Global climate anomalies caused by excessive emissions of carbon dioxide are a common problem faced by all mankind. Various solutions have been proposed by people from all walks of life to slow the rate of deterioration, or if possible, completely solve this problem. In Haloworld, we have developed a synthetic biology answer to this serious environmental problem by genetically modifying a strain Halomonas TD from the Turpan Salt Lake in Xinjiang, China, and designing an industrial control software for further scale-up production. Besides, we have developed a locus screening program that can be used to screen for insertable lucuses on the genome, in order to help researchers to achieve more stable intracellular expression of genes related to product synthesis. Hopefully, by deeply digging the potential of Halomonas to solve this specific environmental problem, we could bring the world a Haloworld!

Problem

Nowadays, in order to solve the problem of excessive CO₂ emissions, there are two main directions. The first one is to control carbon emissions, and the second is to increase the conversion and utilization of carbon dioxide. In the second direction, there are currently three main methods of carbon dioxide conversion and utilization: synthesis gas fermentation utilization, electrochemical catalysis utilization, and enzyme catalysis utilization. However, the utilization of synthetic gas fermentation requires explosion-proof engineering and high safety requirements during the production process. Additionally, it has drawbacks such as a long fermentation cycle and low yield. On the other hand, electrochemical catalysis utilization and enzyme catalysis utilization are still facing challenges including high production costs, significant energy consumption, and poor selectivity for product types.

Solution

In our preliminary investigations, we learned that the derivatives produced by the electrochemical catalysis of carbon dioxide include a large amount of acetate, a small amount of formate and some carbonates. Based on what we learned in class , many microorganisms naturally have the ability to use acetate as a carbon source. For example, in Escheria coli.,it could change acetate into acetyl-CoA through acyl coenzyme A synthetase. Thus, a thought came to our mind——we could combine electrochemistry with synthetic biology and make full use of CO₂-derived electrolytes (CDE) to produce a large amount of products that could bring benefits to people's life.

Conclusion

We selected the technical approach of "electrochemistry +synthetic biology" for biomanufacturing. After we made the decision,we contacted a company in Shenzhen that specializes in electrochemical conversion of CO₂,shared our thought with them and asked if they could provide us with CDE. In return, if it proves that we could make the concept works, we will build a long-term relationship with them.

Problem

Since the primary products of electrochemical catalysis are acetate and formate , most industrial microorganisms such as Escherichia coli and Saccharomyces cerevisiae are intolerant to this high salt environment and have poor growth conditions. This drastically reduced the number of microorganisms we can choose from.
At this point, some articles about the next generation industrial biotechnology （NGIB） caught our eyes, especially those based on halophilic bacteria, mentioning rapid growing Halomonas TD inoculated under high salt concentration and alkaline pH^[1][2].The alkaline and halophilic characteristics of Halomonas TD make it highly tolerant to CDE and give it a significant inherent advantage in utilizing CDE as a carbon source, which meets our requirements perfectly.
However, research shows Halomonas TD01 shows lower tolerance and utilization of acetate compared to glucose. When grown in a minimal medium containing up to 75 g/L sodium acetate (NaAc), the cell growth rate of Halomonas TD01 decreases by 56.14%^[3]. The laboratory has utilized adaptive laboratory evolution technology to screen TD80, resulting in a strain that exhibits enhanced metabolism and tolerance to acetate.

At this stage, CDE contains a significant amount of formate and only a minimal quantity of bicarbonate remaining. On the one hand, unused formate must undergo complex sewage treatment processes before they can be discharged, otherwise they will cause substantial environmental pollution. On the other hand, successfully utilizing formate can increase the carbon conversion rate from CDE to the final product. Hence, our objective is to explore the potential of Halomonas TD in absorbing and utilizing formate. However, after our research and testing, we found that Halomonas TD lacks the necessary genes for the tetrahydrofolate (THF) cycle, rendering it unable to effectively utilize formate.

