Codon-Optimized Myoglobin Expression in Trichoderma

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Solution Overview

Problem

Current methods for producing myoglobin, a heme-protein essential for oxygen transport and nutritional benefits, face challenges in scalability and sustainability, particularly in fungal hosts like Trichoderma reesei, where high expression levels and efficient secretion are needed for food product applications.

Innovation Solution

A method involving codon optimization of the myoglobin gene for Trichoderma reesei, insertion into a plasmid, and expression with additional enzymes from the porphyrin synthesis pathway, along with heme-iron supplementation, to enhance heme group production and secretion of recombinant bovine myoglobin into the culture media.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Quantity of substance

If myoglobin is produced in yeast using recombinant technology, then myoglobin expression is achieved, but scalability is difficult

Engineering Contradiction:

Improvemyoglobin expression levelVSAvoidscalability

Core Design Contradiction:

Quantity of substanceVSProductivity

Solution Approach 1:

The patent changes the host organism parameter from yeast to Trichoderma reesei fungus, which naturally secretes large amounts of cellulase into the culture media. This parameter change enables scalable production while maintaining high myoglobin expression levels, as T. reesei's secretory machinery can efficiently export recombinant proteins to the extracellular environment where they can be easily harvested

Inventive Principle:

Principle #35Parameter changes

2Productivity

If myoglobin is produced in Trichoderma reesei, then scalability is improved, but expression level and secretion efficiency need enhancement

Engineering Contradiction:

ImprovescalabilityVSAvoidmyoglobin expression level

Core Design Contradiction:

ProductivityVSQuantity of substance

Solution Approach 1:

The patent applies codon optimization to the myoglobin gene sequence before insertion into the T. reesei expression system. This preliminary action adapts the gene coding sequence to match the fungal host's preferred codon usage patterns, ensuring efficient translation and high expression levels of myoglobin in Trichoderma reesei

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The patent uses a plasmid vector as an intermediary carrier to deliver the codon-optimized myoglobin gene into Trichoderma reesei. The plasmid contains appropriate promoters, terminators, and selection markers that facilitate stable integration and high-level expression of the myoglobin gene in the fungal host

Inventive Principle:

Principle #24Intermediary (Mediator)

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 significantly increases the yield and stability of recombinant myoglobin in Trichoderma reesei, making it suitable for use in plant-based food products that mimic the flavor and nutritional benefits of meat, while being scalable and sustainable.

Implementation Method 1

co-expression of porphyrin pathway enzymes, such as ALAS, ALAD, PBGD, UROD, and/or a ferrochelatase

Methodology Applied

Scientific EffectPorphyrin synthesis pathway:

Implementation Method 2

enhance heme group production and secretion of recombinant bovine myoglobin

Methodology Applied

Scientific EffectHeme biosynthesis:

Data Source

PatentUS20230340077A1Production of myoglobin from trichoderma using a feeding media

Publication Date: 2023.10.26 LUYEF BIOTECHNOLOGIES INC

AI summary

A method to express myoglobin in a fungus is described. The method includes optimizing a codon for a fungal host (e.g., Trichoderma reesei), inserting the optimized codon into a plasmid (e.g., a pTrEno plasmid or one of its derivatives), and ultimately collecting the secreted myoglobin from a feeding media in response to the myoglobin being expressed extracellularly. Optionally, the myoglobin is purified.