Microbial Export of 2′ Fucosyllactose via Heterologous Transporters
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Solution Overview
Problem
Current methods lack an efficient way to increase the export of 2′ fucosyllactose from microbial cells, particularly in yeast, where a sugar efflux transporter for this compound is not well established.
Innovation Solution
Genetically engineered microbial cells are developed with heterologous nucleic acid molecules encoding a transporter protein, GDP-mannose-4,6-dehydratase, GDP-4-keto-6-D-deoxymannose epimerase-reductase, and 2-β-L-fucosyltransferase, facilitating the production and export of 2′ fucosyllactose, using SetA, Sugar porter, or SWEET transporters.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If a microbial cell is engineered to produce 2′FL through heterologous genes, then 2′FL production capability is improved, but export efficiency of 2′FL remains insufficient
Solution Approach 1:
The patent divides the 2′FL production system into two functional modules: (1) intracellular synthesis pathway genes (gmd, gmer, futB, lacZ) and (2) extracellular export pathway (sugar efflux transporter). This segmentation allows independent optimization of production and export functions, resolving the contradiction by adding the transporter component specifically addressed in claim 1.
Solution Approach 2:
The patent employs a sugar efflux transporter that can handle multiple sugar substrates, making it a multi-functional protein that not only exports 2′FL but also maintains cellular sugar homeostasis. This universal transporter approach resolves the export efficiency limitation while avoiding the need for multiple specialized transporters.
2Quantity of substance
If E. coli is used for 2′FL production, then biosynthetic pathway is established, but lack of efficient sugar efflux transporter limits export
Solution Approach 1:
The patent introduces a sugar efflux transporter as an intermediary component between the intracellular synthesis pathway and the extracellular medium. This mediator protein facilitates the transfer of 2′FL from the cytoplasm to the growth medium, resolving the export limitation in E. coli systems described in the background.
Solution Approach 2:
The engineered microbial cell becomes self-sufficient by integrating both the biosynthetic pathway and the export mechanism within a single cellular system. The cell autonomously produces 2′FL through heterologous genes and simultaneously exports it via the expressed sugar efflux transporter, eliminating the need for separate extraction procedures.
3Quantity of substance
If yeast is used as host, then metabolic route to GDP-fucose is established, but no reported sugar efflux transporter exists for 2′FL
Solution Approach 1:
The patent performs preliminary identification and selection of suitable sugar efflux transporter candidates from transporter families (SetA, Sugar porter, SWEET) before implementing them in the yeast system. This preliminary screening approach ensures reliable export functionality is established prior to full-scale production, addressing the unreliability of export in yeast.
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 engineered cells significantly increase the export of 2′ fucosyllactose, with the method allowing for its production and identification of effective nucleic acid sequences that enhance export, demonstrating improved production efficiency.
Implementation Method 1
at least one heterologous nucleic acid molecule encoding a transporter protein that facilitates the export of 2′ fucosyllactose from the microbial cell
Implementation Method 2
at least one heterologous nucleic acid molecule encoding a GDP-mannose-4,6-dehydratase (EC 4.2.1.47)
Implementation Method 3
at least one heterologous nucleic acid molecule encoding a GDP-4-keto-6-D-deoxymannose epimerase-reductase (EC 1.1.1.271)
Implementation Method 4
at least one heterologous nucleic acid molecule encoding a 2-L-L-fucosyltransferase (EC 2.4.1.69)
Data Source
AI summary
Microbial cells genetically engineered with a heterologous nucleic acid sequence that increases export of 2′ fucosyllactose are disclosed. Methods of increasing export of 2′ fucosyllactose from a microbial cell and for identifying a heterologous nucleic acid sequence that increases export of 2′ fucosyllactose from a microbial cell are also disclosed.