Part I: Creation possibilities of polyploid minimalist Escherichia coli (PMEC)
Cell factories refer to genetic modification in microbial chassis cells to produce target compounds in microbial cell factories, which have the advantages of green and clean/renewable/gentle reaction conditions compared to chemical synthesis, and have now become an important core of the global key strategy of green manufacturing.
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Fig 1. The “cell factory” and the “production line” with different divisions of labor
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The use of rapidly developing synthetic biology is an effective strategy for building cell factories. The core of synthetic biology is to design and construct new biological systems or new life forms with physiological functions to achieve sustainable green biomanufacturing, and three levels of cell factory construction have been proposed: standardized components/dynamic regulation and the construction of new life systems. Meanwhile, the creation of new life systems is precisely as the ultimate goal of synthetic biology. Current research includes: 1. Integration of 16 chromosomes to construct monochromosomal yeast cells; 2. Creation of artificial diploid E. coli by Crisper technology; 3.High-threonine-producing strain TH-103Z pioneered by Prof. Liang Quanfeng’s group, which has achieved the world’s highest yield so far reported.
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Fig 2. Gene editing in Escherichia coli chassis cells using the crisper technique（Photo credit: Tianyuan Su. A CRISPR-Cas9 Assisted Non-Homologous End-Joining Strategy for One-step Engineering of Bacterial Genome.Sci Rep.2016）
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In current synthetic biology research, minimalist strains are often used as the starting research object because they can bring: lower genome complexity and other excellent characteristics. However, no researcher has proposed how to combine the advantages of prokaryotic streamlining with those of eukaryotic polyploidization. Therefore, we sought the guidance of Prof. Liang Quanfeng from the State Key Laboratory of Microbial Technology to learn how to introduce polyploidization on the basis of prokaryotic organisms in the hope of creating and exploring the properties of this new cell system.
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Fig 3.Prof. Quanfeng Liang
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Synthetic biology is all about To Build to Understand. in this project, our team SDU-CHINA asked the following question: is it possible to create a new polyploid streamlined prokaryotic cell? What is the phenotype of this cell? Can it have the advantages of both? What insights can it bring to evolution? , and used these questions as a guide to direct the design and progress of our project.
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Fig 4. Designing and constructing polyploid Escherichia coli by fine-tuning FtsZ expression levels
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After communicating with Prof. Liang, we learned that by regulating the intensity of ftsz gene expression, we could change the morphology of the bacterium as well as its DNA content, thus making the creation of a new chassis cell-polyploid streamlined E. coli possible.
Part II: Awareness of microbial systems when designing new chassis cells
In order to solve the problems that may be encountered in constructing chassis cells such as stability, robustness and so on. Our team interviewed Prof. Xiaoying Bian from the State Key Laboratory of Microbial Technology, Shandong University.
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Fig 5.Prof. Xiaoying Bian
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The most important thing for a cell factory is the design. Feynman once said, “What I cannot create, I cannot understand.” Similarly, for us to know how to design a cell, we need to understand it first. We have to understand how it works in its natural state in order to design a better system. So it is designed on the basis of understanding its basic biological principles. It has not only the scientific attributes of basic science, but also very important applied attributes.
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Fig 6. Metabolic network of DGF-298
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After the exchange with Prof. Xiaoying Bian, we gave feedback on our project. We are improving the adaptability, yield, etc. by polyploidization of E. coli, and after the copy number is increased, the protein expression is generally elevated. But there are two things to keep in mind - stability and balance.Stability can be understood as a drastic environmental change that kills the cells, but slowly changing the environmental conditions, the microorganism adapts to the environment with its rapid rate of evolution. The higher the copy number the higher the yield, which is only realized within a certain range. So to achieve an overall increase in yield, a balance needs to be taken into account. After multiplication of the genome, it is also necessary to observe in multiple generations of cells whether stable inheritance of good traits after multiplication will occur.
