Sterol Acyltransferase Mutations for Selective 7-DHC Esterification

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Solution Overview

Problem

Existing yeast strains used in vitamin D3 production have unspecific sterol acyltransferase enzymes that lead to diverse steryl ester pools, including unwanted intermediates like zymosterol, lanosterol, and lathosterol, which are toxic and reduce productivity, necessitating improved specificity and activity for 7-DHC production.

Innovation Solution

Introduction of specific amino acid substitutions in the ARE2 and ARE1 sterol acyltransferases, particularly at positions corresponding to residues 11, 281, 366, 442, 551, 554, 572, 624, 626, and 636, enhances the enzymes' specificity and activity for 7-DHC esterification, reducing unwanted intermediates and increasing productivity.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Adaptability or versatility

If sterol acyltransferase enzymes (Are1p and Are2p) are used for ester formation of sterols in yeast, then steryl esters are stored in lipid bodies, but the enzymes act unspecifically leading to diverse steryl ester pool including side-products like zymosterol and lanosterol

Engineering Contradiction:

Improvesubstrate specificityVSAvoidester formation specificity

Core Design Contradiction:

Adaptability or versatilityVSManufacturing precision

Solution Approach 1:

The patent applies parameter changes by introducing specific amino acid substitutions at defined positions in the sterol acyltransferase enzyme sequence. These substitutions modify the enzyme's substrate binding properties and catalytic specificity, enabling it to preferentially esterify 7-DHC over other sterol intermediates like zymosterol and lanosterol, thus resolving the contradiction between versatility and precision

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The patent creates modified versions of the native Are1p and Are2p enzymes by copying their sequence and introducing specific mutations. These copied and modified enzymes retain the core function of ester formation while gaining improved substrate specificity for 7-DHC, allowing selective storage of desired steryl esters in lipid bodies

Inventive Principle:

Principle #26Copying

2Quantity of substance

If diverse steryl esters are stored in lipid bodies, then sterol intermediates are sequestered, but side-products like zymosterol and lanosterol accumulate which are toxic and reduce productivity

Engineering Contradiction:

Improvesterol storage capacityVSAvoidtoxicity of side-products

Core Design Contradiction:

Quantity of substanceVSObject-generated harmful factors

Solution Approach 1:

The patent applies the extraction principle by using the modified sterol acyltransferase enzymes to selectively extract and esterify only the desired 7-DHC substrate from the mixture of sterol intermediates. This selective esterification removes 7-DHC from the toxic side-product pool and directs it into storage as specific steryl esters in lipid bodies, preventing accumulation of toxic compounds

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The patent converts the potential harm of unspecific esterification that would lead to toxic side-product accumulation into a benefit by engineering enzymes with enhanced specificity. The modified enzymes now preferentially process 7-DHC, transforming what was previously a source of toxicity into a productive pathway for vitamin D3 precursor accumulation

Inventive Principle:

Principle #22Blessing in disguise (Convert harm into benefit)

3Productivity

If amino acid substitutions are introduced in sterol acyltransferase enzymes, then specificity and activity towards 7-DHC is enhanced, but enzyme structure is modified

Engineering Contradiction:

Improve7-DHC productionVSAvoidenzyme sequence complexity

Core Design Contradiction:

ProductivityVSDevice complexity

Solution Approach 1:

The patent applies local quality by introducing amino acid substitutions only at specific, localized positions in the enzyme sequence rather than throughout the entire protein. These targeted changes at defined positions modify local substrate binding properties to enhance 7-DHC specificity while preserving the overall enzyme structure and function

Inventive Principle:

Principle #3Local quality

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 enzymes significantly increase the specificity and activity for 7-DHC, enhancing its production by up to 12-times compared to non-modified yeast strains, leading to higher 7-DHC yields and reduced accumulation of side-products.

Implementation Method 1

The present invention is related to modified sterol acyltransferase enzymes with improved activity and/or specificity towards acylation of the vitamin D3 precursor 7-dehydrocholesterol (7-DHC)

Methodology Applied

Scientific EffectEnzyme catalysis: Enzyme

Data Source

PatentUS12630852B2Modified sterol acyltransferases

Publication Date: 2026.05.19 DSM IP ASSETS BV

AI summary

The present invention is related to modified sterol acyltransferase enzymes with improved activity and/or specificity towards acylation of the vitamin D3 precursor 7-dehydrocholesterol (7-DHC) to be used in biotechnological production of vitamin D3. The invention further relates to a host strain expressing said modified enzymes and their use in a process for production of vitamin D3 or derivatives and/or metabolites thereof.