1. Introduction
Understanding how life adapts to extreme environments is not only a fundamental scientific question but also an opportunity to unlock new biotechnological resources. Extremophiles—organisms that thrive under high temperature, salinity, acidity, or pressure—have evolved unique biochemical systems that challenge the traditional limits of life. These organisms offer invaluable insights into molecular adaptation, enzyme stability, and evolutionary resilience, while also providing a treasure trove of biocatalysts for industrial and environmental applications.
Our project focuses on thermophilic microorganisms from hot spring ecosystems in Kangding, Sichuan Province, aiming to elucidate the molecular mechanisms underlying thermal tolerance, particularly the role of polyamine metabolism. Through metagenomic sequencing and functional gene mining, we seek to identify novel polyamine biosynthetic pathways and analyze their contribution to cellular stability under extreme temperature stress. By integrating synthetic biology and computational approaches, our ultimate goal is to construct a robust microbial platform for sustainable, high-temperature biomanufacturing.
2. Background Investigation
2.1 Extremophiles and the Limits of Life
Extreme environments—such as deep-sea hydrothermal vents, polar ice caps, hypersaline lakes, and volcanic hot springs—represent ecosystems where temperature, pH, or chemical composition exceeds the tolerance of most organisms. Yet, extremophiles not only survive but flourish in such niches, employing unique molecular adaptations. For instance, Methanopyrus kandleri 116 can grow at 122 °C [1], while Picrophilus torridus tolerates pH −0.06 [2]. These examples illustrate how life continuously redefines its boundaries. Studying extremophiles enhances our understanding of biochemical resilience and reveals potential for biotechnological innovation—such as thermostable enzymes ("extremozymes"), specialized metabolites, and adaptive biomolecules.
2.2 Polyamines: Molecular Guardians of Life Stability
Polyamines (PAs)—including putrescine, spermidine, and spermine—are small, positively charged molecules that play essential roles in DNA stabilization, translation, and oxidative stress response [3]. In extremophiles, unusual long-chain polyamines such as thermospermine or thermopentamine are often observed, contributing to the stabilization of nucleic acids and membranes at high temperatures (Table 1) [4].
Polyamines form strong electrostatic interactions with DNA and RNA, preventing thermal denaturation and maintaining transcriptional fidelity [5]. They also act as antioxidants, as the accumulation of polyamines and the upregulation of their biosynthetic genes promote proline accumulation, thereby alleviating oxidative stress [6]. Therefore, deciphering polyamine metabolism in thermophiles not only enriches our understanding of molecular adaptation but also provides a foundation for the design of thermostable biotechnological systems.
Table 1 Natural linear polyamine structural formula
2.3 Thermophilic Microorganisms and Heat Resistance Mechanisms
Thermophiles—organisms growing optimally above 50 °C—exhibit distinctive genome compositions, protein structures, and membrane lipids that ensure stability under extreme conditions. Among their adaptations, polyamine biosynthesis is a key determinant of thermal tolerance. In the DNA double helix, the minor and major grooves are two primary spatial depressions between base pairs that provide binding sites for various molecules. Polyamine binding sites have been observed in both the minor and major grooves of DNA [7].Thermophilic enzymes, or thermozymes, remain catalytically active between 80–113 °C, offering industrial advantages such as reduced contamination risk and increased process efficiency [8].
Harnessing these enzymes in metabolic engineering could revolutionize industrial biocatalysis, biofuel production, and pharmaceutical synthesis—especially under conditions where conventional mesophilic systems fail.
2.4 Metagenomics and Functional Gene Mining
Metagenomics allows direct access to genetic information from environmental samples without the need for cultivation. High-throughput sequencing and bioinformatic analysis enable the reconstruction of microbial community structures and the discovery of novel functional genes. Advances in third-generation sequencing (e.g., SMRT, Oxford Nanopore) have made it possible to assemble near-complete genomes from complex microbial consortia, leading to the discovery of new enzymes and biosynthetic pathways [9,10].
For instance, in a hot spring located in Tibet, China, researchers successfully isolated a novel thermophilic bacterial strain, which was designated as Thermus caldifontis sp. YIM 73026T. This strain exhibits unique biological characteristics, including the ability to utilize a wide range of organic carbon sources. Genomic analysis revealed an abundance of carbohydrate-active enzyme genes, suggesting its potential for decomposing plant residues [11]. In the shallow hot springs of Sakhalin Island, Russia, scientists isolated the first strain of Tenuifilum thalasicum gen. nov., designated 38H–strT. This strain demonstrates a remarkable ability to degrade proteins, particularly keratin. Its genome encodes multiple hydrolytic enzyme genes that are closely associated with its protein-degrading capability [12].