Solution

To facilitate the establishment of formatotrophic growth, we divided the formate assimilation pathway into three metabolic modules:
(1) C1 module is the tetrahydrofolate (THF) cycle, consisting of formate-THF ligase, methenyl-THF cyclohydrolase and methylene-THF dehydrogenase, together converting formate into methylene-THF;
(2) C2 module is glycine assimilation, consisting of the endogenous enzymes of the GCS (GcvT, GcvH , GcvP andLpd), that condenses methylene-THF with CO₂ and ammonia to give glycine;
(3) C3 module is the glycine cleavage system, consisting of serine hydroxymethyltransferase (glyA) and serine dehydratase, together condensing glycine with another methylene-THF to generate serine and finally pyruvate.^[4]
Based on our literature research, we choose the C1M of Methylobacterium extorquens AM1 and overexpress the endogenous C2 and C3 modules in Halomonas TD^[5][6][7]. Meanwhile, taking into account the homology between Vibrio. Natriegens and Halomonas TD, we take a bold innovative step by introducing the entire formate assimilation pathway from Vibrio. Natriegens.^[8]

Conclusion

Following the introduction of formate assimilation pathways, Halomonas TD80 now have the ability to utilize most of the components found in CO₂-derived electrolytes for growth and production, leaving only bicarbonate, which is easily recyclable, non-toxic, and present in low quantities. Having successfully optimized the efficiency and tolerance of Halomonas TD in utilizing CO₂-derived electrolytes, we proceed to explore its production pathways and assess its potential for large-scale production.

Reference

[1] Chen G Q, Jiang X R. Next generation industrial biotechnology based on extremophilic bacteria[J]. Current opinion in biotechnology, 2018, 50: 94-100.
[2] Jiangnan C, **aoning C, **nyi L I U, et al. Engineering Halomonas spp. for next generation industrial biotechnology (NGIB)[J]. Synthetic Biology Journal, 2020, 1(5): 516.
[3]Zhang J, et al. Substrate profiling and tolerance testing of Halomonas TD01 suggest its potential application in sustainable manufacturing of chemicals. J Biotechnol 316, 1-5 (2020).
[4] Kim S, Lindner S N, Aslan S, et al. Growth of E. coli on formate and methanol via the reductive glycine pathway[J]. Nature chemical biology, 2020, 16(5): 538-545.
[5] Yishai O, Bouzon M, Doring V, et al. In vivo assimilation of one-carbon via a synthetic reductive glycine pathway in Escherichia coli[J]. ACS synthetic biology, 2018, 7(9): 2023-2028.
[6] Turlin J, Dronsella B, De Maria A, et al. Integrated rational and evolutionary engineering of genome-reduced Pseudomonas putida strains promotes synthetic formate assimilation[J]. Metabolic Engineering, 2022, 74: 191-205.
[7] Claassens N J, Bordanaba-Florit G, Cotton C A R, et al. Replacing the Calvin cycle with the reductive glycine pathway in Cupriavidus necator[J]. Metabolic Engineering, 2020, 62: 30-41.
[8] Tian J, Deng W, Zhang Z, et al. Discovery and remodeling of Vibrio natriegens as a microbial platform for efficient formic acid biorefinery[J]. Nature Communications, 2023, 14(1): 7758.

Abstract:

Because Halomonas TD is a non-type strain that has only been discovered in recent years, there is a lack of corresponding gene regulation tools, which makes it difficult to genetically modify it. Thus, we decided to develop a dynamic control tool for Halomonas TD. Current dynamic control methods are mainly thermal regulation, optogenetic tools and quorum sensing-based collaborative dynamic control. Because optogenetic tools will face the problem of poor uniformity in the actual production process, and we ultimately want our project to be actually put into production, thus we chose to build a thermalsensitive bio-switch(T-switch)

Problem

At the moment (until we started this project), there are no thermosensitive bio-switch for Halomonas TD, so it is difficult to dynamically regulate Halomonas TD using temperature. However, in our research, we found that thermosensitive bio-switch suitable for E. coli was poorly characterized in Halomonas TD, so directly copying the thermosensitive bioswitch in E. coli to Halomonas TD is not a feasible method.

