Engineered Microorganisms for High-Yield LNT and LNnT Production
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Solution Overview
Problem
Current methods for producing lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT) are laborious, costly, and yield inefficient due to chemical synthesis requiring multiple steps and enzymatic synthesis being affected by unfavorable equilibrium product distributions and regioselectivities.
Innovation Solution
Genetically modified microorganisms engineered with specific transgenes encoding β 1,3-N-acetylglucosaminyltransferase and β 1,3- or β 1,4-galactosyltransferase, integrated into the LacZYA locus with suppressed LacZ and LacA expression, and supplemented with transgenes for LacY and UDP-sugar pyrophosphorylase to enhance lactose uptake and intracellular UDP-galactose production, enabling efficient biotechnological synthesis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If chemical synthesis is used to produce LNT, then product purity can be achieved, but the process requires multiple steps of protection and deprotection leading to low productivity and high cost
Solution Approach 1:
The patent replaces chemical synthesis methods with a biotechnological system using genetically modified microorganisms. The microorganisms express glycosyltransferases that catalyze the formation of LNT through biochemical pathways, eliminating the need for multiple chemical protection and deprotection steps while maintaining product purity and significantly improving productivity
Solution Approach 2:
The genetically modified microorganisms autonomously produce LNT through their metabolic pathways. The cells take up lactose and nucleotide-activated sugars from the medium and convert them to LNT intracellularly, with the product being secreted into the medium, eliminating the need for external intervention in each synthesis step
2Ease of manufacture
If enzymatic synthesis is used to produce LNT, then the process is simpler than chemical synthesis, but unfavorable equilibrium product distributions and regioselectivities reduce manufacturing precision
Solution Approach 1:
The patent introduces specific glycosyltransferase enzymes with defined substrate specificities into the microorganism. Each enzyme catalyzes a specific reaction at a specific position, ensuring that LNT is produced with the correct structure and avoiding unwanted byproducts, thereby improving product specificity while maintaining process simplicity
Solution Approach 2:
The patent optimizes the intracellular concentrations of nucleotide-activated sugars (UDP-galactose, UDP-N-acetylglucosamine) by introducing additional biosynthetic pathways. This changes the metabolic parameters to favor LNT production over alternative products, improving both yield and specificity
3Device complexity
If standard genetically engineered microorganisms are used, then the system is simpler to construct, but they lack the necessary enzymes and metabolic pathways for efficient LNT production
Solution Approach 1:
The patent combines multiple functional elements into a single genetically modified microorganism system: lactose uptake capability, nucleotide-activated sugar biosynthesis pathways, and glycosyltransferase enzyme expression are all integrated into one cell system, enabling efficient LNT production while maintaining reasonable system complexity
Solution Approach 2:
The microorganism is pre-equipped with the complete biosynthetic machinery needed for LNT production before the actual synthesis begins. This includes pre-installed enzymes for nucleotide sugar synthesis and glycosyltransferases for LNT formation, allowing immediate high-yield production when substrates are provided
4Manufacturing precision
If extraction from breast milk is used to obtain HMOs, then natural composition is preserved, but the process is laborious and yields are insufficient for research needs
Solution Approach 1:
The genetically modified microorganisms autonomously produce LNT and other HMOs through their metabolic pathways, secreting the products into the culture medium. This self-producing capability eliminates the need for laborious extraction from breast milk while producing sufficient quantities for research and commercial applications
Solution Approach 2:
The patent creates a biotechnological copy of the natural HMO production system. Instead of extracting HMOs from their natural source (breast milk), the system replicates the biosynthetic pathways in microorganisms, producing identical or structurally similar compounds through engineered metabolic routes
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 process achieves high-yield, cost-effective production of LNT and LNnT, allowing for scalable and efficient synthesis of these oligosaccharides, with improved yields and reduced reliance on chemical or enzymatic methods.
Implementation Method 1
a first transgene encoding β 1,3-N-acetylglucosaminyltransferase
Implementation Method 2
a second transgene encoding β 1,3-galactosyltransferase (in the case of the synthesis of lacto-N-tetraose)
Implementation Method 3
β 1,4-galactosyltransferase (in the case of the synthesis of lacto-N-neotetraose)
Implementation Method 4
the microorganism contains another transgene encoding LacY
Implementation Method 5
a further transgene encoding a UDP-sugar pyrophosphorylase (USP)
Data Source
Figure 1
Figure 2
Figure 3~4
AI summary
The invention relates to primarily genetically engineered microorganisms for the in-vivo synthesis of lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT) and the fucosylated derivatives thereof, and to uses of said microorganisms in methods for producing lacto-N-tetraose and lacto-N-neotetraose and the fucosylated derivatives thereof.