In our project, metagenomic analysis of Kangding hot spring samples is used to identify novel polyamine synthesis enzymes, providing insights into thermophilic metabolism and biotechnological application potential.
2.5 Research Region and Sampling Background
Kangding, located at the eastern edge of the Tibetan Plateau, hosts one of China's most geothermally active zones, with more than 50 identified hot springs ranging from 22–89 °C. This region provides an ideal natural laboratory for exploring thermophilic microbial diversity and adaptation. Samples were collected and analyzed using metagenomic sequencing to identify polyamine-related genes and assess community structure across temperature gradients.
3. Stakeholder Analysis
Our project's development is closely tied to multiple stakeholder groups. Understanding their perspectives ensures responsible research and meaningful social integration.
Academic Community:
Researchers in microbiology, biochemistry, and synthetic biology gain new insights into thermophilic adaptation and polyamine function, facilitating fundamental discoveries and new research directions.
Industry:
Industrial biotechnology, bioenergy, and food fermentation sectors could benefit from heat-resistant enzymes and microbial strains, reducing cooling costs and improving process safety. Potential applications include high-temperature fermentation, enzyme production, and waste valorization.
Local Community:
Collaboration with Kangding local authorities and communities promotes sustainable utilization of geothermal resources. Educational outreach helps demystify synthetic biology and supports local environmental stewardship.
Government and Policy Makers:
The project aligns with China's "Green Biomanufacturing" and "Carbon Neutrality 2060" strategies. By developing bio-based, energy-efficient production systems, it supports national priorities for sustainability and innovation.
4. Value-sensitive and Ethical Reflection
We recognize that technological innovation must be balanced with ethical responsibility and social acceptance. Therefore, we adopted a value-sensitive design (VSD) approach throughout our research.
1.Safety and Biosafety:
All laboratory procedures comply with institutional biosafety regulations. Engineered strains are contained within biosafety level 1 (BSL-1) facilities, ensuring environmental and human safety.
2.Transparency and Open Science:
We commit to data sharing and open communication of results, following FAIR (Findable, Accessible, Interoperable, Reusable) data principles. Collaboration with peer institutions ensures reproducibility and accountability.
3.Environmental Responsibility:
Our work aims to reduce the environmental footprint of industrial bioprocesses. By promoting heat-tolerant biocatalysts, we lower cooling energy demands and contamination-related waste.
4.Public Engagement:
We recognize that public perception of synthetic biology is shaped by trust and understanding. We therefore engage in science communication through seminars, online dissemination, and cooperation with local schools to foster public literacy in biotechnology.
5. Reflection and Conclusion: Potential and Challenges
Through continuous dialogue with stakeholders, we reflected on the scientific and societal dimensions of our work.
From an industrial perspective, thermotolerant Bacillus strains with enhanced polyamine metabolism could substantially improve bioproduction efficiency. They can operate at high temperatures, reduce energy consumption, and increase system robustness. Yet, large-scale deployment will require investment in process optimization and regulatory evaluation.
From an academic perspective, our findings contribute to understanding molecular adaptation to extreme environments and open avenues for synthetic biology of extremophiles. Ethical challenges, including data transparency and dual-use concerns, must be addressed through rigorous oversight and community dialogue.
For local communities, the project encourages sustainable resource use and economic opportunities linked to biotechnology. However, we must continue fostering trust and dispelling misconceptions about genetic engineering through active communication.
From a policy standpoint, the research supports China's sustainable development and low-carbon goals. Nevertheless, complex regulations on genetically modified microorganisms (GMMs) demand careful compliance and risk assessment.
Overall, our Human Practices activities underscore that responsible innovation in synthetic biology requires continuous reflection, inclusive participation, and transparent governance. By integrating scientific discovery with ethical awareness and societal engagement, we aim to advance not only biotechnology but also the shared vision of a sustainable and inclusive future.
6.Reference
[1] Ken Takai, Kentaro Nakamura, Tomohiro Toki, et al. Cell proliferation at 122°C and isotopically heavy CH4 production by a hyperthermophilic methanogen under high-pressure cultivation. [J]. The Proceedings of the National Academy of Sciences (PNAS). 2008, 105(31): 10949-10954.