Solution

We have modified the thermosensitive bio-switch into two parts, that is, one plasmid originally carried all the related genes, and now we modified it to have two plasmids with different copy number carrying the regulatory protein and reporter gene respectively, and we successfully realized the application of the thermosensitve bio-switch in Halomonas TD. In addition to this, we mutated the regulatory protein and replace the promoter of regulator protein and reporter gene, to obtain a series of T-switches with a range of various expression intensity for broader application.

Conclusion

We selected several CI857 mutants that result in different expression intensity of the T-switch, and got several promoter-replaced groups that could bring positive change to the fold change and leakage of the fluorescence intensity between 30℃ and 37℃. Thus with all of the experiment results, we provide a series version of T-switch with different level of expression intensity that could be use for dynamic control in Halomonas TD. We uploaded parts related to these for future iGEMer to use them to realize their own goals, and you could find them here in our part page.

PHA series:P34HB

Problem

The current industrial-scale production method for P34HB mainly relies on microbial fermentation, which is primarily limited by the molar ratio of 4HB. Increasing the 4HB molar ratio results in a decrease in the melting temperature and apparent fusion heat of the copolymer, as well as an improvement in its resistance to polymer deformation. Therefore, enhancing the 4HB molar ratio is of great significance for the modification of P34HB. [1][2]

We introduced the 4hbd-sucD-ogd-orfZ gene cluster into pSEVA341 and pSEVA321 respectively to synthesize P34HB. Experimental results indicated that the 4HB molar ratio achieved by introducing the pSEVA321 was higher than that of the pSEVA341. However, the dry weight decreased somewhat compared to the wild TD80.

Solution

In order to increase the 4HB molar ratio while stabilizing cell dry weight, we investigated the significant impact of pSEVA341 on cell dry weight, and believed that the addition of chloramphenicol during the fermentation process affected cell growth. Consequently, we decided to adjust the screening pressure and knock out cysNC in Halomonas TD80, a gene encoding the key enzyme in the sulfate assimilation pathway, thereby blocking the supply of sulfur sources for methionine synthesis. Simultaneously, we incorporated cysNC into the pSEVA321 backbone to facilitate the screening of strains that had been transformed with the pSEVA321-194-4hbd-sucD-ogd-194-orfZ.

Refernce

[1]Chen, X.B., et al., Engineering Halomonas bluephagenesis TD01 for non-sterile production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate). BIORESOURCE TECHNOLOGY, 2017. 244: p. 534-541.

[2]Huong, K.H., C.H. Teh and A.A. Amirul, Microbial-based synthesis of highly elastomeric biodegradable poly(3-hydroxybutyrate-co-4-hydroxybutyrate) thermoplastic. INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES, 2017. 101: p. 983-995.

Amino acid derivative：Tyrian Purple

Problem

Tyrian purple is a precious dye with a long history. First extracted by the Phoenicians in the 16th century BC from the slime of sea snails, it was used in the ancient world to dye the clothing of elites and royalty due to its deep, vibrant purple color and fade-resistant properties. The manufacturing process of this dye is very complex and expensive, requiring large quantities of sea snails, so it is expensive, sometimes even more valuable than gold. In modern times, the status quo of tyrian purple production has changed. Due to the scarcity of its natural sources and the unsustainability of the production process, people began to look for alternative production methods. For example, one company uses a microbial fermentation process to produce tyrian purple, which not only improves production efficiency, but also reduces environmental impact. In addition, there are some patented technologies, such as the biosynthesis method of Zeno (Suzhou) Biotechnology Co., Ltd., which synthesizes tyrian purple dye in E. coli through genetic engineering technology, which is conducive to alleviating the pressure of chemical production and enhancing the environmental protection of the production process. However, prior to starting our project, there was no literature on the production of tyrian purple using Halomonas TD with CDE as carbon source.

Solution

After reading an amount of papers, we decided to introduce some pathway related genes into Halomonas TD to use trptophan to produce tyrian purple.

Because Stth and tnaA both could catalyze trptophan, it would create a large amount of by-products instead of our target product Tyrian purple. Thus, we used the thermal regulation that we've created above to separate the action of these two enzymes.During our experiments,we surprisingly found that other scientists thought about this method too[5]. We were excited to see that it's a right direction and we brought up thoughts that was same with some real scientists.

