Modified Lactose Permease for Oligosaccharide Production
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Solution Overview
Problem
Current methods for producing lactose-containing oligosaccharides are not efficient, and there is a need for a more effective method to enhance productivity, particularly using microorganisms with mutant lactose permease or by modifying the lactose permease protein.
Innovation Solution
A microorganism with a modified lactose permease protein, where the amino acid residue at position 319 is replaced with L-aspartic acid or L-glutamic acid, or a homologous protein with at least 80% identity, is used to enhance the production of lactose-containing oligosaccharides through recombinant DNA transformation and incorporation into the chromosome.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional lactose permease is used in fermentative production, then the process is simple and cost-effective, but the productivity of lactose-containing oligosaccharide is insufficient
Solution Approach 1:
The patent applies parameter changes by modifying the amino acid sequence of lactose permease at specific positions (319, 320, 321) to alter its functional properties. By changing amino acid residues at these positions, the microorganism's ability to transport lactose is enhanced, thereby improving oligosaccharide productivity without requiring complete redesign of the production system
Solution Approach 2:
The patent implements local quality by making targeted modifications at specific positions (319-321) of the lactose permease protein rather than altering the entire protein structure. This localized modification approach allows precise optimization of lactose transport function while maintaining the overall stability and other functions of the permease protein
2Productivity
If lactose uptake is mediated by wild-type lactose permease, then the system is stable and well-characterized, but the ability to efficiently utilize lactose substrate is limited
Solution Approach 1:
The patent changes specific parameters of the lactose permease protein by substituting amino acid residues at positions 319, 320, and 321 with specific alternatives (e.g., position 319 with Asp, Glu, Lys, or Arg). These parameter changes enhance the protein's ability to transport lactose while the patent verifies that the modified protein maintains sufficient structural stability and function
Solution Approach 2:
The patent employs feedback by measuring and comparing the oligosaccharide production efficiency of modified microorganisms against the parent strain. The amino acid substitutions are selected and optimized based on observed performance feedback, where variants showing improved lactose utilization and oligosaccharide production are identified and further developed
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 significantly increases the efficiency of lactose-containing oligosaccharide production, specifically producing 2′-fucosyllactose, by improving the microorganism's ability to utilize lactose as a substrate, leading to higher yields compared to parent strains.
Implementation Method 1
it is known that the uptake of lactose from outside of microbial cells is mediated by lactose permease (Non-Patent Literature 3)
Data Source
AI summary
The purpose of the present invention is to provide a more efficient method for producing a lactose-containing oligosaccharide by fermentation production. According to the present invention, a microorganism capable of producing a protein having the activity of a lactose permease that is modified in such a manner that a specific amino acid residue is substituted by another amino acid residue is used, whereby it becomes possible to produce a lactose-containing oligosaccharide, e.g., 2'-fucosyllactose, more efficiently compared with a case where a microorganism capable of producing a protein having the activity of the lactose permease of the wild type is used.