Recombinant Yeast Tyrosol Production via Metabolic Pathway Engineering

Solution Overview

Problem

Current methods for producing tyrosol and hydroxytyrosol are costly, environmentally unfriendly, and face challenges in large-scale industrial production due to high raw material costs and the presence of endotoxins in Gram-negative bacteria, which are toxic to humans.

Innovation Solution

A recombinant Saccharomyces cerevisiae strain is developed by introducing tyrosine decarboxylase and alcohol dehydrogenase genes, along with a PDC1 knockout cassette and 4-hydroxyphenylacetic hydroxylase genes, to enhance tyrosol and hydroxytyrosol production, bypassing the ethanol synthesis pathway and utilizing a safer yeast strain for industrial production.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If E. coli is used for tyrosol production, then production capability is achieved, but endotoxin contamination occurs which is toxic to humans

Engineering Contradiction:

Core Design Contradiction:

ProductivityVSObject-affected harmful factors

Solution Approach 1:

The patent extracts the harmful endotoxin component by replacing the Gram-negative E. coli host with a Gram-positive yeast host (Saccharomyces cerevisiae or Pichia pastoris), which naturally lacks endotoxins in its cell wall structure, thereby eliminating the toxicity problem while maintaining production capability

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The patent introduces an intermediary organism (yeast) that serves as a safe mediator between the production process and human application, providing the necessary enzymatic functions for tyrosol synthesis without introducing harmful endotoxins

Inventive Principle:

Principle #24Intermediary (Mediator)

2Productivity

If chemical synthesis methods are used, then production can be achieved, but raw material costs are high and environmental friendliness is compromised

Engineering Contradiction:

Core Design Contradiction:

ProductivityVSObject-affected harmful factors

Solution Approach 1:

The patent replaces complex chemical synthesis mechanisms with biological enzymatic mechanisms, using genetically engineered yeast to catalyze the conversion of tyrosine to tyrosol through decarboxylation, thereby eliminating the need for expensive and environmentally harmful chemical reagents and multiple synthesis steps

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

Solution Approach 2:

The patent changes the fundamental parameter of the synthesis system from chemical to biological, utilizing metabolic pathways and enzyme catalysis in yeast cells to achieve tyrosol production under mild, environmentally friendly conditions with lower raw material costs

Inventive Principle:

Principle #35Parameter changes

3Stability of the object's composition

If ethanol synthesis pathway is active in yeast, then yeast metabolism is normal, but tyrosol production is limited

Engineering Contradiction:

Core Design Contradiction:

Stability of the object's compositionVSProductivity

Solution Approach 1:

The patent segments the metabolic pathways by introducing a dedicated tyrosol biosynthesis pathway parallel to the ethanol pathway, with specific enzymes (tyrosine decarboxylase and alcohol dehydrogenase) dedicated to tyrosol production, allowing independent optimization of tyrosol yield without disrupting overall yeast metabolic stability

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The patent performs preliminary genetic engineering to knockout or downregulate the ethanol synthesis pathway genes before introducing the tyrosol production pathway, pre-conditioning the yeast metabolism to favor tyrosol production while maintaining cellular stability

Inventive Principle:

Principle #10Preliminary action

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 enhances tyrosol production, converts it to hydroxytyrosol, and provides a safer, more stable, and environmentally friendly biological method suitable for large-scale industrial production, reducing costs and contamination risks.

Implementation Method 1

introducing a tyrosine decarboxylase (TryDC) PcAAS encoding DNA sequence

Methodology Applied

Scientific EffectDecarboxylation:

Implementation Method 2

introducing an alcohol dehydrogenase (ADH) encoding DNA sequence

Methodology Applied

Scientific EffectReduction: Reduction

Implementation Method 3

transforming with hydroxylase genes able to produce hydroxytyrosol

Methodology Applied

Scientific EffectHydroxylation:

Data Source

PatentUS11634720B2Yeast producing tyrosol or hydroxytyrosol, and construction methods thereof

Publication Date: 2023.04.25

AI summary

The technical field of preparation of organic compounds, and particularly to yeasts producing tyrosol or hydroxytyrosol and construction methods thereof. PcAAS and ADH-encoding DNA sequences are introduced into the yeast strain BY4741, to obtain a PcAAS-ADH recombinant yeast producing tyrosol. A PDC1 knockout cassette and a TyrA expression cassette are introduced into the PcAAS-ADH recombinant yeast to obtain a PcAAS-ADH-ΔPDC1-TyrA recombinant yeast producing tyrosol. A HpaBC encoding DNA sequence is introduced into the PcAAS-ADH-ΔPDC1-TyrA recombinant yeast, to obtain a PcAAS-ADH-HpaBC-ΔPDC1-TyrA recombinant yeast producing hydroxytyrosol. The construction of a tyrosol or hydroxytyrosol biosynthesis pathway in the yeast strain BY4741 enhances the production of tyrosol or hydroxytyrosol.