Invertase-Expressing Microbial Strains for Single-Enzyme Sucrose Use
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Solution Overview
Problem
Existing microbial strains require multiple genes and systems to utilize sucrose as a carbon and energy source, leading to inefficiencies and increased costs in large-scale biosynthetic production processes, particularly for human milk oligosaccharides, and sucrose utilization by engineered E. coli strains is often less productive than native strains.
Innovation Solution
A genetically modified cell expressing a single heterologous enzyme, such as SacC Agal or Bff, is translocated to the extracellular or periplasmic space to hydrolyze sucrose into fructose and glucose, enabling efficient utilization of sucrose as the main or sole carbon and energy source without the need for additional transporters or phosphorylation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If multiple genes and systems are introduced into microbial strains to enable sucrose utilization, then sucrose can be used as carbon and energy source, but the complexity of the genetic system increases and productivity decreases
Solution Approach 1:
The patent extracts and utilizes the native sucrose utilization system already present in certain E. coli strains (such as the sucrose phosphorylase system), rather than introducing multiple foreign genes. This approach leverages the endogenous capability of some strains to metabolize sucrose, thereby reducing genetic system complexity while maintaining sucrose utilization functionality.
Solution Approach 2:
The patent employs a single heterologous enzyme (invertase/sucrose hydrolase) that performs multiple functions: it hydrolyzes sucrose into glucose and fructose, and the resulting sugars are then utilized by the cell's existing metabolic pathways for both energy production and carbon source. This multi-functional approach eliminates the need for separate transporters and phosphorylation systems.
2Adaptability or versatility
If multiple genes and systems are introduced into microbial strains to enable sucrose utilization, then sucrose can be used as carbon and energy source, but the productivity of engineered strains becomes less than native strains
Solution Approach 1:
The patent enables the engineered microbial strain to utilize sucrose through a streamlined system where a single heterologous enzyme (invertase) hydrolyzes sucrose, and the cell's own native metabolic pathways handle the subsequent utilization of glucose and fructose. This self-service approach, leveraging endogenous pathways, improves productivity by reducing the metabolic burden associated with expressing multiple foreign genes and systems.
3Ease of manufacture
If sucrose is used as carbon source in large-scale biosynthetic production, then cost-effectiveness and sustainability improve, but the need for multiple gene systems increases process complexity
Solution Approach 1:
The patent extracts and utilizes the native sucrose utilization system already present in certain E. coli strains (such as the sucrose phosphorylase system), rather than introducing multiple foreign genes. This approach leverages the endogenous capability of some strains to metabolize sucrose, thereby reducing genetic system complexity while maintaining sucrose utilization functionality.
Solution Approach 2:
The patent changes the metabolic parameter by introducing a single heterologous enzyme (invertase) that alters the sucrose hydrolysis pathway, enabling the cell to utilize sucrose through a simplified route that produces glucose and fructose, which are then processed by existing metabolic pathways. This parameter change reduces the number of required genetic components while maintaining sucrose as a cost-effective carbon source.
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 reduces the need for multiple gene systems, minimizes metabolic overflow, and enhances productivity by using sucrose as a cost-effective and sustainable carbon source, improving large-scale biosynthesis processes.
Implementation Method 1
which is capable of hydrolysing non-phosphorylated sucrose into fructose and glucose
Data Source
AI summary
The present disclosure relates to a genetically modified cell capable of utilizing sucrose as energy and carbon source following the expression of a single heterologous enzyme, which upon expression is translocated from the cytosol and which is capable of hydrolysing non-phosphorylated sucrose into fructose and glucose.The identification of efficient enzymes capable of hydrolysing sucrose in its non-modified form, and which on its own enable the cell to utilize sucrose as a, or as the main and/or sole, carbon and/or energy source, i.e., without the need for multi-gene sucrose utilizing systems comprising several other heterologous polypeptides, such as other enzymes and/or transporters, is highly advantageous, since it allows for cost-effective use of sucrose in large scale production processes.