To identify the pre-existing problems of DHA supplements and from a wide perspective, omega-3 supplements in general, we approached a nutritionist, lecturer at HKBU and representative of the Hong Kong based nutrition company Negimenforall: Ms. Queenie Lam. By touching upon Ms. Lam’s expertise in nutrition, we were able to further understand DHA Deficiency’s prevalence in both Hong Kong and the global scale and of DHA’s nutritional importance in building a healthy diet.

Ms. Lam described the DHA deficiency as a “silent killer”, emphasizing the lack of attention to the particular phenomenon. Within Hong Kong, she expressed how there was a rise in potential industrial malpractice that may undermine the extraction yield and the health benefits DHA supplements could bring citing this SCMP article. She also explained that the use of genetically modified organisms for DHA extraction was undesirable for consumers, pointing mainly to unknown long-term health effects as the deterrent.

Ms. Lam kindly published samples of our recipes onto the @negimenforall Instagram page, through which we were able to reach a wider audience.

Considering the cited concerns of consumers and the growing neglect of DHA in daily diets, Ms. Lam affirmed the potential benefits our project could bring to address these problems.

In light of our pursuit to understand some of the literature we were basing our project upon, we reached out to Research Assistant Professor Jee Joon Foo at the National University of Singapore. We first gave a quick presentation regarding our project scope and explained the specific parts of our chassis. As an expert in the field of synthetic biology, Professor Foo gave us feedback regarding our chassis selection as we were still considering the sort of yeast model we would use in our project. Additionally, he explained that the processes we were intending to take were theoretically doable although questions regarding the feasibility within the given time were raised.

From our meeting with Professor Foo, we were able to reaffirm the scope, content and intentions of our project. Additionally, through the recommendations he provided regarding the choice of the chassis (taking into consideration factors such as literature published related to the chassis, survivability etc.) we were able to narrow our choice down to Y. lipolytica.

Our team was honored to reach out to Henry Hei Ning LAM who is a professor for the School of Engineering at the Hong Kong University of Science and Technology (HKUST).

The consultation with Professor Henry helped us to enhance our Yeast Growth Model. He shared with us that tools like COBRA (Constraint-Based Reconstruction and Analysis) can already predict the basic growth and metabolic characteristics of Y. lipolytica, and these models could be used for scaling up the process and integrating with a reactor model.

Regarding the modeling of promoter strength, Professor Henry suggested that we change the rate constants of the relevant reactions to simulate the impact of promoter strength. Additionally, he emphasized the need for wet lab data to calibrate and validate this approach. Professor Henry also advised us to start with a base model, and add our reactions but cautioned that this approach may not be highly accurate, as we would not be able to optimize the reaction rates.

Incorporating the feedback from Professor Henry, we decided to explore the use of COBRA and other existing models to understand the basic growth characteristics of Y. lipolytica. We will also decided to focus on developing a batch growth model, which will simulate the dynamics of nutrients, biomass, and product formation.

We also took the approach of exploring the method of building our model from scratch. Notably, we decided on simplifying the model and focusing on a smaller set of reactions, rather than the complexity of tools like COBRA. Additionally, we decided to seek out former iGEM students recommended by Professor Henry to seek their guidance and support as we continued our modeling work.

By implementing these changes based on Professor Henry's feedback, we strived to develop a more robust and informed modeling approach for our project, leveraging both existing tools and our customized models.

Based on the discussion with Professor Yong Lai who is a professor in the School of Engineering at the Hong Kong University of Science and Technology (HKUST), we have learned several key insights regarding the integration and expression of the PUFA synthase cluster in Y. lipolytica:

The main gene integration method in Y. lipolytica is non-homologous end joining rather than homologous recombination, which is less efficient. Professor Lai suggested exploring two rounds of transformation to improve integration efficiency. He also highlighted the importance of checking existing publications to understand the novelty of the project, noting that the higher specificity of the PUFA synthase could be a key advantage.

Regarding the integration strategy, Professor Lai suggested exploring random integration using a linearized DNA fragment. He also discussed the challenges of integrating the large PUFA synthase subunits, which may be difficult due to their length. To address this, he recommended amplifying the sequences from the algae through PCR and potentially exploring codon optimization or mutations.

Professor Lai also provided several alternative strategies, such as using a yeast artificial chromosome for the recombination and exploring Gibson assembly.

For the expression analysis, Professor Lai highlighted the potential challenges of using Western blot for large proteins and suggested exploring alternative methods such as LCMS or real-time PCR. He also discussed strategies to improve expression, such as knocking out the PEX10 gene to prevent protein degradation.

Overall, the discussion with Professor Lai provided valuable insights and practical suggestions to address the key challenges in the integration and expression of the PUFA synthase cluster in Y. lipolytica. We could now consider these recommendations to refine their experimental approach and improve the likelihood of success in their project.

We reached out to Professor Karl Tsim, who is the Chair Professor in the Division of Life Science at the Hong Kong University of Science and Technology (HKUST). We were able to receive valuable insight into technical challenges, information on potential investors, and regulations to be aware of.

