Short-Chain Monooxygenase Expression in Microbes Using Folding Chaperones

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Solution Overview

Problem

Soluble diiron monooxygenases are difficult to functionally express in industrially relevant host cells, hindering the conversion of inexpensive short alkanes like methane or ethane into valuable industrial products such as methanol or ethanol.

Innovation Solution

The functional expression of a monooxygenase synthetic polynucleotide in microorganisms, comprising a monooxygenase coding region linked to a promoter, and optionally a protein folding chaperone, to facilitate the expression of soluble diiron monooxygenases, along with dehydrogenase enzymes, enabling the conversion of alkanes into industrial products.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Adaptability or versatility

If soluble diiron monooxygenases are expressed in industrially relevant host cells, then the conversion of short alkanes into industrial products becomes possible, but the expression difficulty and functional stability worsen

Engineering Contradiction:

Improveability to convert short alkanes into industrial productsVSAvoidfunctional expression stability

Core Design Contradiction:

Adaptability or versatilityVSReliability

Solution Approach 1:

The patent introduces protein folding chaperones as intermediary molecules that facilitate the proper folding and stabilization of soluble diiron monooxygenases in heterologous host cells. The chaperones act as mediators between the expressed enzyme and the cellular environment, ensuring proper conformation and functional stability without requiring modification of the enzyme itself or the host cell machinery.

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The patent modifies expression parameters including the use of inducible promoters to control expression timing and level, optimization of cultivation conditions, and adjustment of enzyme concentration. These parameter changes enable the system to overcome expression difficulties while maintaining functional stability of the monooxygenase in industrially relevant hosts.

Inventive Principle:

Principle #35Parameter changes

2Productivity

If sugar is used as a raw material for biological fermentations, then the production of industrial products is enabled, but the production cost increases

Engineering Contradiction:

Improveproduction of industrial productsVSAvoidproduction cost

Core Design Contradiction:

ProductivityVSEase of manufacture

Solution Approach 1:

The patent fundamentally changes the substrate parameter from sugar to short alkanes (methane, ethane). This parameter change enables the use of cheaper, abundant natural gas resources instead of expensive sugar feedstocks, directly reducing production costs while maintaining the ability to produce industrial chemicals through engineered microbial pathways.

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The patent introduces heterologous gene pathways that copy and adapt natural alkane activation mechanisms into industrially relevant host cells. By expressing soluble diiron monooxygenases and associated metabolic pathways, the system replicates the functionality of native alkane-metabolizing organisms in hosts optimized for industrial production, enabling cost-effective manufacturing.

Inventive Principle:

Principle #26Copying

3Adaptability or versatility

If the range of substrates is expanded to include short alkanes, then the economic viability improves, but the complexity of the enzymatic pathway increases

Engineering Contradiction:

Improverange of substratesVSAvoidenzymatic pathway complexity

Core Design Contradiction:

Adaptability or versatilityVSDevice complexity

Solution Approach 1:

The patent employs soluble diiron monooxygenases that exhibit broad substrate specificity, enabling a single enzyme system to process multiple short alkanes (methane, ethane, and other C1-C4 hydrocarbons). This multi-functionality reduces the need for separate specialized enzymes for each substrate, thereby simplifying the overall pathway complexity while expanding substrate range.

Inventive Principle:

Principle #6Universality (Multi-functionality)

Solution Approach 2:

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

Enables the efficient conversion of alkanes into industrial products like methanol or ethanol, reducing production costs and expanding the range of products that can be produced by engineered microorganisms.

Implementation Method 1

Some chemical reactions are catalyzed by either inorganic catalysts or certain enzymes... enzymes are advantageous catalysts if the alternative process requires expensive or energy-intensive conditions

Methodology Applied

Scientific EffectEnzymatic catalysis: Enzyme

Implementation Method 2

Naturally occurring bacteria that can activate methane use dioxygen to convert methane to methanol. As an example, an enzyme capable of performing this reaction belongs to the class known as soluble diiron monooxygenases

Methodology Applied

Scientific EffectMonooxygenase reaction: Oxidation