Docosahexaenoic acid (DHA, C22:6) is an essential nutrient required for human health and development, which can only be obtained through dietary intake. It contributes to specific and crucial cell membrane properties such as regional fluidity and protein localization, accounting for approximately 40% of the total fatty acid in the human brain (Tawhari et al, 2022).

The problem of DHA deficiency is severe and widespread, particularly in regions with limited access to DHA-rich food sources. This deficiency poses a serious risk to various vulnerable groups, such as pregnant women, infants, and children, who require adequate DHA for brain development and overall health. The severity of the problem underscores the importance of global DHA accessibility. ^[1]

DHA Health Benefits

Data based on research from Start et al. (2016)^[2]

DHA, one type of omega-3 fatty acid, is usually included in the following products in the market:

• Infant Formula
• Nutritional Supplements
• Food & Beverage

According to NHI, DHA provides substantial health benefits. It is crucial for brain and cognitive function, eye health, heart health, pregnancy and infant development, mental well-being, and reducing inflammation associated with chronic diseases.^[1] Increasing DHA intake, either through diet or supplements, can provide significant improvements to overall health and well-being, making it an important nutrient for people of all ages.^[3]

Consequently, FDA approved health claims for EPA and DHA omega-3 consumption in 2019, driving up its market demand and raising awareness of DHA. Until today, DHA is still a growing market with a projected CAGR ranging from 6.76% to 9.2% in the next decade.^[4][5]

Fish oil has traditionally been a common source of DHA in the market. However, it is prone to pollution, lacks stability in terms of quality, and its production is expected to decline rapidly due to overfishing and the impact of global warming, which will be discussed below in detail. Other challenges for DHA from fish oil include:

Other challenges for DHA from fish oil include:

• Complex and expensive purification process involved for obtaining pure products
• Unfriendly to vegetarians
• Unwanted odor

DHA can also be derived from microalgae, which is its natural origin. Commercialized DHA-producing, heterotrophic microalgae species include Schizochytrium sp. and Crypthecodinium cohnii. However, the industrial-scale fermentation of microalgae for DHA production still faces certain challenges including high production costs and low efficiency, resulting in microalgae being a minor source of DHA in the market ^[6]. Typically, with the same amount of DHA, the market price of microalgal oil is 2-5 folds higher than fish oil.

On the other hand, traditional microalgae farms with pond cultivation, for photosynthetic species, require a significant land use, restricting the application

Gap in the Field

Though the DHA market has such an increasing demand, the supply side is not optimistic. In 2020, Colombo et al. warned that global warming is predicted to reduce the de novo synthesis of DHA by algae, which forms the base of aquatic food chains, leading to a decrease in the amount of DHA transferred to fish and shrimp. [7] The researchers estimated that, depending on the climate, increasing water temperature will result in a 10 to 58% loss of global DHA by 2100. In the worst scenario, DHA availability could decline to levels where 96% of the global population do not have access to sufficient DHA.

Based on data from literature and market reports, we have projected the outlook of the market and identified a significant gap for an alternative DHA production method.

The projections are based on the following assumptions:

• The global market for DHA is currently growing at a rate of 9% per year. ^[8]
• The amount of DHA available from marine organisms is projected to decrease by 58% of current levels by the year 2100. This equates to an annual decrease of approximately 1.07%. ^[7]
• The current global market size for DHA is $3,821.4 million. ^[8]
• The DHA market is segmented, with 20% coming from algae sources and 80% coming from fish sources.
• The growth rate for the algae-based DHA segment is assumed to be 7% per year. ^[7]

In order to address the gap between the increasing market demand and decreasing market supply as well as the global problem of DHA deficiency, the iGEM HKUST Team 2024 DHA Express is working on efficient DHA production through oleaginous yeast chassis Yarrowia lipolytica, a safe, commonly-used organism for industrial fatty acid production.

We plan to synthesize the PUFA synthase gene cluster (around 20kb in total) originally from Schizochytrium sp. (ATCC 20888) and integrate it into the genome of Y. lipolytica strain Po1f, which can achieve a relatively high lipid content, growth rate, and growth density, boosting the production ^[11]. The PUFA synthase gene cluster consists of 3 open reading frames (ORF) corresponding to different protein subunits, which will assemble together to become one large enzyme in the cytoplasm ^[12]. A PPTase from the same organism will also be introduced to activate the enzyme.

