Biosynthesis | A ray of hope for the environment and biotechnology

Biosynthesis | A ray of hope for the environment and biotechnology
Protein Synthesis
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Biosynthesis | A ray of hope for the environment and biotechnology

Contents

What is Biosynthesis?

It’s an enzyme-catalyzed process in which simple reactants (substrates) are converted into complex products inside living organisms or by the help of living organisms. In this process, the substrates are transformed, modified, or polymerized to form large-sized products known as macromolecules. This conversion and modification include a series of reactions called a biosynthetic pathway. These biosynthetic pathways can occur inside or outside the organism’s body with enzymes (biocatalysts). Production of the cell membrane components (phospholipids and membrane proteins), nucleotides and proteins are typical examples of biosynthetic pathways. The Biosynthesis process is usually an anabolic process requiring monomers, substrates, precursor compounds, enzymes, co-enzymes and energy. 

In general, Biosynthesis is the process of production of biomolecules or natural compounds through enzyme-catalyzed reactions using cellular metabolic machinery. Generally, a series of enzyme-catalyzed reactions are involved in the production of a single biomolecule. Biosynthesis can also be used to synthesize chemicals by utilizing substrate in vitro (outside the living organism) or in vivo (inside a living cell such as E. coli) with enzymes’ help recombinant technology. 

Biosynthesis
Figure 1: utilization of cell components for biosynthesis.
https://commons.wikimedia.org/wiki/File:Ribosome_mRNA_translation_en.svg

Difference between biosynthesis and chemical synthesis

Chemical synthesis or chemosynthesis is the formation of complex products by utilizing simpler substances in the absence or presence of chemical agents known as catalysts. Biosynthesis forms large organic compounds by utilizing smaller substrates in a living system by following a metabolic pathway. 

Why is Biosynthesis essential?

The production methods used in the chemosynthesis are generally hazardous to our surrounding environment and other living beings. Hence, there is a need for environmentally safe, cost-effective and method of synthesis. A variety of biological systems such as fungi, bacteria, diatoms, plants and even human cells can convert simpler substrates into biomolecules via enzyme-catalyzed reactions and metabolic pathways. It is advantageous to use Biosynthesis instead of chemical synthesis because it is environmentally safer and enormous. Several biosynthetic methods are currently in use for small scale production and industrial production of molecules. 

Some applications of Biosynthesis

Cell factories for the Biosynthesis of insulin

A variety of pharmaceutical products are synthesized and marketed by companies including antibodies and several proteins which are required in large amounts. Out of which therapeutic monoclonal antibodies are the most marketed product followed by hormones and growth factors.

Presently insulin is produced generally in yeast and E. coli. Earlier, insulin was isolated from the porcupine and bovine pancreas. the expression of insulin in yeast and E. coli provided greater yield in a short span of time.

Why are microorganisms like E. coli essential for insulin biosynthesis?

The reasons for preferring E.coli for insulin biosynthesis are as follows:

  • – Faster reproduction rate (population doubles in every 20 minutes)
  • – It contains antibiotic resistance genes which restrict the growth of unwanted bacteria.
  • – Easy to handle
  • – Low maintenance cost
  • – High profitability

Previously, people use to take insulin from the pancreatic cells of the cows and pigs. The insulin obtained from pig and cow slightly differs from human insulin, and the harvesting cost is too high. It would take a ton of pig pancreatic tissue to produce a few ounces of insulin. Yeast (Saccharomyces cerevisiae) is also used for commercial insulin production like E. coli. But the production rate of insulin produced in yeast is significantly less as compared to the E. coli

Gene transfer of insulin from human to E. coli

The mRNA transcript is taken out from the insulin-producing pancreatic cells (β-cells of Islet of Langerhans). The reverse enzyme transcriptase binds to the mRNA and forms a single strand of cDNA (complementary DNA). The DNA polymerase then further polymerized cDNA to form a double-stranded DNA. The Copies of DNA are then prepared by performing polymerase chain reaction (PCR). The amplified DNA is transferred to a plasmid (extracellular circular DNA) of E. coli. The DNA/gene insertion process is accomplished by cutting and ligating the plasmid with restriction enzymes and DNA ligase. This plasmid has antibiotic resistance genes for tetracycline and ampicillin. 

