Millipede Hydroxynitrile Lyase Expression in E. coli
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Solution Overview
Problem
The low expression levels of (R)-hydroxynitrile lyase in yeast and the lack of an E. coli expression system for millipede-derived hydroxynitrile lyase genes make large-volume preparation difficult, and no cloning methods have been established for hydroxynitrile lyase genes from millipede species other than Chamberlinius hualienensis.
Innovation Solution
Cloning of hydroxynitrile lyase genes from various millipedes using degenerate primers with conserved sequences, followed by expression in cultured insect cells and specific E. coli strains to produce millipede-derived hydroxynitrile lyase enzymes, which are then used to manufacture optically active cyanohydrins.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If (R)-hydroxynitrile lyase is expressed in yeast, then the enzyme can be produced, but the expression level is very low making large-volume preparation difficult
Solution Approach 1:
The patent changes the host organism parameter from yeast to E. coli, and further optimizes by selecting specific E. coli strains (SHuffle T7, Origami B(DE3)) with enhanced disulfide bond formation capabilities. This parameter change resolves the low expression level issue by achieving significantly higher enzyme production in the new host system
Solution Approach 2:
The patent uses E. coli, a simple and inexpensive model organism with rapid growth characteristics, replacing the more complex yeast system. This allows for quick, large-scale production of the enzyme, enabling large-volume preparation that was previously difficult with yeast expression
2Adaptability or versatility
If hydroxynitrile lyase genes from millipede species other than Chamberlinius hualienensis are desired, then new gene sources must be explored, but no cloning methods have been established for these species
Solution Approach 1:
The patent develops a universal cloning approach using degenerate primers based on conserved amino acid sequences that can amplify HNL genes from multiple millipede species simultaneously. This universal method eliminates the need to develop species-specific cloning protocols, making the process adaptable to diverse gene sources while maintaining ease of manufacture
Solution Approach 2:
The patent uses degenerate primers designed from conserved amino acid sequences as intermediaries to bridge the gap between diverse millipede species and the cloning process. These primers serve as a universal mediator that can bind to and amplify HNL genes from different species without requiring species-specific knowledge
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
Enables the easy preparation of millipede-derived (R)-hydroxynitrile lyase with strong specific activity and heat stability, facilitating the production of optically active cyanohydrins, which are crucial intermediates in drug and fine chemical manufacturing.
Implementation Method 1
A method of reacting an aldehyde or ketone and a cyanide donor with a hydroxynitrile lyase has been proposed as a cyanohydrin manufacturing method
Data Source
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AI summary
Provided are a method for obtaining an HNL gene and HNL derived from a millipede other than Chamberlinius hualienensis, and preparing a practically useable amount of HNL; and a method for producing optically active cyanohydrin using this HNL. A method for producing a millipede-derived HNL gene. A method that includes the selection of a gene having a base sequence that encodes a conserved amino acid sequence TAX1DIX2G (SEQ ID NO: 15) or VPNGDKIH (SEQ ID NO: 16) of millipede-derived HNL from genes present in an organism belonging to the Diplopoda. A protein having an amino acid sequence of any of (1)-(3) and having HNL activity. (1) An amino acid sequence listed in any of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 83, 85, 87, or 89; (2) an amino acid sequence having amino acids deleted, substituted, and/or added in an amino acid sequence of (1); or (3) an amino acid sequence having 90% or greater identity to an amino acid sequence of (1). A method for preparing optically active cyanohydrin by causing this millipede-derived HNL to act on a reaction solvent that contains an aldehyde or the like and a substance that generates hydrogen cyanide or the like.