Introduction

Team Cornell 2024 aims to synthetically manufacture Ursolic Acid within yeast using a continuous bioreactor, which will provide a more sustainable and efficient pathway compared to existing manufacturing methods. Ursolic Acid is a natural product that has been discovered to have numerous medically beneficial properties. Preliminary studies have shown therapeutic potential for cancer, liver disease, and obesity, among other benefits [1]. It is currently obtained through extraction from fruits, such as loquats and apples, which is environmentally taxing [2]. Furthermore, apples represent a significant part of Ithaca, NY’s local agriculture, where Cornell University is based. In 2022, Tompkins County reported nearly 200 acres of apple-bearing land use [3]. Current research shows that a biological pathway exists within yeast to produce this compound, but it has only been done at a lab scale.

Taking this as inspiration, Cornell can engineer the genetic elements involved both upstream and downstream of the mevalonate (MVA) pathway of yeast to not only introduce ursolic acid synthesis into yeast, but to optimize its yield through genetic knockouts to increase the cytosolic acetyl-CoA pool. Integrating the alpha amyrin α-amyrin synthase gene into the yeast genome via the Ura3 homology site on the pRS306 plasmid will allow us to complete the former, while the latter will be completed by knocking out the mitochondrial porin POR2 gene through a PCR-mediated knockout utilizing the pFA6a-kanMX6 plasmid. We are using two separate plasmids to run these experiments in parallel and reduce metabolic strain on the yeast during transformation. We will additionally be integrating a third plasmid to produce a fusion protein consisting of a signal peptide sequence and a terpene-binding protein for extracellular delivery of ursolic acid to isolate it outside of yeast cells.

While this step is being completed, product development can work in parallel to design a continuous bioreactor for manufacturers to produce this compound more efficiently by removing downtime between production batches. An integrated biosensor will also monitor the concentration of ursolic acid within the bioreactor and alter the system conditions to maintain a high output of ursolic acid. The feedback from both the sensor and probes monitoring the environmental conditions (temperature, pH, glucose concentration, dissolved O2 concentration) will allow relative autoregulation of the reactor. The bioreactor will also combat the high energy cost typically found within batch reactors which will overall allow pharmaceutical companies to save money, lower their Environmental Factor (E-Factor) and Process Mass Intensity (PMI). By combining synthetic biology methods with industrial scale-up we can impact oncology by maximizing the production of a natural product in order to get its positive effects into the hands of manufacturers and ultimately patients.

Genetic Design

Basic Components

The basic components of our projects which include plasmids, promoters, terminators, ribosome binding sites and markers for our project are listed below. These form the backbone of our genetic circuits.

1. pRS306: A plasmid backbone with URA3 marker and ampicillin resistance
2. pFA6a-kanMX6: A plasmid backbone with ampicillin and kanamycin resistance
3. BBa_K530008: (TDH3) Constitutive promoter
4. BBa_K165002: Kozak sequence (Yeast RBS site)
5. BBa_K2926005: TPs1 terminator
6. BBa_K5059000: α-amyrin synthase gene from Malus domestica
7. BBa_K5059001: α-amyrin synthase gene from Malus domestica with TDH3 promoter, Kozak sequence (RBS), and TPS1 terminator

In regards to genetic design, we are utilizing two plasmids - pRS306 and pFA6a-kanMX6 each serving a distinct purpose in our overall project.

pRS306 is a well-characterized plasmid used in yeast due to the presence of a Ura3 site, which provides opportunities for integration into the yeast genome. We are inserting the alpha amyrin α-amyrin synthase into the pRS306 plasmid via Gibson Assembly, then transforming the plasmid into yeast cells. This will introduce ursolic acid production into S. cerevisiae.

pFA6a-kanMX6 is a plasmid commonly used in yeast due to the presence of the kanMX selectable marker which confers resistance to G418, an antibiotic that is standard for eukaryotic selection. We are knocking out POR2, a mitochondrial porin, that has been shown to increase the yield of ursolic acid [1].

Last but not least, we are designing the encapsulation to integrate ꞵ-cyclodextrin with ursolic acid to form a two-part complex that improves the water solubility of ursolic acid. This is based on previous research which determined that ursolic acid, when encapsulated with ꞵ-cyclodextrin, became much more water soluble, which is crucial for its bioavailability as a drug [4].

Both plasmid designs are shown below.

     

Wet Lab

Approach

To introduce ursolic acid production in S. cerevisiae, we are inserting the exogenous ⍺-amyrin synthase gene from Malus domestica into the yeast genome through the Ura3 homology site found in pRS306. Yeast naturally produces the precursor for ⍺-amyrin, 2-3-oxidosqualene, but lacks the enzyme, ⍺-amyrin synthase, necessary for its conversion into ⍺-amyrin. Thus, to complete the metabolic pathway, inserting the ⍺-amyrin synthase gene allows S. cerevisiae to produce ⍺-amyrin which is converted to ursolic acid.

