HUMAN PRACTICES
Design
Following last year's project, our BUCT-China team has been focusing on the use of synthetic biology and tissue engineering techniques to realize the laboratory culture of meat, and to provide the necessary technical support for the project to move towards industrial production in the future. Based on the analysis of the structure of traditional cultured meat, we divided the whole project into three sections (Figure 1) in order to simulate the three-dimensional structure of meat as much as possible, the first section is to realize the production of scaffold material PHFA through biosynthesis. The second section is to make the synthesized PHFA into microspheres suitable for cell growth by double-emulsion volatilization method, and add collagen and RGD to simulate the in vivo environment to provide attachment and growth conditions for muscle cell growth in vitro; the third section is to realize the production of meat with three-dimensional structure by differentiation and culture of muscle cells. We hope to find a complete production line of cell-cultured artificial meat and propose a solution for improving food safety, food hygiene and standardized food production.
Figure1 the sections of our project
Most animal cell cultures have an appositional growth characteristic, and only a large enough surface area can produce a large number of cells, while scaffold systems can provide the necessary attachments and carriers for satellite cell proliferation and differentiation. Therefore, research on scaffold systems is directed toward the development of materials that are edible or inedible but easy to peel, flexible, mechanically stretchable, and can be attached by cells. However, there are few edible materials suitable for constructing scaffold systems for network branching, so the development of scaffold materials with excellent properties, safe and non-toxic, and high biocompatibility is our primary problem.
Inspired by the fat fraction in traditional cultured meat, we focused on fatty acid substances, and after the screening, we finally find the PHFA [1], polyhydroxy fatty acid ester, polyester material with medium and long chain fatty acids as monomers, which has good mechanical properties and mechanical strength [2] and has the material properties to be made as a scaffold. At the same time, because its structure is very similar to that of PHA, we speculate that it may have similar properties to PHA, that as high biocompatibility, safe and non-toxic, and environmentally friendly. Therefore, we can make it into a scaffold system to provide the appropriate environment for the growth of satellite cells. Also, as a fatty acid, it can improve the taste of cultured meat and provide energy and nutrition to the consumer.
The biosynthesis of PHFA in this project is a non-natural pathway. In order to obtain polyhydroxy fatty acid esters (PHFA) with a longer carbon skeleton, we designed the following pathway for the synthesis of PHFA (Figure 2), in which steps 1 and 2 are natural pathways that already exist in E. coli, and the hydroxylation reaction in step 3 has been proven to be feasible in a large amount of literature, so the polymerization reaction of hydroxy fatty acids is the key step in this pathway, and therefore the study and optimization of steps 4 and 5 are the key topics to achieve the construction of the whole metabolic pathway.
Figure2 reaction pathways of biosynthesis PHFA
Based on last year's study, we initially constructed a CoA ligase/acyltransferase pathway with the exogenous addition of hydroxy fatty acids as substrate, but the degree of product polymerization and product yield obtained were not high, and almost no observable product appeared, indicating that this production method is not yet able to provide sufficient amount and high-quality material for subsequent construction of scaffolds, so in order to meet the requirements of subsequent construction of scaffold systems, we must improve the original metabolic pathway.
Our work this year is divided into two parts: firstly, we will screen and optimize the key enzymes in the polymerization step of the process, and focus more on the improvement of product yield and product polymerization, while using more characterization means to verify our products; secondly, because of the competition between hydroxylase P450 and CoA ligase. We want to construct a metabolic pathway for whole-cell synthesis of PHFA, and we need to construct a logical regulatory system that sequentially switches the expression of P450 enzymes and CoA ligases on the metabolic pathway to achieve the emergence of periodic oscillations of related enzymes, where fatty acid hydroxylation precedes the reaction of fatty acids with CoA ligases.
Therefore, in order to further improve the yield of PHFA products and product polymerization, our team analyzed the whole biological reaction process and found that the problems might be mainly focused on the following three aspects: the transmembrane of the substrate, the activity of CoA ligase and acyltransferase, and the metabolic activity of bacteria, and proposed the solution for these problems.