Professor Tsim shared his experiences working with a local Hong Kong-based company that isolated DHA from fish meat. He affirmed the well-established health advantages of DHA for brain development, cardiovascular health, and stroke prevention.

In terms of the technical challenges, Professor Tsim highlighted that the enzymatic pathways involved in DHA production are quite complex, making recombination in yeast a significant hurdle. He pointed out that we would need to thoroughly investigate whether yeast can truly outperform other approaches before pursuing that route. Additionally, we would need to focus on efficient purification of the DHA extract.

To attract potential investors down the line, the professor suggested we explore opportunities for intellectual property protection. Investors would also be interested in the investment-to-profit ratio and timelines. Differentiating factors could include optimizing the ratio of DHA compounds produced that offer us advantages.

On the business side, Professor Tsim advised us to reach out to potential manufacturers in mainland China that utilize DHA as a raw material for their products, as the Hong Kong market currently lacks major DHA suppliers.

Regarding regulations, the professor indicated that there are relatively few restrictions on DHA in Hong Kong, primarily requiring heavy metal testing and ensuring the product does not contain Western medicines like aspirin.

Overall, the discussion with Professor Tsim provided valuable guidance to refine the project's scope, technical priorities, and potential commercialization pathways. Our team decided to follow the advice, including the project direction.

We also reached out to Professor Banfield, a cell biologist in the Division of Life Science at the Hong Kong University of Science and Technology (HKUST). In this outreach, we had the opportunity to discuss several key considerations regarding enzyme expression and complex purification. The primary objective was to ensure we have a reliable way to measure the expression of the target enzymes, as the commonly used His-tag may not be compatible with the available antibodies. Alternative options, such as the HA tag and Myc epitope, were explored as potential solutions.

To start the investigation, we proposed expressing each gene individually in yeast to better understand the system. This approach would allow us to consider strategies for purifying the complex itself, potentially using affinity chromatography with the combination of tag 1 and tag 2 or peptide sequences.

One potential challenge Professor Banfield pointed out was the concern about the transformation process, particularly if the same marker is used for multiple integrated genes. To address this, the use of auxotrophic markers and the recycling of markers through the incorporation of loxP sites were suggested as possible solutions.

Potential issues, such as cryptic splice sites and the impact of suboptimal codons near the 5' end of the gene, were also discussed. These factors could potentially lead to premature termination or detachment of the RNA polymerase, resulting in problems during expression.

Lastly, the challenges associated with yeast transformation, particularly with large plasmids, were addressed. The recommendation was to consider separating the genes into smaller units, as yeast may have difficulty maintaining and expressing large plasmids. The order of integration and potential toxicity issues were also discussed, with the suggestion of trying different permutations to identify the optimal approach.

This outreach has provided valuable insights and guidance on the various considerations and potential challenges involved in enzyme expression and complex purification, which will be instrumental in refining our experimental approach and ultimately advancing our research goals which is greatly helpful insight to us.

We are honored to have NNU iGEM instructor——Professor Ye Chao to share his valuable experience with us which are greatly beneficial to a clear direction of our team’s project.

Professor Ye Chao suggested several avenues for improvement, including the use of a MATLAB-based COBRA toolbox in the genome-scale metabolic models, setting appropriate objective functions and constraints, and exploring machine learning techniques for parameter estimation. He also gave detailed suggestions of other types of models to consider including the ME model and the Enzyme-constraint model.

The feedback received from Professor Ye Chao will be invaluable in refining and enhancing our computational framework, ensuring that it provides a robust and versatile tool for the scientific community.

We were able to reach out to Professor Joseph, a synthetic biology professor from the Hong Kong University of Science and Technology (HKUST), One of the primary concerns discussed was the strain selection and cultivation process. It was noted that the strain development can be a time-consuming endeavor, often taking months to achieve the desired characteristics. The importance of proper biological safety protocols was emphasized.

The discussion also delved into the potential for protein secretion from the cells. While the codon bias was not seen as a significant concern, we focused more on ensuring proper organelle.

Regarding the Y. lipolytica plasmid, we discussed the potential differences compared to other systems and the importance of understanding the inductive or repressive mechanisms involved.

The concept of gene knockouts was also discussed, with the suggestion of exploring the use of repressive promoters and evaluating the conditions, such as redox and pH, that could influence the desired outcomes. We also highlighted the potential use of resources like the Saccharomyces Genome Database (SGD) to gather information on genetic interactions, physical interactions, and synthetic lethality.

Throughout the discussion, we considered various aspects of the project, including the production and secretion of the desired compounds, the growth characteristics and culturing densities of Yarrowia lipolytica, and the potential pathways involved in the synthesis of long-chain hydrocarbons.

By engaging with Professor Joseph and leveraging his expertise, we were able to gain a deeper understanding of the challenges and opportunities inherent in the project. This collaboration has helped to refine the project's direction and inform the necessary adjustments to ensure the successful development and implementation of the desired microbial engineering solutions.