Recombinant Transporter for GDP-Fucose Membrane Export
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Solution Overview
Problem
Current methods for producing GDP-fucose are laborious, costly, and inefficient, particularly due to the difficulty in transporting this nucleotide-activated sugar across the membrane of recombinant bacteria, leading to accumulation in the cytosol and hindering large-scale production.
Innovation Solution
A method involving a recombinant prokaryotic microorganism host cell transformed with a nucleic acid sequence encoding a transporter protein fused with a signal sequence, enabling GDP-fucose to be transported across the cellular membrane, thereby producing it in the periplasm or growth medium, using eukaryotic transporter proteins like SLC35 C1 from Homo sapiens.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If GDP-fucose is produced in recombinant bacteria using endogenous pathways, then production occurs in the cytosol, but the GDP-fucose accumulates in the cytosol and cannot be efficiently transported across the membrane, hindering large-scale production
Solution Approach 1:
The patent introduces a heterologous nucleic acid sequence encoding a transporter protein that mediates the transport of GDP-fucose from the cytosol across the cellular membrane to the periplasm or extracellular medium. This intermediary transporter protein resolves the transport difficulty by providing a dedicated molecular pathway for GDP-fucose export, enabling efficient large-scale production.
2Quantity of substance
If chemical synthesis methods are used to produce GDP-fucose, then production can occur, but the process is very time and cost intensive and appears very inefficient for technical application
Solution Approach 1:
The patent replaces the mechanical/chemical synthesis system with a biological enzymatic system. By using recombinant bacteria with endogenous enzymatic pathways for GDP-fucose synthesis, the method substitutes time-intensive chemical synthesis with more efficient biological production, significantly reducing production time and cost while maintaining high yields.
3Ease of operation
If transporter protein is fused to signal sequence for membrane transport, then GDP-fucose can be transported to periplasm or medium, but requires transformation with heterologous nucleic acid sequence
Solution Approach 1:
The patent segments the production system into distinct functional components: the endogenous GDP-fucose synthesis pathway in the cytosol, the heterologous transporter protein with signal sequence for membrane translocation, and the periplasmic or extracellular collection compartment. This segmentation allows GDP-fucose to be produced and then actively transported to a separate compartment, facilitating easy isolation while the transformation complexity is managed through modular genetic construction.
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 an efficient, cost-effective, and scalable production of GDP-fucose, facilitating its isolation and further processing for applications in glycosylated oligosaccharides and other glycoconjugates, avoiding the limitations of chemical synthesis and cytosolic accumulation.
Implementation Method 1
a signal sequence which transports the protein through a cellular membrane of the host cell
Implementation Method 2
the GDP-fucose is transported over the membrane; and thereby providing the GDP-fucose in the periplasm of said recombinant prokaryotic microorganism cell or the medium of its growth
Data Source
Figure 1A~1B
Figure 2A~2B
Figure 3A~3B
AI summary
The present invention relates to a method for producing a nucleotide-activated sugar in a recombinant prokaryotic microorganism host cell, wherein the recombinant prokaryotic microorganism is transformed to express an eukaryotic transporter protein for the nucleotide-activated sugar. The present invention also relates to the recombinant prokaryotic microorganism used in the method and its use for the production of nucleotide-activated sugars.