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Fig 7.Communication with Prof. Xiaoying Bian
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This interview provided many lessons for us to build polyploids and hinted at the parts we should focus on.
Part III: Explanation of the unpredictable properties of brand-new chassis cells
After successfully designing and constructing the polyploid, we conducted a series of analyses of the external morphology and internal phenotype of the new prokaryotic minimal chassis cells and found that they brought about a number of phenotypes beyond our predictions. Firstly, during our chassis cell robustness validation, we found that the strain showed good resistance under perturbed conditions such as pH/temperature, but poor tolerance to acetate. In addition, polyploid E. coli showed large differences in mass doubling time when glycerol or glucose was used as the carbon source, respectively.
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Fig 8.Robustness changes in polyploid streamlined E. coli
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To learn more about this strain and to improve its potential as a whole chassis cell, we sought the advice of Prof. Su-Meng Wang of Qingdao Agricultural University, whose research interests had involved the construction of the polyploid high-yield chassis strain MG1655. Mr. Wang pointed out that the deletion of more genes adapted to specific ecological niches in the starting strain DGF-298 may result in phenotypic changes, while the complex metabolic network in the cell is more difficult to predict after polyploidization. For specific phenotypes, it is necessary to analyze the changes in the expression levels of the corresponding downstream RNAs or proteins. For example, for its improved temperature tolerance, it is possible to focus on analyzing the changes in the expression levels of heat shock proteins (HSPs), which are related to the repair of protein folding.
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Fig 9.Researcher Su-Meng Wang
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From this, we recognized the need to analyze specific pathway up- or down-regulation changes by transcriptomic or metabolomic changes and to model the corresponding gene-scale metabolic networks for this analysis.
Part IV: Modeling of gene-scale metabolic networks in prokaryotic polyploids
In order to establish a gene-scale metabolic network model, we found Prof. Sun Lei, a professor in bioinformatics at Shandong University, for an interview and study.
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Fig 10.Mr. Sun Lei
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In the communication with Mr. Sun Lei, Mr. Sun Lei recognized our idea of analyzing genes through metabolic network combined with transcriptome data. He also gave us some advice on how to combine the transcriptome data. Mr. Sun Lei told us that the effect of genes on metabolic network is a combination of regulation, not only to look at the effect of individual genes on metabolic network, but also to look at the effect of multiple genes on the metabolic network at the same time, and then we came up with the idea of using multi-objective optimization to solve this problem, which is to use the multiple reactions encoded by multiple genes as the objective function at the same time to solve the problem.
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Fig 11. Integration of transcriptomic data with the GSSM of DGF-298
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As for our fermentation modeling, Mr. Sun Lei gave us advice on the problem of fewer data points that we could have pre-trained based on other people’s data. And you can use interpolation to increase the data points in the sample, these two points can significantly optimize the learning effect of the model.
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Fig 12.Online communication with Mr. Sun Lei
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Part V: Production of downstream high value-added products inspired by the comparison of different chassis cells
In order to explore the applied properties of prokaryotic minimal polyploidy, we interviewed Associate Professor Tingting Guo of Shandong University to examine the direction of prokaryotic minimal polyploid Escherichia coli, inspired by the chassis cells of the lactic acid bacteria industry.
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Fig 13.Prof. Tingting Guo
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E. coli is less acid tolerant than lactic acid bacteria. So it is possible to explore the acid-tolerant properties in Lactobacillus and apply them to E. coli to increase the acid tolerance. For example, Lactobacilli can pump hydrogen ions out of the cell or convert them through metabolic pathways, and so on. These pathways can then be borrowed into E. coli to improve the tolerance of E. coli.
Secondly, the metabolic pathways of polyploid E. coli can be optimized by learning the strategies and methods of synthetic biology in Lactobacillus to improve the synthesis efficiency and yield of target products. In addition, some unique enzymes and metabolic pathways in Lactobacillus can be introduced into your chassis cells to broaden their synthesis capabilities.