The solubility of long-chain hydroxy fatty acids is very low in aqueous solutions. Also, the permeability of hydroxy fatty acids through the cell membrane is low because the outer layer of the outer membrane of E. coli consists mainly of lipopolysaccharides, which are the main barrier for the transport of hydrophobic compounds to E. coli. Restricted by the substrate transmembrane, the intracellular biotransformation of hydroxy fatty acids may be slow. Based on this analysis, we proposed to design an enhanced expression of E. coli's own natural fatty acid transport protein FadL.
Figure3 plasmid spectrum containing fadL
Figure4 plasmid spectrum containing fadD
Regarding the low degree of product polymerization, we speculated that it might be due to the insufficient activity of the relevant enzymes in the synthesis pathway, resulting in the inability to synthesize the polymerized product efficiently even if there is a sufficient concentration of hydroxy fatty acids in the system. Therefore, we analyzed CoA ligase and acyltransferase, which are key enzymes in the synthesis pathway. For the acyltransferase WS2, the catalytic activity is less affected by different substrates due to its broad substrate spectrum. Therefore, we focused our improvement on CoA ligase. The catalytic process of CoA ligase requires the participation of ATP and the generated hydroxylated lipoic CoA is in the high-energy state, and we speculated that this step of the reaction may be the key rate-limiting step in both reactions. Further, through literature research, we analyzed and screened the CoA ligase, hoping to obtain a CoA ligase with stronger catalytic activity for hydroxy fatty acids, and constructed the following plasmid profile (Figure 4).
Based on the proteomics, we know that there is substrate competition between hydroxylase P450 and lipid CoA ligase, which means that they compete for medium and long-chain fatty acids (Figure 5), and we also speculate that the catalytic efficiency of the p450 enzyme is lower than that of CoA ligase, which may cause the fatty acids to be acted upon by acyltransferase first and not recognized by hydroxylase, resulting in the inability to complete the hydroxylation reaction efficiently even if there are sufficient intracellular concentrations of fatty acids, and thus the inability to polymerize hydroxy fatty acids to produce a sufficient amount of PHFA.
Figure5 possible competitive reaction between hydroxylase and CoA synthetase
Further, we also wanted to improve polymer production by reducing the impact of cell growth hindrance and metabolic flow perturbation due to intermediate product accumulation, and while researching the literature, genetic oscillatory circuits inspired us.
It was found that dynamic regulation of microbial metabolic pathways could reduce the influence of exogenous pathways, facilitate the maintenance of cell growth, and balance metabolic flow, thus reducing costs and achieving high yields, high substrate conversion rates, and high production intensity. The dynamic regulation system can effectively avoid the problems of intermediate metabolite accumulation, cofactor imbalance, and impaired cell growth caused by traditional modification strategies, and has promising applications in improving our PHFA production capacity.
Given that our team aims to construct a non-natural pathway that enables the dynamic regulation of the ab initio synthesis of polyhydroxy fatty acid esters from glucose, such a dynamic regulatory system may be able to address the possible excessive accumulation of toxic intermediates in this metabolic pathway and redirect the cellular metabolic flow toward the synthesis of target products to minimize the production of unnecessary substances and thus better synthesize PHFA.
Figure6 dynamically regulated gene circuit
Based on this inference, we designed a dynamic regulation strategy to regulate gene expression, so that two groups of genes, TesA gene (encoding thioesterase), p450 gene (encoding hydroxylase) and fadD gene (encoding lipoic CoA synthase), WS2 gene (encoding wax ester synthase), could be expressed periodically alternately. The periodic alternate expression of these genes enables E. coli to complete PHFA synthesis in a rhythmic manner, which is conducive to reducing the consumption of material and energy. We designed the following circuit, hoping to construct the following dynamic regulatory system in our designed non-natural synthetic PHFA pathway (Figure 6).
However, since the above gene circuit cannot visually detect the dynamic regulation of the system, we will use red-green fluorescent protein to characterize the results and design a feasible system (Figure 7) as well as construct a plasmid profile (Figure 8).
