Acetohydroxy Acid Synthase Variant for L-BCAA Production

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Solution Overview

Problem

Current methods face challenges in producing L-branched-chain amino acids on an industrial scale due to the complexity of parallel biosynthesis pathways and the limited understanding of acetohydroxy acid synthase, a crucial enzyme in their biosynthesis.

Innovation Solution

Development of an acetohydroxy acid synthase variant with specific amino acid substitutions in the large subunit, such as at positions 96 and 503, to enhance the enzyme's activity, allowing for increased production of L-branched-chain amino acids in microorganisms like Corynebacterium glutamicum.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If acetohydroxy acid synthase is used in parallel biosynthesis pathways, then branched-chain amino acids can be produced, but it is difficult to produce a single kind of branched-chain amino acid on an industrial scale

Engineering Contradiction:

Improveproduction yield of L-branched-chain amino acidsVSAvoidspecificity of single amino acid production

Core Design Contradiction:

ProductivityVSAdaptability or versatility

Solution Approach 1:

The patent applies local quality by introducing specific amino acid substitutions at particular positions (e.g., T96S, W503Q) in the acetohydroxy acid synthase large subunit to modify the enzyme's properties. This localized modification approach allows the enzyme to maintain its catalytic function while improving production efficiency and reducing feedback inhibition, enabling selective production of specific L-branched-chain amino acids.

Inventive Principle:

Principle #3Local quality

Solution Approach 2:

The patent employs parameter changes by modifying the amino acid sequence parameters of acetohydroxy acid synthase through site-directed mutagenesis. Specific substitutions such as Thr96→Ser and Trp503→Gln alter the enzyme's kinetic parameters and regulatory properties, thereby changing the production characteristics to favor single amino acid synthesis while maintaining high productivity.

Inventive Principle:

Principle #35Parameter changes

2Reliability

If acetohydroxy acid synthase small subunit (IlvN protein) is modified to release feedback inhibition, then enzyme activity is improved, but there is a serious lack of relevant studies on the large subunit

Engineering Contradiction:

Improveenzyme activity and feedback inhibition releaseVSAvoidknowledge gap in large subunit research

Core Design Contradiction:

ReliabilityVSLoss of information

Solution Approach 1:

The patent applies the inversion principle by shifting the research focus from the traditionally studied small subunit (IlvN protein) to the large subunit of acetohydroxy acid synthase. Instead of continuing to modify only the small subunit, the invention systematically investigates and modifies the large subunit, thereby inverting the conventional research approach and filling the knowledge gap while achieving improved enzyme performance.

Inventive Principle:

Principle #13The other way round (Inversion)

3Productivity

If acetohydroxy acid synthase catalyzes decarboxylation of pyruvate and condensation with pyruvate or 2-ketobutyrate, then acetolactate or acetohydroxybutyrate is produced, but the process is complex and difficult to control for single amino acid production

Engineering Contradiction:

Improveproduction rate of amino acid precursorsVSAvoidcomplexity of biosynthesis pathway

Core Design Contradiction:

ProductivityVSDevice complexity

Solution Approach 1:

The patent applies feedback control by engineering acetohydroxy acid synthase variants with reduced feedback inhibition from L-valine, L-leucine, and L-isoleucine. The specific amino acid substitutions (e.g., T96S, W503Q) modify the enzyme's regulatory sites, allowing the biosynthesis pathway to proceed more efficiently without being strongly inhibited by end-product accumulation, thereby simplifying control for single amino acid production.

Inventive Principle:

Principle #23Feedback

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 modified acetohydroxy acid synthase variant significantly increases the yield of L-branched-chain amino acids, facilitating their large-scale production by improving the microorganism's ability to produce these essential compounds.

Implementation Method 1

The acetohydroxy acid synthase catalyzes decarboxylation of pyruvate and a condensation reaction with another pyruvate molecule to produce acetolactate

Methodology Applied

Scientific EffectDecarboxylation:

Implementation Method 2

The acetohydroxy acid synthase catalyzes decarboxylation of pyruvate and a condensation reaction with another pyruvate molecule to produce acetolactate

Methodology Applied

Scientific EffectCondensation reaction:

Implementation Method 3

a method for producing an L-branched-chain amino acid, which includes: culturing the microorganism producing the L-branched-chain amino acid in a medium; and recovering the L-branched-chain amino acid from the microorganism or cultured medium thereof

Methodology Applied

Scientific EffectFermentation: Fermentation

Data Source

PatentUS11248220B2Acetohydroxy acid synthase variant, microorganism comprising the same, and method of producing l-branched-chain amino acid using the same

Publication Date: 2022.02.15 CJ CHEILJEDANG CORP

AI summary

The present disclosure relates to a novel acetohydroxy acid synthase, a microorganism comprising the same, or a method for producing an L-branched-chain amino acid using the same.