Choosing a Target
Due to the nature of the steroid biosynthetic pathway we essentially had two options for the target class of steroid molecule.
The first being Corticosteroids, specifically Hydroxycortisone aka Cortisol, a steroid present in all vertebrate cells. It has incredibly broad uses and is the preferred starting material for synthesis of drugs with potent anti-inflammatory, abortive, or antiproliferative effects. It was also synthesized successfully via de novo fermentation in yeast in 2002, by several members of the team who’s research inspired the PoPPY project [1]. This would have been an ambitious project with real world implications based off of a clear roadmap. An all around excellent iGEM project (and one that we still recommend to future teams! Check out our Biobricks!) We had already decided to leverage our research from Souza et al [2]. to determine how a mammlianized sterol composition compared with ergosterol when channeling flux through our network of recombinant mammalian proteins to effect steroid titer. This would have already been a novel and interesting piece of state of the art synthetic biology in its own right, however there was a second option.
The other class of steroid molecules are the sex hormones, Testosterone and Estradiol being chief among them. While biotransformation, both with mycobacterium and yeast are currently the predominant manufacturing paradigm for sex hormone production, they require cholesterol or androstenedione as a key starting material (KSM).[3] There is no recorded record of Testosterone ever being synthesized de novo from a simple carbon source in the literature, and Estradiol is 1 enzymatic transformation downstream of Testosterone. [4] So why take on such an ambitious, and seemingly or perhaps likely impossible task? Unbridled optimism? Hubris? Naivete? While the answer to all of those questions is likely partially yes, it is primarily because we as a team are seeking to maximize impact.
Impact of Sex Hormones
The impact that comes with using the tools of biology to engineer a process for radical body autonomy. We are an international team at BOKU-Vienna 2023 and all of us support the rights of individuals to live their lives in a healthy body that reflects who they are inside. Whether that be an aging or infirm cisgender man or woman seeking the endocrine support they need to be vivacious, pain free, and hearty or a transgender or intersex person seeking gender affirming care. Sex hormones including Testosterone and Estradiol have experienced repeated, and current, [6,7] and longest lasting shortage durations for a variety of reasons, including manufacturing issues, supply chain complexity and disruptions, that could be addressed if our project were successful. [3] A de novo process for their fermentation could provide real and lasting relief for this repeated issue, as it would provide the opportunity to dramatically open the market and supply these critically undersupplied generic drugs.
However, many of these critical needs and opportunities addressed by our project have in truth been somewhat post-hoc. The critical truth is that iGEM is about more than Engineering Success, it is a platform. One of the largest in the scientific world. As an Austrian-American who was raised in the US, I felt compelled to use it to call out the distressing trends in LGBTQ+ rights taking place in my other home country. The Human Rights Campaign has declared a present “National State of Emergency for LGBTQ+ Americans'' beginning in the summer of 2023[5].
The opportunity to use this platform to not just draw attention to this issue but provide some concrete path to action has been chief among the motivating factors for this project to have taken the form that it has. We hope that this path to a potential process benefits all people of the world, but wish it to be known that any life lost to deaths of despair due to misaligned hormones, whether they be due to hypogonadism induced depression or gender dysphoria is a tragedy and one that can be prevented with compassion, knowledge, understanding and cooperation.
You are not alone. You are loved. You can help each other, help each other, help each other.
Call to Action - Corticosteroids
We would also like to highlight that during our project it has become clear that hormonal drug shortages are an equally pressing problem for Corticosteroids! Shortages of IV hydrocortisone (brand name Solu-Cortef injection, Pfizer) are also leading to critically undersupplied emergency rooms and preventable deaths from Sepsis!
We stand by our choice to prioritise research into novel biomanufacturing pathways for sex hormones for the reasons stated above, in addition to the opportunities afforded by the generic status of Testosterone, and potentially lapsing IP rights that will be discussed in other sections.
However we will officially make a call to future synthetic biologists to creatively utilise our research and Biobrick parts to improve manufacturing and access to Corticosteroids!
References References Choosing a Target
[1] Szczebara, F., Chandelier, C., Villeret, C. et al. Total biosynthesis of hydrocortisone from a simple carbon source in yeast. Nat Biotechnol 21, 143–149 (2003). https://doi.org/10.1038/nbt775
[2] Cleiton M. Souza, Tatjana M.E. Schwabe, Harald Pichler, Birgit Ploier, Erich Leitner, Xue Li Guan, Markus R. Wenk, Isabelle Riezman, Howard Riezman, A stable yeast strain efficiently producing cholesterol instead of ergosterol is functional for tryptophan uptake, but not weak organic acid resistance, Metabolic Engineering, Volume 13, Issue 5, 2011, Pages 555-569, ISSN 1096-7176, https://doi.org/10.1016/j.ymben.2011.06.006.
[3] Homeland Security and National Affairs (USA), The health and national security risks of drug shortages, accessed September 2023, https://www.hsgac.senate.gov/wp-content/uploads/2023-06-06-HSGAC-Majority-Draft-Drug-Shortages-Report.-FINAL-CORRECTED.pdf
[4] Fernández-Cabezón L, Galán B and García JL (2018) New Insights on Steroid Biotechnology. Front. Microbiol. 9:958. doi: 10.3389/fmicb.2018.00958
[4] Krishika Sambyal, Rahul Vikram Singh,Production aspects of testosterone by microbial biotransformation and future prospects,Steroids,Volume 159,2020,108651,ISSN 0039-128X, https://doi.org/10.1016/j.steroids.2020.108651.
