Microbial Cell Producing Oligosaccharides Without Exogenous Lactose
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Solution Overview
Problem
The existing fermentative production processes for human milk oligosaccharides (HMOs) face challenges such as the rearrangement of lactose to lactulose during thermal treatment, leading to contamination with undesired by-products, and the high cost and potential contamination risks associated with using lactose derived from the dairy industry.
Innovation Solution
A genetically engineered microbial cell capable of producing lactose or an oligosaccharide with a galactose-β1,4-glucose moiety without exogenously added lactose, utilizing a mixed monosaccharide feedstock as the carbon and energy source, and expressing specific genes for glucose and fructose transport, UDP-galactose biosynthesis, and glycosyltransferase activities.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If lactose is used as substrate in fermentative production processes, then HMOs can be produced efficiently, but lactose rearranges to lactulose during thermal treatment causing contamination with undesired by-products
Solution Approach 1:
The invention extracts and eliminates the problematic step of exogenous lactose addition from the production process. By engineering the microbial cell to produce lactose endogenously through expressed genes (β-galactosidase, galactose-1-phosphate uridyltransferase, UDP-galactose 4-epimerase), the harmful thermal treatment and lactulose formation are avoided entirely, while maintaining efficient HMO production.
Solution Approach 2:
Instead of adding lactose externally and having it convert to HMOs, the invention inverts the approach by enabling the microbial cell to synthesize lactose internally from basic carbon sources (glucose, galactose) through expressed enzymatic pathways. This reversal eliminates the need for thermal treatment of exogenous lactose and prevents lactulose contamination.
2Productivity
If lactose derived from the dairy industry is used, then fermentative production can proceed, but there are high costs and potential contamination risks
Solution Approach 1:
The microbial cell is engineered to be self-sufficient in producing lactose through endogenous synthesis pathways. By expressing genes for β-galactosidase, galactose-1-phosphate uridyltransferase, and UDP-galactose 4-epimerase, the cell converts basic carbon sources (glucose, galactose) into lactose internally, eliminating dependence on external dairy-derived lactose and its associated contamination risks.
Solution Approach 2:
The invention introduces enzymatic intermediaries (expressed genes for lactose synthesis enzymes) that mediate the conversion of simple carbon sources into lactose within the cell. This enzymatic mediation replaces the need for direct use of dairy-derived lactose, ensuring product purity while maintaining production capability.
3Ease of manufacture
If exogenously added lactose is used as substrate, then HMO production can be achieved, but the process involves thermal treatment causing lactose rearrangement
Solution Approach 1:
The invention performs preliminary action by engineering the microbial cell to produce lactose endogenously before HMO synthesis occurs. The expressed enzymes (β-galactosidase, galactose-1-phosphate uridyltransferase, UDP-galactose 4-epimerase) continuously supply fresh lactose to the HMO production pathway, eliminating the need for subsequent thermal treatment that would cause rearrangement and compromise product integrity.
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 the efficient production of HMOs without the need for exogenous lactose, reducing contamination risks and production costs, while maintaining the integrity and quality of the desired oligosaccharides.
Implementation Method 1
possesses at least one glucose transporter for translocating glucose from the culture medium into the cytoplasm of the microbial cell
Implementation Method 2
a UDP-galactose biosynthesis pathway for intracellular biosynthesis of UDP-galactose
Implementation Method 3
at least one galactosyltransferase that is able to galactosylate free intracellular glucose to intracellularly produce lactose
Implementation Method 4
Fermentative production of oligosaccharides by total fermentation utilizing a mixed feedstock
Data Source
AI summary
Disclosed are genetically engineered microbial cells for the production of oligosaccharides comprising a galactose-β1,4-glucose moiety at their reducing end, wherein said microbial cells are able to produce said oligosaccharides in the absence of exogenously added lactose, and a method of producing said oligosaccharides using said microbial cells.