LacY Permease Variants for Oligosaccharide Production
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Solution Overview
Problem
Microbial cells face lactose killing when transferred from a lactose-limited to a lactose-containing growth medium, leading to rapid internalization of lactose and decreased productivity in the fermentative production of oligosaccharides, particularly those with a lactose moiety at their reducing end, such as Human Milk Oligosaccharides.
Innovation Solution
Genetically engineered bacterial cells with variants of the E. coli lactose permease LacY that exhibit reduced transport activity for lactose, reducing lactose internalization and making them less sensitive to lactose killing, thereby enhancing the production of oligosaccharides by maintaining a stable intracellular lactose pool for biosynthesis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If microbial cells are transferred from lactose-limited to lactose-containing growth medium to produce oligosaccharides, then oligosaccharide production is enabled, but lactose killing occurs leading to cell death and decreased productivity
Solution Approach 1:
The patent applies parameter changes by modifying the transport activity of the lactose permease LacY through amino acid substitutions (e.g., L292R, L293R, L294R, L293H). These mutations reduce the transport activity of LacY, thereby reducing the rate of lactose internalization and preventing the harmful effects of lactose killing while still allowing sufficient lactose uptake for oligosaccharide biosynthesis.
2Quantity of substance
If lactose transport activity is increased to provide sufficient substrate for oligosaccharide biosynthesis, then substrate availability is improved, but lactose killing is exacerbated leading to rapid cell death
Solution Approach 1:
The patent modifies the transport parameter of lactose permease by introducing specific amino acid mutations that reduce transport activity. This creates an optimized parameter range where lactose internalization is sufficient for biosynthesis but below the threshold that triggers lactose killing, thereby resolving the contradiction between substrate availability and cell survival.
Solution Approach 2:
The patent ensures continuous oligosaccharide production by maintaining a stable, reduced-rate lactose internalization that avoids the oscillating cell death and regeneration cycles associated with wild-type LacY. The mutated permease provides sustained, controlled lactose uptake that supports continuous biosynthesis without triggering harmful feedback effects.
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
The use of LacY variants in microbial cells results in increased resistance to lactose killing, improved growth kinetics, and higher productivity of oligosaccharides, including Human Milk Oligosaccharides, by reducing lactose internalization and maintaining a stable intracellular lactose pool for biosynthesis.
Implementation Method 1
The E. coli lactose permease LacY is a membrane protein that transports lactose across the cell membrane
Implementation Method 2
variants of the E. coli lactose permease LacY which possess a reduced transport activity for lactose as compared to the E. coli wild-type lactose permease LacY
Implementation Method 3
The lacZ gene encodes a β-galactosidase (LacZ) which hydrolyzes lactose
Data Source
AI summary
Disclosed are genetically engineered microbial cells for the production of an oligosaccharide of interest, wherein said microbial cells possess a variant of the E. coli lactose permease LacY which exhibits a reduced transport activity for lactose, and methods for producing an oligosaccharide by using said genetically engineered microbial cells.