[5] https://www.hrc.org/campaigns/national-state-of-emergency-for-lgbtq-americans
[6] Testosterone Transdermal System, 3/13/2023 , https://www.ashp.org/drug-shortages/current-shortages/drug-shortage-detail.aspx?id=925&loginreturnUrl=SSOCheckOnly
[7] Testosterone Cypionate Injection, 9/5/2023, https://www.ashp.org/drug-shortages/current-shortages/drug-shortage-detail.aspx?id=592
Testosterone – function and its importance for the well-being of humans
Note: In discussing sex hormones and how they affect the development of the human body, gendered language is an inevitabilty. In the following text, the terms 'male' and 'female' bodies, refer to the effects on development caused by hormones produced in the testies and ovaries respectively. The iGEM 2023 Team wishes it to be known that we respect the gender identies and expression of all readers regardless of gender assigned at birth, hormonal, chromosomal or anatomical differences.
Testosterone, classified as an androgen hormone, is responsible for the development of primary male sexual characteristics such as spermatogenesis, increasing sex drive and growth of male reproductive tissues such as the epididymis, seminal vesicles, glands, and penis[1]. Furthermore, testosterone act as a regulator of secondary male characteristics which include: growth of male pattern hair, voice changes, and both the growth and development of skeletal muscles[2]. According to Kelly and Jones (2013), testosterone is able to regulate metabolism of lipids, carbohydrates and proteins which facilitate muscle growth and fat distribution in the male body. As one of the most abundant sex hormones in the male body, testosterone contributes to many different physiological processes in various organs and organ systems, e.g. erythropoiesis.[3]
Testosterone is synthesised in the female body as well, but in smaller amounts (15-70 ng/dl) in comparison to concentrations of 265-923 ng/dl of testosterone in the blood of an average male[4]. Moreover, testosterone is required for the health and well-being of females. Testosterone acts as a precursor of estradiol, one of the most potent sex hormones in females. However, testosterone has an independent effect on the female reproductive system, bones, breast, mood and vasculature[5]. Interestingly, testosterone acts both directly and indirectly (when converted to estradiol) on the cardiovascular system[6]. Considering the structure of testosterone, it is classified as an androstanoid which has 17β-hydroxy and 3-oxo groups (marked with red on Figure 1), together with unsaturation at C4‒C5 bond[7]. Testosterone is a lipophilic molecule as are all other androgen hormones, meaning they are contained within hydrophobic subcellular environments[8]-
In the male body, Leydig cells which are part of the male gonads, are responsible for synthesizing testosterone from a precursor called cholesterol. Intermediates in this process are so called weak-acting androgens, dehydroepiandrosterone (DHEA) and androstenedione (AD). Synthesis of androgen hormones in the gonads, both in male and female bodies, are regulated by secretion of the same hormone called gonadotropin-releasing hormone (GnRH). The Hypothalamus synthesizes GnRH which stimulates the release of both luteinizing (LH) and follicle-stimulating (FSH) hormone from the pituitary glands. LH acts as a stimulator of the synthesis of testosterone in ovarian and testicular tissues.[9] Adrenal glands in both male and female bodies can produce testosterone from weak-acting androgens if they are synthesized in high levels[10].
Most of the testosterone circulating in the blood stream binds to sex-hormone-binding-globulin and albumin[11]. Binding of testosterone to these transport proteins increases half-life of testosterone and ensures equal distibution of it in the body[12]. Testosterone levels in tissues such as bones, muscles and prostate glands, are significantly smaller, than the amounts found in blood. Furthermore, testosterone and dihydrotestosterone can bind to specific androgen receptors and regulate expression of proteins respectively.
The importance of testosterone for human well-being proves the fact that it deserves it's historic spot on the World Health Organization list of Essential Medicines. Essential medicines are defined as medicines which may be required to meet the priority health care needs of a population and should be available in the health care system at all times[13]. Usual ways of testosterone administration are transdermal, subcutaneous and topical. Testosterone is available in the form of cypionate injections, ethanate injections, propionate injection solutions, subcutaneous pellets, topical gels, topical creams and transdermal patches[14].
Medical Conditions Treated
Testosterone can be used in the treatment of various medical conditions such as male hypogonadism, pre and postmenopausal syndrome in women and potentially in breast cancer treatment[15]. Testosterone therapy is used for treating females which are experiencing androgen deficiency in premenopause or postmenopause[16]. Hypogonadism (HG) is defined as the medical condition in which the body does not synthesise sufficient amounts of testosterone because of the decreased activity of gonads[17]. Studies have shown that hypogonadism affects 10-30% of the male population[18].
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Levels of androgen decrease both in men and women throughout their lifespan, which could result in androgen defficiency. Symptoms of androgen defficiency include: dysphoric mood (anxiety, irritability, depression), lack of well being, physical fatigue, bone loss, muscle loss, changes in cognition, memory loss, insomnia, hot flashes, rheumatoid complaints, bodily pain, breast pain, urinary complaints, incontinence, as well as sexual dysfunction[19]. It is well-known that breast cancer is an estrogen sensitive type of cancer. Since testosterone has an antagonistic effect on estrogen, it has great potential to be used in the treatment of breast cancer and other estrogen sensitive diseases such as breast pain, chronic mastitis and endometriosis[20].
Testosterone and its analogues (e.g. oxandrolone) are applied in the treatment of diabetes[21] (highly associated with HG) and various types of wasting syndromes – acquired immune deficiency sindrome (AIDS), anorexia, alcholism and in treatment of patients with severe burns, muscle or bone injury, osteoporosis, certain types of anemias[22][23][24].
Furthermore, testosterone is used as a masculinizing hormone in gender-affirming treatment because of its function to induce secondary male characteristics and supress female secondary sex characteristics[25].
Steroid hormones are one of the most marketed drugs because of a variety of physiological roles in human bodies [26], which makes them the second largest category of drugs next to antibiotics [27] with an annual global market over $10 billion [28] . Need for testosterone specifically has increased over the past few years, which makes testosterone one of the most widely prescribed drugs in the United States[29]. However, patients who need testosterone for their medical condition may experience testosterone shortage. According to the Homeland Security and Governmental Affairs of the United States[30], hormonal therapeutics shortage duration is the highest among other therapeutic categories (1201 days). Even though most of the hormonal therapeutics including testosterone are classified as generic drugs. Drug shortage is not only a problem in the United States, 14 of OECD countries are experiencing shortage of some steroid hormones [31].