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Fig 14.Communication with Prof. Tingting Guo
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Part VI: Research on the environmental impact and economic benefits of the carbon source product PHB
Why is the outlet of the chassis cell PHB?
After an interview with Prof. Tingting Guo, which inspired our idea about the regulation of the pathway for chassis cells to produce high value-added products, and combined with the aforementioned experiments that revealed the up-regulation of the metabolic pathway of carboxylic acid compounds in polyploid minimalist strains through the transcriptome, we turned our attention to plastics: macromolecules made of polyalkanes as the backbone.
Plastic is one of the indispensable materials in the modern chemical industry, however, the large number of waste plastic products, their wide distribution and difficulty in recycling, these characteristics have led to a global “white pollution” problem. According to statistics, the demand for plastic products in China remains high (Fig 15), but our recycling and disposal of waste plastics is only about 30%.
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Fig 15.Changes in the Production of Plastic Products in China, 2013-2023
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Against this background, the relevant state departments have intensively issued policies to strengthen plastic pollution control, promote biodegradable plastics and encourage the healthy and sustainable development of the biodegradable plastics industry.
Since the introduction of the “plastic ban” and dual-carbon policy in 2020, China’s production and sales of biodegradable plastics have shown significant growth. With the in-depth promotion of relevant policies, the continuous substitution of biodegradable plastics in common applications such as disposable shopping bags, disposable tableware and express packaging is becoming more and more prominent, and the acceptance of the society towards them is also increasing. In the future, degradable plastics are expected to realize wide application in more downstream fields.
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Fig 16.Changes in Production and Sales of Degradable Plastics 2018-2023
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Degradable plastics are mainly divided into three categories: photo-degradable plastics, bio-degradable plastics and photo-/biological double-degradable plastics, among which bio-degradable plastics have excellent degradation performance, and the production process and cost are in the middle of the three, which is the mainstream degradable plastics types at present. However, after market research, PLA (polylactic acid) and PBAT (polybutylene terephthalate) are the most widely used biodegradable plastics in the market at present, instead of PHB (polyhydroxybutyrate), which is the object of our research. The reasons for the limited marketing of PHB are: the production of PHB depends on glucose as a carbon source and the fermentation process is extremely harsh, which leads to high production costs; the production efficiency is low due to technical limitations; and no ideal production strain has been found yet. Therefore, we hope to break through the technical and cost constraints of PHB by conducting synthetic biology experiments, and then improve its market application.
Specific analyses are as follows:
Part VII: Introduction of dynamic cascade regulation lines
After the market and environmental research, and the up-regulation of carboxylic acid metabolic pathway expression through transcriptome sequencing data, we hope to select PHB as the next high-value-added product to be produced, as it is proved that the production of PHB using the new minimal polyploid prokaryotic chassis cells is of potential high economic benefits. In order to broaden the understanding of PHB metabolic pathway and production characteristics, we invited a group of professors from Taishan Academy and participated in a focus meeting hosted by them, with the participation of professors Mingyu Wang/Yu Shen/Wei Zhang from the State Key Laboratory of Microbial Technology and the School of Life Sciences.
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Fig 17.Online meetings hosted by a panel of professors from the Tarzan Academy
Fig 18.Mingyu Wang,Executive Director of the Taishan Academy Professors’ Group
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In the meeting, Mr. Wang pointed out that if the production of PHB is targeted to a carbon source, it is necessary to pay attention to its competition with the TCA reaction, i.e., the production of PHB and the TCA cycle, which is an important physiological process, occur at the same time. In bacteria, the production of PHB and the TCA cycle, an important physiological process, compete for the utilization of acetyl coenzyme A. Such a process affects the production of PHB products in practice.
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Fig 19.Competition between TCA response and PHB production
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Since then, we have continued to review the literature, and found that dynamic regulatory cascades may be the key to solving this problem: based on the quorum sensing (QS) system and the principle of cascade regulation, we have investigated the order and time interval of gene expression in the metabolic pathway, and finally realized the construction of a self-induced dynamic temporal cascade in E. coli, which is expected to solve the problem of the competition of carbon and carbon backbone substrates in the production pathway. The finalization of the self-induced dynamic cascade in E. coli is expected to solve the competition for carbon substrates in the production pathway.
