HUMAN PRACTICES
Description
According to the statistics, cultured meat products have surpassed $5 billion in size and are expected to reach $6.4 billion by 2023. In recent years, there have been dozens of listed companies involved in the field of "cultured meat", and many listed companies have already started to lay out [1]. As a meat substitute, cell cultured meat has become one of the world's top ten breakthrough technologies in 2019.
（1）Insufficient supply. As the global population approaches 8 billion, traditional agriculture and livestock will consume more and more natural resources to meet the demand. It is expected that by 2030, the supply gap of meat products in China will reach 38.04 million tons [5].
（2）Industrial production.
Initially, the proportion of processed meat products in China is still low; Secondly, the concentration of the meat processing industry and the openness of the circulation links are not enough, which leads to the low industrialization level of the whole industrial chain. Simultaneously, the regulatory standards of domestic processing enterprises still need to be improved[6].
Based on the limitations of traditional meat production, people pay their attention to providing protein sources that does not need the participation of traditional livestock and poultry breeding industry. Statistics show that the scale of cultured meat replacement food is expected to reach $6.4 billion by 2023. However, in the initial stage of the industrialization of cell cultured meat, there are still many difficulties and technical barriers to overcome.
Figure1 Domestic pork supply gap from 2010-2020
China's meat consumption reached 88.296 million tons in 2018, and the supply gap of Chinese meat products is expected to reach more than 38 million tons by 2030. In this gap, the penetration rate of artificial meat will reach 5%, which is also a 100 billion level market.
Figure2 Global artificial meat industry market size statistics and growth forecast, 2020-2025
Artificial meat will account for 10% of the meat market over the next decade, and the global artificial meat market will rise to $140 billion.
（1）Limitations of the industrial production of cell cultured meat
If the cost of cell cultured meat is to be close to or even lower than that of traditional meat, it takes even millions of liters to produce it, this involves a series of engineering and technical problems not often encountered in the laboratory, which need to be solved by combining knowledge in multiple fields[7].
（2）Industrial problems that the project can solve
The synthesis of PHFA by using an autonomous dynamic regulation system is beneficial to solve the problems of low yield and low product polymerization, as well as the imbalance of cell metabolic flow and energy flow, growth arrest, the simulation of toxic intermediates, and reduction of energy consumption. Then the material is made into a scaffold system for cell growth.
Microspheres can levitate animal cells that need to be grown against the wall like traditional microbial cells [7]; and they are easy for cell growth and material circulation. Compared with 3D printing, they can ensure maximum yield increase, reduce labor and time input, and reduce the cost of large-scale tissue culture of artificial meat.
Cell cultured meat is one of the symbols of the future of food, and we can apply big data statistics and artificial intelligence technology to this field. We can understand the needs of consumers through big data statistics, design product formulas more in line with consumer preferences and acceptance to produce a cell culture artificial meat product with good quality, excellent senses, and healthy nutrition[8].
（1）The perspective of food safety
The FAO and the WHO says that 40 percent of foodborne diseases due to food insecurity occur in children under the age of five, causing 125,000 child deaths each year. Global food trade currently stands at $1.6 trillion, or about 10 percent of the world's annual trade. FAO and WHO are working together to assist countries in preventing, managing, and responding to risks in food supply chains and improving safety in food production and import to reduce the emergence and spread of antimicrobial resistance in food chains and the natural environment [9].
Cultured meat can be produced and packaged in a safe and controlled environment, and it can partly solve the problem of livestock diseases and the transmission of disease-causing microorganisms to humans in animal husbandry. A known practice of increasing antibiotic resistance that is expected to kill 10 million people a year by 2050 [10]. Cell-cultured meat, like natural meat, is made up of muscle cells and stem cells. It can even regulate the amount of fat and other essential nutrients in cultured meat by regulating the number of fat cells added to it[7].
（2）The perspective of resources and environment
According to the FAO of the United Nations, 30 percent of the world's land area is devoted to farming, which accounts for 18 percent of greenhouse gas emissions from human activities. Figures show that assuming 10 percent of the world's animal meat was replaced by artificial meat, it would reduce 176 million tonnes of carbon dioxide emissions and 8.6 billion cubic meters of water.
In the era of synthetic biology, new biotechnology is being linked to the pressing issues of ecological civilization, carbon neutrality, and sustainable development.Therefore, we need to carry out engineering transformation, and that's what we're doing in synthetic biology.
Animal ethics
From a bioethical perspective, the development and production of cell cultured meat can help to ensure the sustainable development of human society. The acquisition of traditional meat products requires the end of animal life, and the production of cell cultured meat will reduce the human cognitive "life-killing" behavior from the bioethical dimension[13]. It is believed that with the establishment of market access rules and regulatory rules, cultured meat will gradually enter the legislative stage, which promotes its healthy and sustainable development of it to a large extent[12].
Introducing the dynamic regulating to the sequential control could reduce the turbulence, assisting cell growth, and making the metabolic circus balanced. Thus, a reliable and efficient system with a high yield could be achieved. To come up with the goal, an independent dynamic regulation system based on E. coli is designed, synthesizing polyhydroxy fatty acids to make an advanced scaffold of porous microsphere simulating the in vivo environment, which promotes the flesh-forming growth of animal muscle cells in vitro. We are BUCT-China, looking forward to exploring the automated and industrialized production line for cultured meat. The project would through new light on avoiding the public health issues caused by food, contributing to the regulations of food production, and inducing more strict safety standards in the industry. Meanwhile, improving the nutritional value of human food and reducing global carbon emissions, looking ahead, serves as a firm source of unique protein for intergalactic colonialization.
