Alpha-2,6-Sialyltransferase Production of 6′Sialyllactose
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Solution Overview
Problem
The production of sialylated di- and/or oligosaccharides, such as 6′sialyllactose, is challenging due to difficulties in controlling stereochemistry, forming specific linkages, and availability of feedstocks, leading to the need for alternative production methods like metabolic engineering of microorganisms.
Innovation Solution
A newly identified alpha-2,6-sialyltransferase is used in metabolically engineered cells to produce 6′sialylated disaccharides and/or oligosaccharides, preferably 6′sialyllactose, through a pathway that enables efficient production, purification, and high yields.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If chemical or (chemo-) enzymatic synthesis methods are used to produce sialylated di- and/or oligosaccharides, then the production process can be performed, but control of stereochemistry, formation of specific linkages, and availability of feedstocks becomes extremely difficult
Solution Approach 1:
The patent replaces chemical synthesis methods with biological synthesis using metabolically engineered microorganisms. The sialyltransferase enzyme catalyzes the formation of specific alpha-2,6-sialyl linkages with high stereospecificity, eliminating the need for complex chemical protection/deprotection steps and achieving precise control of stereochemistry and linkage formation through enzymatic specificity rather than chemical reagents
Solution Approach 2:
The patent employs metabolically engineered microorganisms that possess endogenous pathways for synthesizing sialic acid and related sugars. The engineered cells self-produce the necessary feedstocks (CMP-NeuNAc, lactose, etc.) through their metabolic pathways, eliminating the need for external supply of complex feedstocks and enabling autonomous production of 6′sialyllactose
2Productivity
If traditional chemical synthesis methods are used, then production can proceed, but time consumption and production costs increase significantly
Solution Approach 1:
The patent implements continuous production through metabolically engineered microorganisms that can continuously synthesize 6′sialyllactose as they grow and metabolize. The engineered cells maintain continuous enzymatic activity for sialylation, converting substrates to product throughout the cultivation period, eliminating the need for repeated batch processing and isolation steps required in chemical synthesis
Solution Approach 2:
The patent performs preliminary metabolic engineering of the microorganism to establish complete biosynthetic pathways before production. The cells are pre-engineered with necessary enzymes and pathways for sialic acid synthesis, CMP-NeuNAc formation, and sialyltransferase activity, so that once cultivation begins, production proceeds immediately without needing to assemble complex reagents and conditions
3Productivity
If chemical synthesis methods are employed, then sialylated di- and/or oligosaccharides can be produced, but production costs become prohibitively high
Solution Approach 1:
The patent uses simple, inexpensive microorganisms (such as E. coli or yeast) as production platforms instead of complex chemical reagents and catalysts. The microbial cells are grown on cheap carbon sources and nutrients, and their rapid growth and reproduction provide continuous production capacity at low operational costs, making the process economically viable for large-scale production
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 method allows for the efficient, time- and cost-effective production of high amounts of 6′sialylated disaccharides and/or oligosaccharides, addressing the challenges of traditional synthesis methods.
Implementation Method 1
Sialyltransferases are distinguished due to the glycosidic linkages that they form, e.g., into a-2,3-, a-2,6- and a-2,8-sialyltransferases. All of these sialyltransferases transfer the sialic acid residue from cytidine 5'-monophosphate sialic acid (e.g., CMP-NeuNAc) to a variety of acceptor molecules
Data Source
AI summary
Newly identified alpha-2,6-sialyltransferases are disclosed herein. The disclosure also provides for production of sialylated di- and/or oligosaccharides and relates to the use of the sialyltransferases in such methods and cells. The disclosure is also in the technical field of synthetic biology and metabolic engineering. More particularly, the disclosure is in the technical field of metabolically engineered cells and use of the cells in a cultivation or incubation. The disclosure describes a metabolically engineered cell and a method by cultivation or incubation with the cell for production of 6′sialylated disaccharide and/or 6′sialylated oligosaccharide. Furthermore, the disclosure provides for purification of the 6′sialylated disaccharide and/or 6′sialylated oligosaccharide from the cultivation or incubation.