Engineered Microbial Cells for (6E)-8-Hydroxygeraniol Fermentation
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Solution Overview
Problem
Current methods for producing (6E)-8-hydroxygeraniol through microbial fermentation face challenges due to limitations in microbial hosts' ability to express necessary enzymes and overcome feedback inhibition, leading to suboptimal yields.
Innovation Solution
Engineered microbial cells are developed to express non-native geranyl diphosphate diphosphatase and geraniol-8-hydroxylase, with increased activity of upstream pathway enzymes and reduced activity of enzymes consuming precursors, along with feedback-deregulated HMG-CoA reductase, to enhance (6E)-8-hydroxygeraniol production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional microbial fermentation methods are used for producing (6E)-8-hydroxygeraniol, then the production process is simple, but the yield is suboptimal due to limitations in enzyme expression and feedback inhibition
Solution Approach 1:
The biosynthetic pathway is segmented into distinct enzymatic steps, each catalyzed by a separately engineered enzyme. The pathway is divided into: (1) mevalonate pathway enzymes for precursor synthesis, (2) geranyl diphosphate synthase for intermediate formation, (3) geranyl diphosphate diphosphatase for geraniol production, and (4) geraniol-8-hydroxylase for final product formation. This segmentation allows independent optimization of each step to overcome feedback inhibition and expression limitations.
Solution Approach 2:
The invention employs parameter changes by modifying enzyme properties through heterologous expression and genetic engineering. Specific parameters changed include: expressing plant-derived enzymes (geranyl diphosphate diphosphatase from Perilla frutescens and geraniol-8-hydroxylase from Phaseolus angularis) with different kinetic properties than native microbial enzymes, adjusting enzyme expression levels through promoter engineering, and modifying HMG-CoA reductase to overcome feedback inhibition by introducing feedback-deregulated variants.
2Productivity
If feedback inhibition is present in HMG-CoA reductase, then the natural regulatory mechanism is maintained, but the production of (6E)-8-hydroxygeraniol is limited
Solution Approach 1:
The feedback inhibition mechanism is extracted or removed from the HMG-CoA reductase enzyme. The invention uses feedback-deregulated variants of HMG-CoA reductase that have been engineered to lack the normal feedback inhibition response to downstream terpenoid products. This allows the enzyme to continue catalyzing the rate-limiting step of the mevalonate pathway without being downregulated by product accumulation, thereby maintaining high production rates.
Solution Approach 2:
The invention introduces intermediary elements between the feedback signal and the HMG-CoA reductase enzyme. Feedback-deregulated variants serve as intermediaries that receive the feedback signal from downstream products but do not transmit the inhibitory effect to the enzyme's catalytic activity. This intermediary mechanism allows the pathway to maintain flux despite the presence of feedback signals.
3Productivity
If non-native enzymes are expressed in microbial cells, then the desired biosynthetic pathway is enabled, but the expression efficiency is limited by host compatibility
Solution Approach 1:
The invention employs universal expression systems that can accommodate multiple heterologous genes from different organisms. The engineered microbial host (Saccharomyces cerevisiae or Yarrowia lipolytica) is equipped with expression vectors and regulatory elements that can efficiently transcribe and translate plant-derived enzymes (geranyl diphosphate diphosphatase from Perilla frutescens and geraniol-8-hydroxylase from Phaseolus angularis) alongside native yeast enzymes, enabling multi-species pathway functionality in a single host.
Solution Approach 2:
The invention optimizes expression parameters by using strong constitutive or inducible promoters (such as GAL1, TDH3, or PGK1 promoters in yeast), optimizing codon usage for the host organism, adjusting growth conditions (temperature, pH, aeration), and using optimized translation initiation sequences. These parameter changes enable efficient expression of non-native enzymes that would otherwise be poorly expressed in the microbial host.
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 engineered microbial cells significantly increase (6E)-8-hydroxygeraniol titers, achieving levels greater than 100 μg/L in culture medium, overcoming natural production limitations and feedback inhibition.
Implementation Method 1
Engineered microbes for production of (6E)-8-hydroxygeraniol by fermentation
Data Source
AI summary
The present disclosure describes the engineering of microbial cells for fermentative production of (6E)-8-hydroxygeraniol and provides novel engineered microbial cells and cultures, as well as related (6E)-8-hydroxygeraniol production method.