Brief History of Testosterone Manufacturing
Modern androgen therapy began in 1935, when Lacquer and his coworkers extracted TS from 100 kg of bull testis and managed to isolate 10 mg of purified 17β-hydroxy-4-androstene-3one and called it ‘testosterone’ [32][33]. Independently that same year the labs of Adolf Butenandt in Göttingen and Leopold Ruzicka in Basel managed to chemically synthesise the drug from precursor molecules, and physicians began administration soon after [34]. The first synthetic route developed for the total synthesis of testosterone was the process of hydrochrysene approach by Johnson et al., characterised as a multi-step reaction with 55% yield at final [35]. However, chemical synthesis of testosterone was difficult because of its complex chemical structure and asymmetric centres [36]. Furthermore, chemical cleavage for the modification of basic steroid ring structures is not appropriate because steroid ring structures are highly sensitive to it [37]. Compared to a biological approach synthetic methods also have certain drawbacks: lower yield, multistep reactions, non-specificity and creation of hazardous by-products [38]. Usage of various reagents for chemical synthesis such as sulphur trioxide, pyridine, and selenium dioxide, is considered unsafe for the environment [39].
Testosterone and its derivatives have been reported from microbial transformation (Table 1) in fungi, yeasts, and bacteria over the past half century. These biotransformations include various reactions like hydroxylation, methoxylation, esterification, acylation, halogenation, isomerization, dehydrogenation/reduction, and hydrolyzation or sidechain cleavage at all carbon atoms of the four basal ring structure of steroids, with exception of C-10 and 13 [40]. Some Mycobacterium sp. Mutants have been used for a single step process for the synthesis of testosterone from sterols by microbial transformation [41][42]. Microbial transformations are an eco-friendly and effective alternative for the chemical synthesis of testosterone. Furthermore, microbial transformation gives higher yield and can be conducted in ambient pressure and temperature conditions [43][44].
However, microbial biotransformations rely on high quality pharmaceutical key starting materials (KSMs) such as purified cholesterol, AD, or plant based equivalents. Cholesterol, which is extracted from animal tissues or byproducts, must be purified to remove any microbial contamination or misfolded proteins that could cause contamination or spread disease [45]. Recently proprietary plant based cholesterol alternatives have been developed, but must be chemically modified from complex mixtures of plant sterols [46]. These KSMs can require large amounts of time, energy, infrastructure, and inputs to ensure that they meet the rigorous pharmaceutical standards and thus increase costs and supply chain complexity, which can lead to the observed shortages [47].
| Microorganism | Substrate | Product |
|---|---|---|
| Mycobacterium sp | Cholesterol | Testosterone |
| Lactobacillus bulgaricus | Cholesterol, AD | Testosterone |
| Yeast | AD | Testosterone |
| Aspergillus flavus | Progesterone | Testosterone, testolacton |
| Zoosporic fungi | Progesterone | Testosterone, testolacton |
The main objective of iGEM 2023 Vienna Team is to address the problem of hormonal agents shortage. Therefore, our goal is to establish a de-novo synthesis of steroid hormone in yeast Pichia pastoris which does not implement complex chemical synthesis processes and complex, non eco-friendly and expensive intermediates. Our process implements fermentation of simple carbon sources which finally leads to our target molecule in the biochemical pathway – testosterone. Benefits of de-novo fermentation of steroid hormones would be: decrease cost of steroid hormones production process and steroid hormones itself, improve access to hormonal agents and reduce barriers for people to receive life-affirming and gender-affirming treatment.
TestosteroneReferences
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[2] Nassar, G.N., Leslie, S.W. Physiology, Testosterone.: StatPearls. Treasure Island (FL): StatPearls, (2023)
[3] Kelly, D.M., Jones, T.H. Testosterone: a metabolic hormone in health and disease. J Endocrinol. 217 (2013).
[4] Medical News Today, Typical testosterone levels in males and females, accessed October 2023 https://www.medicalnewstoday.com/articles/3230853
[5] Labrie, F., Luu-The, V., Labrie, C., Simard, J. DHEA and its transformation into androgens and estrogens in peripheral target tissues: intracrinology. Front Neuroendocrinol 22(3), 185–212 (2001).
[6] Dos Santos, R.L., Da Silva, F.B., Ribeiro, R.F., Stefanon, I. Sex hormones in the cardiovascular system. Horm Mol Biol Clin Invest 18(2):89–103 (2014).
[7] Chemical Entities of Biological Interest, Testosterone, accessed October 2023, https://www.ebi.ac.uk/chebi/searchId.do?chebiId=CHEBI:17347
[8] Holst, J.P., Soldin, O.P., Guo, T., Soldin, S.J. Steroid hormones: Relevance and measurement in the clinical laboratory. Clin. Lab. Med. (2004).
[9] Schally, A.V., Arimura, A., Kastin, A.J. et al. Gonadotropinreleasing hormone: one polypeptide regulates secretion of luteinizing and follicle-stimulating hormones. Science. ,173:1036–1038 (1971).
[10] Clark, B.J., Prough, R.A., Klinge, C.M. Mechanisms of Action of Dehydroepiandrosterone. Vitam Horm. ,108:29-73 (2018).
[11] Clark, B.J., Prough, R.A., Klinge, C.M. Mechanisms of Action of Dehydroepiandrosterone. Vitam Horm., 108:29-73 (2018).
[12] Holst, J.P., Soldin, O.P., Guo, T., Soldin, SJ. Steroid hormones: Relevance and measurement in the clinical laboratory. Clin. Lab. Med. (2004).
