Diterpene Alcohol Synthase for C-20 Terpenoid Production

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Solution Overview

Problem

There is a need for highly efficient enzymes that can catalyze the step 2 reaction in the manufacture of C-20 terpenoid alcohols, particularly for abienol, sclareol, and manool, and enzymes that are not limited to producing only one C-20 terpenoid alcohol.

Innovation Solution

A method involving a polypeptide with diterpene alcohol synthase activity, capable of converting geranylgeranyl pyrophosphate into copalyl diphosphate or labda-13-en-8-ol diphosphate, and further converting these into C-20 terpenoid alcohols, using specific amino acid sequences or their variants that exhibit high identity to sequences such as those shown in SEQ ID NOs: 3 to 10 or 34, encoded by nucleic acid sequences in SEQ ID NOs: 1, 2, 16, 17, or 35, to produce manool, sclareol, and abienol.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If conventional enzymes are used for C-20 terpenoid alcohol production, then the production process is established, but the production efficiency and substrate versatility are insufficient

Engineering Contradiction:

Improveproduction efficiencyVSAvoidenzyme performance consistency

Core Design Contradiction:

ProductivityVSReliability

Solution Approach 1:

The patent applies parameter changes by modifying amino acid residues in the enzyme sequence (specifically positions 117, 118, and 185) to optimize catalytic activity and substrate acceptance. This systematic variation of molecular parameters enables the enzyme to achieve both high productivity across multiple substrates and consistent performance, resolving the technical contradiction between production efficiency and reliability.

Inventive Principle:

Principle #35Parameter changes

2Adaptability or versatility

If specific enzymes are designed for single C-20 terpenoid alcohol production, then production specificity is achieved, but enzyme versatility is limited

Engineering Contradiction:

Improvesubstrate acceptance rangeVSAvoidproduct specificity

Core Design Contradiction:

Adaptability or versatilityVSManufacturing precision

Solution Approach 1:

The patent implements universality by engineering an enzyme that can accept multiple substrates (CPP and LPP) and produce different C-20 terpenoid alcohols (manool, sclareol, abienol) with high efficiency. The modified enzyme maintains product specificity through controlled stereochemistry while achieving broad substrate acceptance, thus resolving the contradiction between versatility and manufacturing precision.

Inventive Principle:

Principle #6Universality (Multi-functionality)

3Productivity

If natural plant sources are used for C-20 terpenoid alcohols, then product authenticity is maintained, but production scalability and efficiency are insufficient

Engineering Contradiction:

Improveproduction scalabilityVSAvoidproduction process complexity

Core Design Contradiction:

ProductivityVSEase of manufacture

Solution Approach 1:

The patent replaces mechanical extraction from natural plant sources with a biocatalytic system using engineered enzymes. This substitution enables scalable production through controlled enzymatic reactions in bioreactors, significantly improving productivity while simplifying the manufacturing process compared to traditional extraction methods, thus resolving the contradiction between scalability and ease of manufacture.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

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 method enables the efficient manufacture of C-20 terpenoid alcohols, such as manool, sclareol, and abienol, by utilizing polypeptides with diterpene alcohol synthase activity, enhancing production efficiency and versatility in producing multiple C-20 terpenoid alcohols.

Implementation Method 1

converting geranylgeranyl pyrophosphate into copalyl diphosphate (CPP) or labda-13-en-8-ol diphosphate (LPP) and converting CPP or LPP into at least one C-20 terpenoid alcohol, wherein said conversion is carried out by a polypeptide exhibiting diterpene alcohol synthase activity

Methodology Applied

Scientific EffectEnzyme catalysis: Enzyme

Data Source

PatentUS20250002947A1Recombinant manufacture of c-20 terpenoid alcohols

Publication Date: 2025.01.02 ISOBIONICS BV

AI summary

Disclosed is a method for the manufacture of at least one C-20 terpenoid alcohol comprising the steps of converting geranylgeranyl pyrophosphate into copalyl diphosphate (CPP) or labda-13-en-8-ol diphosphate (LPP) and converting CPP or LPP into at least one C-20 terpenoid alcohol, wherein said conversion is carried out by a polypeptide exhibiting diterpene alcohol synthase activity capable of converting CPP into manool, LPP into sclareol and/or LPP into abienol, and wherein said polypeptide comprises an amino acid sequence as specified in the claims. The invention further relates to the aforementioned polypeptide exhibiting diterpene alcohol synthase activity as well as a fusion protein comprising said polypeptide, a polynucleotide encoding it, a vector or gene construct comprising said polynucleotide, a host cell comprising said vector or gene construct. a non-human transgenic organism comprising the polynucleotide, vector, gene construct or host cell, as well as uses thereof for the manufacture of at least one C-20 terpenoid alcohol.