N-Terminal Truncated Sialyltransferase for Higher 3'-Sialyllactose Yield
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Solution Overview
Problem
Existing methods for producing 3′-sialyllactose using recombinant microorganisms face challenges in achieving high productivity due to the limited activity of α2,3-sialyltransferase, which transfers a sialic acid residue to lactose, and the specific regions affecting this activity are not well understood.
Innovation Solution
A microorganism with a protein sequence deleted by 20 to 37 amino acids at the N-terminus, such as SEQ ID NO: 2, exhibits enhanced α2,3-sialyltransferase activity, leading to improved 3′-sialyllactose productivity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If a recombinant microorganism is used to produce 3′-sialyllactose, then the production method is economically rational, but the productivity is limited due to the activity of α2,3-sialyltransferase
Solution Approach 1:
The invention changes the parameter of the protein sequence by deleting 20-37 amino acids from the N-terminus of α2,3-sialyltransferase, which optimizes the enzyme's catalytic activity and substrate binding affinity, thereby significantly improving 3′-sialyllactose productivity
Solution Approach 2:
The invention extracts and removes the N-terminal region (20-37 amino acids) of the α2,3-sialyltransferase protein that was identified as affecting its activity, resulting in a truncated enzyme with enhanced functionality for 3′-sialyllactose production
2Reliability
If the N-terminus of the amino acid sequence is cleaved to express sialyltransferase, then the expression and activity change, but it is not clarified which region is specifically cleaved to obtain useful activity
Solution Approach 1:
The invention segments the N-terminal region into specific deletion ranges (20-37 amino acids) and systematically tests each segment to identify the precise region whose removal optimizes enzyme activity, thereby filling the knowledge gap on specific cleavage requirements
Solution Approach 2:
The invention changes the protein sequence parameter by deleting specific N-terminal amino acid residues (20-37 positions), which optimizes the enzyme's structural and functional properties, clarifying which region must be removed for useful activity
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 modified microorganism significantly enhances 3′-sialyllactose production by optimizing the activity of the α2,3-sialyltransferase, allowing for higher yields of this important oligosaccharide.
Implementation Method 1
transferring a sialic acid residue of the CMP-sialic acid to lactose added from the outside using an α2,3-sialyltransferase to produce 3′SL
Implementation Method 2
synthesizing cytidine-5′-monophosphate (hereinafter, also referred to as 'CMP')-sialic acid from an inexpensive carbon source such as glycerol or glucose within cells
Data Source
AI summary
The present invention relates to a microorganism having an enhanced activity of a protein consisting of an amino acid sequence in which N-terminus 20 to 37 amino acid residues in the amino acid sequence represented by SEQ ID NO: 2 are deleted, where the microorganism has improved productivity of 3′-sialyllactose as compared with that of a parent strain.