     

To increase ursolic acid production, we are knocking out the mitochondrial porin POR2, a voltage-dependent anion channel that transports acetyl-CoA from the cytoplasm into the mitochondria. By turning off that shuttle down, there will be an increase in the acetyl-CoA pool for the mevalonate pathway to produce ursolic acid. The knockout will be performed using pFA6a-kanMX6 and a PCR-mediated knockout method [2].

To confirm whether or not the AAS insertion is successful, we will be utilizing Ni-NTA affinity chromatography, which is applicable due to the presence of 6x-His tags on the AAS insert. To confirm the POR2 knockout, we will grow the transformants on growth media containing G418, an antibiotic that the kanMX gene provides resistance to.

Once the genetic alterations are confirmed to be successful, we will extract the ursolic acid through a signal peptide system as [5]. A terpene-binding protein will be fused with a signal peptide sequence derived from the Suc2 gene, which encodes for invertase. Invertase is exported out of cells through co-translational translocation, so combining its signal peptide sequence with a terpene-binding protein provides an opportunity for selective terpene secretion. Although the researchers only tested this construct with squalene secretion, we believe that ursolic acid is an intriguing candidate for this innovative transport method since the researchers successfully secreted beta-carotene, a terpene, using this method, suggesting that the Suc2-tSPF construct can be suitable for other terpenes.

The signal peptide system will be integrated through a third plasmid that encodes for the lipid-binding domain of the supernatant protein factor tSPF and has the signal peptide sequence attached to tSPF. We will use pMV-LEU2 as our backbone to facilitate the integration of the system into the yeast cell. We are utilizing a third plasmid to integrate this signal peptide to reduce the metabolic burden that could potentially occur if we tried integrating this system with one of the other two plasmids.

     

Modeling

The wet lab subteam used computational modeling to direct our approach to the optimization of UA production that was shown above. We employed Michaelis-Menten kinetics to analyze the substrate-level interactions within the metabolic pathway. This model allowed us to assess the efficiency of different variants of α-amyrin synthase, an enzyme crucial for ursolic acid production. By comparing the kinetics of the MdOSC1 variant from domesticated apples and the EjAS variant from loquats, we determined that MdOSC1 is significantly more efficient due to its higher catalytic efficiency and lower substrate affinity.

     

Furthermore, stochastic simulations using Michaelis-Menten kinetics revealed that manipulating individual enzymes in the pathway is not a practical approach. The high specificity required for such modifications would be challenging to achieve within the timeframe of the iGEM competition.

To overcome the limitations of enzyme-level manipulation, Team Cornell turned to global metabolome analysis. This approach allowed us to consider the global metabolic perspective, identifying hubs, bottlenecks, and potential points of intervention for optimizing ursolic acid production.

Based on our findings, Team Cornell proposes knocking out the POR2 gene, which would disrupt the transport of acetyl-CoA into the mitochondrial matrix. By increasing the availability of acetyl-CoA for other cytosolic processes, this intervention could potentially redirect more of this metabolite towards ursolic acid synthesis.

     

Hardware

Contemporary means of ursolic acid (UA) biomanufacturing involve the use of batch bioreactors and expensive testing kits. As such, Oncurex seeks to develop a novel and more efficient system to produce UA. Our hardware is to be tailored for industrial scale-up applications while also integrating an electrochemical biosensor to monitor levels of produced UA on the outflow to quantify the performance of our reactor.

     

We want our Continuously Stirred Tank Reactor (CSTR) system to be more efficient in terms of both energy and throughput. We will prove this through mathematical and computational modeling. The methodology and results of this modeling can be found on the Modeling page on our wiki page. First, we attempted to identify the key biological reactions and pathways occurring in the bioreactor, such as the production of UA, as well as other reactions that could potentially compete with this production. Based on this, we conducted computational modeling using Visimix to simulate the mixing state within the reaction, which is crucial for evaluating the reaction conditions. Additionally, since it is necessary to maintain the yeast’s reaction environment at 37°C, we also calculated the Biot number of the bioreactor to model the ratio of conduction and convection.

     

In terms of biosensors, the primary purpose of this electrochemical biosensor is to monitor the concentration of UA in the reaction output in real-time, which is crucial for assessing reaction efficiency and controlling industrial production yield. This sensor will be integrated in the reactor itself, eliminating the need for discrete (and expensive) equipment and allowing for real-time response to UA production. This will also increase efficiency compared to batch reactors by ensuring that one “bad apple” doesn’t ruin the batch – that is, by ensuring that one parameter falling below ideal can be corrected in real-time, we can prevent large losses of product. Lastly, using the bioreactor and models built this year, we modeled an industrially scaled version of our bioreactor using dimensionless numbers to match heat transfer, fluid flow, and determine the material of the scaled reactor. By taking a dimensionless number approach, we were able to understand the characteristics of our bioreactor not only at the lab-scale and industry-scale, but also at any scale in-between as well.