DHA Express – Integrating the most powerful DHA synthesis system into the most promising microbial factory

In order to increase the accessibility of DHA, the industry that is making profit from the supplement has to find more cost-effective methods to produce DHA on a larger scale and through less demanding ways. And this, more specifically, leads to the requirement of a higher yield and productivity, in simple words meaning that either the manufacturing process needs to be faster, yield needs to be higher or the product needs to be purer, which facilitates the purification, while reduces the generation of byproduct and thus waste of substrates. These represent the main challenges currently met by the industry. Besides, substrate and operation costs are considered as significant in industrial manufacturing, so efforts towards reducing these costs are also highly valued.

Why Yarrowia lipolytica?

Therefore, we introduce the PUFA synthase system into a well established chassis Yarrowia lipolytica, an oleaginous, unconventional yeast species. Yarrowia lipolytica is very popular among the industry, especially for obtaining food-related products, and is the mostly-used organism for lipid and fatty acid production. It can accumulate lipids up to 90% of the cell while achieving a considerable growth rate and final biomass of more than 100 g/L, making it well-suited for the industrial requirements. Potentially applying the efflux strategy can even exceed the limit of cellular accumulation. [Cases reported] Its availability and mature fermentation technology set the basis for the application. Also it is regarded as safe even for food uses and has well established genetic tools, making it a perfect platform for supporting DHA synthesis under the help of synthetic biology. Expert in Yarrowia industrial application Prof. Rodrigo Ledesma-Amaro from Imperial College London further pointed out the chassis’s advantage to be able to grow on low-cost substrate and even waste, due to the organisms’ unique metabolism and tolerance to a wider range of environmental conditions including pH, which can lower the substrate and operation cost to a large extent.

Why the PKS Pathway?

PUFA synthase from certain organisms can directly synthesize DHA from acetyl-CoA and malonyl-CoA, which is considered as a more specific pathway for DHA synthesis and consumes less NADPH compared to others ^[9-10]. For DHA synthesis itself, we identified the PUFA synthase pathway in Schizochytrium sp. representing the most efficient one so far (please refer to the Design page for more details). However, the DHA-producing Schizochytrium strain, though commercialized, still accounts for only a limited percentage of the market, and from our interviews of both the academia and industry experts in dha-producing Schizochytrium, Prof. CHEN Zhi from China Agriculture University and founder of ALDEHA, we know that the productivity from the organism is actually meeting a barrier for lack of genetic tools, and many times its improvement is largely inspired by researches in yeast, especially in our chassis Yarrowia lipolytica.

The DHA specificity and modular nature of the enzyme offers us abundant space for further improvements. By introducing and potentially designing the key machine into a well-established microbial factory, we can harness the great advantage of synthetic biology to achieve flexible control of the whole process, while setting a basis for introducing future designs efficiently. While traditionally in microalgae, though the yield can still be boosted through genetic engineering, the production can be constrained to limited flexibility in design and understanding of the host organism ^[6]. With everything designed and operated under full control throughout the process, our product is likely to have a more stable and consistent quality compared to other existing sources. Moreover, as many synthetic pathways include a PKS system and share similarities, our research can also potentially benefit the production of other valuable polyketides. For more details, please refer to the Contribution page.

Our project aims to combine these 2 systems to create the potentially most powerful DHA-synthesis factory so far.

DHA Express – Steps towards a controlled but powerful bioproduction platform

Combining the best options does not necessarily mean the best outcome. So strategies must be adopted to make it happen. One of the most promising features of Yarrowia lipolytica is it has relatively mature synthetic biology tools for us to further improve and achieve higher, offering much more flexibility. And that is also what we are trying to do in our project via metabolic engineering.

Our engineering strives to achieve a synergetic effect with combined strategies, together with a meticulous biocontainment measure. These include:

• Minimize burden and maximize the desired product — By expanding the toolbox for controlled and tunable expressions under a preferred condition for Yarrowia lipolytica bio-production
• Ensure the redox power supply — By replenishing rate-limiting NADPH from NADP⁺ with enzyme GapC introducing an altered metabolic pathway
• Promote the overall accumulation — By eliminating peroxisomes where fatty acids are oxidized and degraded
• Towards continuous production — By exploring potential product efflux mechanisms and design possibilities.

The primary aim of the project is to explore an alternative solution in response to the growing market gap of DHA and existing challenges of the industry, hoping to bring DHA to more people in need sustainably and efficiently through our efforts.