Part VIII: Multidimensional Sensor Design for Cooperative Fermentation in LSTM Models
In order to better design the hardware, the hardware group had many exchanges with Prof. Wang Xia of Shandong University Taishan Academy, we had a long time communication and practice exchanges on how to design a more reasonable monitoring device as well as better combining the fermenter with the monitoring device in order to get better experimental data and images, etc. When designing the control core device, we need to stabilize the voltage of the core board microcontroller at 3.3V, and the microcontroller comes with a voltage that can’t be stabilized all the time. In the design of the control core device, we need to stabilize the voltage of the core board microcontroller at 3.3V, and the voltage that comes with the microcontroller can not be stabilized at 3.3V all the time, so we need an external regulator module. In the sensor’s operating range, we need to construct the voltage reference required by the ADC module according to the sensor’s operating voltage and linear equations, so we need to add 100uf capacitors and so on during the design process of the PCB board in order to control its ultimate accuracy.
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Fig 20.Hardware blueprint
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We also went deep into the laboratory for actual measurement and deduction, and finally determined the final program of this hardware group. In the process of connecting the microcontroller and the sensor, in order to prevent it from being affected by the environment of the fermentation tank, we placed it in the exhaust emission place for processing, Mr. Wang Xia gave us a lot of suggestions by using her rich theoretical and practical ability, which benefited us a lot and improved our understanding of the project and the ability of comprehensive application.
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Fig 21.Prof. Xia Wang
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Part IX: Looking to the future of synthetic biology chassis design
Through the market research, professor interviews, wet experiments, hardware design and other work carried out in the early stage, we have completed a more complete project content and achieved certain results. Considering that we need to have knowledge of cutting-edge synthetic biology applications and challenges, we are eager to communicate with and learn from frontline researchers and experts. We were fortunate to invite Ms. Chunyan Zhan, a technical expert of Beijing Tsingke Biotech Co., Ltd, who has rich experience in gene synthesis and sequencing, and Director Li of BGI Genomics Co., Ltd, to communicate with us in online manner.
IX.1 Interaction with Ms. Chunyan Zhan,Beijing Tsingke Biotech Co., Ltd. - Close to lab specifics: on sequencing efficiency and cost control, ultra-long gene fragment synthesis
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Fig 22.Ms.Chunyan Zhan
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Firstly, we introduced the project “Construction of polyploid Escherichia coli based on the minimal chassis cell DGF298 and regulation of chromosome number and size of the cell by regulating the expression level of FTSZ gene, and then exploring its potential application in biomanufacturing” to Ms. Chunyan Zhan, and introduced the construction method of polyploid E. coli . Ms. Chunyan Zhan affirmed our approach and pointed out that similar gene expression regulation techniques are promising for industrial applications.After that, we exchanged with Ms. War on some experimental specific operations and the frontier development of synthetic biology.
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Fig 23.Application of “gene factories”
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First of all, in gene sequencing and synthesis, the current challenges of our project are mainly how to improve the accuracy of gene synthesis and reduce the cost of sequencing. In this regard, Ms. Chunyan Zhan shared their experience of using one-generation sequencing and NGS technology to double-validate the synthesized products, emphasizing the complementary nature of the two technologies in validating ultra-long gene fragments. Regarding the improvement of sequencing efficiency, we also discussed how to improve sequencing efficiency by interrupting long fragments for splicing. Regarding the synthesis of ultra-long gene fragments, Ms.Zhan instructed us how to optimize the DNA sequence to avoid complex secondary structures and improve the accuracy of synthesis.
