Engineered Bacterial Strains for Low-Byproduct HMO Production
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Solution Overview
Problem
Existing methods for producing oligosaccharides, such as Human Milk Oligosaccharides (HMOs), face challenges in reducing the formation of unwanted by-products like phosphoglycerol and phosphoethanolamine-conjugated oligosaccharides, which complicate purification and increase production costs due to the need for additional purification steps.
Innovation Solution
Genetically engineering bacterial cells to delete or render dysfunctional the genes encoding phosphoglycerol transferase I and II, phosphoethanolamine transferase, and OPG biosynthesis protein C, thereby preventing the formation of these by-products and optimizing energy expenditure for increased production of desired oligosaccharides.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If the osmoregulated periplasmic glucan biosynthetic machinery is left intact, then the cell can maintain normal osmotic regulation and produce periplasmic oligosaccharides, but unwanted by-products like phosphoglycerol and phosphoethanolamine-conjugated oligosaccharides are formed, complicating purification
Solution Approach 1:
The patent applies the extraction principle by deleting specific genes (opgB, opgE, opgC) from the bacterial genome that encode enzymes responsible for conjugating phosphoglycerol, phosphoethanolamine, and succinyl groups to oligosaccharides. This removes the harmful by-product formation capability while preserving the core OPG biosynthetic machinery for maintaining osmotic regulation.
Solution Approach 2:
The patent segments the OPG biosynthetic machinery into essential functions (osmotic regulation) and non-essential functions (by-product formation). By selectively deleting only the genes responsible for harmful conjugations (opgB, opgE, opgC) while keeping other OPG-related genes intact, the system maintains osmotic regulation capability while eliminating by-product formation.
2Manufacturing precision
If additional purification steps are added to remove by-products, then the purity of desired oligosaccharides increases, but production costs and process complexity increase
Solution Approach 1:
The patent applies preliminary action by preventing by-product formation at the source through genetic modification of the biosynthetic machinery. By deleting the genes encoding phosphoglycerol transferase, phosphoethanolamine transferase, and succinyl transferase, the cell cannot produce harmful by-products during oligosaccharide synthesis, eliminating the need for subsequent purification steps.
3Object-generated harmful factors
If metabolic energy is redirected away from OPG synthesis, then by-product formation decreases, but the cell's ability to maintain osmotic regulation may be compromised
Solution Approach 1:
The patent extracts only the harmful enzymatic activities (phosphoglycerol transferase, phosphoethanolamine transferase, succinyl transferase) from the OPG biosynthetic pathway while preserving the core glucan synthesis machinery. This selective removal allows metabolic energy to be redirected away from by-product formation without compromising the essential osmotic regulation function.
Data Source
AI summary
The present disclosure relates to the production of oligosaccharides, especially Human milk Oligosaccharides (HMOs) using a genetically engineered cell which has decreased or total loss of function of phosphoglycerol transferase I and II and/or phosphoethanolamine transferase and/or glucans biosynthesis protein C to reduce oligosaccharide by-products and/or increase oligosaccharide production.