Our iGEM project focuses on developing microbial strains capable of producing glycoproteins decorated with glycan structures, particularly Man3GlcNAc2, which is essential for therapeutic proteins. We have worked on both yeast and E. coli systems for glycoprotein production, as well as introducing new strategies to enhance glycosylation efficiency using archaeal enzymes. We are contributing several engineered parts, including oligosaccharyltransferase and glucocerebrosidase genes that can be helpful to other iGEM teams, which have been optimized for expression in various microbial hosts. This collection of parts will be useful for research into glycosylation and the production of therapeutic enzymes.
We are in the process of engineering both yeast and E. coli to produce glycoproteins with the Man3GlcNAc2 structure. Yeast strains will be optimized to limit excessive glycosylation, while E. coli has been modified to express a functional glycosylation pathway, providing a cost-effective alternative for the production of glycoproteins.
Both yeast and E. coli have been used to express GCase, an enzyme used for Gaucher disease treatment. Proper glycosylation in yeast systems ensures the stability and functionality of GCase. Meanwhile, our bacterial system leverages fast growth and lower costs for industrial-scale production of the enzyme.
We propose the use of archaeal oligosaccharyltransferase to create more efficient glycosylation systems in bacteria. These enzymes are known for their robustness under extreme conditions, making them ideal candidates for enhancing glycosylation processes in microbial systems.
We have described the parts for other iGEM teams to use in the glycosylation pathway. With all the proteins documented, it becomes easier to build more efficient glycosylation methods using the Gibson Assembly cloning technique.
| Name | Nickname | Type | Description | Specie | Length (bp) |
|---|---|---|---|---|---|
| BBa_K5428000 | Gout-A | Basic | Oligosaccharyltransferase | S. acidocaldarius | 2306 |
| BBa_K5428001 | Gout-B | Basic | Oligosaccharyltransferase | L. major | 2615 |
| BBa_K5428002 | Gout-C | Basic | Glucocerebrosidase | E. coli | 1621 |
| BBa_K5428003 | Gout-D | Basic | Glucocerebrosidase | S. cerevisiae | 1602 |
| BBa_K5428004 | Gout-E | Basic | Oligosaccharyltransferase | M. fervidus | 2624 |
| BBa_K5428005 | Gout-F | Basic | Oligosaccharyltransferase | M. voltae | 2815 |
| BBa_K5428006 | Gout-G | Basic | Oligosaccharyltransferase | T. brucei | 4934 |
| BBa_K5428016 | Gout-H | Composite | Operon of Glycosylation | _ | 7120 |
| BBa_K5428010 | Gout-I | Basic | Glycosyltransferase | S. cerevisiae | 609 |
| BBa_K5428011 | Gout-J | Basic | Glycosyltransferase | S. cerevisiae | 712 |
| BBa_K5428012 | Gout-K | Basic | Glycosyltransferase | S. cerevisiae | 1350 |
| BBa_K5428013 | Gout-L | Basic | Glycosyltransferase | S. cerevisiae | 1512 |
| BBa_K5428014 | Gout-M | Basic | Phosphomannomutase | S. cerevisiae | 1437 |
| BBa_K5428015 | Gout-N | Basic | Phosphomannomutase | S. cerevisiae | 1371 |
The oligosaccharyltransferase (OST) from Sulfolobus acidocaldarius emerged as our key candidate in our bacterial glycoprotein production system. Archaea, known for synthesizing structurally distinct glycans, possess OSTs with broader sugar substrate specificity compared to their eukaryotic counterparts. We focused on archaeal OSTs that process glycans structurally similar to those found in humans, particularly those bearing a GlcNAc moiety as the lipid-linked sugar. Among the three selected species, S. acidocaldarius synthesizes a glycan closely resembling the human Man₃GlcNAc₂ core, with only minor variations. Notably, S. acidocaldarius incorporates a sulfoquinovose residue on the second GlcNAc in its Man₂GlcNAc₂ backbone. Despite this difference, we believe that such structural modifications might not affect the biological activity of our glycoproteins.