[13] World Health Organization, WHO Model List of Essential Medicines - 23rd list, 2023, accessed September 2023, https://www.who.int/publications/i/item/WHO-MHP-HPS-EML-2023.02
[14] Shorr, R.I., Hoth, A.B., Rawls, N. Drugs for the Geriatric Patient, W.B. Saunders, 1166-1282 (2007).
[15] DrugBank Online, Testosterone – use, mechanism of action, accessed Septmeber 2023, https://go.drugbank.com/drugs/DB00624
[16] Glaser, R., Dimitrakakis, C. Testosterone therapy in women: myths and misconceptions. Maturitas. 74(3),230-4 (2013).
[17] Kumar, P., Kumar, N., Thakur, D.S., Patidar, A. Male hypogonadism: Symptoms and treatment. J Adv Pharm Technol Res. 1(3), 297-301 (2010).
[18] Mulligan, T., Frick, M.F., Zuraw, Q.C. et al. Prevalence of hypogonadism in males aged at least 45 years: The HIM study. Int J Clin Pract. ,60:762–9 (2006).
[19] Davey, D.A., Androgens in women before and after the menopause and post bilateral oophorectomy: clinical effects and indications for testosterone therapy. Womens Health 8, 437-44 (2012).
[20] Glaser, R., Dimitrakakis, C. Testosterone therapy in women: myths and misconceptions. Maturitas. 74(3),230-4 (2013).
[21] Hackett, G. Type 2 Diabetes and Testosterone Therapy. World J Mens Health. 37(1), 31-44 (2019).
[22]Orr, R.; Fiatarone Singh, M. The anabolic androgenic steroid oxandrolone in the treatment of wasting and catabolic disorders: Review of efficacy and safety. Drugs 64, 725–750 (2004).
[23] Church, J.A. Oxandrolone treatment of childhood hereditary angioedema. Ann. Allergy. Asthma. Immunol. 92, 377–378 (2004).
[24] Herndon, D.N.; Tanksley, J.D.; Jennings, K.; Klein, G.L.; Mlcak, R.P.; Clayton, R.P.; Crites, N.N.; Hays, J.P.; Andersen, C.; et al. Five-year outcomes after long-term oxandrolone administration in severely burned children: A randomized clinical trial. Shock Augusta Ga, 367–374 (2016).
[25] Unger, C.A. Hormone therapy for transgender patients. Transl Androl Urol. 5(6), 877-884 (2016).
[26] Fernandes, P.,Cruz, A., Angelova, B., Pinheiro, H.M., Cabral, J.M.S. Microbial conversion of steroid compounds: recent developments, Enzyme Microb. Technol. 32, 688–705 (2003).
[27] Tong, W.Y., Dong, X. Microbial biotransformation: recent developments on steroid drugs, Recent Pat. Biotechnol. 3 (2), 141–153 (2009).
[28] Fernández-Cabezón, L., Galán, B., García, J.L., New Insights on Steroid Biotechnology, Frontiers in Microbiology 9, (2018).
[29] Handelsman, D. Global trends in testosterone prescribing, 2000–2011: expanding the spectrum of prescription drug misuse. Med J Aust. , 199: 548–551 (2013).
[30] Homeland Security and National Affairs (USA), The health and national security risks of drug shortages, accessed September 2023, https://www.hsgac.senate.gov/wp-content/uploads/2023-06-06-HSGAC-Majority-Draft-Drug-Shortages-Report.-FINAL-CORRECTED.pdf
[31] Chapman, R., Dedet S.G., Lopert R. Shortages of medicines in OECD countries, OECD Health Working Paper 137, OECD Publishing, Paris (2022).
[32] David, K., Dingemanse, E., Freud, J., Laquer, E. Über krystallinisches männliches Hormon aus Hoden (Testosterone), wirksamer als aus Harn oder aus Cholesterin bereitetes Androsteron, Hoppe-Seyler’s Z. Physiol. Chem. 233 (5–6), 281–283 (1935)
[33] Nieschlag, E., Nieschlag, S. The history of testosterone and the testes: from antiquity to modern times, Testosterone, Springer, Cham, 1–19 (2017).
[34] Nieschlag, E., Nieschlag, S. Testosterone deficiency: a historical perspective. Asian J Androl. 16, 261 (2014).
[35] G. Guevara, Y.O. Flores, L.F. de las Heras, J. Perera, J.M.N. Llorens, Metabolic engineering of Rhodococcus ruber Chol-4: A cell factory for testosterone production, PLoS one 14 (7) (2019).
[36] Al-Aboudi, A., Mohammad, M.Y., Musharraf, S.G., Choudhary, I.M., Rahman, A. Microbial transformation of testosterone by Rhizopus stolonifer and Fusarium lini, Nat. Prod. Rep. 22, 1498–1509 (2008).
[37] Bensasson, C.S., Hanson J.R., LeHuerou, L. The microbiological hydroxylation of 3,5-cycloandrostanes by Cephalosporium aphidicola, Phytochemistry 52 1279–1282, (1999).
[38] Sambyal, K., Singh, R.V. Production aspects of testosterone by microbial biotransformation and future prospects, Steroids 159 (2020).
[39] Weber, A., Kennekke, M., Klages, U., Nickisch, K., Rhode, R. Process for the production of 17-oxosteroids via the fermentative oxidation of 17-hydroxysteroids by Mycobacterium, US Patent 5,472;854 (1995).
[40] Tong, W.Y., Dong, X. Microbial biotransformation: recent developments on steroid drugs, Recent Pat. Biotechnol. 3 (2), 141–153 (2009).
[41] Egorova, O.V., Nikolayeva, V.M., Sukhodolskaya, G.V., Donova, M.V., Transformation of C19-steroids and testosterone production by sterol-transforming strains of Mycobacterium sp, J. Mol. Catal. B Enzym. 57, 198–203 (2009).
