Psicose Epimerase Expression in Corynebacterium for High Yield
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Solution Overview
Problem
Current methods for producing psicose, a rare sugar used as a diet sweetener, face challenges due to low enzyme activity and heat stability of Psicose epimerase derived from Agrobacterium tumefaciens, and the use of non-GRAS recombinant E. coli, which limits its application in the food industry.
Innovation Solution
Development of a gene expression cassette and vector for Corynebacterium sp. that includes a promoter and regulating sequence to express Psicose epimerase with high stability and activity, using a GRAS microorganism, enabling high-yield production of psicose.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If Psicose epimerase derived from Agrobacterium tumefaciens is used for psicose production, then the production method is established, but the enzyme activity and heat stability are low
Solution Approach 1:
The patent optimizes the amino acid sequence of Psicose epimerase by introducing specific mutations (e.g., Q179L, D181N, N182I) to improve enzyme activity and heat stability. This parameter change at the molecular level directly addresses the low reliability and productivity issues of the original enzyme from Agrobacterium tumefaciens.
2Reliability
If recombinant E. coli is used to express Psicose epimerase, then production is achieved, but the microorganism is not GRAS and thus unsuitable for food industry
Solution Approach 1:
The patent uses Corynebacterium glutamicum as an intermediary host organism to express Psicose epimerase. This GRAS microorganism serves as a safe mediator that can be used in food industry applications, replacing the non-GRAS E. coli while maintaining production capability through proper expression system design.
3Reliability
If Corynebacterium glutamicum is used as host, then GRAS status is achieved, but the expression level of Psicose epimerase is low due to limited promoter choices
Solution Approach 1:
The patent employs a multi-functional expression system in Corynebacterium glutamicum that incorporates multiple promoter options (native C. glutamicum promoters and heterologous promoters from E. coli and other sources). This universal approach allows the system to achieve both GRAS status and high expression levels by selecting the most appropriate promoter for the specific application.
4Productivity
If E. coli promoters are used in Corynebacterium glutamicum, then expression can be induced, but the activity is low due to low permeability of inducer and absence of gene expression inhibitor
Solution Approach 1:
The patent adapts and modifies E. coli promoter sequences to function effectively in Corynebacterium glutamicum. By copying the essential regulatory elements and optimizing them for the C. glutamicum cellular environment, the system achieves proper inducer response and gene expression regulation while maintaining the benefits of using a GRAS host organism.
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 solution achieves high-yield, stable expression of Psicose epimerase in Corynebacterium sp., ensuring efficient conversion of fructose to psicose with improved enzyme activity and heat stability, suitable for industrial food applications.
Implementation Method 1
a method of producing psicose by using the psicose epimerase produced by the transformed GRAS microorganism or a composition including the psicose epimerase
Data Source
AI summary
A gene expression cassette capable of producing psicose at high yield with high stability, a GRAS (Generally recognized as safe) microorganism, a method of producing the enzyme by using the GRAS microorganism, and a method of producing the psicose by using the GRAS microorganism and enzyme are provided.