We also aim to create a platform that is applicable to not only DHA production but also other cases, please refer to our Contribution page for more potential applications.

Reference:
[1] Oliver, L., Dietrich, T., Marañón, I., Villarán, M.C., Barrio, R.J. (2020). Producing Omega-3 Polyunsaturated Fatty Acids: A Review of Sustainable Sources and Future Trends for the EPA and DHA Market. Resources 2020, 9, 148. https://doi.org/10.3390/resources9120148
[2] Ken D. Stark, Mary E. Van Elswyk, M. Roberta Higgins, Charli A. Weatherford, Norman Salem. Global survey of the omega-3 fatty acids, docosahexaenoic acid and eicosapentaenoic acid in the blood stream of healthy adults. Progress in Lipid Research, Volume 63, 2016, Pages 132-152. https://doi.org/10.1016/j.plipres.2016.05.001
[3] Omega-3 Fatty Acids - Health Professional Fact Sheet. https://ods.od.nih.gov/factsheets/Omega3FattyAcids-HealthProfessional/ ​​
[4] LLOYD A. HORROCKS, YOUNG K. YEO. HEALTH BENEFITS OF DOCOSAHEXAENOIC ACID (DHA). Pharmacological Research, Volume 40, Issue 3, 1999, Pages 211-225. https://doi.org/10.1006/phrs.1999.0495
[5] Docosahexaenoic Acid (DHA) Market Size, Growth Report, 2032. Business Research Insights, 03 June 2024. https://www.businessresearchinsights.com/market-reports/docosahexaenoic-acid-dha-market-110648
[6] Socio-economic assessment of Algae-based PUFA production. PUFAChain, 2017. https://www.pufachain.eu/fileadmin/download/Socio-economic_assessment_of_Algae-based_PUFA_production.pdf
[7] Colombo, S. M., Rodgers, T. F., Diamond, M. L., Bazinet, R. P., & Arts, M. T. (2020). Projected declines in global DHA availability for human consumption as a result of global warming. Ambio, 49(4), 865-880. https://doi.org/10.1007/s13280-019-01234-6
[8] DHA from Algae Market Size Projected to Surpass US$ 720.44 million by 2031, by 2031, with Increasing CAGR of 7.05%. Research Industry Network, 04 Jan 2024. https://www.linkedin.com/pulse/dha-from-algae-market-size-projected-x2pyf/
[9] Gemperlein, K., Dietrich, D., Kohlstedt, M. et al. Polyunsaturated fatty acid production by Yarrowia lipolytica employing designed myxobacterial PUFA synthases. Nat Commun 10, 4055 (2019). https://doi.org/10.1038/s41467-019-12025-8
[10] Guo P, Dong L, Wang F, Chen L and Zhang W (2022), Deciphering and engineering the polyunsaturated fatty acid synthase pathway from eukaryotic microorganisms. Front. Bioeng. Biotechnol. 10:1052785. https://doi.org/10.3389/fbioe.2022.1052785
[11] Young-Kyoung Park, Rodrigo Ledesma-Amaro. What makes Yarrowia lipolytica well suited for industry? Trends in Biotechnology. Volume 41, Issue 2, 2023, Pages 242-254, https://doi.org/10.1016/j.tibtech.2022.07.006
[12] Hauvermale, A., Kuner, J., Rosenzweig, B. et al. Fatty acid production in Schizochytrium sp.: Involvement of a polyunsaturated fatty acid synthase and a type I fatty acid synthase. Lipids 41, 739–747 (2006). https://doi.org/10.1007/s11745-006-5025-6
[13] Qiao, K., Wasylenko, T., Zhou, K. et al. Lipid production in Yarrowia lipolytica is maximized by engineering cytosolic redox metabolism. Nat Biotechnol 35, 173–177 (2017). https://doi.org/10.1038/nbt.3763
[14] Qin J, Kurt E, LBassi T, Sa L and Xie D (2023). Biotechnological production of omega-3 fatty acids: current status and future perspectives. Front. Microbiol. 14:1280296. https://doi.org/10.3389/fmicb.2023.1280296
[15] Xue, Z., Sharpe, P., Hong, SP. et al. Production of omega-3 eicosapentaenoic acid by metabolic engineering of Yarrowia lipolytica. Nat Biotechnol 31, 734–740 (2013). https://doi.org/10.1038/nbt.2622