Codon-optimized fungal cellobiohydrolase 2 expression in yeast

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Solution Overview

Problem

Current methods for converting lignocellulosic biomass into ethanol are hindered by the recalcitrance of biomass and the inefficiency of heterologous cellulase expression in yeast, particularly in achieving consolidated bioprocessing (CBP) systems that can efficiently and cost-effectively convert cellulosic substrates to ethanol.

Innovation Solution

The heterologous expression of wild-type and codon-optimized variants of cellobiohydrolases from fungal organisms such as Cochliobolus heterostrophus, Gibberella zeae, Irpex lacteus, Volvariella volvacea, and Piromyces sp. in Saccharomyces cerevisiae, enhancing the specific activity of expressed cellobiohydrolases for improved cellulose hydrolysis and ethanol production.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Adaptability or versatility

If heterologous cellulase expression is implemented in yeast to achieve consolidated bioprocessing, then the integration of cellulase production and ethanol fermentation is improved, but the efficiency of cellulose hydrolysis deteriorates due to low specific activity of expressed enzymes

Engineering Contradiction:

Improveconsolidated bioprocessing capabilityVSAvoidcellulose hydrolysis efficiency

Core Design Contradiction:

Adaptability or versatilityVSProductivity

Solution Approach 1:

The patent applies codon optimization to change the nucleotide sequence parameters of fungal cbh2 genes to match yeast codon preferences, thereby improving translation efficiency and specific activity of expressed cellobiohydrolases while maintaining the consolidated bioprocessing capability

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The patent creates a composite enzymatic system by co-expressing multiple cellulase components (endoglucanases, cellobiohydrolases, and β-glucosidases) from different fungal sources in a single yeast host, achieving synergistic effects that improve overall cellulose hydrolysis efficiency

Inventive Principle:

Principle #40Composite materials

2Adaptability or versatility

If fungal cbh2 genes are heterologously expressed in yeast, then the ability to utilize cellulosic substrates is improved, but the cost-effectiveness deteriorates due to low expression efficiency and high enzyme production costs

Engineering Contradiction:

Improvecellulosic substrate utilizationVSAvoidcost-effectiveness

Core Design Contradiction:

Adaptability or versatilityVSEase of manufacture

Solution Approach 1:

Codon optimization changes the genetic parameters of fungal cbh2 genes to enhance expression levels in yeast, reducing the metabolic burden and production costs while maintaining cellulosic substrate utilization capability

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The engineered yeast strain produces its own cellulase enzymes internally through heterologous gene expression, eliminating the need for external enzyme addition and reducing manufacturing costs associated with separate enzyme production and purification steps

Inventive Principle:

Principle #25Self-service

3Adaptability or versatility

If wild-type fungal cellobiohydrolases are expressed in yeast, then the heterologous expression system is established, but the specific activity deteriorates compared to native fungal enzymes

Engineering Contradiction:

Improveheterologous expression capabilityVSAvoidspecific activity

Core Design Contradiction:

Adaptability or versatilityVSReliability

Solution Approach 1:

The patent optimizes codon usage parameters and potentially other genetic parameters of fungal cbh2 genes to match yeast cellular environment, thereby restoring and enhancing the specific activity of heterologously expressed cellobiohydrolases to levels comparable to or exceeding native fungal enzymes

Inventive Principle:

Principle #35Parameter changes

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 enables efficient and cost-effective conversion of cellulosic substrates to ethanol by augmenting cellulose hydrolysis, thereby facilitating the goal of achieving a consolidated bioprocessing system capable of utilizing cellulosic substrates without externally added enzymes.

Implementation Method 1

Cellobiohydrolases (CBHs) are exo-acting enzymes that liberate cellobiose units from the non-reducing ends of cellulose chains

Methodology Applied

Scientific EffectHydrolysis: Hydrolysis

Implementation Method 2

The heterologous expression of wild-type and codon-optimized variants of cellobiohydrolases from fungal organisms... enhancing the specific activity of expressed cellobiohydrolases for improved cellulose hydrolysis

Methodology Applied

Scientific EffectEnzyme catalysis: Enzyme

Data Source

PatentUS10196622B2Heterologous expression of fungal cellobiohydrolase 2 genes in yeast

Publication Date: 2019.02.05 STELLENBOSCH UNIVERSITY

AI summary

The present invention provides for heterologous expression of polypeptides encoded by wild-type and codon-optimized cbh2 genes from the organisms Cochliobolus heterostrophus, Gibberella zeae, Irpex lacteus, Volvariella volvacea, and Piromyces sp. in host cells, such as the yeast Saccharomyces cerevisiae. The expression in such host cells of the corresponding genes, and variants and combinations thereof, result in improved specific activity of the expressed cellobiohydrolases. Thus, such genes and expression systems are useful for efficient and cost-effective consolidated bioprocessing systems.