[42] Fernández-Cabezón, L., Galán, B., García, J.L., New Insights on Steroid Biotechnology, Frontiers in Microbiology 9, (2018)..
[43] Fernandes, P.,Cruz, A., Angelova, B., Pinheiro, H.M., Cabral, J.M.S. Microbial conversion of steroid compounds: recent developments, Enzyme Microb. Technol. 32, 688–705 (2003).
[44] Tong, W.Y., Dong, X. Microbial biotransformation: recent developments on steroid drugs, Recent Pat. Biotechnol. 3 (2), 141–153 (2009).
[45] Cordenpharma.com, Facile & Scalable Synthesis of Plant-Based Cholesterol Botanichol in GMP Grade, accessed September 2023, https://cordenpharma.com/facile-scalable-synthesis-of-plant-based-cholesterol-botanichol-in-gmp-grade
[46] Jiang, Yq., Lin, Jp. Recent progress in strategies for steroid production in yeasts. World J Microbiol Biotechnol 38, 93 (2022).
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[48] Hung, B., Falero A., Llanes, N., Pérez, C., Ramírez, M.A. Testosterone as biotransformation product in steroid conversion by Mycobacterium sp., Biotechnol. Lett. 16, 497 (1994).
[49] Kumar, R., Dahiya, J.S., Singh, D., Nigam, P. Biotransformation of cholesterol using Lactobacillus bulgaricus in a glucose-controlled bioreactor, Bioresour. Technol. 78 (2), 209–211 (2001).
[50] Singer, Y., Shity, H., Bar, R. Microbial transformations in a cyclodextrin medium.Part 2.Reduction of androstenedione to testosterone by Saccharomyces cerevisiae, Appl. Microbiol. Biotechnol. 35, 731-737 (1991).
[51] Eman Mostafa, M., Zohri, A.A. Progesterone side-chain degradation by some species of Aspergillus flavus group, Folia Microbiol. 45, 243-247 (2000).
[52]Khallil, A.M., Eman Mostafa,M. Microbial transformation of progesterone by some zoosporic fungi, J. Basic Microbiol. 36, 225–229 (1996).
Biomanufacturing
Properties of our bioprocess called „De-novo fermentation of steroid hormones in yeast Pichia pastoris from simple carbon sources“
Bioprocessing implements microbial, plant, and/or animal cells or the components of these cells to manufacture new products or eliminate harmful waste from the environment[1].
- Biopharmaceuticalswhich involve therapeutic proteins, polysaccharides, vaccines, antibiotic drugs, and diagnostics.
- Specialty products and industrial chemicalssuch as environmental antibiotics, value-added food and agricultural products, fuels, chemicals and fibers from renewable resources.
- Environmental-management aidswhich include products and services used to control or remediate toxic wastes[2], e.g. microbial cells are used to remove or reuse carbon, nitrogen, phosphorus and micropollutants from the wastewater in combination with production of bioenergy and products. Therefore, microbial cells are sufficient for the development of a circular economy in the treatment of the wastewater by turning waste products into useful process inputs[3].
Every bioprocess consists of different unit operations conducted in the optimal environment required for specific cells to grow, divide and synthesize the molecule(s) of interest[4]. Generally, bioprocesses involving steroid hormone production have been developed by traditional approaches based on the isolation of microorganisms that produce the desired product or bio catalyze a specific process. After the successful isolation, physical or chemical mutagenesis and selection should be implemented in order to improve the desired microbial strain[5].
However, our process is not based on this traditional approach via strain development through directed evolution. Certain bioprocesses have been developed by recombinant DNA technology techniques in favor of the construction of microbial cell factories with improved robustness and versatility[6]. The design of microbial cell factories, genetically modified cells capable of specific biosynthesis (ex: Steroids), via the application of engineering principles such as abstraction, modularity, and the DBTL (Design/Build/Test/Learn) Cycle is the goal of both the iGEM competition and our 2023 Vienna team. This approach to strain development is referred to as Synthetic Biology[7].
According to Fernandez-Cabezon et al. (2018) steroid synthesis bioprocesses can be classified into three different groups:
- bioprocesses for production of steroid intermediates from natural sterols
- bioprocesses for modification and/or functionalization of steroidal molecules
- de novo biosynthesis of steroids[8].
Upstream - Fermentation
The bioprocess that we aim to implement in yeast Pichia pastoris [TA2] is de-novo fermentation of steroid hormones from simple carbon sources. Fermentation can be defined as the process of anaerobic fragmentation of organic compunds by metabolic processes occuring in microorganisms. Broadly speaking, fermentation is the term for chemical changes of a substrate performed by respective microorganisms in order to synthesize a useful product. Therefore, fermentation can be carried out under anaerobic (absence of oxygen) or aerobic (presence of oxygen) conditions, and standard temperatures and pressures. Fermentation is widely used in bioprocess engineering and in some cases it may be the only possible way to perform a specific or complex chemical transformation of raw materials. Fermentation processes are usually carried out at ambient or near ambient conditions because they employ enzymes from microorganisms which act as biological catalysts (substances which reduce the energy barrier for a reaction to take place)[9].
Other benefits of using enzymes include their high specificity, enantioselectivity and regioselectivity for the desired product, allowing the fine grained control of molecular products[10]. Under optimal fermentation conditions, the rate of reproduction of microorganisms, such as yeast and bacteria is rapid, accumulating large quantities of biomass and/or desired product. Properties of yeast such as large surface to volume ratio and small size allow rapid diffusion of substrates into the cell, and the desired product out of the cell[11]. Using fermentation for the synthesis of testosterone has many advantages such as: avoiding lengthy synthesis processes, harsh reaction conditions, multi-step reactions and production of unwanted industrial by-products[12][13].
