Get help from the best in academic writing.

The Potential of Algae as Bioreactors for Protein Expression

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.
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 ?-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 ?-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.
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:
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
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

Mapping and Understanding Genes of Human DNA

The Human Genome Project is a worldwide project and to understand the genomic sequence of human DNA. The main goal for the Human Genome Project is to obtain a complete 3 billion DNA sequencing base pairs and the location of human genes. The genetic information that is stored in human as DNA sequence because even though all the genome in humans is the same, but the sets of chromosomes which replicate make us different from others. The Human Genome Project was launched in 1990 in order to the complete genomic sequence of a human being. Understanding the structure of DNA is crucial before proceeding the project. As James Watson and Francis Crick mentioned in the article, Nature, “Molecular Structure of Nucleic Acids: A Structure for Deoxyribose Nucleic Acid.” Human genes are made up of four nitrogenous base pairs adenine, thymine, cytosine and guanine arranged in long twisted DNA. The Human Genome Project promised to bring benefit to medical field and treat genetic diseases. The Human Genome Project brought the technologies to a new level for scientists and gave more opportunities for researchers to discover the relationship between human gene with different drugs and/or diseases.
Firstly, before the Human Genome Project proceeded to obtain the DNA sequence in the human body, they first completed the genome sequence in E. coli in animals to get an idea of how genes are regulated. E. Coli are group of harmless bacteria found in the intestines of humans and animals (NIH). One of the finest discoveries of James Watson and Francis Crick was the structure of DNA model and published on April 25, 1953 in Nature article. DNA structure has to have complementary pairs of one purine and one pyrimidine. Adenine and guanine which are purines always pairs with nitrogenous bases thymine and cytosine which are pyrimidine. “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material” (Nature, pg 737). This discovery of DNA structure by Watson and Crick helped the scientists to proceed the Human Genome Project.
Secondly, since the Human Genome was a massive project that needed funding to complete the project. The Human Genome Project was funded by the National Institute of Health and Department of Energy which brought scientists together from different countries to team-up on this massive task of sequencing DNA of first human genome (NHGRI). United States distributed their work on sequencing of the Human Genome Project to the United Kingdom, France, Germany, Japan and China to complete the project within the time frame Congress anticipated. US Congress anticipated that it would cost approximately $3 billion but it ended up costing $2.7 billion (NHGRI). Francis Collins was appointed as the head of the National Human Genome Research Institute. (NHGRI). The project was completed in 2003, two years earlier than estimated time frame.
Since, the Human Genome Project was publicly funded to all the scientists that were involved in this project to obtain the first DNA sequence of a human body. Back in those days, there were not many advanced technologies present that would have helped scientists to complete the project faster. Scientists across the globe used multiple techniques including well-known electrophoretic technique. Frederick Sanger who invented electrophoretic technique and won a Nobel Prize in Chemistry in 1980, which was advanced technology back in the 1970s (BiteSize Bio). This electrophoretic technique helped scientists to speed the sequencing process and it was a more effectual technique to sequence the DNA with the help of glass capillary electrophoretic technique. Glass capillary electrophoretic technique is unique in such a way that if there is an error in computer when obtaining the result, the computer will not proceed to obtain additional nucleotide which could had delayed the project. Glass capillary electrophoretic technique helped scientists to store the DNA result in a computer for further studies of the relationship between human genome and drugs. This technique was effective that it significantly shortened the time to obtain the genomic sequence of one individual.
