Intro

duction

This year 2023 OUC-China focuses on the challenges of green and low-carbon production of medium-chain fatty acids and their derivatives. In view of the current low utilization rate of kitchen waste oil, the complex synthesis process of medium-chain fatty acids, and the difficulty of purification. 2023OUC-China uses M. aphidis XM01 newly discovered from Hainan mangrove forests to regulate its metabolic pathway to synthesize medium-chain fatty acids and express the corresponding enzymes to catalyze the preparation of medium-chain α olefins and 10-hydroxycapric acid, which will have high industrial value.

Problem 1: The urgency of green and efficient production of medium-chain fatty acids.

Global warming has a significant impact on humanity, and fossil fuels —— coal, oil and gas – are by far the leading cause of global climate change, accounting for 75% of global greenhouse gas emissionsabove, accounts for nearly 90% of all CO2 emissions. The greenhouse gases emitted envelop the Earth and trap the sun's heat, contributing to global warming and climate change. Global warming is now faster than at any time on record. Over time, warming temperatures are changing weather patterns and disrupting nature's normal balance. This poses many risks to humanity and all other forms of life on Earth. Increasing carbon emissions have become an urgent problem. Countries around the world have successively set dual carbon targets that meet the national conditions of each country, of which green manufacturing technology is the key to solving the negative impact of manufacturing resource consumption and the environment ^[3].



Figure 1 Global CO2 emissions

Medium-chain fatty acids (MCFAs) have both the stability of long-chain fatty acids and the solubility of short-chain fatty acids due to their carbon chain lengths between short and long chains. Therefore, MCFAs as an important organic chemical raw materials, in food, medicine, agriculture, chemical and other fields have a wide range of applications, medium chain fatty acid triglycerides, can reduce the weight of hypertriglyceridemia patients, body fat mass, but also reduce triglycerides, LDL cholesterol and apolipoprotein B, apolipoprotein C2, apolipoprotein C3 levels, but also can alleviate insulin resistance and early metabolic disorders, improve insulin sensitivity, for celiac leakage, steatorrhea, Effective nutritional adjunct for patients with chronic pancreatic insufficiency, bile duct obstruction and other related conditions. Medium-chain fatty acids have been shown to have good immunomodulatory effects and can promote the body's immune function by regulating the expression of immune cells and cytokines. In daily life, medium-chain fatty acids can be used as components in shampoos and cosmetics to improve oil secretion. The application market of medium-chain fatty acids is broad, and the market demand is on the rise.



Figure 2 Application of MCFA s

However, the current two synthesis processes of medium-chain fatty acids, natural material extraction and chemical synthesis, bring a lot of pollution to the environment: biomass extraction pathways have limited sources and low concentrations, such as coconut and palm kernel extracted MCFAs account for 7.9%-15% of the total fatty acids, and the yield is low. Carbon emissions increase, the process consumes more energy, the synthesis reaction is violent, the risk factor is high, and the reaction conditions change to generate more by-products. It is easy to discharge a large amount of oxidation tail gas and waste acid water to cause environmental pollution, and the biological treatment technology is immature and the yield is low. Therefore, it is urgent to solve the problem of green synthesis of medium-chain fatty acids.



Figure 3 Dilemma of medium-chain fatty acid synthesis

Problem 2: Large reserves of kitchen waste oil and low utilization rate.

Medium-chain fatty acids are mainly extracted from petroleum and other substances, and have few natural resources to meet the growing needs of people today. Therefore, starting from the current low utilization rate of kitchen waste oil, the feasibility of its recycling is explored. Edible oil is an indispensable ingredient for food cooking, according to the United Statesagriculture According to USDA, the global edible vegetable oil production in 2018 was 203.38 million tons, a year-on-year increase of 2.3%; in 2019, the global edible vegetable oil production was 203.91 million tons, a year-on-year increase of 0.3%. Among them, the vast majority of edible oil is converted into kitchen waste oil, kitchen waste oil reserves are large, but the recycling rate is low, in addition to biodiesel, functional chemicals and other ways, there is no better biological recycling method, its industrial utilization prospects are huge.



Figure 4 Global food waste oil change data

Problem 3: Food waste oil contains more harmful substances.

These kitchen waste oils contain macromolecular hydrophobic organic compounds with low biochemical properties. Entering the water body will cause deterioration of water quality and eutrophication, and significantly reduce the treatment efficiency of the biological treatment unit of the sewage treatment plant. Due to repeated high-temperature frying, a certain amount of benzopyrene carcinogens are generated. Among them, Benzo(a)pyrene, B(a)P) is the first chemical environmental carcinogen discovered by humans, one of the three major carcinogens determined by the World Health Organization, is a refractory to degrade three (carcinogenic, teratogenic and mutagenic) toxic and harmful compounds. When these substances enter the environment or are ingested by the human body, they will cause serious environmental pollution and health threats.



Figure 5 Benzopyrene structure

We use Moesziomyces aphidis XM01 to produce MEL mannose erythritol from kitchen waste oil, and by hydrolyzing it, a large amount of medium-chain fatty acids can be obtained. Due to the coexistence of different fatty acid pathways in the XM01 strain, a large number of MEL formations are formed and the accumulation of intracellular lipids is caused. So we plan to engineer it to achieve carbon flow reconstruction, reduce the generation of intracellular oil droplets, and increase the synthesis of MEL.