           In the next step, the insertion of plasmids back into the E. coli is required; this process is known as transformation. The cell membrane of the E. coli is made porous and prepared for the uptake of plasmid by introducing calcium chloride into the cell containing medium. After this, plasmids were introduced in the medium and plasmids were taken up by cells after giving heat or electric (electroporation) shock to the cells. After electroporation, there are possibilities of getting two types of cells:

– Cells without plasmid

– Cells with a desired recombinant plasmid containing insulin gene

How to identify the desired E. coli cells with recombinant plasmid?

The recombinant plasmids, taken up by E. coli cells, produce antibiotic resistance in the E.coli cells. By this virtue, the desired cells are distinguished among the types above of E. coli cells obtained after electroporation. The recombinant plasmid containing cells will survive in the ampicillin and tetracyclin containing medium. However, the cells without plasmid will not survive in this medium.

Biosynthesis | A ray of hope for the environment and biotechnology
Figure 3: Schematic representation of plasmid transfer and clone identification. https://commons.wikimedia.org/wiki/File:Artificial_Bacterial_Transformation.svg

Insulin Production

The recombinant E. coli cells were then isolated, identified and then transferred to a large fermenter for their large scale production. Nutrient and growth media are prepared by adding optimum amounts of salt, sugar, nitrogen and water for E. coli. Ampicillin is also added to the growth medium to restrict the growth of unwanted cells and microbial contamination. The E. coli cells are expected to double their number in every 20-30 minutes. E. coli cells divide for several days to increase their number. After this, several chemical agents are added into the medium that initiates the insulin production by removing the insulin gene’s repressor protein resulting in the insulin gene’s activation.

Furthermore, some other chemicals agents are added to trigger the insulin production in the E. coli cells. After a few hours, E. coli cells produce a considerable amount of insulin. The broth is taken out from the fermentation tank, and cells are harvested and separated from the broth through centrifugation. Several chemical agents were added to disrupt E. coli cells’ cell membrane to release insulin from the cells. Insulin is then purified and crystallized by adding Zinc before distributing into the market.

Why is synthetic insulin necessary?

The recombinant insulin is meeting demands globally. This insulin production process made insulin affordable and available in a much better way compared to the previously used methods. Recombinant insulin gave freedom to subjects for living an everyday life without worrying much about their blood sugar levels. Biosynthesis of recombinant insulin provides a ray of hope for the production of other recombinant hormones required in various physiological conditions.

Biosynthesis of fuels

What are Biofuels?

Biofuels are generally produced from biomass such as plants and animal waste. It may have the potential to be used as a substitute for fossil fuels. Unlike fossil fuels (such as coal, petroleum, natural and natural gas), biofuels are considered a good source of green and renewable energy. They are generally environmentally friendly and cost-effective, biofuels have a promising future as it can be used as a substitute of fossils fuels in context to the rising petrol prices and a possible shortage of fossil fuels soon. 

Biosynthesis | A ray of hope for the environment and biotechnology
Figure 4: Concept of biosynthesis of renewable energy https://commons.wikimedia.org/wiki/File:Logo_Renewable_Energy_by_Melanie_Maecker-Tursun_V1_bgGreen.svg

Production of biofuels

The biofuels are synthesized by the action of microorganisms (generally bacteria and fungi). Bioethanol is synthesized by the fermentation of carbohydrates, while biodiesel (ester) is obtained from the fermentation of oils and fats. The biofuels obtained from the substances mentioned above are utilized by the fermentation of carbohydrates, while biodiesel (ester) is obtained from the fermentation of oils and fats. The biofuels obtained from the substances mentioned above are utilized to produce a significant amount of energy, and they also contribute towards environmental safety as they affect the environment to a minimal extent. 

