Fusion Polypeptide Enhances 7-DHC Production Efficiency
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Solution Overview
Problem
The expression of cholesterol 7-desaturase in heterologous organisms is low, limiting the efficient conversion of cholesterol to 7-dehydrocholesterol (7-DHC), a precursor for Vitamin D3 production.
Innovation Solution
A fusion polypeptide comprising cholesterol 7-desaturase and ferredoxin reductase, linked by flexible or rigid linkers, is expressed in microbial cells to enhance the enzyme complex's activity for 7-DHC production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If cholesterol 7-desaturase is expressed in heterologous organisms, then 7-DHC production is achieved, but the enzyme activity is very low
Solution Approach 1:
The patent combines cholesterol 7-desaturase with ferredoxin reductase into a fusion polypeptide. This merging of two enzyme functions into a single polypeptide chain creates an enzyme complex that maintains proper folding and electronic structure, thereby significantly improving the catalytic activity and reliability of cholesterol 7-desaturase in heterologous expression systems while maintaining high 7-DHC production efficiency
2Reliability
If fusion polypeptide with ferredoxin reductase is used, then enzyme activity is improved, but protein structure complexity increases
Solution Approach 1:
Rather than adding separate components, the patent merges ferredoxin reductase and cholesterol 7-desaturase into a single fusion polypeptide with a defined linker sequence. This approach improves enzyme activity through proper complex formation while keeping the structural complexity manageable through rational linker design that maintains flexibility and proper spatial arrangement of functional domains
Solution Approach 2:
The linker sequence in the fusion polypeptide acts as an intermediary element that connects the ferredoxin reductase domain and the cholesterol 7-desaturase domain. This intermediary structure allows proper folding and electronic interaction between the two functional domains while maintaining overall protein stability and solubility, thus improving enzyme activity without excessive structural complexity
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 fusion polypeptide significantly increases the production of 7-DHC, achieving concentrations greater than 700 mg/L from 800 mg/L cholesterol, improving the efficiency of Vitamin D3 precursor synthesis.
Implementation Method 1
a second moiety comprising a ferredoxin reductase
Implementation Method 2
cholesterol 7-desaturase is a Rieske oxygenase originally identified in insects and also observed in multiple other species. It has the unique ability to irreversibly catalyze the oxidation of cholesterol to 7-dehydrocholesterol (7-DHC)
Implementation Method 3
It has the unique ability to irreversibly catalyze the oxidation of cholesterol to 7-dehydrocholesterol (7-DHC)
Data Source
AI summary
The present disclosure relates to fusion proteins including an iron-sulfur protein (e.g., cholesterol 7-desaturase (C7D)) and a ferredoxin reductase (e.g., KshB) that can efficiently convert cholesterol to 7-dehydrocholesterol (7-DHC) in vivo. Also disclosed herein are engineered microbes (e.g., E. coli) expressing the fusion proteins and methods of use thereof.