In addition, we also use E. coli as a chassis to design relevant gene circuits to produce α-olefins and 10-hydroxycapric acid, two high value-added medium-chain fatty acid derivatives. Through communication with relevant experts, we found that benzopyrene contained in kitchen waste oil is often a major problem in the treatment process, so we designed a red light system to secrete the corresponding enzymes to complete the degradation of the substance.



Figure 6 Our solution

1. We hope to contribute to the dual carbon of mankind in the form of green and low-carbon production

The resource main line theory and product life cycle theory of green manufacturing establish the fundamental way and fundamental route of green manufacturing, that is, to optimize the whole process of product life cycle, which requires that green manufacturing technology should have the following 6 aspects of principles (referred to as 6R principle)^{[1, 2]}. This project has improved and pursued in reuse, recycle, recovery, redesign, and remanufacturing, and finally achieved green manufacturing.

2. We hope to contribute to the solution of the current global food and oil crisis by recycling kitchen waste oil

Our project leverages the knowledge of synthetic biology to enable the biomanufacturing of medium-chain fatty acids and is expected to increase yields. This green and low-carbon form of production and manufacturing adds strength to the current dual carbon of all mankind. On 19 May 2022, the World Food Programme warned that humanity could face the "biggest food crisis since World War II", with up to 1.7 billion people severely affected by the energy and food crises and the number of severely hungry people worldwide rising from 80 million to 323 million. At present, many of the raw materials for the production of medium-chain fatty acids are precious grain oil or petrochemical raw materials, so we transfer raw materials to kitchen waste oil, and use kitchen waste oil as raw materials to produce medium-chain fatty acids and derivatives to achieve waste utilization. In 2021, the total global UCO (used cooking oil) production was about 6.4 million tons, of which China produced about 1.86 million tons, accounting for up to 29%, so our project is important to the world.

3. We hope to use the knowledge of synthetic biology to increase the yield of medium-chain fatty acids and carry out the production and manufacturing of high value-added medium-chain fatty acid derivatives, in order to achieve more economic benefits

Why take a synthetic biology approach?

After determining the green synthesis of medium-chain fatty acids, after trying to reconstruct the metabolic pathways of organisms based on synthetic biology, it was found that its harmfulness and environmental impact have significant advantages over chemical methods and natural synthesis. Therefore, the method of synthetic biology is also convenient for the subsequent use of metabolites, the production of more diversified industrial raw materials, and the use of synthetic biology methods to adjust, so that we can regulate the production rate of industrial raw materials and better meet the needs of industrialization.

The Science

Zhu et al. modified the fatty acid synthase in Saccharomyces cerevisiae to improve fatty acid synthesis, and directed the evolution of the membrane transporter TPO1 [8], which increased the tolerance of Saccharomyces cerevisiae to fatty acid toxicity and constructed a cell factory that can efficiently produce medium-chain fatty acids. He et al. successfully constructed the alkBGT system in Escherichia coli ^[9].

Our Inspiration

Within iGEM, we were inspired by a team that iGEM10_TU_Delft 2010 to treat alkanes with the Alkane Hydroxylase System, and we used a similar alkBGT system to produce 10-hydroxydecanoic acid from capric acid and increase overall production efficiency by building a two-enzyme complex system.

Why this project?

In terms of environment, the global dual carbon plan makes related manufacturing industries need technological innovation to achieve green production, low-carbon or even zero carbon emissions, so biosynthesis provides a new idea. Economically, the multiple effects of medium-chain fatty acids make the market demand large, although biosynthesis can achieve zero carbon emissions, but there is a problem that the yield is not high enough, and our carbon flow reconstruction of engineered bacteria is expected to solve this dilemma. At the same time, in order to achieve higher added value, we design circuits to produce derivatives of medium-chain fatty acids α olefins and 10-hydroxycapric acid, thus adding more economic benefits.

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[2] Stark, R. , Seliger, Günther, & Bonvoisin, Jérémy. (2017). Field of research in sustainable manufacturing. Springer International Publishing.

[3] LIU Peiji,LIU Fei,WANG Xu et al.Theoretical and technical system and new framework of green manufacturing[J].Journal of Mechanical Engineering,2021,57(19):165-179.

[4] Yangyang Li, Yiying Jin, Jinhui Li, Yixing Chen, et al. Current Situation and Development of Kitchen Waste Treatment in China[J]. Procedia Environmental Sciences, 2016, 31:40-49.

[5] Talha Ahmad, Tarun Belwal, Li Li, Sudipta Ramola, et al. Utilization of wastewater from edible oil industry, turning waste into valuable products: A review[J]. Trends in Food Science & Technology, 2020, 99:21-33.

[6] He Q F. Synthesis of ω-hydroxyl fatty acids catalyzed by metabolically engineered Escherichia coli [D]. East China university of science and technology, 2019.

[7] Zhu Z, Hu Y, Teixeira P G, et al. Multidimensional engineering of Saccharomyces cerevisiae for efficient synthesis of medium-chain fatty acids [J]. Nature Catalysis, 2020, 3(1) : 64-74.

[8] He Q F. Synthesis of ω-hydroxyl fatty acids catalyzed by metabolically engineered Escherichia coli [D]. East China university of science and technology, 2019.

[9] Zhu Z, Hu Y, Teixeira P G, et al. Multidimensional engineering of Saccharomyces cerevisiae for efficient synthesis of medium-chain fatty acids [J]. Nature Catalysis, 2020, 3(1) : 64-74.