Conclusion

After detecting the content of 6-Br-Trp and tyrian purple in the fermentation sample, we were surprised to find that their content increased greatly.

Reference

[1] Lee, J., Kim, J., Kim, H., Park, H., Kim, J. Y., Kim, E. J., Yang, Y. H., Choi, K. Y., & Kim, B. G. (2022). Constructing multi-enzymatic cascade reactions for selective production of 6-bromoindirubin from tryptophan in Escherichia coli. Biotechnology and Bioengineering, 119(10), 2938-2949.

[2] Yishai, O., Bouzon, M., Döring, V., & Bar-Even, A. (2018). In vivo assimilation of one-carbon via a synthetic reductive glycine pathway in Escherichia coli. ACS Synthetic Biology, 7(9), 2023-2028.

[3] Du, J., Yang, D., Luo, Z. W., & Lee, S. Y. (2018). Metabolic engineering of Escherichia coli for the production of indirubin from glucose. Journal of Biotechnology, 267, 19–28.

[4] Lee, J., Kim, J., Song, J. E., Song, W.-S., Kim, E.-J., Kim, Y.-G., Jeong, H.-J., Kim, H. R., Choi, K.-Y., & Kim, B.-G. (2021). Production of Tyrian purple indigoid dye from tryptophan in Escherichia coli. Nature Chemical Biology, 17(1), 104–112.

[5] Li, F., Chen, Q., Deng, H., Ye, S., Chen, R., Keasling, J. D., & Luo, X. (2024). One-pot selective biosynthesis of Tyrian purple in Escherichia coli. Metabolic Engineering, 81, 100–109.

Proteins SOD and phaP

Problem

After domestication, Halomonas can utilize acetate, a product of electrochemistry, as a carbon source for biomass production. However, the natural metabolic pathways within Halomonas have a limited capacity to produce a diverse range of metabolites, particularly those with high added value.

Solution

To address this limitation, we introduced synthetic pathways for the production of heterologous proteins Meta, Amalase, SOD, and phaP, and increased the concentration of urea, an inexpensive nitrogen source, in the culture medium.

Conclusion

The fermentation broth was centrifuged to obtain the supernatant and precipitate after cell disruption, and they were subjected to SDS-PAGE, and the following results were obtained after shooting: from left to right, the protein results obtained by fermentation under two carbon sources, sodium acetate and glucose, were phaP, TD80 control, SOD, Amalase, Meta and sodium acetate and glucose. It can be seen that compared with the control group, the TD80 after the introduction of protein synthesis pathway genes can produce phaP and SOD proteins, and the protein produced with glucose as the carbon source is more than that with sodium acetate as the carbon source.

Locus screening program

Problem

For industrial production hosts, genomic expression offers advantages over plasmid-based systems, such as improved genetic stability. However, a key challenge remains in identifying optimal locations for integrating synthetic pathways within the E. coli MG1655 genome, which contains around 4,000 genes (4.14 Mbp). Gene expression is location-dependent (Sousa et al., 1997)[1], as nearby genes tend to show correlated transcriptional activity. few studies have systematically organized methods for selecting chromosome positions that optimize gene expression efficiency without disrupting natural metabolism. traditional locus screening methods are often inefficient, with the goal being to select appropriate-length loci that lack functional sequences while exhibiting high transcriptional activity. Additionally, locus screening is time-consuming, with each parameter iteration taking approximately 14 hours.

Solution

We developed a locus screening program that identifies the desired loci on the genome through a five-step filtering process. This program integrates an improved KMP algorithm and the VWZ-curve promoter prediction method, significantly enhancing both efficiency and accuracy.[2]

Conclusion

The initial screening of genomic loci takes about 2 hours, primarily due to the need to search for transcription factor binding sites and promoters across the genome. After the first round, this information is stored, allowing subsequent parameter iterations to take approximately 10 minutes per round, continuing until around 10 suitable loci are identified. This new method improves efficiency by over 80% compared to previous screening methods, with some genomes showing more than a 90% increase in efficiency. The selected loci have been experimentally validated, resulting in the identification of multiple sites with over a tenfold increase in expression.

