Transgenic Yeast Lignin-Modifying Enzyme Secretion

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Solution Overview

Problem

Current methods for lignin deconstruction, particularly the production of lignin-modifying enzymes, face challenges such as reliance on aggressive chemical treatments, difficulty in tuning processes for valuable intermediate breakdown products, and limited genetic engineering capabilities in basidiomycete hosts, leading to inefficient enzyme production and secretion.

Innovation Solution

Development of transgenic organisms capable of expressing lignin-modifying enzymes, specifically using yeast strains like S. cerevisiae with genetic knockouts and expression cassettes that include inducible promoters and surface display proteins to enhance secretion and activity of enzymes like heme peroxidases.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If inorganic catalytic means are used for lignin deconstruction, then lignin breakdown is achieved, but aggressive chemical treatment is required and tuning for valuable intermediate products is difficult

Engineering Contradiction:

Improvelignin deconstruction efficiencyVSAvoidaggressive chemical treatment

Core Design Contradiction:

ProductivityVSObject-affected harmful factors

Solution Approach 1:

The patent replaces inorganic catalytic means (chemical system) with biological enzymes (biological system). Specifically, lignin-modifying enzymes such as peroxidases and laccases are used to degrade lignin under mild conditions, eliminating the need for aggressive chemical treatments while maintaining or improving deconstruction efficiency and enabling better control over intermediate product formation

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

Solution Approach 2:

The patent changes the operational parameters from harsh chemical conditions to mild biological conditions. By using enzymes that operate at neutral pH, moderate temperatures, and atmospheric pressure, the system achieves lignin deconstruction without aggressive chemical treatment, and enzyme specificity allows for tuning reaction conditions to capture valuable intermediate breakdown products

Inventive Principle:

Principle #35Parameter changes

2Productivity

If basidiomycete hosts are used for enzyme production, then lignin-modifying enzymes can be produced, but genetic engineering capabilities are limited and secretion is inefficient

Engineering Contradiction:

Improveenzyme production capabilityVSAvoidgenetic engineering complexity

Core Design Contradiction:

ProductivityVSDevice complexity

Solution Approach 1:

The patent uses Saccharomyces cerevisiae (baker's yeast) as a universal host platform that can produce diverse lignin-modifying enzymes from different fungal sources. The yeast's well-characterized genetics, ease of transformation, and efficient secretion machinery make it a multi-functional platform for producing peroxidases, laccases, and other lignin-degrading enzymes that were originally from basidiomycete fungi

Inventive Principle:

Principle #6Universality (Multi-functionality)

Solution Approach 2:

The patent introduces an intermediary organism (S. cerevisiae) that bridges the gap between basidiomycete enzyme production capabilities and ease of genetic engineering. The yeast serves as a mediator that can be easily genetically manipulated to produce basidiomycete-derived enzymes, combining the enzymatic capabilities of basidiomycetes with the genetic tractability of yeast

Inventive Principle:

Principle #24Intermediary (Mediator)

3Productivity

If conventional expression systems are used, then enzyme production is achieved, but secretion efficiency is low and productivity is limited

Engineering Contradiction:

Improveenzyme secretion rateVSAvoidsecretion efficiency

Core Design Contradiction:

ProductivityVSEase of manufacture

Solution Approach 1:

The patent applies preliminary actions to optimize secretion before actual enzyme production. This includes pre-induction of secretion pathways, optimization of signal peptide sequences for efficient ER targeting, and conditioning of growth media to prepare the yeast for high-level protein secretion. These preliminary steps ensure that when enzyme production is induced, secretion efficiency is already maximized

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The patent employs dynamic control of enzyme production and secretion through inducible promoters and regulated expression systems. Expression levels can be dynamically adjusted by controlling inducer concentration, allowing optimization of the balance between intracellular enzyme accumulation and extracellular secretion. This dynamic control enables the system to adapt secretion rates to match production rates, maximizing overall productivity

Inventive Principle:

Principle #15Dynamics

Data Source

PatentUS20230313257A1Methods and Systems to Secrete Lignin-Modifying Enzymes and Uses Thereof

Publication Date: 2023.10.05 THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIV

AI summary

Embodiments of the invention are generally directed to lignin-modifying enzymes and systems and methods of their manufacture. In many embodiments, yeast strains, including S. cerevisiae, are used to produce and secrete lignin-modifying enzymes Further embodiments are directed to methods to screening peroxidase-producing yeast strains, including S. cerevisiae. Additional embodiments are directed to an expression vector or cassette encoding for a protein of interest and one or more proteins to allow for surface to display of the protein of interest.