Biosynthesis | A ray of hope for the environment and biotechnology
Figure 5: possible approaches for the production of biofuels.
https://commons.wikimedia.org/wiki/File:Types_and_generation_of_biofuels.png

Types of biofuels

Bioethanol: It has a promising application for its use in internal combustion engines. The cost-effective production of bioethanol enables it to be used as a replacement of conventional car fuels such as petrol and gas oil. 

Synthesis of bioethanol

The synthesis of bioethanol takes place in 4 steps:

– Biomass production by the fixation of atmospheric carbon dioxide.

– Biomass is then converted into food (usually glucose/starch), further utilized in fermentation.

The fermentation of biomass is conducted by microorganisms (yeast or bacteria), resulting in the production of ethanol concentration.

– Further processing and purification of this ethanol concentrate yield bioethanol and several by-products. The end product can be further used for various purposes, such as electrical energy, heat, other chemical compounds etc.

Biodiesel: Plant, animal and kitchen waste can be converted into biodiesel by following a series of experimental technologies. The process initiates by performing chemical reactions at lower temperatures, resulting in the production of esters. Esters are sweet/pleasant smelling substances, and they can be solid or liquid and are soluble in organic solvents because of their non-polar/hydrophobic nature. The ester produced now can be easily converted into biodiesel and glycerine. The glycerine formed in the process is a by-product and can be potentially used in cosmetics, lubricants and mouthwashes.

Biodiesel formed by this method doesn’t require any further modifications; hence it can be used directly in a pure state and mixed with gas oil for motors, burners and diesel engines. Large-scale biodiesel use for automobile and industrial use can help the world fight the fossil fuel crisis, health hazards due to air pollution, and greenhouse emission problems shortly. It is biodegradable, and hence it is non-toxic.

Biosynthesis in the light of biotechnology

Synthesizing compounds by chemical methods is a well-substantiated process for the large scale producing various biomolecules. However, chemical synthesis has some drawbacks such as multistep reactions, unstable reaction intermediates, complicated process control etc. Biosynthesis provides a promising and alternative approach to overcome these challenges, but the process’s efficiency is the central question of concern.

Design-construction-evaluation-optimization (DCEO) biotechnology provides an approach for developing efficient cell factories in association with conceptual techniques for designing pathways to perform Biosynthesis. Furthermore, DCEO can modify and optimize the existing pathway/process for the production of desired biochemical. DCEO biotechnology is a promising approach for the establishment of bio-refineries in the future.

Conclusions

Microorganisms provide so many benefits such as Biofuels, insulin and other hormones and biomolecule production. The use of biodiesel is essential for fighting the fossil fuel crisis of the future. Biofuels are beneficial for the environment because of their negligible toxicity. The recombinant insulin is advantageous because it can be produced without sacrificing a lot of animals and damaging the ecosystem. The process is cost-effective, and significantly less area is required for growth, harvesting and purification of recombinant protein form microorganisms. 

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About Dr. Abdullah Arsalan

Biosynthesis | A ray of hope for the environment and biotechnologyI am Abdullah Arsalan , Completed my PhD in Biotechnology. I have 7 years of research experience. I have published 6 papers so far in the journals of international repute with an average impact factor of 4.5 and few more are in consideration. I have presented research papers in various national and international conferences. My subject area of interest is biotechnology and biochemistry with special emphasis on Protein chemistry, enzymology, immunology, biophysical techniques and molecular biology.

Let's connect through LinkedIn(https://www.linkedin.com/in/abdullah-arsalan-a97a0a88/) or Google scholar(https://scholar.google.co.in/citations?user=AeZVWO4AAAAJ&hl=en).

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