Industrial Production of Hormones

Gurpreet Kaur Bamrah, Shoolini University, Himachal Pradesh

Hormones are a member of the signaling molecules present in multicellular organisms. They are coined as chemical messengers that are secreted into the bloodstream (endocrine hormones) or are secreted directly into the ducts of the target organ (exocrine hormones). The role of hormones is to control and coordinate different activities of the body, affecting diverse processes such as growth and development, reproduction, metabolism, sexual characterization, maintaining salt concentrations, and homeostasis.

The need for commercial production of hormones was realized with the growing demand for peptide drugs to treat degenerative diseases. For example, calcitonin, a hormone secreted from the parafollicular cells of the thyroid gland, is used to maintain calcium homeostasis inside our body. It is also being used to treat various metabolic bone diseases, especially Paget’s disease, and hypercalcemic shocks.

Industrially produced hormones are also required in the agriculture sector. Hormones like Abscisic acid (ABA) help delay the plant’s growth rate, improve plant tolerance to various environmental stresses, etc. Plant hormone products retrieved from plant extractions show extremely low yields because of their chemical complexity, which is overcome by mechanized production in bioreactors and fermenters.

BASIC OUTLINE OF INDUSTRIAL-SCALE PRODUCTION OF HORMONES

Hormones, medicines, and vaccines today are synthesized using recombinant DNA technology (rDNA). Microorganisms can nearly convert any carbon form into the desired product, of which, only a handful of cases have had a successful transition to industrial-scale processing. At the core bioconversion level, the leading cause for this futile implementation is concerned with the strain performance in the industrial bioreactor. The environment of industrial bioreactors is drastically different from laboratory-scale cultures in shake flasks. To overcome this problem, strains are developed that are fit to maintain aggressive conditions inside the bioreactor and can scale up the production.

• Strains with the desired genotype and phenotype composition are isolated at laboratory scale through omics approach for process and pathway optimization.
• With rDNA technology, strains with the desired gene to be expressed are cultivated.
• The culture is transferred from a flask to an inoculum preparation reactor to a production bioreactor.
• Microbial cells are harvested through centrifugation or filtration. The product is extracted through precipitation.
• The product is down streamed. It passes through high-resolution purification filters followed by formulating the product (adding buffers, protectants) and lyophilization.
• Last, the product is checked for quality, packaged, and marketed to biopharmaceutical companies.

MICROBIAL PRODUCTION OF HORMONES

Diverse microbial species possess the ability to produce hormones and other secondary metabolites. Most of these microorganisms are present in the soil. Inoculating the soil with bacteria and certain fungi has significantly increased the growth per hectare of crops, reduced wastage of resources, promoted fruit ripening, and leaf drop, accelerated seed germination and budding process, increased resistance to pathogens and natural disasters with lowering the cost of production. Microbial production products possess higher bioactivity and purity. Some of the microbial-assisted hormone products are:

I. Microbial production of phytohormones:

Common phytohormones like auxin, gibberellic acid (GA), ABA, ethylene, and cytotoxin are known to us as chemical messengers that coordinate the cellular process inside the plants. These hormones are not only found in higher plants but also algae and plant-associated bacteria and fungi.

ABA is the most abundant phytohormone present in plants. Industrial production of this hormone is facilitated using fungi like Cercospora rosicola and Botrytis cinerea which can produce a liquid culture of ABA. In vitro mechanism of production involves using some of the potent plant pathogenic fungi thus inducing plant-pathogenesis. Indole-3-acetic acid (IAA) is a common type of auxin which is synthesized from L-Tryptophan (L-TRP) and metabolized by plant soil. This IAA is formed by two mechanisms:

• TRP deaminated to indole-3-pyruvic acid (IPyA) decarboxylated to indole-3-acetaldehyde (IAAld) oxidized to IAA
• TRP decarboxylated to indole-3-acetamide hydrolyzed to IAA

IPyA is the most apparent intermediate in IAA production from L-TRP by alpha-keto glutarate-depended transaminase (AKGT). Any parameters affecting the activity of transaminase would affect the yield of auxins. AKGT is extracted and purified from cell-free extracts of rhizobacterial strains along with Festuca octoflora grass. Rhizobacterium converts substrate L-TRP to IAA in a culture medium using ion-suppression reverse phase HPLC. Another evidence of commercial IAA production using Douglas fig inoculated with ectomycorrhiza, Pisolithus tinctorius using TRP as substrate. IAA-derived culture medium is testified using thin-layer chromatography, ELISA assay followed by unequivocal identification by gas chromatography-mass spectroscopy. The latter model has shown a tremendous increase in the overall nutrition of the plant, including an increment in the root length. ABA is mainly responsible to prevent abscission and enhancing fruit development.