[2] Christa Schleper, Gabriela Puehler, Ingelore Holz, et al. Picrophilus gen. nov., fam. nov.: a Novel Aerobic, Heterotrophic, Thermoacidophilic Genus and Family Comprising Archaea Capable of Growth around pH 0. [J]. Journal of Bacteriology. 1995, 177(24): 7050-7059.
[3] Syed Sarfraz Hussain, Muhammad Ali, Maqbool Ahmad, et al. Extremophiles: from abyssal to terrestrial ecosystems and possibly beyond. [J]. Biotechnology Advances. 2011, 29(3): 300-311.
[4] Akihiko Sakamoto, Masatada Tamakoshi, Toshiyuki Moriya, et al. Polyamines produced by an extreme thermophile are essential for cell growth at high temperature. [J]. The Journal of Biochemistry. 2022, 172(2): 109–115.
[5] Michael, Anthony J. Polyamine function in archaea and bacteria. [J]. Journal of Biological Chemistry. 2021, 293(48): 18693–18701.
[6] Radyukina, N. L., Kartashov, A. V., et al. Functioning of defense systems in halophytes and glycophytes under progressing salinity. [J]. Russian Journal of Plant Physiology. 2007, 54: 806–815.
[7] Juan Pelta, Françoise Livolant, Jean-Louis Sikorav, et al. DNA Aggregation Induced by Polyamines and Cobalthexamine. [J]. Journal of Biological Chemistry. 1996, 271: 5656–5662.
[8] Jumpei Hayakawa, Yoshihide Kondon, Morio Ishizuka, et al. Cloning and Characterization of Flagellin Genes and Identification of Flagellin Glycosylation from Thermophilic Bacillus Species. [J]. Bioscience, Biotechnology, and Biochemistry. 2009, 73(6): 1450-1452.
[9] John Eid, Adrian Fehr, Jeremy Gray, et al. Real-time DNA sequencing from single polymerase molecules. [J]. Science. 2009, 323(5910): 133-138.
[10] Thomas P Niedringhaus, Denitsa Milanova, Matthew B Kerby, et al. Landscape of next-generation sequencing technologies. [J]. Analytical Chemistry. 2011, 83(12): 4327-4341.
[11] Inam Ullah Khan, Neeli Habib, Firasat Hussain, et al. Thermus caldifontis sp. nov., a thermophilic bacterium isolated from a hot spring. [J]. Systematic and Evolutionary Microbiology. 2017, 67(8).
[12] Olga A. Podosokorskaya, Tatiana V. Kochetkova, Andrei A. Novikov, et al. Tenuifilum thalassicum gen. nov., sp. nov., a novel moderate thermophilic anaerobic bacterium from a Kunashir Island shallow hot spring representing a new family Tenuifilaceae fam. nov. in the class Bacteroidia. [J]. Systematic and Applied Microbiology. 2020, 43(5).
Integrated Human Practices
"Human Practices is the study of how your work affects the world, and how the world affects your work." ——Peter Carr, Director of Judging
Throughout the course of our project, we have continuously contemplated how to be responsible both locally and globally and how to engage with the world in a manner where our work can impact the world, and in turn, the world can guide us. Our project introduces a chassis cell construction strategy that excavates from a high-temperature environment and is used for efficient polyamine production cell factories. If we can achieve our intended goal, it can save nearly 20 % of the production cost for the polyamine production companies in our region every year, thereby improving their economic viability. Furthermore, this contribution aligns with the United Nations Sustainable Development Goal 9 (Industry, Innovation, and Infrastructure) and aligns with our nation's "dual carbon" policy while also contributing to Goal 12 (Responsible Consumption and Production). Moreover, there is significant potential for our project to be promoted globally, aligning with the concept of a low-carbon lifestyle, as virtually everyone is influenced by polyamines in some way in their lives. This is how we aim to truly be responsible both locally and globally.
As we progressed with our project, we actively engaged with individuals outside of our team, reflecting on and improving our project. This interaction allowed us to truly practice integrated Human Practices (integrated human practice) by engaging with the world and continually enhancing our project.).
Forming a team and choosing our project
1) Build a diverse Team
Our story begins with life, and after a journey, it will return to life. Synthetic biology has added vibrant colors to this experience.