1. Modeling Stability of OST PglB with Engineered Sequons Our project leveraged computational modeling to evaluate the structural stability of the OST PglB enzyme when glycosylation sequons were modified in E. coli. The model demonstrated that PglB retains stability despite these modifications, ensuring that the system can reliably facilitate glycoprotein production. This finding is crucial for future teams seeking to engineer glycosylation pathways in bacterial systems, as it confirms the robustness of PglB in handling sequence alterations without compromising function.
2. Bioinformatics: Identifying Sec-Dependent Glycosylation Targets We performed a detailed bioinformatics study to map all E. coli proteins secreted through the Sec pathway that could potentially be glycosylated by our engineered OST. By identifying these glycosylation targets, we’ve created a valuable database for future teams interested in glycosylation. This resource can be used as a starting point for further experimental work, facilitating the design of glycosylated proteins and reducing time spent on target identification. 3. Engineering a Comprehensive Glycosylation Operon We successfully engineered a glycosylation operon containing four key glycosyltransferases from yeast responsible for assembling the glycan chain. To ensure efficient production of the sugar precursor UDP-mannose (UDP-Man), we also incorporated the manB and manC genes. This operon is designed for flexibility, with regions for homology-directed integration into the IS5 intergenic site of E. coli via the lambda red system. Additionally, the operon contains a selectable marker for kanamycin resistance, which can be excised using FRT recombination sites, leaving the system marker-free for further genetic engineering. For increased versatility, we also designed primers to amplify the operon for cloning into standard vectors, such as pRSFDuet-1, enabling its use in different bacterial strains and experimental setups. 4. Pioneering the Use of Archaeal OSTs in Bacteria Our team is the first to propose the use of archaeal oligosaccharyltransferases (OSTs) in bacterial glycosylation pathways. Archaeal OSTs have shown unique substrate specificities and efficiencies, making them attractive candidates for expanding the range of glycans that can be transferred in bacterial hosts. By introducing archaeal OSTs into E. coli, we aim to broaden the toolkit available for protein glycosylation, paving the way for new applications in synthetic biology and biopharmaceutical production. 5. Production of Therapeutic GCase in Yeast and Bacteria Glucocerebrosidase (GCase) is a therapeutic enzyme used in the treatment of Gaucher's disease, and our project explored its production in both yeast and bacterial systems. By engineering yeast and E. coli to produce GCase with the necessary glycosylation patterns, we contribute to the development of more affordable and accessible therapeutic proteins. Our work provides a foundation for future teams to optimize and scale up the production of glycosylated enzymes for medical use. 6. Employing Low-Order Eukaryotic OSTs for Bacterial Glycosylation In addition to using archaeal OSTs, we explored OST enzymes from low-order eukaryotes, specifically Leishmania major and Trypanosoma brucei. These organisms have OSTs that are simpler than their counterparts in higher eukaryotes, yet still capable of glycosylating proteins in a bacterial context. By incorporating these OSTs into E. coli, we open new possibilities for glycoengineering in bacteria, allowing for the transfer of complex glycans that were previously difficult to achieve in prokaryotic systems. 7. Fine-Tuning Glycosylation with Alpha-1,2-Mannosidase To precisely control the structure of the glycan chains, we introduced Trichoderma reesei alpha-1,2-mannosidase into our system. This enzyme allows us to perform sugar trimming, reducing the complexity of the glycan and controlling its final structure. This step is crucial for applications that require specific glycan patterns, as it enables the fine-tuning of glycosylation profiles in engineered proteins. 8. Engineering Central Glycan Structures: Man3GlcNAc2 The core achievement of our project is the engineering of E. coli and yeast to produce the central glycan structure Man3GlcNAc2. This sugar structure is a key component of many human glycans and serves as a foundational building block for more complex glycans. By engineering both yeast and bacteria to produce this structure, we have created a platform that future teams can modify to generate a wide range of human glycans. This system has the potential to revolutionize the production of glycoproteins in microbial systems, enabling the synthesis of therapeutic glycoproteins with human-like glycosylation patterns. 9. Future Applications and Modularity Our project lays the groundwork for future teams to take glycoengineering in new directions. The flexibility of our glycosylation operon and the introduction of diverse OSTs and enzymes offer modular tools that can be adapted to various experimental needs. Whether it's producing therapeutic proteins with tailored glycosylation, studying glycan function, or developing new synthetic biology applications, the components we've developed are designed to be accessible and easy to modify.