Gibberellins are another class of important phytohormones that participates in the growth and development of plants such as germination, cell elongation, development of flowers and, stimulates higher development of xylem and phloem in leguminous plants, thus making them an asset in agriculture for increasing crop yield. GA3, the most important class for GA out of 136 varieties, is produced by bacteria, algae, fungi, and plants. For industrial production of GA3, ascomycetous fungi Gibberella fujikoria LPB06 and Fusarium moniliforme LPB03 are used due to their higher concentrations and citric pulp as substrate. This extraction makes use of different bioreactors and varied fermentation techniques like solid-state fermentation (SSF), submerged fermentation (SmF), and semisolid state fermentation (SSSF).

Cytokinin activates dormant buds, delays senescence, and affects cell division, is reported to be produced by 11 bacteria and 4 funguses. Out of these, Azotobacter chroococcum is the prolific producer of cytokine. Using adenine (ADE) as a precursor and isopentyl alcohol increases the production of this hormone under aerated conditions, which is further identified and quantified using HPLC/UV spectroscopy.

II. Production of hormones using rDNA technology:

From making a prenatal diagnosis of genetic diseases to manufacturing pharmaceutical commodities, rDNA technology has modernized the purpose of bioremediation and treating illnesses. Tremendous advancement has been made with this technology that is seen in the application of hormone production at the industrial scale. A plasmid vector is isolated from the host organism, the gene of interest which is cut using restriction enzymes cleaves a part of the plasmid vector. The gene is inserted along and ligated using DNA ligase. This plasmid is then inserted back into the host and allowed to replicate. The desired gene is expressed, the product is extracted and purified through a downstream process.

Insulin is a hormone used to regulate blood sugar levels in the body. With the growing number of type-2 diabetes patients, the need for an alternate method for the large-scale production of insulin was demanded. Initially, insulin was derived from purified bovine and porcine pancreas, but it was soon discouraged because of its peak inactivity time. It took 3-4 hours after injecting for the insulin to work. People started developing allergies and also raised some ethical concerns. Using rDNA technology, genes encoding human insulin were cloned and expressed in E. coli and Saccharomyces cerevisiae. The latter is preferred and predominates the large-scale production market of insulin. Sequences encoding for insulin chains A and B are inserted into two different E. coli strains. These cells are cultured separately into large-scale fermenter vessels. Purification methods, including affinity chromatography, remove the cells from the product formed. Human insulin thus comprises a modified amino acid sequence.

The gene for human growth hormone (hGH) is isolated from the human pituitary gland. The mRNA of hGH is used to create a complementary DNA strand (cDNA) for the gene. The First 24 amino acids of the cDNA are removed by restriction endonuclease and are joined to synthetically made first 24 hGH amino acids by T4 DNA ligase. This is done because bacteria cannot express the region of the gene that is not translated by humans. Expression vector phGH407 derived from plasmid vector PBR322 is used as carrier vector, further, the hGH gene is ligated into a restriction site just downstream of Lac; promotor/operator region of the expression vector. The recombinant plasmid is then introduced into a suitable host like E. coli and mass-produced using fermenter technology and purification methods.

III. Using plants for the production of hormones:

Certain plant seedlings like Arabidopsis thaliana and Nicotiana tabacum have been used to produce sex hormones like progesterone, testosterone, and oestradiol. Recent use of ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) detected the presence of all the androsteroids in Digitalis purpurea L., Nicotiana tabacum L., and Inula helenium L.  Testosterone, along with epitestosterone and androstenedione, was first isolated from the pollen of Scotch pine, Pinus silvestris.

CONCLUSION

Various studies and methodology reported here reveal the ability of specific microorganisms, the technology used, and soil microflora to derive hormones from precursors provided in pure culture. Inoculation with specific organisms may prove beneficial in affecting crop yield, development of pharma important hormones like insulin which is in high demand of the times and, essential hormones that are used in treatments like hormone replacement therapies, genital affirmation surgeries, breast augmentation, and infertility. Lab-scale to large-scale production of hormones or any pharmaceutical products requires a thorough assessment of the sample, high purity-based cultures, and all the materials and methods that operate to up-scale the production like the use of high-end fermenters.

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Also read: Genetic switch: How does it help to control plant growth?

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