The design of our project is mainly divided into three parts:
The first part is the construction of a biocompatible microsphere scaffold. We will achieve efficient production of polymeric materials by improving the pathway of non-natural biosynthetic PHFA. Then, double emulsion volatilization was used when porous microspheres with a large specific surface area were prepared and collagen and RGD were added to simulate the in vivo environment to provide attachment and growth conditions for the in vitro growth of muscle cells and make them a part of the muscle tissue.
The second part is to make synthetic PHFA into cells suitable for cell growth by double emulsion volatilization and add collagen and RGD to simulate the in vivo environment, to provide attachment and growth conditions for muscle cells growth in vitro
The third part is the production of meat with a 3-dimensional structure through the differentiation and culture of muscle cells
In terms of technological innovation,
(1) We changed the key polymerase activity to increase the degree of polymerization of PHFA. We found a more active FadD enzyme and according to the NMR spectra showed that the polymerization of the product increased from 2 to 5 and 6, proving that the improvement of acyl-CoA was beneficial to increase the yield of PHFA.
(2) Based on synthetic biology, we constructed an engineered bacterium to utilize hydroxy myristoleic acid to synthesize product the new material PHFA(poly hydroxyl fatty acid) through polymerization of hydroxy fatty acid.
(3) Due to the high cost and high technical requirements of 3D printing technology, which is not conducive to the use of industrial expansion and scale, we innovatively proposed the use of microsphere culture cells to replace it, which is a qualitative leap we made to realize the early large-scale promotion of cell cultured meat.
Figure3 Technological innovation of scaffolds
To address the effects of metabolic flow perturbations, we integrated the metabolic pathways of synthetic PHFA into an engineered bacteria, adopting a whole-cell approach to synthesize PHFA to avoid the transfer of intermediates and to enable the entire process from glucose to the production of the product polyhydroxy fatty acid ester (PHFA) to be performed in one cell, resulting in the more efficient production of PHFA [11]. To enable engineered bacteria to produce the target compound PHFA with maximum yield and production capacity during production, we decided to employ a dynamically controlled strategy of building regulatory elements and designing genetic pathways to regulate the flow of matter and energy.
The natural biological clock (a genetic oscillator) in living organisms inspired us to design autonomous dynamic regulatory systems that allow genes to be expressed periodically. The circadian clock consists of a set of rhythmically expressed genes and their encoded proteins. When the rhythm gene is activated, the corresponding proteins are produced by transcription and translation. When this protein concentration reaches a certain point, the feedback acts on the starting point of the gene, causing its concentration to oscillate in a 24-hour cycle [14].
Figure4 PHFA synthetic system
On the basis of last year's research, we have preliminarily constructed the CoA ligase/acyl transferase pathway with the addition of hydroxy fatty acids as substrates from exogenous sources. It is unfortunate that the degree of product polymerization and product yield is not high. And almost no observable products appear, indicating that the synthetic pathways cannot provide sufficient and high-quality materials for the subsequent construction of the scaffold. In order to meet the requirements of the subsequent construction of the scaffold system, we must improve the original metabolic pathways. The first is to develop and optimize the key enzymes of the polymerization step in the process, focusing more on the improvement of product yield and product polymerization, while using more characterization methods to verify our products. Secondly, due to there being a competitive relationship between hydroxylase P450 and CoA ligase, we need to build a logical regulatory system. In that case, if we want to build a metabolic pathway for whole-cell synthesis of PHFA, we need to switch the expression of the P450 enzyme and CoA ligase on the metabolic pathway to achieve the purpose of periodic oscillation of related enzymes, which can lead to the hydroxylation of fatty acids precedes the reaction of fatty acids with CoA ligases to achieve the goal of hydroxylation of fatty acids after polymerization.
Although 3D bioprinting can mix cells with scaffold biomaterials and layer them together to form meat tissue, this technology is not very suitable for the scaling ratio needed to grow cell-cultured meat. Future innovations in cell-sheet tissue engineering methods or the design of small tissue-building units and assembling them into tissue structures may effectively address this challenge. In view of the above problems, we plan to use biodegradable polyester microspheres to build scaffold materials to provide a better cell culture environment for cell-cultured meat, because microspheres have a lower cost compared with 3D printing, and they are commonly used in tissue engineering. Microspheres have relatively large specific surface areas and are easy to be modified. The combination of collagen and RGD improves its affinity to biological cells, mimics cellular adhesion in vivo, and can effectively transport substances, thus laying the foundation for the industrialization of cell culture meat.
Figure5 The microspheres served as the injectable microcarriers
The global population is growing, and so is the demand for agriculture and animal husbandry. Expanding animal breeding will cause infectious diseases which are detrimental to public health, and will also cause environmental pollution, resource overexploitation, excessive greenhouse gas emissions as well as food safety problems. In response to the possible future food security crisis, and to regulate the current food production, “cell-cultured meat” has come into being. We will jump out of this vicious circle, to subvert the traditional diet structure and dietary sources with a new vision and new technology.
