Truncated Linalool Synthase Expression in Brewing Yeast
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Solution Overview
Problem
Current methods for producing monoterpenes like linalool in brewing yeast are limited by the instability of plant-derived linalool synthases due to plastid targeting sequences, which prevent functional expression, and result in subthreshold concentrations in beer, necessitating the identification of gene sequences that can produce linalool above flavor detection levels without these sequences.
Innovation Solution
Integration of N-terminally truncated gene sequences from plant linalool synthases into brewing yeast, specifically using pseudo-mature forms predicted by algorithms like ChloroP, to stabilize and enhance linalool production, combined with modifications to the mevalonate pathway genes to increase precursor availability, allowing for production of linalool and geraniol at detectable levels in beer.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If plant-derived linalool synthases with plastid targeting sequences are expressed in brewing yeast, then linalool production is attempted, but the plastid targeting sequences cause instability and prevent functional expression
Solution Approach 1:
The invention removes the plastid targeting sequences from the N-terminus of plant linalool synthases, retaining only the mature catalytic domain. This extraction of the problematic targeting sequence eliminates the instability issue while preserving the enzyme's linalool-producing function in brewing yeast.
Solution Approach 2:
The linalool synthase gene is segmented into distinct functional regions: the N-terminal plastid targeting sequence is separated and removed, while the mature catalytic domain is retained and expressed independently in yeast. This segmentation allows the enzyme to function without the destabilizing targeting sequence.
2Quantity of substance
If plant linalool synthases are expressed in brewing yeast, then linalool production is attempted, but the concentrations remain subthreshold for flavor detection
Solution Approach 1:
By removing the unstable plastid targeting sequences, the invention enables reliable functional expression of the linalool synthase catalytic domain in yeast, which subsequently produces linalool at concentrations exceeding human flavor detection thresholds.
Solution Approach 2:
The invention changes the expression parameters by using truncated gene sequences that encode only the mature catalytic domain without plastid targeting sequences. This parameter change stabilizes the enzyme and increases linalool production to detectable levels.
3Reliability
If truncated linalool synthase sequences are used to stabilize expression, then functional expression improves, but additional modifications to mevalonate pathway genes are required to increase precursor availability
Solution Approach 1:
The invention extracts and removes the unstable plastid targeting sequences from linalool synthase genes, enabling functional expression in yeast. While this alone improves stability, the patent also modifies mevalonate pathway genes to increase precursor availability, accepting the additional complexity to achieve high-level linalool production.
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 approach enables brewing yeast to produce linalool and geraniol at concentrations significantly higher than human flavor detection thresholds, potentially eliminating the need for hops in beer production, reducing costs and variability, and providing a sustainable alternative for flavor and aroma compounds.
Implementation Method 1
modifications to the mevalonate pathway genes to increase precursor availability
Implementation Method 2
linalool synthases, which convert geranyl pyrophosphate to linalool
Data Source
AI summary
The present disclosure provides a genetically modified host cell capable of producing linalool (or 3,7-dimethylocta-1,6-dien-3-ol).