Chimeric Enzymes for Lignin Monomer Conversion

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Solution Overview

Problem

Lignin, a key component of lignocellulosic biomass, is underutilized in biofuel production due to its inherent heterogeneity and recalcitrance, limiting the ability to selectively upgrade its monomers into value-added products.

Innovation Solution

Development of chimeric enzymes comprising a cytochrome P450 polypeptide and a catechol 1,2-dioxygenase polypeptide, which are used to dealkylate aromatic substrates such as guaiacol, allowing for the conversion into catechol and subsequently cis,cis-muconic acid, facilitating the efficient use of lignin-derived products.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Use of energy by moving object

If lignin is used as a substrate for biofuel production, then energy density is improved, but selectivity and ease of upgrading monomers to value-added products deteriorates due to heterogeneity and recalcitrance

Engineering Contradiction:

Improveenergy densityVSAvoidease of upgrading monomers

Core Design Contradiction:

Use of energy by moving objectVSEase of operation

Solution Approach 1:

The invention segments the complex lignin degradation process into two distinct enzymatic functions: a cytochrome P450 monooxygenase component for selective monomer release and a catechol 1,2-dioxygenase component for ring cleavage. This segmentation allows each enzyme to perform its specific function efficiently, overcoming the heterogeneity and recalcitrance of lignin while maintaining high energy density utilization.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The invention creates a composite enzymatic system by fusing two different enzyme components (cytochrome P450 monooxygenase and catechol 1,2-dioxygenase) into a single chimeric enzyme. This composite structure enables the enzyme to perform multiple catalytic functions sequentially, transforming lignin monomers through selective release and ring cleavage to produce value-added products like muconic acid, thereby improving ease of upgrading while maintaining energy density.

Inventive Principle:

Principle #40Composite materials

2Productivity

If conventional enzymes are used for lignin depolymerization, then monomer release is achieved, but selective upgrading to value-added products deteriorates due to recalcitrance

Engineering Contradiction:

Improvemonomer releaseVSAvoidselective upgrading

Core Design Contradiction:

ProductivityVSManufacturing precision

Solution Approach 1:

The invention merges two previously separate enzymatic functions into a single chimeric enzyme: the cytochrome P450 monooxygenase function for selective monomer release and the catechol 1,2-dioxygenase function for ring cleavage. This merging ensures that monomer release and selective upgrading occur in a coordinated manner, improving both productivity and manufacturing precision simultaneously.

Inventive Principle:

Principle #5Merging (Combining)

Solution Approach 2:

The chimeric enzyme combines two different catalytic domains into one protein structure, enabling sequential catalysis where the P450 domain releases monomers and the dioxygenase domain immediately processes them for ring cleavage. This composite design ensures high productivity in monomer release while maintaining high selectivity for value-added product formation, overcoming the recalcitrance of lignin.

Inventive Principle:

Principle #40Composite materials

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 chimeric enzymes enable the efficient conversion of lignin-derived substrates into valuable products like catechol and cis,cis-muconic acid, overcoming the limitations of lignin's recalcitrance and heterogeneity, thereby enhancing the utilization of lignin in biofuel production.

Implementation Method 1

chimeric enzymes comprising a cytochrome P450 polypeptide and a catechol 1,2-dioxygenase polypeptide, which are used to dealkylate aromatic substrates such as guaiacol

Methodology Applied

Scientific EffectEnzyme catalysis: Enzyme

Implementation Method 2

contacting a material containing the aromatic substrate with a chimeric enzyme to generate a dealkylation product

Methodology Applied

Scientific EffectDealkylation:

Implementation Method 3

conversion into catechol and subsequently cis,cis-muconic acid

Methodology Applied

Scientific EffectRing cleavage:

Implementation Method 4

catechol 1,2-dioxygenase polypeptide

Methodology Applied

Scientific EffectOxidation: Oxidation

Data Source

PatentUS10087425B2Chimeric enzymes for conversion of lignin-derived chemicals

Publication Date: 2018.10.02 UNIVERSITY OF GEORGIA RESEARCH FOUNDATION INC

AI summary

Disclosed herein are enzymes useful for the dealkylation of aromatic substrates, including the conversion of guaiacol or guaethol to catechol. Methods of converting aromatic substrates found in lignin-based feedstocks such as pyrolysis oil into products such as catechol are also disclosed. Also presented herein are methods for rapidly evolving and optimizing genetic regions.