Secondly, regarding cost control and fragment synthesis stability, we discussed how to optimize the process to reduce the cost while ensuring the synthesis quality. Ms. Chunyan Zhan suggested us to optimize each experimental link, including raw material selection, equipment maintenance, operation standardization, and emphasized the importance of process management system.
Finally, we discussed the future development direction of synthetic biology chassis design. Ms. Chunyan Zhan looked forward to the prospect of intelligent and automated technologies in synthetic biology, and emphasized the importance of standardization and modular design.
Overall, through the in-depth communication with Ms. Chunyan Zhan of Beijing Tsingke Biotech Co., Ltd, we have gained valuable suggestions and inspirations. We realized that the future of synthetic biology will rely more on technological innovation and intelligence. In terms of experimental operation, we learned how to improve sequencing efficiency, optimize the synthesis of ultra-long gene fragments, and how to control costs through process management. This exchange not only provided new ideas for our project, but also pointed the way for our future research direction.
IX.2 In-depth discussion with Director Director of BGI Genomics Co.,Ltd Qing-ou Research Institute, Hanbo Li. Frontiers: creating new life systems, DNA stores genetic information
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Fig 24.Hanbo Li, Director of BGI Genomics Co., Ltd Qing-ou Research Institute
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In order to obtain cutting-edge knowledge in the field of synthetic biology, solve experimental bottlenecks, and promote the progress of our project, we communicated with Director of BGI Genomics Co., Ltd Qing-ou Research Institute,Mr. Li.We first introduced the method of constructing polyploid E. coli by regulating the expression level of FTIC gene. That is, when the gene is highly expressed, the cell divides faster and the cell contains one chromosome; when the gene expression is suppressed, the cell divides slowly in a filamentous manner and contains two chromosomes. Dean Li acknowledged this and further inquired about the seed strain of E. coli we used and whether it had been streamlined. At the same time, he emphasized the importance of using standardized chassis cells for scientific research and industrial applications.
We shared the technical details of the chromosome content of polyploid cells verified by staining and flow cytometry, and our experimental results that the chromosome content of polyploid cells is indeed higher than that of haploid cells. Mr. Li was very interested in our concept of “Polyploid Minimalist Escherichia Coli (PMEC)”.
On the issue of gene expression regulation, we discussed the up-regulation of metabolic pathway expression levels found by transcriptome sequencing, and introduced the establishment and optimization of online network models. We found that the expression levels of threonine synthesis pathway, amino acid synthesis pathway and sulfate compound synthesis pathway were upregulated. In this regard, Dean Li suggested that we further investigate the specific functions of these pathways in polyploid cells and explore whether these pathways can be optimized by further gene editing to improve the productivity of cell factories.
We also explored the issue of fermentation potential, and we demonstrated the performance of polyploid E. coli in a simulated fermentation process and introduced a model-based simulation of PHB yield. We redirected the flow of carbon metabolism by introducing a dynamic regulatory circuit and balanced the relationship between cell growth and product synthesis. Dean Li appreciated that we analyzed the production potential of polyploid E. coli by simulating the fermentation process. He suggested us to consider the effects of different environmental factors (e.g., temperature, pH, carbon source, etc.) on the fermentation process in future experiments and try to optimize these conditions through dynamic regulation to achieve more efficient biosynthesis.
Regarding sequencing efficiency and cost control, Mr.Li shared UWG’s experience in sequencing platform construction, including the use of different optical reading methods to reduce cost and increase speed. For the synthesis of ultra-long gene fragments, Dean Li introduced the sequencing-while-synthesizing technology, emphasizing the improvement of accuracy and efficiency. For stability and cost control, Dean Li suggested that we pay attention to each step in the synthesis process, especially the chemical synthesis of small fragments and the process of linking them into large fragments.
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Fig 25.Sequences of bases that can store genetic information
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Overall, through this exchange with Mr.Li, we have gained feedback and suggestions specific to our research, especially cutting-edge insights and inspirations on creating new life systems and storing genetic information in DNA. We look forward to applying these valuable suggestions to our research in order to promote the deeper development of the project.