Recombinant Ustilago maydis for Itaconic Acid Production

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Solution Overview

Problem

Current methods for producing itaconic acid using filamentous fungi, such as Aspergillus terreus, face challenges including difficulty in controlling morphology in fermentation systems and feedback inhibition, while alternative hosts like Ustilago strains are uncharacterized and inefficient.

Innovation Solution

Identification of a unique biosynthesis pathway in Ustilago maydis using aconitate-delta-isomerase (ADI) and trans-aconitate decarboxylase (TAD) enzymes, which convert cis-aconitate to itaconic acid, allowing for high-yield production by overexpressing these enzymes in recombinant host cells.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If Aspergillus terreus is used for itaconic acid production, then the biochemical pathway is well-characterized and CAD enzyme is available, but morphology control in fermentation systems becomes difficult

Engineering Contradiction:

Improvebiochemical pathway characterizationVSAvoidmorphology control

Core Design Contradiction:

ReliabilityVSEase of operation

Solution Approach 1:

The invention extracts the key enzymatic functions (cis-aconitate decarboxylase and trans-aconitate decarboxylase) from the complex Aspergillus terreus system and implements them in a simplified yeast system (Ustilago maydis or Saccharomyces cerevisiae), thereby eliminating the morphology control problems while retaining the biochemical pathway functionality

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The invention changes the host organism parameter from filamentous fungus to yeast, which fundamentally alters the morphology and fermentation characteristics while maintaining the ability to produce itaconic acid through engineered enzymatic pathways

Inventive Principle:

Principle #35Parameter changes

2Ease of manufacture

If Aspergillus terreus is used for itaconic acid production, then established methods are available, but feedback inhibition limits production yield

Engineering Contradiction:

Improveestablished methodsVSAvoidproduction yield

Core Design Contradiction:

Ease of manufactureVSProductivity

Solution Approach 1:

The invention applies feedback principle by engineering the pathway to include trans-aconitate decarboxylase that converts trans-aconitate (an intermediate) to itaconic acid, thereby preventing accumulation of intermediates that would otherwise cause feedback inhibition and limiting production yield

Inventive Principle:

Principle #23Feedback

Solution Approach 2:

The invention introduces cis-aconitate decarboxylase activity beforehand to convert cis-aconitate to trans-aconitate, preparing the substrate in advance for the subsequent trans-aconitate decarboxylase step, thereby optimizing the overall pathway efficiency and preventing metabolic bottlenecks

Inventive Principle:

Principle #10Preliminary action

3Ease of operation

If alternative hosts like Ustilago strains are used, then morphology control improves, but the biosynthesis pathway is uncharacterized and production efficiency is low

Engineering Contradiction:

Improvemorphology controlVSAvoidbiosynthesis pathway characterization

Core Design Contradiction:

Ease of operationVSReliability

Solution Approach 1:

The invention performs preliminary characterization of the Ustilago maydis pathway by identifying and validating the presence of cis-aconitate decarboxylase and trans-aconitate decarboxylase enzymes, thereby establishing a reliable and characterized biosynthesis pathway in this alternative host system

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The invention demonstrates that the identified enzymatic pathway in Ustilago maydis can be universally applied to other yeast systems (such as Saccharomyces cerevisiae) through heterologous expression, making the solution broadly applicable and highly reliable across different host platforms

Inventive Principle:

Principle #6Universality (Multi-functionality)

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 enables efficient production of itaconic acid in Ustilago maydis and other hosts, overcoming the limitations of traditional methods by providing a high-yield and characterized pathway for this valuable biobased chemical.

Implementation Method 1

Identification of a unique biosynthesis pathway in Ustilago maydis using aconitate-delta-isomerase (ADI) and trans-aconitate decarboxylase (TAD) enzymes, which convert cis-aconitate to itaconic acid

Methodology Applied

Scientific EffectIsomerization:

Implementation Method 2

Identification of a unique biosynthesis pathway in Ustilago maydis using aconitate-delta-isomerase (ADI) and trans-aconitate decarboxylase (TAD) enzymes, which convert cis-aconitate to itaconic acid

Methodology Applied

Scientific EffectDecarboxylation:

Implementation Method 3

Currently, most ITA is produced by fermentation of Aspergillus strains

Methodology Applied

Scientific EffectFermentation: Fermentation

Data Source

PatentUS10829791B2Means and methods for itaconic acid production

Publication Date: 2020.11.10 RWTH AACHEN UNIV

AI summary

The present invention relates to a method of producing itaconic acid. Further the present invention relates to nucleic acids encoding an aconitate-delta-isomerase (ADI) and trans-aconitate decarboxylase (TAD) and uses of such nucleic acids. Provided is additionally a recombinant host cell engineered to overexpress nucleic acids of the present invention. Furthermore an expression cassette and a vector are provided which include the respective nucleic acid.