Recombinant E. coli for Lacto-N-Neotetraose Production
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Solution Overview
Problem
Current methods for producing lacto-N-neotetraose, such as microbial fermentation, require optimization to achieve higher yields and meet industrialization requirements, as existing strains like Bacillus subtilis have limited titers and specificity issues.
Innovation Solution
A recombinant Escherichia coli expressing β-1,4-galactosyl transferase derived from Aggregatibacter actinomycetemcomitans, β-1,3-acetyl glucosamine transferase from Neisseria meningitidis, and UDP-glucose 4 epimerase from E. coli, with specific gene knockouts and vector combinations, is used to enhance precursor supply and produce lacto-N-neotetraose efficiently.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If microbial fermentation is used to synthesize lacto-N-neotetraose, then the production process is simplified and substrates are cheap, but the yield and titer are limited
Solution Approach 1:
The patent changes the enzymatic parameters by introducing a novel ß-1,4-galactosyl transferase enzyme with optimized catalytic properties. This enzyme exhibits enhanced activity and specificity compared to existing enzymes, enabling higher production titers while maintaining the simplicity of microbial fermentation processes
Solution Approach 2:
The patent employs UDP-galactose as a key intermediary substrate that feeds into the ß-1,4-galactosyl transferase reaction. By optimizing the supply and utilization of this intermediary compound, the system achieves improved lacto-N-neotetraose yield while maintaining efficient microbial fermentation
2Quantity of substance
If existing microbial strains are used for lacto-N-neotetraose production, then the process is cost-effective, but the production titer and specificity are insufficient
Solution Approach 1:
The patent creates a recombinant microbial strain that copies and integrates the optimized ß-1,4-galactosyl transferase enzyme into its metabolic pathway. This engineered strain reproduces high-specificity enzyme catalysis while achieving superior production titers compared to wild-type strains
Solution Approach 2:
The patent modifies the enzymatic parameters of the microbial system by introducing a novel ß-1,4-galactosyl transferase with optimized kinetic properties. This enzyme exhibits enhanced catalytic efficiency and substrate specificity, simultaneously improving both production titer and reaction reliability
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 recombinant E. coli strain achieves a lacto-N-neotetraose yield of 12.14 g/L in a 3 L fermentation tank, demonstrating improved production capacity and industrial applicability.
Implementation Method 1
The synthesis of the lacto-N-neotetraose by the microbial fermentation requires a key ß-1,4-galactosyl transferase capable of catalyzing the conversion of a precursor lacto-N-triose II
Implementation Method 2
a gene Aa-β-1,4-GalT encoding the ß-1,4-galactosyl transferase are co-expressed... which uses lacto-N-triose II and UDP-galactose as substrates to produce lacto-N-neotetraose
Implementation Method 3
Currently, microbial fermentation is mainly used to synthesize the lacto-N-neotetraose
Data Source
AI summary
Disclosed are a construction method and application of a microorganism capable of realizing high production of lacto-N-neotetraose, belonging to the field of microbial genetic engineering. Coding genes of ß-1,3-acetyl glucosamine transferase, ß-1,4-galactosyl transferase and/or UDP-glucose 4 epimerase are over-expressed on the basis of a strain which is previously constructed by the team and is subjected to related-gene knockout, thus enabling the strain to have a synthesis capability of producing the lacto-N-neotetraose. The present disclosure accurately regulates the carbon flux of a metabolic pathway and relieves the metabolic stress by screening the high-efficiency ß-1,4-galactosyl transferase gene and regulating the expression of IgtA, Aa-β-1,4-GalT and galE in a lacto-N-neotetraose synthesis pathway in a combined manner. In a shake flask experiment, the lacto-N-neotetraose production capacity of Escherichia coli is 0.91 g/L. The lacto-N-neotetraose yield in a 3 L fermentation tank reaches 12.14 g/L. Therefore, the microorganism has an industrial application prospect.