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scientific strategies overview

Hormone Production Strategies Considered

Below are the methods of producing hormones I looked into before deciding that a plant based bioproduction system (a genetically modified hormone producing plant) would be most accessible and safest.

Extracting Hormones from Animal Urine

Feminizing hormones can be extracted from the Urine of pregnant mares and used for HRT such as brand name Premarin. According to the Transgender Care Health Archive, "Premarin contains a number of different (conjugated) estrogens and is obtained through the extraction process. Compounds derived from such a process not only subject animals to unduly harsh conditions, but may contain additional substances that create a potential for unknown or adverse effects. In our observation, there is a greater likelihood of mood swings and depression seen in patients using naturally derived estrogens as compared to synthesized preparations such as estradiol."

Creating a Cybernetic Testicle or Ovary

Testicular cells and ovarian cells express high levels of the enzymes involved in converting cholesterol into the sex hormones estradiol (ovarian cells) and testosterone (testicular cells). One potential method of producing hormones would be to design a bioreactor that could culture and maintain these mammalian cells in a substrate from which hormones could be extracted. This however would be cost prohibitive. Mammalian cells are more difficult to culture than plant cells, have higher risk of contamination from viruses and bacteria that can infect humans, and can't provide their own food source and amino acids through photosynthesis. "such expression systems are expensive, difficult to scale up and there are safety concerns due to potential contamination with pathogenic organisms or oncogenic DNA sequences."

Schillberg, S., Fischer, R., & Emans, N. (2003). Molecular farming of recombinant antibodies in plants. Cellular and Molecular Life Sciences (CMLS), 433-445.

Developing a Cheap Microbial Bioreactor

Several studies have demonstrated the ability of specific bacterial, and fungal strains to produce Androstadienone and Testosterone from Cholesterol or Phytosterol Substrates (the plant sterols sitosterol and stigmasterol). Conversion efficiency rates as high as 55% conversion have been recorded for testosterone and as high as 83% conversion from sitosterol to Androstadienone. Androstadienone is a precursor for both testosterone and estradiol. However, a plant based sytem would still have the advantage of providing it's own energy and being less expensive to maintain.

Sripalakit, P., Wichai, U., & Saraphanchotiwitthaya, A. (n.d.). Biotransformation of various natural sterols to androstenones by Mycobacterium sp. and some steroid-converting microbial strains.Journal of Molecular Catalysis B: Enzymatic, 49-54.

Zhang, Xiao-Yan, Yong Peng, Zhong-Rui Su, Qi-He Chen, Hui Ruan, and Guo-Qing He. "Optimization of Biotransformation from Phytosterol to Androstenedione by a Mutant Mycobacterium Neoaurum ZJUVN-08." J. Zhejiang Univ. Sci. B Journal of Zhejiang University SCIENCE B: 132-43. Print.

Egorova, O., Nikolayeva, V., Sukhodolskaya, G., & Donova, M. (n.d.). Transformation of C19-steroids and testosterone production by sterol-transforming strains of Mycobacterium spp. Journal of Molecular Catalysis B: Enzymatic, 198-203.

Extracting and Concentration Phytosterols

There are several sterols in the plant kingdom that have estrogenic effects in the body. It would be conceivable to grow plants with high levels of these "estrogen emulators" such as black cohosh, licorice, or dong quai, to extract and concentrate phytoestrogens. Unfortunately, previous studies have found that phytoestrogens bind weakly to estrogen receptors and have a diminished effect compared to synthesized estradiol. Because of this, the dosage required to achieve results comprable to those achieved with synthetic estradiol is toxic, and prohibitively large. "Phytoestrogens work by weakly binding with estrogen receptors, giving in some cases very mild feminizing effects. However, the doses required to achieve any effects at all are prohibitively large and toxic. (FAQ: Hormone Therapy for M2F Transsexuals) Most sources do not recommend that trans women use black cohosh, dong quai, milk thistle, or any other phytoestrogenic herb as a replacement for hormone therapy, even as a low-dose measure, because of their inefficacy. Because of the way that phytoestrogens compete with estrogen for receptors, using them in addition to hormone therapy may also be counterproductive."

Cascio, J. (2002, June 8). HRT Self-Medication: Information Accuracy and Risks of HRT | Trans Health. Retrieved December 16, 2015, from http://www.trans-health.com/2002/hrt-self-medication/

