Complex HMO Fermentation With In-Medium Enzymatic Recycling
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Solution Overview
Problem
Existing methods for producing complex human milk oligosaccharides (HMOs) face challenges such as high costs, complex chemical processes, enzymatic reaction mixtures, and difficulties in exporting HMOs from cells, leading to excess lactose interference and purification issues.
Innovation Solution
A hybrid method combining fermentation and enzymatic reactions in the fermentation medium, using genetically modified cells with side-product recycling and transglycosidase enzymes to produce complex HMOs, reducing the need for pure starting oligosaccharides and minimizing lactose levels.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If enzymatic synthesis is used to produce complex HMOs, then the HMOs can be produced with specific structures, but the reaction produces a mixture of donor, acceptor, third oligosaccharide and side-products requiring extensive purification
Solution Approach 1:
The patent combines the enzymatic synthesis reaction and the purification step into a single integrated system by expressing both the transglycosidase enzyme and the glycosidase enzyme in the same host cell. This allows the reaction mixture to be processed in situ, converting side-products back into substrates and simplifying purification while maintaining structural specificity.
Solution Approach 2:
The host cell performs self-purification by using the co-expressed glycosidase enzyme to convert side-products (lactose) back into substrates (galactose and glucose) within the cell. This self-service mechanism reduces the need for external purification steps while maintaining high product specificity.
2Productivity
If separately produced and purified donor and acceptor substrates are used in enzymatic process, then the reaction can proceed efficiently, but the cost of the process increases
Solution Approach 1:
The host cell is engineered to perform multiple functions: producing the transglycosidase enzyme for synthesis, producing the glycosidase enzyme for side-product conversion, and metabolizing substrates. This multi-functionality eliminates the need for separate production and purification steps for donor and acceptor substrates, reducing costs while maintaining reaction efficiency.
Solution Approach 2:
The cell uses its own metabolic pathways to generate the required substrates (galactose and glucose) from side-products, eliminating the need for external provision of purified substrates. This self-service approach reduces manufacturing costs while maintaining productivity.
3Stability of the object's composition
If side-product (lactose) is released from donor substrate in enzymatic process, then the reaction equilibrium is maintained, but extensive purification is required to remove the large amount of lactose
Solution Approach 1:
The patent converts the harmful side-product (lactose) into a beneficial substrate by expressing glycosidase enzyme that hydrolyzes lactose back into galactose and glucose. This allows the side-product to be reused in the reaction, maintaining equilibrium while eliminating the need for extensive purification.
Solution Approach 2:
The system implements a feedback loop where the glycosidase enzyme continuously converts accumulated lactose side-products back into substrates, which are then reused by the transglycosidase reaction. This feedback mechanism maintains reaction equilibrium while minimizing side-product accumulation and purification needs.
4Productivity
If fermentation route is used for producing complex HMOs, then production can be scaled industrially, but the HMOs face challenges in exporting from the cell into the medium
Solution Approach 1:
The patent extracts the synthesis reaction from the cell interior to the cell exterior by using cell surface display or secretion of the transglycosidase enzyme. This allows the complex HMO synthesis to occur outside the cell where export is not an issue, while still using the cell's metabolic pathways for substrate provision and maintaining industrial scalability.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach allows for higher yields of complex HMOs with reduced costs and lactose levels, overcoming the challenges of enzymatic equilibrium and cell export issues, achieving nearly full conversion of substrates to desired oligosaccharides.
Implementation Method 1
an enzyme with transglycosidase activity to produce a third oligosaccharide
Implementation Method 2
cultivating a genetically modified cell capable of producing a first oligosaccharide in a culture medium which is supplied with a carbon source
Data Source
AI summary
The present disclosure relates to a method for producing oligosaccharides where the method combines a fermentation process and an enzymatic reaction in the medium of the fermentation, thereby enabling the formation of the oligosaccharide in the fermentation medium of an ongoing fermentation. The method is in particular suitable for the generation of complex oligosaccharides.