Codon-Optimized Sucrose Isomerase Expression in E. coli
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Solution Overview
Problem
The mass production of rare disaccharides like trehalulose and isomaltulose is hindered by insufficient enzyme production and low expression levels in native or heterologous organisms, making it challenging to meet commercial demands due to high production costs.
Innovation Solution
The nucleotide sequences encoding for sucrose isomerase from Pseudomonas mesoacidophila MX45 and isomaltulose synthase from Pantoea dispersa UQ68J are modified to enhance expression levels in E. coli, resulting in higher production of recombinant enzymes, which are then used for bioconversion of sucrose into trehalulose and isomaltulose, respectively.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If native or heterologous organisms are used for enzyme production, then the enzymes can be produced, but the expression levels are insufficient and production costs are high
Solution Approach 1:
The patent applies parameter changes by modifying the nucleotide sequence of the enzyme genes to optimize codon usage patterns for E. coli expression. Specifically, the genes encoding sucrose isomerase and isomaltulose synthase were redesigned with codons that match E. coli's preferred codon table, thereby increasing translation efficiency and protein expression levels by 14-19% of total cellular protein.
2Quantity of substance
If conventional enzyme production methods are used, then enzymes can be produced, but production time is excessive and costs are high
Solution Approach 1:
The patent reduces production time by changing the genetic parameters of the enzyme-encoding genes. The modified nucleotide sequences with optimized codons enable faster and more efficient protein synthesis in E. coli, thereby reducing the fermentation time and increasing enzyme production quantity within shorter periods.
3Productivity
If recombinant enzymes are used for bioconversion, then sucrose can be converted to rare disaccharides, but the conversion efficiency is limited by low enzyme expression
Solution Approach 1:
The patent enhances conversion efficiency by changing the genetic parameters of the enzyme genes to achieve higher expression levels. The codon-optimized nucleotide sequences result in increased enzyme quantities (14-19% of total cellular protein), which directly improves the bioconversion efficiency of sucrose to trehalulose and isomaltulose.
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 increases enzyme expression by 14% to 19% of total cellular protein, reducing production time and costs, and achieves efficient conversion of sucrose to trehalulose or isomaltulose, with immobilized enzymes showing 70-80% protein retention and high purity of the products.
Implementation Method 1
Both disaccharides: isomaltulose and trehalulose can be produced at an industrial scale through isomerization of sucrose using Sucrose Isomerases (SIase) which bio-convert sucrose into both isomaltulose and trehalulose with trace amounts of glucose and fructose.
Implementation Method 2
immobilized enzymes showing 70-80% protein retention
Data Source
AI summary
The present invention is directed towards genetic modification of native gene encoding for sucrose isomerase and isomaltulose synthase to substantially increase the expression level of these enzymes and use of said enzymes in a process to produce rare disaccharides such as isomaltulose and trehalulose. Also disclosed in the present invention is expression constructs comprising the modified genes and a host cells to express the same.