In our case we are employing an aerobic fermentation of simple carbon sources in the yeast Pichia pastoris. Since Pichia pastoris is classified as a methylothropic yeast, methanol could also be used as the carbon-energy source. Benefits of using methanol as a simple carbon source include: very high cell densities, low price and effective induction of expression. Methanol can act both as growth substrate and inducer. However, high levels of methanol can have an inhibiting effect on the growth of cells – substrate inhibition effect[14]. Methanol is also a toxic and highly flammable substance, special care must be taken in designing production facilities that work with methanol as a feedstock. However industry standard practices have been developed to reduce these risks.
Based on the research from Duport et al. (1998), biosynthesis of pregnenolone, precursor of testosterone, was reproduced in the yeast Saccharomyces cerevisiae from a simple carbon source. Engineering of pregnenolone synthesis was achieved by disrupting the Δ22-deasturase gene (ERG5), introduction of the Arabidopsis thaliana Δ7-reductase gene (DHCR7) and coexpression of bovine side chain cleavage cytochrome P450 (CYP11A), adrenodoxin (ADX), and adrenoxodin reductase (ADR)[15]. Cytochrome P450s are heme-containing monooxygenases, which catalyze a variety of oxidative reactions[16]. Therefore, the biosynthesis of pregnenolone has to be conducted in aerobic conditions[17]. Biosynthesis of testosterone in the yeast Pichia pastoris also employs cythochrome P450s, for this reason the testosterone biosynthesis should also take place in aerobic conditions.
Proof of Concept
Our process focuses on a proof of concept study for the engineering of a testosterone producing yeast Pichia pastoris. This alone is already a very ambitious goal, given that this has never before been documented and engineering of metabolic process of similar complexity in the yeast S. Cerevisae, the 13 step enzymatic process of hydrocortizone bioproduction, took approximately 10 years and a well funded professional research team (1992-2002)[18]. [TA3] We are optimistic however that with access to 20 additional years of literature and new technologies, the inital proof of concept should not take nearly that long. Once biosynthesis is verified the yeild can be improved via further metabolic engineering and traditional strain development. In a linear process involving repeated rounds of both mutagenesis and selection of individual parental strains, strain development can select progeny with improved phenotypes and yeild through directed evolution[19]. Combined with exploring additional genetic modifications described in literature we are confident that a strain with commercial yeilds can be acheived. To this end, we have provided a simple description of what the rest of our process could look like based literature review and discussion with scale up experts. We hope that this can serve to provide context to future researchers or iGEMers in doing process design and scale up research.
Segments of our bioprocess called de-novo fermentation of steroid-hormones which should need future development and optimization include: fermentation (upstream) and recovery, isolation, purification and polishing of the product (downstream). Further improvement and optimization of these parts of the bioprocess would allow us to commercialize testosterone production in the yeast Pichia pastoris. For the fermentation process, we should determine optimal growth conditions (pH, temperature, ionic strength, medium composition, pO2) as well as the optimal feeding strategy.
Downstream Processes
Both recovery and purificiation of products from fermentation broth are crucial for commercializing any biomanufacturing process. Complexity of downstream processing is dependant on the properties of the product. Since fermentation broth has highly complex chemical properties and pharmaceuticals products (i.e. hormonal agents) must achieve high-purity during downstream processing, purification processes require many steps and account for 60% of the total costs of the bioprocess. An advantegous property of yeast is the size of their cells, which are larger than bacteria cells, therefore they can be recovered more easily from a fermentation broth[20].
Main objectives of the isolation and purification steps are:
- Separation of insoluble products (macromolecules, insoluble particles and biomass)
- Isolation or concentration of product and removal of most of the water
- Purification or removal of contaminating agents (DNA, proteins, RNA, viruses)
- Final product preparation – drying or crystallization[21].
Isolation is defined as the separation of the desired product of our bioprocess from the bulk of the organism that produces it. If the desired product is released from the microbial cell factory into the medium, isolation is performed by a solid-liquid separation step such as centrifugation, microfiltration, or ultrafiltration[22]. If the product is localized in the cellular compartments such as the cytoplasmic or periplasmic space, isolation processes get more complex. In our case, as testosterone is a lipophilic molecule and thus stored in either intermembrane space, aggrated in intracellualr compartments or lipid droplets. Then cells have to be lysed firstly by enzymatic, chemical, or mechanical methods (or a combination) then the product has to be separated from the cells and other medium components[TA4] [TA5] [23]. Separation method is chosen based on the molecular weight and the type of our molecule(s) of interest, in our case a proper method would be liquid-liquid extraction with a solvent like chloroform. The liquid extractant should be selective, nontoxic, inexpensive and immiscible with the fermentation broth[24]. Chloroform is a non-polar solvent which has all of the named properties, which could extract lipophilic testosterone from the cell lysate.
The next step in our downstream process would be purification of testosterone which include removal of contaminating agents like yeast DNA, RNA or proteins, and viruses. This could be done with some choromatographic methods like affinity chromatograhpy and HPLC (High Performance Liquid Chromatography). In the end our product needs final prepration like drying and crystallization.
Bioprocess References
[1] Doran, P. M. ,Editor(s): Pauline M. Doran, Bioprocess Engineering Principles (Second Edition),Academic Press, 255-332 (2013).
[2] National Research Council (US) Committee on Bioprocess Engineering. Putting Biotechnology to Work: Bioprocess Engineering. Current Bioprocess Technology, Products, and Opportunities 4. (1992).
[3] Nielsen, P.H. Microbial biotechnology and circular economy in wastewater treatment. Microb Biotechnol 5, 1102-1105 (2017).
[4] Mears, L., Stocks, S.M., Albaek, M.O., Sin, G., Gernaey, K.V., Mechanistic fermentation models for process design, monitoring, andcontrol. Trends Biotechnol 35, 914–924 (2017).
[5] Fernández-Cabezón, L., Galán, B., García, J.L., New Insights on Steroid Biotechnology, Frontiers in Microbiology 9, (2018).