Synthetic biology is an emerging discipline that encompasses wide-ranging intersections between life sciences and various other fields. We firmly believe that confining synthetic biology solely within the realm of biological sciences does not facilitate the true realization of interdisciplinary collaboration. Our team leader had previously participated as a team member in the 2023 iGEM competition with a team primarily led by Sichuan University's College of Biomass Science and Engineering. In an effort to propagate synthetic biology to every corner of this university, renowned for its engineering disciplines, he decided to step out of his comfort zone and take the first step toward this goal. In the Research Group of Qin Jiufu, he found two like-minded teammates, and together, they set out to form a team. Before long, they successfully gathered students with diverse academic backgrounds, forging a united team to embark on the iGEM journey together.
2) Explore Content——Get Inspired
Take microbial samples to extreme environments → find key enzyme elements for polyamine synthesis → construct chassis cells for efficient cell factory for polyamine synthesis.
Get inspired initially
Our journey, born from the intersection of chemical engineering and economics, initially sparked our inspiration. Dr. Kaifeng Du's lecture elucidated the multifaceted prospects inherent in establishing a robust green chemical industry. Within these insights, one thread piqued our curiosity — the use of microbial cell factories for production.
As microorganisms engage in fermentation, complex interactions of metabolic reactions unfold, accompanied by the inevitable rise of thermal stress, impacting the normal growth and expression of microorganisms. To ensure the continuity of optimal production conditions, the introduction of cooling water is crucial. However, as we observed the operation of this cooling mechanism, we uncovered a fact — excessive consumption of energy and capital.
This observation further solidified our determination, making the necessity for innovation abundantly clear. Beyond the domains of traditional cooling practices, an opportunity emerged to harmonize energy efficiency and economic prudence with microbial capabilities. Our focus shifted from mere cooling mechanisms to encompass a broader vision: the seamless integration of sustainable production with microbial prowess. As we navigate this uncharted territory — the enhancement of engineering microbial stress resistance — we are summoned forward.
Narrow "microorganisms" into "thermophilic microorganisms"
Purpose: We decided to engage in a discussion with our inspiring teacher, Dr. Kaifeng Du, with the aim of validating the feasibility of our ideas.
Contribution: Dr. Du confirmed the urgency and feasibility of studying microbial stress resistance. He suggested narrowing the range of extreme microorganisms to specific environments, such as hot springs, volcanoes, and deep seas..
Realization: Extreme environment is the host of most microbial biomass on earth, and extreme microorganisms are defined as organisms that have evolved biological molecular characteristics or physiological strategies. Through our dialogue with Dr. Du, we have received a lot of feedback on this idea. We have conducted in-depth research and analysis on the acquisition methods of several thermophilic microorganisms proposed by Dr. Du. Among them, geothermal energy, as a clean and renewable energy, refers to the heat energy contained in the water flow or rock and soil layer with underground temperature above 25°C or higher than 10°C in the local constant temperature zone. This kind of thermal energy is mainly derived from the long-lived radioactive isotope thermonuclear reaction inside the earth, which is an ongoing natural process and provides a steady stream of energy for the formation of geothermal resources. From the distribution of plate tectonics and geothermal anomalies, China is a country with very rich geothermal resources. It not only has thermal anomalies in tectonic active zones, but also has thermal anomalies in many large and medium-sized sedimentary basins. Due to the particularity of geological structure, these sedimentary basins have better preservation and accumulation of geothermal resources, which provides a broad prospect for the development and utilization of geothermal resources in China.
Kangding City is located in Ganzi Tibetan Autonomous Prefecture, Sichuan Province. The geographical location spans 101° 02' to 102° 30' east longitude and 29° 08' to 30° 46' north latitude. The administrative area is 11,600 square kilometers. According to the long-term monitoring data of Kangding Meteorological Station ( 2616 meters above sea level ), the minimum temperature recorded in this area is − 14.7 °C, the maximum temperature is 29.4 °C, and the average annual temperature is 7.10 °C. Kangding City is located in the Mediterranean-Himalayan tropics, at the junction of the eastern margin of the Qinghai-Tibet Plateau and the Sichuan Basin. The geological tectonic activities are frequent, so the hot spring resources are abundant. The special geological structure makes the geothermal activity in Kangding area significant, and the hot water activity is very strong, covering a variety of medium and high temperature geothermal phenomena such as natural hot springs and high temperature self-spraying hot water wells and spring blooms formed under mining conditions. In the hydrothermal activity area of Kangding, there are 58 hot springs or springs. The water temperature ranges from 22 °C to 89 °C. The flow rate of a single spring varies from 0.3 L / s to 104 L / s, and the total flow rate is about 226 L / s. Therefore, we set the sample collection site in Kangding City, Sichuan Province.
