This year, team SR-CHINA is dedicated to systematically addressing three core challenges in modern infant formula using synthetic biology: nutrient deficiency, protein indigestion, and lactose intolerance. We have engineered E. coli into a versatile "cell factory" capable of efficiently producing three key bioactive molecules: Human Milk Oligosaccharide (2'-FL), trypsin, and β-galactosidase. In total, we have contributed 7 new basic parts and 1 new composite part, and have provided a comprehensive new characterization for 1 existing part. These contributions span the fields of metabolic engineering, protein engineering, and enzyme functional validation.
Core Contributions
Basic Parts
- BBa_25NORE8B (manB, 1371 bp) – Encodes phosphomannomutase, which catalyzes the entry of metabolic precursors into the 2'-FL synthesis pathway.
- BBa_25E1V5RR (manC, 585 bp) – Encodes mannose-1-phosphate guanylyltransferase, used to synthesize the key intermediate GDP-mannose.
- BBa_255UMBLF (gmd, 1119 bp) – Encodes GDP-D-mannose-4,6-dehydratase, which directs mannose derivatives toward the fucose synthesis pathway.
- BBa_25QP8Z04 (fcl, 963 bp) – Encodes GDP-L-fucose synthase, responsible for generating the direct donor for fucosylation, GDP-L-fucose.
- BBa_25XTA19J (futC, 1095 bp) – Encodes a human-derived α-1,2-fucosyltransferase, which catalyzes the final reaction between lactose and GDP-fucose to produce 2'-FL.
- BBa_25FE8YUB (TRYP, 696 bp) – Encodes porcine trypsinogen. After in vitro refolding and activation, this zymogen is converted into highly active trypsin, which can be used to pre-hydrolyze large casein molecules in milk, improving their digestibility.
- BBa_25ZZXY0V (manA, 1,173 bp) – A coding gene sourced from E. coli BL21.
Composite Parts
- BBa_25DDSHWK (manB+manC+gmd+fcl+futC, Gold Medal Part) – A synthetic operon containing five key enzyme genes. This composite part was optimized through systematic Design-Build-Test-Learn (DBTL) cycles, including switching the host chassis and applying protein engineering to the key enzyme FutC (TrxA tag fusion), ultimately achieving efficient de novo synthesis of 2'-FL using only inexpensive glycerol and lactose as substrates.
Benefits for Future iGEM Teams
Our work not only aims to solve infant nutrition issues but, more importantly, provides the iGEM community with a set of fully validated, reusable tools and design principles:
- A Modular 2'-FL Synthesis Toolkit: Our Gold Medal Part (BBa_25DDSHWK) is a "plug-and-play" genetic device for 2'-FL synthesis. Future teams can use it directly to produce 2'-FL or adapt its fucose-synthesis module (manB-manC-gmd-fcl) to synthesize other fucosylated compounds.
- A Key Case Study on Chassis Optimization: We demonstrated that moving the synthesis pathway from E. coli BL21(DE3) to a lacZ-deficient strain, DH5α, increased 2'-FL yield by ~19-fold. This provides a powerful, quantifiable lesson for all future projects using lactose as a substrate on how to eliminate substrate competition by carefully selecting or engineering the host.
- A Validated Strategy for Enhancing Heterologous Protein Function: We showed that adding a TrxA solubility tag to the human-derived FutC enzyme boosted final production by 56%. Our TrxA-FutC fusion gene part offers a validated solution for future teams struggling with the expression of eukaryotic or aggregation-prone proteins in prokaryotic systems.
- A Complete Workflow for Industrial Enzyme Production: Our characterization of trypsinogen (TRYP) and β-galactosidase (LacZ) demonstrates a complete workflow from gene design, codon optimization, inclusion body expression, and protein refolding/activation to functional validation. This serves as a detailed experimental roadmap for future teams aiming to produce and apply industrial enzymes.
- In-depth Characterization of an Existing Part: We performed a comprehensive "gene-to-protein-to-function" three-step validation of the existing part BBa_I732005 (LacZ), greatly enriching its characterization data and making it more reliable and accessible for future teams.
In summary, our project transcends a single application. It provides the broader synthetic biology community with valuable genetic parts, design strategies, and optimization experience, perfectly aligning with iGEM's core vision of "getting, giving, and sharing."