[6] Fernández-Cabezón, L., Galán, B., García, J.L., New Insights on Steroid Biotechnology, Frontiers in Microbiology 9, (2018).
[7] Smolke, C. Building outside of the box: iGEM and the BioBricks Foundation. Nat Biotechnol 27, 1099–1102 (2009).
[8] Fernández-Cabezón, L., Galán, B., García, J.L., New Insights on Steroid Biotechnology, Frontiers in Microbiology. 9 ,958 (2018).
[9] Hocking, MB. Fermentation Processes. In: Modern Chemical Technology and Emission Control. Springer, Berlin, Heidelberg (1985).
[10] Bommarius. A.S., Riebel, B.R. Biocatalysis, 1. vol, 8, Wiley-VCH Verlag GmbH & Co.KGaA, Weinheim, (2008).
[11] Hocking, MB. Fermentation Processes. In: Modern Chemical Technology and Emission Control. 1985, Springer, Berlin, Heidelberg (1985).
[12] Fernandes, P., Cruz, A., Angelova. B., Pinheiro, H.M., Cabral, J.M.S. Microbial conversionof steroid compounds: recent developments, Enzyme Microb. Technol. , 688–705 (2003).
[13] Al-Aboudi, A., Mohammad, M.Y., Musharraf, S.G., Choudhary, I.M., Rahman, A., Microbial transformation of testosterone by Rhizopus stolonifer and Fusarium lini, Nat. Prod. Rep. 22 (2008).
[14] Shuler, M.L., Kargi, F., Bioprocess Engineering Basic Concepts, Prentice Hall. 427 (2001).
[15] Duport, C., Spagnoli, R., Degryse, E., Pompon, E., Self-sufficient biosynthesis of pregnenolone and progesterone in engineered yeast, Nature (1998).
[17] Cook, D.J., Finnigan, J.D., Cook, K., Black, G.W., Charnock, S.J. Chapter Five - Cytochromes P450: History, Classes, Catalytic Mechanism, and Industrial Application. Academic Press. 105, 105 (2016).
[18] Dumas, B., Brocard-Masson, C., Assemat-Lebrun, K. and Achstetter, T. Hydrocortisone made in yeast: Metabolic engineering turns a unicellular microorganism into a drug-synthesizing factory. Biotechnology Journal, 1, 299-307 (2016).
[19] Strohl, W.R., Biochemical engineering of natural product biosynthesis pathways, Metab. Eng. 3., 4-14 (2001).
[20] Shuler, M.L., Kargi, F., Bioprocess Engineering Basic Concepts, Prentice Hall. 329-380; 427 (2001).
[22] National Research Council (US) Committee on Bioprocess Engineering. Putting Biotechnology to Work: Bioprocess Engineering. Washington (DC), Current Bioprocess Technology, Products, and Opportunities 4 (1992).
[23] Minglong, S., Zhang, X.,Zhiming, R.,Meijuan, X, Taowei, Y., Hui L, Zhenghong X., Shangtian, Yang. Efficient testosterone production by engineered Pichia pastoris co-expressing human 17β-hydroxysteroid dehydrogenase type 3 and Saccharomyces cerevisiae glucose 6-phosphate dehydrogenase with NADPH regeneration. Green.Chem.
[24] Shuler, M.L., Kargi, F., Bioprocess Engineering Basic Concepts, Prentice Hall. 329-380; 427 (2001).
Towards Open Source
Possible Futures of HormOWN IP Strategy
The goal of iGEM Vienna 2023 is to establish an open-source de novo fermentation process for steroid hormones (ex: testosterone).
In order to do that, firstly we needed to conduct a patent search to check if our de novo fermentation process for steroid hormones has already been protected by a patent.
How to conduct a Patent Search
During a patent search, we used a free patent database called Espacenet and with the use of keywords, synonyms and different terms found within methods similar to ours. The patents which could be overlapping with our method are listed in Table 1 and Table 2, along with additional information like expiration date and location. After carrying out the Patent Search, we had an interview with Dipl.-Ing. Christian Kögl, an expert in patent law from the Austrian Patent Office. He provided us with additional information and specific details which should be taken into account when performing a Patent Search and also establishing an open-source process.
Requirements for patentability
According to PatG (Austrian Patent Act), there are certain requirements for a technical invention to be granted as a patent: novelty, inventivity, industrial applicability and technical character. Patented inventions should be a concrete technical solution to a specific technical problem. Categories of technical inventions that could be patented include: method, system, apparatus, compound and use of the certain compound. Technical inventions can be protected by the patent for 20 years. In special cases, patent protection can be extended for additional 5 years when a pharmaceutically active compound is protected. This type of Industrial Property Right is called a Supplementary Protection Certificate – time lost during the process of gathering the Marketing Authorisation is compensated with additional 5 years of protection. The benefits of owning a patent are: exclusivity, higher legal security, no legal obligation to pay provisions for trade license, possible income from license fees and 20 years of invention protection. The patent owner is exclusively entitled to commercially manufacture the product/method/system, place it on the market, to offer for sale or use it and to import or own the product according to §22 PatG. Every patent has claims – a combination of technical features protected by the patent (§22a PatG).
Results of our Patent Search
When comparing our de novo fermentation process with the ones that are patented, it can be seen that in most of the cases the claims are not matching with the compounds/organisms that we have been using in our process. Some of the patents are claiming methods or organisms which could be overlapping with ours. However, these patents have expired due to not paying annual fees or expiration of a 20 years protection period. When performing a Patent Search, one should also be careful how the claims of the same patent can vary between different regions, e.g. patent nr. one from Table 1 claims “yeast strains” in Europe, but only the yeast Saccharomyces cerevisiae is claimed in the same patent in the USA. Specifically, this means that we should be aware that our process may have different legal statuses and rights in different regions/countries. Furthermore, collaborators who are wishing to employ our process must perform their own due diligence in order to ensure Freedom to Operate in the respective region/country.
