LNTII Production via Heterologous Transport and YdeA Extraction
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Solution Overview
Problem
Current methods for producing lacto-N-triose II (LNTII) and oligosaccharides with an LNTII skeleton face challenges due to the lack of effective transport proteins in various Escherichia coli strains, and there is a need for efficient methods to produce high-purity LNTII crystals.
Innovation Solution
The use of microorganisms with enhanced activity of YdeA or MdfA proteins improves the productivity of LNTII, while microorganisms with reduced activity of these proteins enhance the production of oligosaccharides with an LNTII skeleton. Additionally, a method involving centrifugation, cation and anion exchange, concentration, and cooling or poor solvent addition is employed to produce high-purity LNTII crystals.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional microbial fermentation methods are used with existing transport proteins, then LNTII can be produced, but the productivity is insufficient due to effective transport protein deficiency in various E. coli strains
Solution Approach 1:
The patent introduces a heterologous transport protein (ProP from Mannheimia succiniciproducens or SetA from Cedecea neteri) as an intermediary to compensate for the insufficient or non-functional native transport proteins in E. coli strains. This external protein mediator enables effective LNTII transport across the cell membrane, resolving the reliability issue of transport protein effectiveness and thereby improving overall productivity.
2Ease of manufacture
If reverse crystallization method is used to obtain LNTII crystals, then crystals can be produced, but the process requires addition of methanol or acetone which complicates the manufacturing
Solution Approach 1:
The patent extracts and utilizes the unique ability of YdeA protein to specifically bind and precipitate LNTII from the fermentation broth without requiring external chemicals like methanol or acetone. This extraction-based approach simplifies the crystallization process by using a biologically derived precipitating agent that can be easily removed, reducing manufacturing complexity while maintaining ease of 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
This approach allows for improved productivity of LNTII and oligosaccharides with an LNTII skeleton, and facilitates the easy production of high-purity LNTII crystals, overcoming the limitations of existing methods.
Implementation Method 1
a method for producing an LNTII crystal from a culture obtained by culturing the microorganism according to the above 1, the method including the following steps (i) to (iv): (i) a step of centrifuging the culture to obtain a supernatant from which bacteria cells are removed; (ii) a step of subjecting the supernatant obtained in the step (i) to cation exchange and anion exchange to obtain a solution from which ions are removed; (iii) a step of concentrating the solution obtained in the step (ii) with an evaporator; and (iv) a step of cooling the solution obtained in the step (iii) or adding dropwise a poor solvent to the solution obtained in the step (iii) to obtain an LNTII crystal
Implementation Method 2
a step of centrifuging the culture to obtain a supernatant from which bacteria cells are removed
Implementation Method 3
a step of subjecting the supernatant obtained in the step (i) to cation exchange and anion exchange to obtain a solution from which ions are removed
Implementation Method 4
a step of concentrating the solution obtained in the step (ii) with an evaporator
Implementation Method 5
a step of cooling the solution obtained in the step (iii) or adding dropwise a poor solvent to the solution obtained in the step (iii) to obtain an LNTII crystal
Data Source
AI summary
The present invention relates to a microorganism having a reduced or inactivated activity of a protein according to any one of the following [1] to [3] and improved productivity of lacto-N-triose II or an oligosaccharide having a lacto-N-triose II (LNTII) skeleton as compared with a parent strain: [1] a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 or 4, [2] a mutant protein having an oligosaccharide transporting activity and consisting of an amino acid sequence in which 1 to 20 amino acids are deleted, substituted, inserted, or added in the amino acid sequence represented by SEQ ID NO: 2 or 4, and [3] a homologous protein having an oligosaccharide transporting activity and consisting of an amino acid sequence having an identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 2 or 4.