Mutated Chitin Synthase for Controlled Oligosaccharide Production
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Solution Overview
Problem
Current methods for producing chitin oligosaccharides with defined architecture and high yield are inefficient, resulting in complex mixtures that require extensive purification, leading to high production costs and limited applications due to the inability to produce chitin oligosaccharides with specific degrees of polymerization effectively.
Innovation Solution
Engineered chitin oligosaccharide synthases are used to produce chitin oligosaccharides with specific degrees of polymerization by site-directed engineering of the enzyme activity, allowing for controlled elongation rounds to yield chitin oligosaccharides with degrees of polymerization of four, five, six, or seven, thereby increasing the percentage of higher DP fractions and improving product specificity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If random chemical or enzymatic depolymerization of chitin is used, then chitin oligosaccharides can be produced, but complex mixtures with undefined architecture are obtained requiring extensive purification
Solution Approach 1:
Instead of breaking down chitin polysaccharides into oligosaccharides (depolymerization), the invention uses chitin oligosaccharide synthases to build up oligosaccharides from monomeric units (UDP-GlcNAc). This synthetic approach allows controlled formation of specific DP oligosaccharides with defined architecture, eliminating the mixture problem inherent in depolymerization methods
Solution Approach 2:
The invention modifies the parameters of the synthase enzyme through site-directed mutagenesis, specifically changing amino acid residues in the transmembrane domain (e.g., R349E mutation). These parameter changes in enzyme structure control the elongation rounds and product release, enabling selective production of specific DP oligosaccharides (DP4, DP5, DP6, DP7) with high manufacturing precision
2Manufacturing precision
If extensive purification is performed to obtain pure chitin oligosaccharides, then product purity increases, but production costs increase enormously
Solution Approach 1:
The engineered synthase enzyme performs preliminary action by selectively producing pure oligosaccharides of specific DP directly during the synthesis process. The enzyme's modified transmembrane domain controls chain elongation and release to yield predominantly one DP product, eliminating the need for subsequent extensive purification steps and reducing production costs
3Manufacturing precision
If native NodC enzymes are used, then short soluble chitooligosaccharides with DP 3-5 are produced, but oligosaccharides with higher DP (6-7) cannot be produced effectively
Solution Approach 1:
The invention changes the parameters of the NodC enzyme by introducing specific mutations in the transmembrane domain (e.g., R349E, R353E, R355E, R358E). These parameter changes extend the enzyme's capability to perform more elongation rounds, enabling production of oligosaccharides with higher DP (6-7) while maintaining control over product specificity
Solution Approach 2:
The engineered synthase achieves multi-functionality by being able to produce a range of specific DP oligosaccharides (DP4, DP5, DP6, DP7) through controlled elongation rounds. The enzyme can be tuned to produce different DP products by adjusting the mutation type, making it a versatile tool for producing various oligosaccharide lengths
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 enables the production of specific chitin oligosaccharides with increased purity and controlled DP, reducing production costs and expanding their applications in various industries by eliminating batch-to-batch variability and improving product specificity.
Implementation Method 1
CHSs utilize the nucleotide sugar donor UDP-GlcNAc and transfer the α-linked GlcNAc sugar in an inverting mechanism onto the non-reducing end of the growing acceptor oligosaccharide
Implementation Method 2
The size of the channel is decreased due to the presence of aromatic residues, enabling important C—H-π interactions and hydrogen bonds interactions
Implementation Method 3
The size of the channel is decreased due to the presence of aromatic residues, enabling important C—H-π interactions and hydrogen bonds interactions
Implementation Method 4
Of specific importance is Trp383 (W in the QxxRW motif), enabling a strong carbohydrate-π stacking interaction and stabilizing translocation
Data Source
AI summary
The present invention relates to mutated chitin oligosaccharide synthases and their usage to produce molecules having useful features. More specifically, the present invention discloses the usage of engineered microorganisms expressing a mutated chitin oligosaccharide synthase to produce chitin oligosaccharides having a degree of polymerization of four, five, six or seven.