Legal regulations of an Open Source Platform
A major step in the creation of an Open Source Platform would be finding a potential collaborator which would be interested in commercializing and implementing our process on a larger scale. When collaborating with companies or other research groups, it is important to transparently define legal obligations of two concerning parties in a contract. It would be beneficial to create a Material Transfer Agreement (MTA) with our collaborator. MTA is a contract which clarifies the terms of exchanging materials such as biological samples or research data. It specifically defines the rights of provider and recipient of materials, e.g. what is being shared, for what use or purpose [1]. Furthermore, it would be necessary to communicate with the Technology Transfer Office (TTO) of our university since the whole research has been conducted in the facilities of the University of Life Sciences and Natural Resources in Vienna. The TTOs are usually active within a university in order to regulate the transfer of technology and knowledge to industry. Another key task of the TTOs is Intellectual Property (IP) management such as promotion of IP awareness, management of IP disclosure, commercialization, maintenance of IP assets and more. In other words, TTOs are responsible for moving innovations from the laboratories to the marketplace and community in order to make a beneficial impact on people's lives [1]. In our case, the potential benefits of open sourcing our process would decrease shortage of hormones, decrease the cost of the production process and therefore the product itself and make hormones easily accessible to the patients in need.
Marketing Approval for Generic Drugs
Since our compound of interest, testosterone, is a generic drug, pharmaceutically regulating agencies such as the FDA Generic Drugs Program would need to perform a thorough pre-approval review to make sure that our testosterone meets certain requirements. Companies who want to put their generic drug on the market must submit an abbreviated new drug application (ANDA) to FDA for marketing approval. Generic drug applications are termed „abbreviated“ because they are not obligated to conduct preclinical and clinical studies to prove their safety and efficacy. Alternatively, generic drug applicants must prove that their drug is bioequivalent to the brand-name drug. In order to get the FDA approval for generic medicines, our drug should meet the following requirements: the active ingredient in the generic medicines is the same as in the brand-name drug/ innovator drug, the generic medicine has the same strength, dosage form and route of administration, the generic medicine is manufactured under the same strict standards as the brand-name medicine, the label is the same as the brand-name medicine's label and the generic medicine is bioequivalent to the brand-name medicine. The FDA inspects the manufacturing plants in order to make sure that the manufacturing process is compliant with the good manufacturing practices. They are also monitoring that all of the steps in the manufacturing chain are ensuring the final product is of high-quality, safety and efficacy. Generic drugs must have a marketing authorisation before putting them on market in Europe as well, granted from the regulatory authority such as the European Medicines Agency (EMA). Prior to the marketing authorisation, EMA has to conduct the evaluation of the generic drug in terms of efficacy, safety and quality.
Freedom to operate
In the process of investigating the possibility of open-sourcing our process of de-novo fermentation of steroid hormones, our team also had an insightful interview with Sara Holland, UK patent attorney with her expertise in synthetic biology and industrial biotechnology. We have been mainly discussing possible legal risks of establishing an open-source process which overlaps with some of the expired patents and the detailed specificities about determining Freedom to Operate. Theoretically, expired patents are free to use, although there is a possibility that there is still some other Intellectual Property Right upstream or downstream of the process we have been trying to commercialize such as unexpired patents in Prior art or patents that are built on the expired patent. Furthermore, proving the Freedom to Operate of a certain manufacturing process is a very demanding and lengthy procedure. The first reason is because there are different claims for the same patent in different regions. Secondly, legal interpretation of those claims by the court vary as well in different regions.
Possible risks of open-source processes/methods
We should also be cautious with every method, equipment and even partial gene sequence we are using in our process because it could also be protected with a patent. After we have proven that any part of our process does not belong to prior art, we can establish an open-source process. Open-sourcing of a process/method can increase legal risks because everything you are doing is clearly exposed to the public and therefore anybody can make arguments and target specific parts of the process with lawsuits. A few possible ways of mitigating these legal risks is to engage patent agents or patent attorneys, generate your own IP and disclose your invention in a defensive publication. Search agents can be engaged in every step of the patent application process such as: submitting patent application, contacting patent office, filling out the administrative work and dealing with difficulties during the patent examination process [6] Defensive publication is a type of publication which is used to prevent other people from generating IP based on your invention by disclosing it in a written form.
Open Source References
[1] U.S. Food and Drug Administration, Abbreviated New Drug Application (ANDA), accessed September 2023, https://www.fda.gov/drugs/types-applications/abbreviated-new-drug-application-anda
[2] U.S. Food and Drug Administration, Generic drugs: Questions & answers, accessed September 2023, https://www.fda.gov/drugs/types-applications/abbreviated-new-drug-application-andahttps://www.fda.gov/drugs/frequently-asked-questions-popular-topics/generic-drugs-questions-answers
[3] European Medicines Agency,Questions and answers on generic medicines , accessed September 2023, https://www.ema.europa.eu/en/documents/medicine-qa/questions-answers-generic-medicines_en.pdf
[4] U.S. Food and Drug Administration, Generic drugs: Questions & answers, accessed September 2023, https://www.fda.gov/drugs/types-applications/abbreviated-new-drug-application-andahttps://www.fda.gov/drugs/frequently-asked-questions-popular-topics/generic-drugs-questions-answers
[5] European Medicines Agency,Questions and answers on generic medicines , accessed September 2023, https://www.ema.europa.eu/en/documents/medicine-qa/questions-answers-generic-medicines_en.pdf
[6] World Intellectual Property Organization, Technology Transfer Organizations, accessed September 2023, https://www.wipo.int/technology-transfer/en/organizations.html
[7] World Health Organization, About MTAs, accessed September 2023, https://apps.who.int/blueprint/mta-tool/about-mtas.html
[8] UpCounsel, What is a Patent Agent: Everything You Need To Know, accessed September 2023, https://www.upcounsel.com/what-is-a-patent-agent