Thirdly, recombinant proteins can be expressed from nuclear, chloroplast, and mitochondrial genomes of some microalgal species. Unlike bacteria, eukaryotic microalgae possess complex post-translational modification pathways, and therefore microalgae can produce glycosylated proteins.

In addition, microalgae can be grown either phototrophically or heterotrophically. Transgenic microalgae are particularly suitable for growth in controlled photobioreactors, in which the culture conditions such as light, temperature, nutrients, and mixing, can be well monitored.

The culture of transgenic microalgae in photobioreactors can also prevent transgenes from escaping into the environment, which may potentially occur in higher plants by the means of pollen. These advantages make microalgae attractive systems for the production of recombinant proteins and other high-value compounds. (Yangmin Gong, 2011)

Conclusion:

Microalgae are vital natural resources that contain important proteins, oils, fatty acids, polysaccharides and other bioactive proteins that are currently commercially available. Also, as a new type of bioreactor, microalgae can produce recombinant proteins. In addition, they are significant in the pharmaceutical industry as they can be used to produce vaccines and antibodies. Microalgae as bioreactors have several advantages over bacteria, yeast, plants, and other systems for recombinant protein production, including low cost, safety, alternative culture methods, and rapid scalability. However, major advances achieved in pharmaceutical protein production with transgenic microalgae are from unicellular green algae Chlamydomonas, indicating that microalgae are not a well-studied group from a biotechnological perspective. The major problem for microalgal protein expression systems is the lack of standard procedures for genetic transformation of commercially important species of microalgae, limited availability of molecular toolkits for genetic engineering of microalgae, and relatively low expression levels of recombinant proteins resulting from several factors.

References:

  1. Beth A. Rasala1,† , Machiko Muto1,† , Philip A. Lee1 , Michal Jager1 , Rosa M.F. Cardoso2 , Craig A. Behnke2 , Peter Kirk3 , Craig A. Hokanson3 , Roberto Crea3 , Michael Mendez2, and Stephen P. Mayfield1 (2010), Production of therapeutic proteins in algae, analysis of expression of seven human proteins in the chloroplast of Chlamydomonas reinhardtii. https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1467-7652.2010.00503.x
  2. Franziska Hempel, Julia Lau, Andreas Klingl, Uwe G. Maier (2011). Algae as Protein Factories: Expression of a Human Antibody and the Respective Antigen in the Diatom Phaeodactylumtricornutum http://europepmc.org/backend/ptpmcrender.fcgi?accid=PMC3229587&blobtype=pdf
  3. Gong, Y., Hu, H., Gao, Y., Xu, X., & Gao, H. (2011). Microalgae as platforms for production of recombinant proteins and valuable compounds: Progress and prospects. Journal of Industrial Microbiology & Biotechnology, 38(12), 1879-1890. doi:10.1007/s10295-011-1032-6
  4. Hempel, F., Bozarth, A. S., Lindenkamp, N., Klingl, A., Zauner, S., Linne, U., . . . Maier, U. G. (2011). Microalgae as bioreactors for bioplastic production. Microbial Cell Factories, 10(1), 81-81. doi:10.1186/1475-2859-10-81
  5. Hempel, F., Bozarth, A. S., Lindenkamp, N., Klingl, A., Zauner, S., Linne, U., . . . Maier, U. G. (2011). Microalgae as bioreactors for bioplastic production. Microbial Cell Factories, 10(1), 81-81. doi:10.1186/1475-2859-10-81
  6. Potvin, G., & Zhang, Z. (2010). Strategies for high-level recombinant protein expression in transgenic microalgae: A review. Biotechnology Advances, 28(6), 910-918. doi:10.1016/j.biotechadv.2010.08.006
  7. Yan, N., Fan, C., Chen, Y., & Hu, Z. (2016). The potential for microalgae as bioreactors to produce

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