Developing our project
1) Stage 1 : Project conception and design period
Action 1: Establish project leadership and core vision-invite researcher Qin Jiufu of Sichuan University as PI.
a) Objective : To establish a solid scientific foundation and clear strategic direction for the project, integrate interdisciplinary resources, and ensure the depth and breadth of research.
b) Contribution : With his deep accumulation in the field of microbial metabolic engineering and synthetic biology, Qin Jiufu laid the core research paradigm of " environmental metagenome mining to guide the construction of efficient cell factories " for the project. He guides us not only to focus on a single gene or product, but also to establish a complete technical chain from environmental samples → functional genes → chassis cells → application expansion. He helped us connect key academic resources and emphasized the importance of combining basic scientific research with potential industrial applications.
c) Realization : Under the overall planning and guidance of Qin researchers, we have constructed a clear research blueprint. He chaired the opening discussion of the project, which ensured the effective integration of multidisciplinary methods such as metagenomics, enzyme engineering, system biology and fermentation engineering. It is based on this high starting point of strategic design that our project can be systematically carried out and has the potential to move from phenomenon insight to technology creation.
Action 2: Consult Li director of Geology Research Institute of Ganzi Prefecture
a) Objective : To ensure that our sample collection activities are scientific and compliant, and to minimize impacts on fragile hot spring ecosystems.
b) Contribution : Director Li introduced the ecological sensitivity and geological characteristics of the sampling area in detail, emphasizing the importance of protecting the native microbial community. He recommended that we adopt a ' minimally invasive, multi-point, low-frequency ' sampling strategy and systematically record in-situ environmental parameters such as water temperature, pH, and ion concentration.
c) Realization : After discussion with Director Li, we have developed a strict ' ethical norms for environmental sample collection '. We not only control the sampling amount to the minimum, but also create an ' identity file ' containing more than ten environmental indicators for each sample. This enables our metagenomic data to be associated with accurate ecological environment information, which greatly improves the depth and reliability of subsequent ecological and evolutionary analysis.
Action 3: Participate in the Frontier Forum on Systems and Evolutionary Biology
a) Objective: To examine the adaptation mechanism of thermophilic microorganisms from the perspective of evolutionary biology, and to find a theoretical framework and innovative ideas for our metagenomic analysis.
b) Contribution: Experts at the forum affirmed strategies for studying extreme environmental microbes from a community and evolutionary perspective. They suggested that we should not only pay attention to the presence or absence of genes, but also analyze the phylogenetic distribution of polyamine synthase genes in the hot spring microbial community, and explore whether there is a horizontal gene transfer event, so as to understand the evolutionary driving force of this trait.
c) Realization: We adopted the advice of experts and strengthened evolutionary analysis in metagenomic analysis. By constructing a phylogenetic tree of polyamine synthase and comparing it with the species tree, we successfully revealed that the gene may have horizontal transfer between some microbial groups, which provides a new evolutionary biological insight for understanding the diffusion of polyamine synthesis ability in extreme environments.
Action 4: Consult Professor Wang Zheng from Beijing University of Chemical Technology
a) Objective: To explore the feasibility and potential of the application of thermophilic microorganisms and the heat-resistant components found in them in industrial biological manufacturing.
b) Contribution: Professor Wang Zheng highly affirmed the value of mining thermophilic chassis cells and pointed out its dual advantages of reducing the risk of bacterial contamination and cooling costs in high-temperature fermentation. He suggested that we should not only pay attention to the target product polyamines, but also evaluate the substrate utilization spectrum, growth rate and genetic manipulation potential of the isolated strains, which are the key traits that determine whether a chassis cell can be applied.
c) Realization: The discussion with Professor Wang strengthened our two-line research strategy of ' developing a thermophilic chassis '. In the isolation and screening of strains, we added evaluation criteria for growth performance and carbon source utilization. The final selected main strains not only had polyamine synthesis ability, but also showed excellent potential as a general thermophilic cell factory, which laid a solid foundation for the industrial application of the project.
