Week 1 HW: Final Project Ideas

The target output I would like to make are the isolated metabolites (mainly bacosides) of Bacopa monnieri that produce the strongest neuroplasticity-associated signaling.

As someone who deals with anxiety and depression, I naturally found the topic interesting of a supplement that can ease the symptoms of both, while also improving neuroplasticity and reversing the damage done by chronic stress. “Bacopa is said to improve or maintain memory, learning retention, concentration, focus, and overall cognitive function and performance” (Shane-McWhorter, 2025). “Bacopa monnieri, also known as Brahmi or Waterhyssop, is a plant used in Ayurvedic medicine for its memory-enhancing properties and control of blood sugar levels. The plant has been studied for its potential in treating Alzheimer’s disease, Parkinson’s disease, attention deficit hyperactivity disorder (ADHD), and depression. Animal studies have shown promising results in reducing symptoms and protecting against neurodegeneration” (Gościniak, 2025). Bacopa is contraindicated in many conditions: bradycardia, thyroid disorders (especially hyperthyroidism), stomach ulcers, asthma, emphysema, urinary tract obstructions, individuals on cholinergic or anticholinergic medications, individuals on antidepressants. “Bacopa can increase acetylcholine and, therefore, may decrease the effect of anticholinergic medications or increase the adverse effects of cholinergic medications that increase acetylcholine (eg, medications for glaucoma or Alzheimer disease ). Bacopa may increase thyroid hormone levels; therefore, people taking thyroid hormone medications should not take bacopa. Bacopa may change the blood levels of medications metabolized by the cytochrome P450 system in the liver (eg, warfarin, some calcium channel blockers, antiseizure medications)” (Shane-McWhorter, 2025).The idea is that if the metabolites that have the strongest neuroplasticity-associated signaling can be isolated and identified, the whole plant can be refined in hopes to have less side effects, being available for more individuals after future clinical studies are done.

My main question: How can engineered yeast be used as a living system to produce specific bacosides from Bacopa monnieri, and how could these compounds be evaluated for their potential neuroprotective and neuroplastic effects?

Proposed chassis: Saccharomyces cerevisiae (baker’s yeast). This yeast is very well studied, has extensive genetic-engineering tools, and has a strong history in metabolic engineering. Yeast is already very good at producing terpenoid precursors, which makes it an attractive chassis. I would use engineered Saccharomyces cerevisiae to produce a selected Bacopa triterpenoid saponin, such as bacopaside I or bacoside A3. The starting materials would primarily be carbon and energy sources supplied to the yeast, along with the metabolic precursors naturally produced by the yeast. The biological processes would include gene expression, enzyme production, and enzymatic conversion of metabolic intermediates through the engineered pathway (Zuo et al., 2025). The desired output would be yeast containing the target Bacopa-derived compound, which could then be extracted and chemically characterized using techniques such as High-Performance Liquid Chromatography (HPLC) (Ganzera, 2004).

Bacosides are triterpenoid saponins. Their biosynthesis ultimately depends on pathways that convert simple cellular metabolites into increasingly complex terpenoid molecules (Zuo et al., 2025). The active constituents and metabolites responsible for the effects of BM could be bacoside A and B, bacopaside I and II, bacopasaponin C, betulinic acid, asiatic acid, loliolide, ebelin lactone, and quercetin. “The mechanistic role of Bacopa monnieri in brain disorders might be related to its ability to modulate neurotransmission, neurogenesis, neuronal/ glial plasticity, intracellular signaling, epigenetics, cerebral blood flow, energy metabolism, protein folding, endoplasmic reticulum stress, neuroendocrine system, oxidative stress, inflammation, and apoptosis” (Vinod et al., 2022). I would initially focus on producing the compound rather than attempting to reproduce the entire Bacopa biosynthetic pathway. This is an important assumption because Bacopa triterpenoid saponins are complex molecules that may require multiple enzymatic steps, and not all of the genes and pathway components may yet be fully characterized.

At a high level, the system would work by introducing genes from Bacopa monnieri into yeast. These genes would encode enzymes involved in converting cellular metabolic precursors into the desired Bacopa compound. The engineered yeast would provide the basic metabolic building blocks, while the introduced enzymes would redirect some of the yeast’s metabolism toward production of the target molecule. DNA assembly methods such as Gibson Assembly could be used to construct the genetic components needed for the pathway. A longer-term goal could be to optimize the engineered yeast so that it produces higher quantities of the target compound. The resulting molecule could then be compared with plant-derived material to determine whether the biologically manufactured compound has the same chemical characteristics and biological activity.

For this project, a desktop liquid-handling robot such as an Opentrons would be a practical automation choice. I would use an Opentrons automated liquid-handling robot to automate the preparation and screening of engineered yeast cultures. The robot could be programmed to accurately transfer small volumes of growth media, yeast cultures, DNA samples, and reagents or assay components between plates or tubes. Opentrons could also help prepare multiple experimental conditions at the same time, such as different yeast strains or different genetic constructs, making it easier to determine which conditions produce the highest amount of the desired bacoside (Opentrons, 2023). I could then use analytical methods such as HPLC to measure which yeast strain produces the greatest amount of the target Bacopa-derived compounds produced by each culture (Ganzera, 2004).

For this project, I would use 1 gram of purified bacoside as a hypothetical research-scale target. This amount could provide material for repeated chemical characterization and laboratory testing, although the exact amount required would depend on the specific experiments (Baell et al., 2026). Chemical identity and purity are particularly important in preclinical research because impurities can affect experimental results.