Another technique was cloning, this technique was used because it provided multiple copies of DNA by cleaving at specific sites of the nucleotide. This technique is also useful for researchers to provide drugs that could possibly cure diseases. PCR is the most common technique used by many researchers in the laboratory. This technique helped scientists to study the DNA sequence of human gene and it provided the information of the diseases. PCR helped amplify DNA and then used Sanger method electrophoresis to visualize the data. (Khan Academy). PCR and cloning techniques go hand in hand. As mentioned above, PCR provides information to the diseases and cloning help with possible treatment to the diseases. Maxam-Gilbert sequencing technique was named after their name Allan Maxam and Walter Gilbert. This technique used by scientists for DNA sequence which helped cut the nucleotide sequence at specific bases using chemical degradation (Britannica.com)
Finally, in 2003, the Human Genome Project achieved their goal of completing first genomic sequence of human DNA which contained 20,000 to 25,000 genes. The project was completed within 13 years less than 2 years Congress estimated. After completing the first human genome DNA sequence, scientists wanted to determine the mechanisms of genome sequence on how to cure diseases that can be life threatening for people by understanding the DNA sequence. Determining how to read genomic sequence gave researchers a better understanding of each individual health need and discover the best treatments for diseases. “In 1993, scientists tested the gene treatment for patients with cystic fibrosis” but unable to treat the patient with the precise medication (NHGRI, CF). Since then scientists knew there is a lot of work needed to be done. Back in those days, there were not many treatments option for cystic fibrosis which increased the dead rate. Cystic fibrosis causes thickening of mucus in airways which causes respiratory congestion and welcomes infected-causing bacteria (Silverthorn, Human Physiology 2015). Cystic fibrosis transmembrane regulator (CFTR) is a protein channel that allows chloride and water to be released in and out from the cells. People with cystic fibrosis disease unable cystic fibrosis transmembrane regulator protein channel to function properly which causes thick mucus in the airways (Silverthorn, Human Physiology 2015). Thus, affecting various organs such as stomach for digestive system and lung damage causes respiratory failure. Scientists are still under research to find the best treatment for cystic fibrosis transmembrane regulator to function normally. This project also opens the door for pharmaceutical industries and discover the best medications for diseases such as multiple sclerosis, cystic fibrosis and other diseases based on the response of human genome to the drugs.
The Human Genome Project was one of the biggest projects that the United States collaborated along with other countries to obtain roughly 20,000 to 25,000 human genes. The research concerning human genome is still continuous and researchers are pursuing to determine how human genome affects a person of certain diseases such as cystic fibrosis, cancer and other diseases. Researchers are also determining how certain drugs will respond to certain diseases due to thousands of protein molecules present in our body. Those thousands of protein molecules are corresponded to each human gene which is why researchers are still working on drugs for certain diseases that are very serious for the body. As I mentioned above, the cause of cystic fibrosis transmembrane regulator protein channel not functioning properly. Someday, the researchers can find the relationship between human genome and drugs to cure the patients with serious diseases with the help of this great Human Genome Project and advanced technologies like CRISPr that can cure patients with life threatening diseases like cystic fibrosis.
Work Cited
“Human Genome Project.” Wikipedia, Wikimedia Foundation, 8 June 2019, en.wikipedia.org/wiki/Human_Genome_Project.
“Human Genome Project FAQ.” Genome.gov, www.genome.gov/human-genome-project/Completion-FAQ.
“Maxam-Gilbert Sequencing: What Was It, and Why It Isn’t Anymore.” Bitesize Bio, 31 Jan. 2018, bitesizebio.com/36696/maxam-gilbert-sequencing/.
“Polymerase Chain Reaction (PCR).” Khan Academy, Khan Academy, www.khanacademy.org/science/biology/biotech-dna-technology/dna-sequencing-pcr-electrophoresis/a/polymerase-chain-reaction-pcr.
Nature News, Nature Publishing Group, www.nature.com/scitable/topicpage/dna-sequencing-technologies-690.
“A Brief History of the Human Genome Project.” National Human Genome Research Institute (NHGRI), www.genome.gov/12011239/a-brief-history-of-the-human-genome-project/.
“Who Was Involved in the Human Genome Project?” Stories, The Public Engagement Team at the Wellcome Genome Campus, 13 June 2016, www.yourgenome.org/stories/who-was-involved-in-the-human-genome-project.
““NIH Fact Sheets – Human Genome Project.” National Institutes of Health, U.S. Department of Health and Human Services, report.nih.gov/NIHfactsheets/ViewFactSheet.aspx?csid=45.
“What Was the Human Genome Project and Why Has It Been Important? – Genetics Home Reference – NIH.” U.S. National Library of Medicine, National Institutes of Health, ghr.nlm.nih.gov/primer/hgp/description.

[casanovaaggrev]