Figure7 the process of dynamic regulation
Figure8 plasmid spectrum of the gene circuit
The core components used in this system (Figure 8) are the CI blocker protein and the PRM' promoter. The promoter p(Bla) is originally a moderately strong constitutive promoter, but the subsequent lac operator can bind to the LacI protein to achieve the effect of blocking the promoter p(Bla). The promoter PRM' is a modified PRM promoter from λ phage (OR3 is eliminated), leaving only OR1 and OR2 binding sites, which can be activated by binding to CI proteins.
Initially, only a small amount of LacI was present in the system, which was unable to block the expression of genes downstream of the p(Bla) promoter, thus allowing the concentration of CI protein in the system to increase. The p(Bla) promoter is blocked by the immediately following lac operator, resulting in a decrease in CI protein synthesis and inability to activate the PRM' promoter, which in turn leads to a decrease in LacI protein expression. In this cycle, gene oscillations are achieved and it is expected that the system will be characterized by the alternating red and green fluorescence results.
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The current study lacks commercial scaffolds that can be used for cultured meat production. Therefore, the establishment of suitable scaffolds for large-scale breeding meat production requires further exploration and optimization of methods and biomaterials, as well as new technologies to enhance the ideal characteristics of scaffolds (such as porosity and ligand availability). The future production of cultured meat may involve the co-culture of multiple cell types to form a structured tissue similar to real meat which requires new scaffolds to support the differentiation of multiple cell types and allows for the spatial heterogeneity of the final product. These scaffolds also should have the characteristics of precise fine-tuning of biomaterial properties, such as stiffness and biochemical properties. However, these methods can only generate about 100-200μm thin layers due to the limitation of diffusion of media infusion. Overcoming the thickness limitation of cultured meat tissue remains a major challenge in this field. The traditional method of seeding cells onto full-size scaffolds to form tissue structures cannot solve this problem.
Last year, our team used 3D printing technology to build cell growth scaffolds, although 3D biological printing can mix cells and scaffold biomaterials to layer them together forming meat tissue. But in fact, 3D biological printing for the development of cultured meat needs a simpler and lower cost technology than 3D printing in tissue engineering, because cultured meat does not require a complex vascular system like natural tissue. Meanwhile, this technology is not suitable for the scaling requirement, and cannot be used for mass industrial production. Future innovations in cell slice tissue engineering methods or designing 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 construct scaffold materials providing a better cell culture environment for cell culture meat. Since microspheres have lower costs compared with 3D printing, and microspheres are commonly used in tissue engineering, the technology is more mature, and it is easier to expand the culture and industrial production.
Figure1 Microspheres
1.1 Using the double lotion volatilization method to prepare Microspheres We use the double lotion volatilization method to prepare microspheres (because the polyhydroxy fatty acid is amphiphilic). The parameters were successfully adjusted so that the medium and long-chain PHFA materials with strong hydrophobicity have appropriate physical and chemical properties. We adopt the method of "water in oil in water". The internal aqueous phase is ammonium bicarbonate, the oil phase is polyhydroxy fatty acid dissolved in the organic solvent, and the external aqueous phase is polyvinyl alcohol. First, the primary emulsion is obtained by mixing the oil phase with the internal water phase, and then the primary emulsion is added to the external water phase to mix evenly to obtain the secondary emulsion, which is stirred to form microspheres. Ammonium bicarbonate is decomposed into ammonia gas and carbon dioxide gas in the preparation process. The above two gases occupy part of the space of primary emulsion droplets, forming a porous structure.
1.2 Parameter regulation and surface modification of microspheres.
By adjusting the size, pore diameter, and particle size of microspheres under different parameters (changing the concentration of ammonium bicarbonate, PVA concentration, and stirring speed), we screened the most suitable microsphere scaffold system for cell attachment and growth. Moreover, the microsphere has a small diameter and a large specific surface area, which can effectively transport materials. The affinity to cells can be improved by surface modification. Adding collagen, RGD, and other substances allows for promoting the surface adhesion and structural stability of cell tissues, which is conducive to accelerating the cell growth rate. Then we can adjust the structural parameters and surface modification of the microsphere according to the cell growth. The surface of microspheres with - NH2 and - OH through the aminolysis of ethylenediamine solution. The aminated microspheres were further cross-linked with glutaraldehyde to make the surface of the microspheres contain bioactive collagen and RGD.
