Mogrosides Biosynthesis via Engineered Yeast
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Solution Overview
Problem
The production of mogrosides, which are natural sweeteners with anti-cancer, anti-oxidative, and anti-inflammatory properties, is hindered by labor-intensive extraction from fruits and the complexity of their chemical synthesis, with limited characterization of the proteins involved in biosynthesis.
Innovation Solution
Engineered host cells with a variant lanosterol synthase enzyme, capable of reduced enzymatic activity, are used to produce mogrol and mogrosides, along with their precursors, by heterologously expressing enzymes like cucurbitadienol synthase, UDP-glycosyltransferase, and epoxide hydrolase, enhancing the mevalonate pathway flux and reducing competition for oxidosqualene.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If labor-intensive extraction from fruits is used to produce mogrosides, then natural mogrosides are obtained with desired properties, but production efficiency is low and costs are high
Solution Approach 1:
The patent replaces mechanical extraction methods with biological synthesis methods using engineered yeast cells. The yeast cells express heterologous genes encoding enzymes from the mogroside biosynthesis pathway, converting substrates into mogrosides through metabolic engineering rather than physical extraction from plant fruits.
Solution Approach 2:
The engineered yeast cells perform self-service by autonomously carrying out the mogroside biosynthesis pathway within their metabolic network. The cells contain the necessary enzymatic machinery to convert provided substrates through multiple metabolic steps to produce mogrosides, eliminating the need for external extraction processes.
2Ease of manufacture
If de novo chemical synthesis is attempted for mogrosides, then production could be achieved, but the structural complexity of mogrosides hinders the synthesis process
Solution Approach 1:
The patent replaces complex chemical synthesis with biological synthesis using engineered yeast cells. The biological system naturally handles the structural complexity of mogrosides through enzymatic reactions, avoiding the need for complex chemical synthesis pathways and multiple protection/deprotection steps that would be required in chemical synthesis.
Solution Approach 2:
The patent changes the synthesis approach from chemical to biological parameters. By using heterologous gene expression in yeast, the synthesis occurs under physiological conditions with enzymatic catalysis, fundamentally changing the parameters of the synthesis process from harsh chemical conditions to mild biological conditions.
3Productivity
If lanosterol synthase activity is reduced to increase mogrol precursors, then flux through mevalonate pathway increases, but cell membrane integrity may be compromised
Solution Approach 1:
The patent uses parameter changes by introducing point mutations in the lanosterol synthase gene (ERG7) to create variants with altered activity. Specific amino acid substitutions modify the enzyme's catalytic properties, reducing its activity toward lanosterol synthesis while maintaining enough activity to preserve cell viability, thereby redirecting metabolic flux toward mogrol precursor production.
Solution Approach 2:
The patent applies partial action by using lanosterol synthase variants with reduced rather than completely abolished activity. The mutant enzymes retain sufficient catalytic function to maintain basic cellular sterol requirements and membrane integrity, while the reduced activity allows increased flux through alternative pathways leading to mogrol precursors.
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 increases the production of mogrol and mogrosides, reducing production costs and complexity, while maintaining cell viability by retaining sufficient lanosterol synthase activity for membrane integrity.
Implementation Method 1
a heterologous polynucleotide encoding a lanosterol synthase with reduced activity as compared to a wild-type lanosterol synthase
Implementation Method 2
enhancing the mevalonate pathway flux
Implementation Method 3
heterologously expressing enzymes like cucurbitadienol synthase
Implementation Method 4
heterologously expressing enzymes like epoxide hydrolase
Implementation Method 5
heterologously expressing enzymes like UDP-glycosyltransferase
Data Source
AI summary
Described in this application are proteins and host cells involved in methods of producing mogrol precursors, mogrol, and/or mogrosides.