Single-Enzyme Sucrose Utilization for Biosynthetic Production

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Solution Overview

Problem

Existing microbial biosynthesis processes face challenges in efficiently utilizing sucrose as a carbon and energy source due to the need for multiple gene systems, leading to inefficiencies and increased costs, and sucrose utilization often results in metabolic overflow and impurities.

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, eliminating the need for multiple gene systems and reducing metabolic overflow.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Adaptability or versatility

If multiple gene systems are used to enable sucrose utilization in microbial cells, then the cell can utilize sucrose as carbon and energy source, but the device complexity increases and manufacturing costs increase

Engineering Contradiction:

Improvesucrose utilization capabilityVSAvoidnumber of gene systems

Core Design Contradiction:

Adaptability or versatilityVSDevice complexity

Solution Approach 1:

The invention extracts and expresses only the essential invertase enzyme (SucC_Agal or Bff) from the complex sucrose utilization pathway, eliminating the need for multiple gene systems. The enzyme is expressed under control of a constitutive promoter and is sufficient to enable sucrose utilization, thereby reducing device complexity while maintaining adaptability.

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The invertase enzyme performs multiple functions: it hydrolyzes sucrose into glucose and fructose, providing both carbon and energy sources for the cell. This single enzyme replaces what would traditionally require multiple gene systems, achieving multi-functionality with one component.

Inventive Principle:

Principle #6Universality (Multi-functionality)

2Adaptability or versatility

If multiple gene systems are used for sucrose utilization, then the cell can process sucrose, but manufacturing costs increase

Engineering Contradiction:

Improvesucrose utilization capabilityVSAvoidmanufacturing cost

Core Design Contradiction:

Adaptability or versatilityVSEase of manufacture

Solution Approach 1:

The invention extracts only the essential invertase enzyme from the complete sucrose utilization pathway. By expressing a single enzyme gene rather than multiple gene systems, the manufacturing cost is significantly reduced while maintaining the ability to utilize sucrose as carbon and energy source.

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The invention uses a simple, inexpensive single-gene expression system with a constitutive promoter, replacing complex multi-gene systems that would require more resources for construction, maintenance, and optimization, thereby reducing overall manufacturing cost.

Inventive Principle:

Principle #27Cheap short-living objects (Disposable)

3Use of energy by moving object

If sucrose is utilized through conventional pathways, then carbon and energy are provided, but metabolic overflow and impurities are generated

Engineering Contradiction:

Improveenergy provision from sucroseVSAvoidmetabolic overflow and impurities

Core Design Contradiction:

Use of energy by moving objectVSObject-generated harmful factors

Solution Approach 1:

The invention extracts the sucrose hydrolysis step from the conventional metabolic pathway and places it extracellularly through secreted invertase. This separates the carbon source provision (sucrose hydrolysis) from the energy metabolism, preventing metabolic overflow and impurities while maintaining energy provision from sucrose.

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The secreted invertase acts as an intermediary enzyme that performs sucrose hydrolysis outside the cell. This mediator approach allows sucrose to be converted into glucose and fructose without entering the cellular metabolic pathway, thereby providing carbon and energy while avoiding metabolic overflow and impurities.

Inventive Principle:

Principle #24Intermediary (Mediator)

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 cost-effective large-scale biosynthesis by utilizing sucrose as a main or sole carbon and energy source, minimizing impurities and metabolic issues, thereby enhancing production efficiency and yield.

Implementation Method 1

which is capable of hydrolysing non-phosphorylated sucrose into fructose and glucose

Methodology Applied

Scientific EffectHydrolysis: Hydrolysis

Data Source

PatentUS20250320535A1Novel technology to enable sucrose utilization in strains for biosynthetic production

Publication Date: 2025.10.16 DSM IP ASSETS BV

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.