Genetically Modifying Plant Pathways

Tobacco has been successfully genetically engineered to produce pharmaceutically valuable molecules such as: human growth hormone, collagen, hemogloben, an ebola vaccine, and a swine flu vaccine. There is a large body of research on this plant, and it is very hearty, making it an ideal chasis organism. Engineering the metabolic pathways of the plant to produce human sex hormones would involve modifying the pre-existing sterol pathways to increase cholesterol accumulation (for which there is precedent) and incorporating genes coding for specific enzymes needed to transform from cholesterol to testosterone and estradiol. Alternatively, sitosterol (instead of cholesterol) could be upregulated to provide raw materials for the production of the sex hormones. Researchers have already had success integrating the gene for the first enzyme involved in the sterol biosynthesis pathway, which transforms cholesterol into pregnenolone. "In the mitochondria of animal steroidogenic tissues, cytochrome P450SCC encoded by the CYP11A1 gene catalyzes the conversion of cholesterol into pregnenolon-the general precursor of all steroid hormones. In this work we study the steroid metabolism in transgenic tobacco plants carrying the CYP11A1 cDNA encoding cytochrome P450SCC from the bovine adrenal cortex. The transgenic plants under investigation markedly surpass the control wildtype plants by size and are characterized by a shortened period of vegetative growth (by rapid flowering); their leaves contain pregnenolone-the product of a reaction catalyzed by cytochrome P450SCC. The level of progesterone in transgenic tobacco leaves is higher than in the control plants of the wild type."

Spivak, S., Berdichevets, I., Litvinovskaya, R., Drach, S., Kartel, N., & Shpakovski, G. (2009). Some peculiarities of steroid metabolism in transgenic Nicotiana tabacum plants bearing the CYP11A1 cDNA of cytochrome P450SCC from the bovine adrenal cortex. Russian Journal of Bioorganic Chemistry Russ J Bioorg Chem, 224-232.

Transgenic Tobacco Bioproduction System

Why a Plant? Why Tobacco?

Plants are ideal for a couple reasons: They produce their own energy source and amino acids (through photosynthesis) and in this way they cut costs. Because they can survive in nutrient substrate with no carbon source (unlike yeast, bacteria, and mammalian cell systems) they are less susceptible to contamination by harmful microorganisms. The graph below, taken from a study on the beniefits of plant based bioproduciton systems compares specific features of a variety of organisms used for bioproduction of pharmaceutical compounds. IMAGE CREDIT Ma, Julian K-C., Pascal M. W. Drake, and Paul Christou. "Genetic Modification: The Production of Recombinant Pharmaceutical Proteins in Plants." Nat Rev Genet Nature Reviews Genetics 4.10 (2003): 794-805. Web. I chose tobacco because of it's heartiness, and it's high success rate with agrobacterium mediated transformation, as well as it's proven ability to manufacture pharmaceutically valuable molecules including human growth hormone, an ebola vaccine, a swine flu vaccine, hemoglobin, human epidermal growth factor, and human homotrimeric collagen among others. Most importantly, it has already successfully been genetically modified to produce pregnenolone and progesterone, the precursors for testosterone and estradiol production.

Previous Research Supporting the Viability of this Method

1Construction and Characteristics of Transgenic Tobacco Nicotiana tabacum L. Plants Expressing CYP11A1 cDNA Encoding Cytochrome P450SCC VIEW

Demonstrates the potential to produce sex hormones in tobacco plants by creating a transgenic tobacco plant expressing Cytochrome P450SCC enzymes coded by Bovine genes.

2Some Peculiarities of Steroid Metabolism in Transgenic Nicotiana tabacum Plants Bearing the CYP11A1 cDNA of Cytochrome P450SCC from the Bovine Adrenal Cortex VIEW

Details of Steroid Metabolism in Transgenic Tobacco Containing Bovine DNA for Cytochrome CYP11A1 (side chain cleaving enzyme necessary for the biosynthesis of sex hormones)

3Biotransformation of various natural sterols to androstenones by Mycobacterium sp. and some steroid-converting microbial strains VIEW

Demonstrates the potential to produce androgens and estrogens directly from plant sterols like Sitosterol and Stigmasterol

4STEROL METHYLTRANSFERASE 1 Controls the Level of Cholesterol in Plants VIEW

Cholesterol is the raw material needed for steroid biosynthesis in mammals. Plant sterols such as sitosterol and stigmasterol have been demonstrated to be viable as alternative raw materials in the process. In the case that reactions beginning with cholesterol rendered a higher efficiency conversion rate than plant sterols, cholesterol accumulation can be increased in plants by modifying the expression of SMT1

5Sterol C-24 Methyltransferase Type 1 Controls the Flux of Carbon into Sterol Biosynthesis in Tobacco Seed VIEW

Demonstrates changes in cholesterol levels specifically in tobacco plants who’s SMT1 gene has been modified

6The presence and activity of progesterone in the plant kingdom VIEW

Documents the presence and activity of the "human" sex hormone progesterone (in important precursor to testosterone and estradiol) in plants.

7Plants as bioreactors: Recent developments and emerging opportunities VIEW

Reviews previous research on plant bioproduction systems.

8Mammalian sex hormones in plants VIEW

Documents the presence and activity of various "mammalian" hormones in the plant kingdom.

Methods and Protocols

Gene Design

Info coming soon

Plant Transformation

Info coming soon

Extraction and Analysis

Info coming soon

Microalgal Bioproduction System

Why a Plant? Why Microalgae?

Information Coming Soon

Previous Research Supporting the Viability of this Method

Information Coming Soon

Methods and Protocols

Information Coming Soon