Codon-Optimized Enzymes for Rare Monosaccharide Production
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Solution Overview
Problem
The production of rare sugars like Psicose and Allose is hindered by low expression levels of enzymes in native and heterologous organisms, leading to high production costs and challenges in mass production due to equilibrium conversion processes between Fructose and Psicose.
Innovation Solution
Genetic modification of the nucleotide sequence encoding D-tagatose 3-epimerase and rhamnose isomerase from Pseudomonas cichorii ST-24 and Pseudomonas stutzeri to enhance expression levels in E. coli, using optimized gene constructs and promoters like T7, resulting in higher yields and more efficient bioconversion processes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If native or heterologous expression systems are used for enzyme production, then the bioconversion process can be established, but the expression levels remain low leading to high production costs
Solution Approach 1:
The patent applies codon optimization by systematically substituting endogenous codons with codons over-represented in highly expressed prokaryotic genes. This parameter change in the gene sequence dramatically increased protein expression levels in E. coli, directly resolving the contradiction between productivity and manufacturing cost by enabling cost-effective mass production of the enzymes.
2Productivity
If the inter conversion between Fructose and Psicose is used, then the bioconversion pathway is established, but the equilibrium process limits large scale and high yield production
Solution Approach 1:
The patent employs a two-enzyme sequential conversion system where D-tagatose-3-epimerase first converts fructose to psicose, followed by rhamnose isomerase converting psicose to allose. This preliminary action of using a second enzyme to consume the intermediate product (psicose) prevents the equilibrium limitation by continuously driving the first reaction forward, enabling high-yield production.
3Productivity
If gene sequence is redesigned with different codons, then protein expression levels are dramatically increased, but the genetic modification complexity increases
Solution Approach 1:
The patent systematically substituted endogenous codons with codons over-represented in highly expressed prokaryotic genes while maintaining the same amino acid sequence. This parameter change in the nucleotide sequence dramatically increased protein expression levels without altering the protein structure, resolving the contradiction between productivity and genetic modification complexity through a well-established molecular biology technique.
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 modified enzymes achieve higher expression levels, reducing production costs and time for converting Fructose to Psicose and Psicose to Allose, with improved enzyme stability and activity, enabling more efficient large-scale production.
Implementation Method 1
The enzyme responsible for bioconversion of ketose to its corresponding epimeric ketose (fructose to psicose) form have been reported from different microorganisms
Implementation Method 2
the enzymes responsible for bioconversion of ketose to aldose (Psicose in to Allose) form have been reported from different microorganisms
Data Source
AI summary
The present invention is directed towards genetic modification of native gene encoding for D-tagatose 3-epimerase and rhamnose isomerase to substantially increase the expression level of these enzymes and use of the enzymes in a process to produce rare monosaccharides such as psicose and allose. Also disclosed in the present invention is expression constructs comprising the modified genes and a host cells to express the same.