Reference

[1]. Sousa, C., V. DeLorenzo and A. Cebolla, Modulation of gene expression through chromosomal positioning in Escherichia coli. MICROBIOLOGY-SGM, 1997. 143: p. 2071-2078.
[2]. Song, K., Recognition of prokaryotic promoters based on a novel variable-window Z-curve method. NUCLEIC ACIDS RESEARCH, 2012. 40(3): p. 963-971.

Motrol

Problem

Traditional genome-scale metabolic models (GEMs) are limited in their ability to accurately predict certain metabolic phenotypes due to the absence of enzymatic kinetic information and constraints on protein resources. These models often have an overly large solution space, reducing their predictive power. Earlier versions of enzyme-constrained GEMs (ec-GEMs) improved upon this by incorporating enzyme turnover numbers (kcat) and molecular weights, but their performance was still hindered by low kcat data coverage, increased computational complexity, and potential numerical issues when predicting low enzyme activities. Additionally, integrating proteomics data can lead to over-constrained models due to uncertainties in enzymatic kinetics, thereby reducing the feasibility of the model.[1][2][4]

Solution

We introduced GO-term analysis to apply partitioned constraints on enzymes, categorizing them based on the function of the reactions they catalyze. For each term, we impose limits on the total enzyme quantity and individual enzyme amounts within a specified fluctuation range.[2] This approach balances precision and efficiency by reducing the overall computational load while constraining the solution space.We introduced deep learning-based tools, ProtParam and DL-kcat, to predict various enzyme parameters (kcat, molecular weight, half-life, hydrophilicity) and normalized them. [1]These parameters were integrated into a single parameter, fakeM, which, along with its inverse, describes the enzyme's demand for total protein resources and the amount of protein required for unit catalytic efficiency. This optimization approach addresses issues related to missing enzyme kinetic and physicochemical data, as well as resolving solutions that fall below the numerical tolerance of linear programming solvers.[3]

Conclusion

The figure shows experimental data (exp) from a single-batch fermentation using 4-stage feeding solutions in a 7 L bioreactor over a 52-hour fed-batch fermentation. The model represents the predicted results using the GO-term based ec-GEM model. The model demonstrates strong predictive performance, with an R^2 value of 0.85. The Percentage Root Mean Square Error (RMSE) between the experimental data and the model's predictions is 13.17%. It is important to note that due to the limited available data, the generalizability of the model's accuracy still requires further validation. However, the results obtained so far demonstrate the potential of this model.

Reference

[1]. Chen, Y., et al., Reconstruction, simulation and analysis of enzyme-constrained metabolic models using GECKO Toolbox 3.0. NATURE PROTOCOLS, 2024. 19(3): p. 629-667.
[2]. Zhang, L.Z., et al., A long-term growth stable Halomonas sp. deleted with multiple transposases guided by its metabolic network model Halo-ecGEM. METABOLIC ENGINEERING, 2024. 84: p. 95-108.
[3]. Bi, X.Y., et al., A Multi-Omics, Machine Learning-Aware, Genome-Wide Metabolic Model of Bacillus Subtilis Refines the Gene Expression and Cell Growth Prediction. ADVANCED SCIENCE, 2024.
[4]. Moreno-Paz, S., et al., Enzyme-constrained models predict the dynamics of Saccharomyces cerevisiae growth in continuous, batch and fed-batch bioreactors. MICROBIAL BIOTECHNOLOGY, 2022. 15(5): p. 1434-1445.

Our products are used in bio-based materials, medical beauty skin care, medical health, biomass dyes and other fields.
Our raw materials (CDE) are provided by our partner company PoweredCarbon, and we integrate the production pipeline with the partner company by combining OEM and CRO models for downstream industrial production.

According to the statistics of downstream factories, we calculated the net carbon emissions for the production line of PHAs, and concluded that for every 1000t-2000t PHA produced, the annual net carbon emissions are 3134.5t-10670t, which greatly reduces the carbon emissions compared with traditional biological/chemical manufacturing.
In the future, our team is also expected to ask Professor Xiaonan WANG of Tsinghua University to calculate the carbon emissions of the production line in detail, and get more credible evidence to prove that our project and ideas are completely environmentally friendly.