Bacillus megaterium CYP11A1 Expression for Steroid Conversion

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Solution Overview

Problem

Current methods struggle to achieve high rates of bioconversion of cholesterol to pregnenolone using recombinant microorganisms due to challenges with expressing insoluble membrane-associated CYP11A1 from mammalian origin in prokaryotic hosts, such as Escherichia coli, as it does not properly fold and maintain enzymatic activity.

Innovation Solution

A genetically engineered Bacillus megaterium strain (MS941) is developed to co-express the insoluble CYP11A1 of bovine origin along with bovine adrenodoxin reductase (AdR) and adrenodoxin (Adx), and its storage capacity is enhanced by modulating polyhydroxyalkanoate granule production, enabling high-rate conversion of cholesterol and its derivatives into pregnenolone and other steroid hormones.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If CYP11A1 is expressed in prokaryotic hosts such as Escherichia coli, then the enzyme can be produced, but it does not properly fold and maintain enzymatic activity due to being an insoluble membrane-associated enzyme

Engineering Contradiction:

Improveenzymatic activityVSAvoidproper folding

Core Design Contradiction:

ProductivityVSReliability

Solution Approach 1:

The patent introduces a membrane scaffold protein (MSP) as an intermediary carrier to express the membrane-associated CYP11A1 enzyme. The MSP forms soluble oligomeric structures that can properly fold and maintain enzymatic activity in prokaryotic hosts, while providing the necessary membrane-like environment for the CYP11A1 enzyme function.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Productivity

If CYP11A1 is expressed in recombinant microorganisms, then bioconversion can be achieved, but the conversion rate remains low due to insolubility and improper folding

Engineering Contradiction:

Improveconversion rateVSAvoidenzymatic stability

Core Design Contradiction:

ProductivityVSStability of the object's composition

Solution Approach 1:

The patent creates a composite enzyme system where CYP11A1 is fused with or associated with membrane scaffold protein oligomers. This composite structure combines the catalytic function of CYP11A1 with the structural stability and solubility of MSP, resulting in improved enzymatic stability and higher conversion rates.

Inventive Principle:

Principle #40Composite materials

Solution Approach 2:

The patent modifies the expression system by changing the host organism to Bacillus megaterium and altering the structural parameters of the enzyme through fusion with MSP. These parameter changes enable proper folding, increased solubility, and enhanced stability of the CYP11A1 enzyme, leading to improved bioconversion performance.

Inventive Principle:

Principle #35Parameter changes

3Productivity

If standard expression systems are used for CYP11A1, then the enzyme can be produced, but high rates of bioconversion cannot be achieved due to solubility issues

Engineering Contradiction:

Improvebioconversion rateVSAvoidexpression difficulty

Core Design Contradiction:

ProductivityVSEase of manufacture

Solution Approach 1:

The membrane scaffold protein serves as a mediator that simplifies the manufacturing process by providing a pre-formed soluble oligomeric structure. This eliminates the need for complex membrane preparation steps and allows straightforward expression in bacterial hosts, making the production process easier while achieving high bioconversion rates.

Inventive Principle:

Principle #24Intermediary (Mediator)

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 allows for efficient conversion of cholesterol and its analogs into pregnenolone and hydroxylated derivatives, surpassing previous systems in product yield and stability, demonstrating improved enzymatic activity and storage capacity for hydrophobic compounds.

Implementation Method 1

CYP11A1 of mammalian origin is an insoluble membrane-associated enzyme... co-expressing into B. megaterium MS941 the insoluble CYP11A1 of bovine origin, bovine adrenodoxin reductase (AdR), and bovine adrenodoxin (Adx)... efficient conversion of cholesterol and its analogs into pregnenolone and hydroxylated derivatives

Methodology Applied

Scientific EffectEnzymatic catalysis: Enzyme

Implementation Method 2

increasing its storage capacity dependent of polyhydroxyalkanoate granules... improved enzymatic activity and storage capacity for hydrophobic compounds

Methodology Applied

Scientific EffectStorage in polyhydroxyalkanoate granules:

Data Source

PatentEP3011045B1Whole-cell system for cytochrome p450 monooxygenases biocatalysis

Publication Date: 2019.10.16 SANOFI SA(FR)

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AI summary

The subject of the present invention is a whole-cell catalysis process for converting substrates of cytochrome P450 monooxygenases of eukaryotic origin into valuable biotechnological products. The subject of the present invention is also microorganisms genetically engineered to achieve those biotransformations with high rates and processes to prepare these microorganism strains.