Engineered Microorganisms for LDFT Production and By-Product Reduction
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Solution Overview
Problem
Existing methods for producing lactodifucotetraose (LDFT) face issues with the accumulation of 3-fucosyllactose (3FL) as a by-product and insufficient conversion of 2'-fucosyllactose (2'FL) due to the limitations of α1,2-fucosyltransferases and α1,3-fucosyltransferases used in microbial fermentation.
Innovation Solution
Development of microorganisms with enhanced α1,2-fucosyltransferase and α1,3-fucosyltransferase activities, specifically through modified proteins with specific amino acid sequences, enabling efficient conversion of 3FL and 2'FL to LDFT, thereby reducing by-product accumulation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If α1,2-fucosyltransferase and α1,3-fucosyltransferase are used in combination for LDFT production, then LDFT can be produced through multiple pathways, but by-products (3FL and 2'FL) accumulate due to insufficient conversion efficiency
Solution Approach 1:
The patent modifies the amino acid sequences of α1,2-fucosyltransferase and α1,3-fucosyltransferase to enhance their catalytic activities. Specific amino acid substitutions optimize the enzymes' ability to convert substrates, thereby improving LDFT production efficiency while reducing by-product accumulation through enhanced conversion rates.
Solution Approach 2:
The patent creates mutant versions of the fucosyltransferase enzymes by copying and modifying their genetic sequences. These engineered enzyme variants exhibit improved catalytic properties compared to the wild-type enzymes, enabling more efficient LDFT synthesis and reduced by-product formation.
2Ease of manufacture
If existing fucosyltransferases are used, then the production process is simple, but conversion of intermediates 2'FL and 3FL to LDFT is insufficient
Solution Approach 1:
The patent modifies enzymatic parameters through amino acid sequence engineering to enhance intermediate conversion efficiency. The engineered fucosyltransferases maintain compatibility with existing production systems while achieving superior conversion of 2'FL and 3FL to LDFT.
3Productivity
If Hp3/4 FT is used to convert 2'FL to LDFT, then some LDFT is produced, but 3FL accumulates as a by-product due to enzyme substrate specificity limitations
Solution Approach 1:
The patent engineers α1,3-fucosyltransferase with modified amino acid sequences to broaden substrate specificity and enhance activity toward 3FL. This enables more efficient conversion of 3FL to LDFT, reducing by-product accumulation while maintaining high LDFT production rates.
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 enhanced microorganisms significantly improve LDFT productivity by rapidly converting intermediates 3FL and 2'FL into LDFT with high efficiency, minimizing by-product formation.
Implementation Method 1
a microorganism having enhanced activities of a protein according to any one of the following [1] to 3 and a protein according to any one of the following 4 to 6 and improved productivity of lactodifucotetraose (LDFT) as compared with a parent strain
Data Source
AI summary
An object is to provide a microorganism having excellent productivity of LDFT. The present invention relates to a microorganism having an enhanced activity of any one of [1] a protein consisting of the amino acid sequence represented by SEQ ID NO: 8 and a mutant protein or a homologous protein thereof, and [4] a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 and a mutant protein or a homologous protein thereof, and improved productivity of lactodifucotetraose (LDFT) as compared with a parent strain.