Chimeric MALT3/MALT4 Transporter for Saccharomyces eubayanus Brewing

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Solution Overview

Problem

Current brewing yeast strains, particularly Saccharomyces eubayanus, struggle to utilize maltotriose as a carbon source, leading to unconsumed sugars, lower ethanol production, and undesirable flavor in beer, as they lack efficient maltotriose transport mechanisms.

Innovation Solution

Development of polypeptides and yeast cells with enhanced maltose/maltotriose transport activity through directed evolution, specifically modifying Saccharomyces eubayanus to express chimeric MALT3/MALT4 proteins that enable maltotriose utilization as a sole carbon source.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Adaptability or versatility

If conventional Saccharomyces eubayanus strains are used for brewing, then the yeast can grow and ferment, but they cannot efficiently utilize maltotriose as a carbon source

Engineering Contradiction:

Improvemaltotriose utilization capabilityVSAvoidethanol production efficiency

Core Design Contradiction:

Adaptability or versatilityVSProductivity

Solution Approach 1:

The patent applies parameter changes by modifying the amino acid sequence of the MALT4 transporter protein through directed evolution. Specific amino acid substitutions (e.g., at positions 468, 503, 504, 505, 508, 512, 522, 534, 536, 538, or 540) are introduced to alter the transporter's substrate specificity and affinity, enabling it to recognize and transport maltotriose efficiently. This molecular-level parameter modification resolves the contradiction by giving the yeast the ability to utilize maltotriose as a carbon source while maintaining high ethanol production efficiency.

Inventive Principle:

Principle #35Parameter changes

2Reliability

If maltotriose is not utilized by the yeast, then the yeast can complete fermentation, but large amounts of unconsumed sugar remain and undesirable flavor is produced

Engineering Contradiction:

Improvefermentation completionVSAvoidunconsumed sugar and undesirable flavor

Core Design Contradiction:

ReliabilityVSObject-generated harmful factors

Solution Approach 1:

The patent converts the previously harmful presence of maltotriose (which caused unconsumed sugar and undesirable flavor) into a beneficial substrate. By engineering the MALT4 transporter to recognize and transport maltotriose, the yeast transforms this unwanted sugar into a usable carbon source that fuels fermentation, thereby eliminating the harmful effects while maintaining reliable fermentation completion.

Inventive Principle:

Principle #22Blessing in disguise (Convert harm into benefit)

3Adaptability or versatility

If new brewing strains are developed through traditional methods, then strain diversity is achieved, but the development process is time-consuming and lacks efficiency

Engineering Contradiction:

Improvestrain diversityVSAvoidstrain development time

Core Design Contradiction:

Adaptability or versatilityVSLoss of time

Solution Approach 1:

The patent applies preliminary action by using computational modeling and in silico directed evolution to predict and identify beneficial amino acid substitutions before performing actual laboratory work. The MALT4 protein sequence is computationally mutated and evaluated to identify promising variants that confer maltotriose utilization, allowing the experimental work to be focused only on validating the most promising candidates, thereby significantly reducing strain development time while maintaining strain diversity.

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

The modified yeast cells can efficiently consume maltotriose, increasing ethanol production and improving beer quality by utilizing maltotriose as a primary carbon source, thereby addressing the limitations of existing strains.

Implementation Method 1

polypeptides capable of acting as maltotiose transporters... polypeptides with enhanced maltose/maltotriose transport activity

Methodology Applied

Scientific EffectActive transport:

Implementation Method 2

yeast cells comprising such polypeptides are provided. The resultant yeast cells are capable of growing on maltose and/or maltotriose... increasing ethanol production

Methodology Applied

Scientific EffectFermentation: Fermentation

Data Source

PatentUS20240084315A1Polypeptide and yeast cell compositions and methods of using the same

Publication Date: 2024.03.14 WISCONSIN ALUMNI RES FOUND

AI summary

Polypeptides comprising maltose/maltotriose transporters are provided. Additionally, polynucleotides, DNA constructs, and vectors encoding a maltose/maltotriose transporter, or yeast cells harboring such polynucleotides are provided. The yeast cell may be a Saccharomyces eubayanus cell modified to increase the expression or transport activity of a maltose/maltotriose transporter at the plasma membrane of the cell. Further, methods are provided for making a fermentation product by culturing any one of the yeast cells described herein with a fermentable substrate. Finally, methods are provided to select for and isolate maltotriose-utilizing strains of Saccharomyces eubayanus.