For the production calculation, I would assume that an engineered yeast strain could eventually produce approximately 10 mg of the target bacoside per liter of culture. This is a hypothetical production rate rather than an established yield for Bacopa compounds. Research has demonstrated mg/L-scale production of plant triterpenoids using engineered microbial systems, making this a reasonable starting assumption for a theoretical calculation. For example, assuming a hypothetical yield of approximately 10 mg of bacoside per liter of culture, the calculation would be: 1,000 mg ÷ 10 mg/L = 100 L Therefore, approximately 100 liters of fermentation culture would be needed to produce 1 gram of bacoside, assuming the hypothetical yield of 10 mg/L and 100% recovery. In reality, the amount of culture needed would be higher because some product would be lost during harvesting, extraction, purification, and other downstream processing. For example, with an overall recovery of 50%: 1,000 mg ÷ (10 mg/L × 0.50) = 200 L So, at this hypothetical production rate, I would estimate needing around 200 liters of fermentation culture to obtain 1 gram of purified bacoside. This calculation also shows why improving the yeast’s production yield would be an important part of the project. If the engineered yeast could produce more bacoside per liter, substantially less fermentation volume would be required.

I would estimate that each fermentation batch would take approximately 5–7 days, including yeast growth, production of the target compound, harvesting, extraction, and purification (Dai et al., 2014). If a small-scale automated system could run 10 parallel 1-liter cultures, each batch would produce approximately 10 mg of bacoside before purification. With 50% recovery, this would result in approximately 5 mg of purified bacoside per batch. To produce 1 gram: 1,000 mg ÷ 5 mg = 200 one-liter culture batches If 10 cultures could be operated simultaneously, this would require approximately: 200 ÷ 10 = 20 batches At approximately 7 days per batch, the total production time would be about: 20 × 7 = 140 days, or approximately 4.5 months.

However, this would be a reasonable estimate for a prototype system, not an industrial manufacturing process. At a larger scale, I would transition from a liquid-handling robot to larger automated bioreactors or fermentation vessels. For example, instead of running hundreds of small cultures sequentially, a larger automated fermentation system could operate several 50–200 liter batches in parallel. This could reduce the manufacturing time substantially. Can yeast produce the correct bacoside, how much can it produce, and can the production process be scaled up efficiently? Answering these questions through small-scale experiments and automated screening would determine whether the proposed biomanufacturing system is feasible.

The largest uncertainty in this estimate is the assumed 10 mg/L production yield. If the engineered yeast produces less bacoside, more fermentation volume and time would be required. If metabolic engineering increases the yield, the same 1 gram target could be produced much faster. Therefore, an important goal of the automation system would be to screen different yeast strains and growth conditions to identify the combination that produces the highest bacoside yield. Another bottleneck is the efficiency of the biosynthetic pathway. Some of the enzymes involved, particularly cytochrome P450 enzymes and glycosyltransferases, may not function efficiently when transferred from Bacopa into yeast. The pathway could also create unwanted intermediate compounds instead of the desired bacoside. I would need to determine which enzymes and pathway combinations produce the highest amount of the desired product while minimizing unwanted byproducts.

References

Baell JB, Pauli GF, Fillmore M, et al. Practical Recommendations for Small-Molecule Characterization in Preclinical Research. 2026 Jun 10. Eli Lilly & Company and the National Center for Advancing Translational Sciences; https://www.ncbi.nlm.nih.gov/books/NBK623671/

Dai, Z., Wang, B., Liu, Y. et al. Producing aglycons of ginsenosides in bakers’ yeast. Sci Rep 4, 3698 (2014). https://doi.org/10.1038/srep03698 Ganzera, Markus, Gampenrieder, Julia, Pawar, Rahul S., Khan, Ikhlas A., & Stuppner, Hermann. (2004). Separation of the major triterpenoid saponins in Bacopa monnieri by high-performance liquid chromatography. Analytica Chimica Acta, 516(1), 149–154. https://doi.org/10.1016/j.aca.2004.04.002

Gościniak, A., Stasiłowicz-Krzemień, A., Szeląg, M., Pawlak, J., Skiera, I., Kwiatkowska, H., Nowak, N., Bernady, K., Trzaskoma, P., Zimak-Krótkopad, O., & Cielecka-Piontek, J. (2025). Bacopa monnieri: Preclinical and Clinical Evidence of Neuroactive Effects, Safety of Use and the Search for Improved Bioavailability. https://doi.org/10.3390/nu17111939

Opentrons. (2023, February). Your Personal Pipetting Robot. Meet the OT-2 Brochure. https://insights.opentrons.com/hubfs/Brochures%20and%20flyers/OT-2%20Brochure%20(1).pdf

Shane-McWhorter, L. (2025, July). Bacopa - special subjects. Merck Manual Professional Edition. https://www.merckmanuals.com/professional/special-subjects/dietary-supplements/bacopa#Evidence_v75590716

Vinod A, Sathianarayanan S, Babu AE, Sadanandan P, Venu AK, Venkidasamy B. Bacopa monnieri for Disorders Affecting Brain: Current Perspectives. Curr Top Med Chem. 2022;22(23):1909-1929. doi: 10.2174/1568026622666220119111538. PMID: 35043757. https://pubmed.ncbi.nlm.nih.gov/35043757/

Zuo, S., Tan, X., Mao, J., Hu, F., Chen, J., Fan, L., Lu, Q., Zhao, Y., Wang, Y., Xiao, Z., Zhang, S., Shan, Y., Liu, J., & Fu, F. (2025). Metabolic Engineering of Yeasts for the Production of the Triterpene Squalene: Current Status and Future Prospective. Microorganisms, 13(11), 2422. https://doi.org/10.3390/microorganisms13112422

Final Project