Figure2 Porous microsphere 1.0 Figure3 Porous microsphere 2.0
1.3 Linker (connecting cells to microspheres)
In order to achieve a better cell attachment rate, the linker should hold the following properties 1) firmly interact with cells and microspheres; 2) Independent and stable existence. From the article, we acknowledge that α5β1 integrins are stably expressed during muscle formation. In the process of mouse myogenesis, various integrins are expressed differently at separate periods and interact with different ECM components respectively, but a few types of integrins retain certain content in the whole process [18]. Although the precise spatiotemporal expression of integrins in chicken muscle formation still needs further study, based on the above examples, we selected α5β1[19] and α2β1[20], which have been verified to be expressed in chicken muscle formation. Apart from that, the selection of supplements for surface modification of microspheres, is shown in Table 1[21]. With considerations of the bio-affinity of materials and experimental feasibility, peptide GRGDSP is selected as our linker. It can form amide bonds with the -OH groups on the microsphere surface, meanwhile having the strongest binding force with α5β1 (see Table 2 for details). According to our experiment, it holds a better performance as a linker compared to collagen.
Figure4 Left: Schematic diagram of the interaction between RGD and integrin [21];
Right: Schematic Diagram of Collagen and Integrin [22]
Table1 Different RGD derivative and Integrins α5β1 Effect [13]
Through a series of cell-related experiments, we hope to comprehensively evaluate the adhesion, growth, and differentiation of cells in the 3D culture (PHFA microsphere)in vitro, and use this as the criteria to optimize the microsphere design (see Part I) and the inoculation of cells to the microsphere as shown below.
2.1 Selection with cell culture
In order to optimize the experimental techniques and improve the efficiency, we chose the C1C12 cell line as our first cell model. After the technique is mature, the muscle cells from the muscle of the chicken are used.
For cell culture, DMEM (1% double antibody, 10%BMS) is used as the culture media, the cells were resuscitated and subcultured for one generation before inoculation on microspheres. The medium was replaced each 48h.
2.2 Cell seeding
We will first utilize the static culture method to demonstrate the feasibility of the scheme and optimize the experimental design based on the following. After the technology is further developed, the suspension culture method is used for quantitative production exploration.
2.3 Cell growth evaluation using fluorescent staining
After the cell-microsphere complex for some time, the cell has to be stained for observing and following growth evaluation.
2.4 Cytotoxicity test & Cell growth evaluation using CCK8 method
The CCK-8 method is utilized to evaluate the cell population of each group at 1d, 3d, and 5d, respectively. Compared with the control group, the growth rate of cells in each experimental group was calculated.
Figure5 CCK8 and microsphere cell co-incubation
2.4 Cell differentiation assay: qPCR
After the cells covered 80% of the surface area, the differentiation medium was replaced, and the total RNA was extracted by the Trizol method on days 1, 3, or 7, respectively. After qualified detection, the RNA was reverse-transcribed into cDNA by a two-step method and then RT-qPCR was performed. The genes to be tested were MyoD, MyoG, and MRF.
2.5 Cross-linking: final product molding (using glutamine transaminase)
Since myosin and other proteins are expressed on the surface of myoblasts, glutamine aminotransferase was selected to cross-link the cell-microsphere complexes together.
In the future, we will develop a microsphere scaffold system with multiple pores Which holds a larger specific area, and the prepared microspheres provided a better culture environment for cell culture meat. We have substantially solved the problems of recycling difficulty, high cost and insufficient stability of 3D printed scaffolders last year. Moreover, cultured meat using a microsphere scaffold have advantages for large-scale production, meanwhile lowering the capital investment. We believe our powerful technics would through new light on the industrialization of cultured meat.
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