2) Stage 2 : Experiment and discovery period
Action 1: Participate in the 15 th China Academic Symposium on Enzyme Engineering
a) Objective: To find the most cutting-edge technical path for functional verification and mechanism analysis of polyamine synthases that we have excavated in the metagenome.
b) Contribution: Enzymology experts recognized our work on structural prediction using AlphaFold, and suggested that we further predict the substrate binding pocket through molecular docking, identify key active site residues, and explain the possible functional differentiation of different synthases at the structural level.
c) Realization: We immediately incorporated molecular docking into our computational biology process. By pairing different polyamine substrates into the predicted enzyme structure model, we successfully distinguished the substrate preference of different synthases from the computational level, and locked the key amino acids that may determine the catalytic specificity. This provides accurate guidance for subsequent enzymatic functional verification and avoids blind experiments.
Action 2: Participate in the National Conference on Computational Biology and Bioinformatics
a) Objective: To learn and apply more advanced multi-omics data integration analysis methods to deeply interpret the global regulatory network of thermophilic bacteria at different temperatures.
b) Contribution: The conference showed us powerful network construction tools such as WGCNA. Experts suggest that we can use it to mine core gene modules that are highly co-expressed with temperature stress from complex transcriptome data, rather than focusing only on individual differential genes.
c) Realization: We abandoned the simple differential expression analysis and adopted the strategy of combining WGCNA and KEGG pathway enrichment analysis. Through this method, we successfully constructed a gene co-expression network under temperature stress, and clearly revealed that there was a highly synergistic regulatory relationship between the ' polyamine synthesis module ' and the ' heat shock protein module ' and ' active oxygen scavenging module ', demonstrating the core position of polyamines in heat adaptation from the system level.
Action 3: Participate in the 8th National Annual Conference on Chemical Engineering and Biochemical Industry
a) Objective: To understand the engineering challenges that may be faced in the process from laboratory shake flask culture to industrial scale fermentation amplification, and to optimize the experimental design in advance.
b) Contribution: The annual meeting report has repeatedly emphasized the ' scale effect ' of the fermentation process, pointing out that dissolved oxygen, mass transfer and shear force are the key control parameters in the amplification process. Experts suggest that we should initially investigate the physiological response of the strain under similar mixing and mass transfer conditions in the laboratory stage.
c) Realization: Inspired, we designed and built a microreactor system to simulate the fermentation tank environment, which was used to cultivate thermophilic strains and re-conduct multi-omics analysis. The obtained data more truly reflect the physiological state of cells in the future industrialization scenario, which makes our engineering data more realistic and provides a forward-looking reference for process amplification.
3) Sage 3: the integration of results and the prospect period
Action 1: Participate in the 24 th National Congress of Plant Genomics
a) Objective: To explore the frontier of functional research of polyamines in higher organisms, especially in plants, and to find cross-border application inspiration for our microbial research.
b) Contribution: The conference report clearly states that polyamines are not only simple metabolites, but also key plant stress resistance signaling molecules, which play a central role in regulating plant response to abiotic stresses such as drought and high salt. Experts have shared successful cases of enhancing crop stress resistance by regulating polyamine synthesis pathways.
c) Realization: These cutting-edge knowledge open up a new application horizon for us. We recognize that the efficient synthesis of plant-derived polyamines ( such as spermidine, spermine ) using our thermophilic chassis cells, or the development of new biostimulants using them as precursors, has great potential to serve green agriculture, which has become an important downstream application direction of our project.
Action 2: Participate in the 3rd ' Thousand Herbs Genome Project ' Symposium
a) Objective: To understand the latest progress in the biosynthesis of medicinal components of traditional Chinese medicine in China, and to explore the application possibility of our thermophilic microbial chassis in the field of synthetic natural products.
b) Contribution: The seminar revealed the great value and challenge of using synthetic biology techniques to reconstruct the synthesis pathway of rare active ingredients in traditional Chinese medicine in microorganisms. Experts pointed out that many synthetic pathways require complex cofactor regeneration and a specific cellular environment, and thermophilic bacteria may provide a unique catalytic microenvironment.
c) Realization: This seminar enabled our project vision to achieve a leap from ' synthesizing a compound ' to ' building a platform '. We expect that the thermophilic chassis we developed may become a new cell factory for the synthesis of high-value herbal natural products in the future with its stable metabolic network and possible unique coenzyme system, providing an innovative technical path for the modernization of traditional Chinese medicine.
