7β-HSDH Mutants for Substrate Inhibition Control
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Solution Overview
Problem
Existing methods for synthesizing ursodeoxycholic acid (UDCA) are inefficient due to the need for multiple enzymes with different cofactor dependencies, substrate inhibition, and complex cofactor regeneration, leading to incomplete reactions and increased costs.
Innovation Solution
Development of novel 7β-hydroxysteroid dehydrogenase (7β-HSDH) mutants from Collinsella aerofaciens, capable of stereospecific reduction of DHCA to 3,12-diketo-7β-CA, combined with cofactor regeneration using formate dehydrogenase (FDH) enzymes, allowing simultaneous or delayed enzymatic conversions in microbial processes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If multiple enzymes with different cofactor dependencies are used for UDCA synthesis, then the enzymatic conversion can be performed, but the process complexity and cost increase
Solution Approach 1:
The patent combines multiple enzymatic functions into a single 7β-HSDH enzyme through domain fusion. The enzyme integrates a 7β-hydroxysteroid dehydrogenase domain for substrate reduction and a formate dehydrogenase domain for cofactor regeneration, eliminating the need for separate enzymes and simplifying the overall process while maintaining high productivity
Solution Approach 2:
The engineered 7β-HSDH enzyme performs multiple functions simultaneously: it catalyzes the reduction of dehydrocholic acid to 3,12-diketo-7β-cholanic acid and regenerates NADPH from NADP+ using formate as a substrate. This multi-functionality reduces process complexity by consolidating what would traditionally require separate enzymatic steps
2Productivity
If substrate inhibition is present in the enzymatic reaction, then the reaction can proceed, but the yield and efficiency decrease
Solution Approach 1:
The patent modifies the enzyme's kinetic parameters through rational design and directed evolution. Mutations were introduced to change the enzyme's substrate binding characteristics and catalytic properties, reducing substrate inhibition effects and improving reaction efficiency at higher substrate concentrations, thereby increasing overall yield
3Duration of action of moving object
If complex cofactor regeneration is required, then the enzymatic reaction can be sustained, but the process complexity and cost increase
Solution Approach 1:
The patent merges the cofactor regeneration function into the main enzymatic protein by fusing the formate dehydrogenase domain directly to the 7β-HSDH domain. This integrated enzyme uses formate as a substrate to regenerate NADPH in situ, eliminating the need for separate cofactor regeneration systems and reducing process complexity
Solution Approach 2:
The engineered enzyme performs self-service by incorporating its own cofactor regeneration capability within the same protein structure. The formate dehydrogenase domain regenerates NADPH from NADP+ using formate, allowing the enzyme to sustain its catalytic activity without external cofactor supplementation or complex regeneration systems
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 7β-HSDH mutants enhance the efficiency and simplicity of UDCA synthesis by reducing substrate inhibition and simplifying cofactor regeneration, leading to improved yield and reduced production costs.
Implementation Method 1
7β-HSDH mutants from Collinsella aerofaciens, capable of stereospecific reduction of DHCA to 3,12-diketo-7β-CA
Implementation Method 2
stereospecific reduction of DHCA to 3,12-diketo-7β-CA
Implementation Method 3
cofactor regeneration using formate dehydrogenase (FDH) enzymes
Implementation Method 4
cofactor regeneration using formate dehydrogenase (FDH) enzymes, allowing simultaneous or delayed enzymatic conversions
Data Source
AI summary
In various aspects and embodiments, the invention provides a nucleic acid molecule comprising a nucleotide sequence encoding a 7β-hydroxysteroid dehydrogenase (7β-HSDH) mutant that catalyzes at least the stereospecific enzymatic reduction of a 7-ketosteroid to the corresponding 7-hydroxysteroid, wherein the mutant has, compared to the wildtype 7β-HSDH of SEQ ID NO:2, a decreased substrate inhibition and/or an altered cofactor usage, and the mutant has, in comparison with the wildtype 7β-HSDH of SEQ ID NO:2, 1 to 15 amino acid additions, substitutions, deletions and/or inversions in the sequence motif VMVGRRE corresponding to positions 36 to 42 of SEQ ID NO:2.