     

Human Practices

Understanding how Cornell iGEM interacts with and learns from the community is always at the forefront of our minds. Our goals for this season were to determine how our project meets the needs of the community while working to ensure that our project was safe, ethical, and centered around sharing our knowledge on synthetic biology and Ursolic Acid with the local Ithaca community and the greater public.

The season started off with a focus on Ursolic Acid and its relationship with cancer treatments. Research into the field and interviews with oncologists and researchers focusing on Ursolic Acid emphasized the potential of Ursolic Acid as an additional treatment to other existing treatments currently available. Our interview with Ursolic Acid specialist Dr. Ran Yin confirmed that Ursolic Acid has several targets on cancer cells that can be maximized for anti-cancer effects, and that we were on the right path in regards to our treatment and target audience. Additional interviews with Dr. Blessing Aderibigbe and Dr. Opeoluwa Oyedeji confirmed that Ursolic Acid not only has several cancer cell targets but can also be applied to a variety of other medical conditions. Ultimately, we decided to focus on breast cancer due to the prevalence of research and the impact it has in both the local and global community. Speaking with oncologists such as Dr. Jini Hyun and Dr. Ragiv Magge additionally confirmed an interest in the administration of Ursolic Acid to cancer patients as a potential viable treatment. Using this information and feedback, we continued to elaborate upon the interest of cancer treatments.

As we aim to understand the various perspectives that both create and form our project, we decided to interview patients who have undergone cancer treatments to understand how our project best fits their understanding. Cancer is an extremely sensitive and personal subject, and Cornell iGEM hoped to be as respectful and mindful as possible when discussing this subject. We elected to submit a protocol to our Institutional Review Board for approval to conduct interviews and surveys with cancer patients either near the end of remission, or declared cancer free. Each question in the interview and survey was designed to be as non-invasive and leading as possible. An interview protocol was additionally written up (detailed in our Integrated Human Practices Section) to be as systematic as possible. The IRB consisted of multiple intended ways to protect the identities and statuses of those involved. From the de-identification protocol, to the inclusion of a consent form along with declaration of risks, Cornell iGEM hoped to be as mindful of the story of the patient as possible.

After interviews with cancer patients, we learned of the vast perspectives on our project, ranging from hesitations of the idea of natural medicines, to rampant enthusiasm at the prospect of a new drug for cancer. Each patient had a story to tell, whether it be an adverse experience with treatment, or lingering side effects. In connection to Oncurex, while the idea of synthetic biology as a whole was confusing, many patients were enthusiastic about the prospect of the project.

As we continued consulting stakeholders and patients, we learned that Ursolic Acid has a place in many other fields of medicine as well. Ursolic Acid and its various derivatives play a heavy role in areas such as cardiology, neurology and endocrinology. Speaking with specialists such as Dr. Ran Yin. Dr. Blessing Aderibigbe and Dr. Opeoluwa Oyedeji elaborated Ursolic Acid’s place within medicine. Using similar mechanisms as us, we hope that other iGEM teams can build upon our work and touch these various fields with different derivatives of Ursolic Acid. As our plasmid parts are novel and have not been used within the iGEM competition before, we hoped to provide a general understanding of Ursolic Acid others can use via the creation of a complete Ursolic Acid handbook comprised of applications, and the unique history Ursolic Acid has in various cultures around the world. We expanded our educational outreach to include a diverse audience beyond those that would be affected by our product. Reaching out to hosting local events such as Sciencenter for little kids, to speaking at larger events such as Splash! geared toward high school students, we hoped to foster an interest in not only our project, but in synthetic biology as a whole. Our team also built upon our previous science education video series from last year, aiming to promote important biology concepts to kids. Additionally, we created a children’s book explaining the concept of synthetic biology, translating it into 5 other languages to reach a much wider audience. Speaking with members of the public as well through Makerfaire and the local farmer’s market allowed us to learn more about the different perceptions of our work.

The business subteam of Cornell supplemented the interviews conducted with analysis of our own, including PEST(LE) and Porter’s Five Forces to better understand where the market as a whole stands. Further SWOT analysis was conducted to envision where our firm would stand in relation to the broader industry. Once we understood the market for the product, we began detailing a potential avenue to achieve the industrial scale-up we hope to achieve. We began with a thorough analysis of our financials: both fixed and variable costs. We then did research into what an industrial scale entailed and scaled up our financial information to represent where we would theoretically be when we entered the market.