Action 3: Consult researcher Ba Zhaoqing, National Institute of Biological Science.
a) Objective: In the final stage of the project, from the perspective of experts from the frontier institutions of synthetic biology, the biosafety strategy and responsible research paradigm of engineering bacteria were finally reviewed and improved.
b) Contribution: Prof. Ba Zhaoqing highly endorses our innovative idea of mining biological elements from extreme environmental microorganisms, and leads us to think deeply about the potential biosafety risks of engineering transformation of non-model thermophiles. He suggested that in addition to conventional physical protection, priority should be given to designing biological protection strategies based on metabolic dependence ( such as auxotrophs ) to achieve a higher level of biocontainment.
c) Realization: The discussion with the researcher greatly improved the biosafety design standards of our project. In the final plan, we not only clarified the physical protection measures, but also included the construction of trophic engineering strains as the core biological protection strategy in the future plan. At the same time, we refer to its recommendations and write a statement of ' forward-looking considerations on the environmental safety of thermophilic engineering bacteria ', which shows our prudent attitude and initiative as young scientists on the potential impact of technology.
Conclusion
Thus, our theme is established as the fire of life in hot springs : decoding the mysteries of polyamine synthesis in thermophilic microorganisms.
In one of the most extreme environments on earth, high-temperature hot springs, there are ancient secrets of life adaptation and reproduction. Our journey of scientific exploration begins here. In order to unravel the mystery of the growth of thermophilic microorganisms in such harsh environments, we went deep into the hot springs and collected environmental samples.
First of all, we adopt a ' face-to-point ' macro strategy. Through metagenomic analysis of the samples, we were surprised to find that polyamine molecules play a crucial role in the microbial community of hot springs. This suggests that polyamines may be the key ' fire of life ' of thermophilic microorganisms against high temperature stress.
This discovery leads us into the stage of molecular mining. Using the BLAST tool, we successfully mined multiple polyamine synthase genes from metagenomic data with different sources but unknown functions. In order to verify their function, we used the advanced AlphaFold to predict the protein structure. Excitingly, these predicted structures are highly similar to the known polyamine synthase structures and strongly support their functions at the computational level.
At the same time, we initiated the microscopic verification of ' from point to volume '. Through the hard culture of multiple media, we isolated a variety of single strains from the samples. We selected a representative thermophilic bacterium as our ' model organism ' and potential ' cell factory '. Its genome sequencing confirmed that it does contain key genetic elements for polyamine synthesis.
Next, we designed a delicate experiment to reveal the core mechanism : this strain was cultured under different temperature gradients and subjected to multi-omics analysis of transcriptome, proteome and metabolomics. This allows us to interpret the global response of cells at different temperatures like a detective with the idea of ' reverse metabolic engineering '. We can accurately locate which genes are activated by high temperature, which proteins are synthesized in large quantities, and which metabolic pathways are reconstructed, so as to reversely deduce the molecular blueprint for the efficient synthesis of polyamines and the co-evolution with temperature.
Finally, we observed the ultrastructure of cells at different temperatures by scanning electron microscopy ( SEM ) and transmission electron microscopy ( TEM ). These visual image evidences, which may reveal the changes of cell morphology, endomembrane system or inclusion body under heat stress, provide the most direct cytological evidence for the protective effect of polyamines.
So far, we have completed a complete and closed-loop scientific exploration story from natural environment ( hot spring ) → community gene ( metagenome ) → single enzyme protein ( structure mining ) → single strain ( isolation and culture ) → system biology ( multi-omics ) → cell phenotype ( electron microscopy ), systematically revealing the core role of polyamines in thermophilic microorganisms adapting to extreme environments.
Under the top-level design and strategic coordination of researcher Qin Jiufu, the high starting point and clear vision of our project have been established. Through extensive dialogues with geologists, evolutionary biologists, industrial biotechnology experts, plant and herbal genomicists, and even senior researchers at the Beijing Institute of Life Sciences, we ensured the comprehensiveness and responsibility of the project from sampling ethics, scientific depth, cross-domain application prospects to biosafety considerations. This network, led by top PI and woven by the wisdom of multi-disciplinary experts, ensures that our research is not only rigorous scientific exploration, but also responsible innovation for a green future.
