Engineered Yeast Sterol Pathways for Ergosterol-Independent Growth
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Solution Overview
Problem
Existing methods fail to provide a commercially viable method for producing non-native sterols, particularly 24-methylenecholesterol, in yeast using a cheap carbon source, and existing yeast strains face growth deficiencies when genes involved in ergosterol synthesis are knocked out, making large-scale production challenging.
Innovation Solution
Genetically modify oleaginous yeast strains like Yarrowia lipolytica by introducing a sterol surrogate, such as tetrahymanol, and heterologous enzymes to bypass ergosterol synthesis, allowing for high-yield production of non-native sterols like 24-methylenecholesterol and campesterol, while using a carbon source like waste vegetable oils.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If genes involved in ergosterol synthesis are knocked out in yeast, then non-native sterol production is improved, but yeast growth is compromised
Solution Approach 1:
The patent introduces a sterol surrogate (such as tetrahymanol) as an intermediary substance that compensates for the loss of ergosterol when genes like ERG4, ERG5, or ERG6 are knocked out. This sterol surrogate acts as a mediator that maintains yeast cell membrane integrity and growth while allowing the cell to redirect metabolic flux toward production of non-native sterols like 24-methylenecholesterol, campesterol, and other phytosterols. The sterol surrogate thus resolves the contradiction by enabling genetic modification for enhanced sterol production without compromising the yeast's ability to grow.
2Productivity
If conventional yeast strains are used for sterol production, then ease of operation is maintained, but productivity for non-native sterols is insufficient
Solution Approach 1:
The patent segments the sterol biosynthesis pathway by introducing heterologous enzymes from different organisms (such as plant or animal sources) into the yeast genome. These foreign enzymes catalyze specific reactions that convert endogenous sterol intermediates into non-native sterols. By dividing the pathway into native yeast steps and introduced heterologous steps, the system achieves high productivity for non-native sterols while maintaining the simplicity of yeast cultivation. The segmentation allows metabolic flux to be directed through specific enzymatic steps that produce the desired non-native sterols in high yield.
3Manufacturing precision
If expensive carbon sources are used, then manufacturing precision of sterol composition is improved, but cost effectiveness deteriorates
Solution Approach 1:
The patent employs parameter changes by utilizing waste vegetable oils as the carbon source instead of expensive conventional sugars. By changing the carbon source parameter from high-cost to low-cost material, the process becomes cost-effective. The patent further controls sterol composition precision by adjusting parameters such as carbon source composition, fermentation conditions, and enzymatic pathway regulation. The heterologous enzymes introduced into the yeast are optimized to work with the alternative carbon source, maintaining manufacturing precision for sterol composition while achieving cost effectiveness through the use of inexpensive waste oils.
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 strains achieve industrially relevant yields of non-native sterols, up to 48 mg/g dry cell weight, enabling the production of sterol mixtures suitable for artificial dietary compositions, particularly for honeybees, and other applications.
Implementation Method 1
introducing a sterol surrogate, such as tetrahymanol, and heterologous enzymes to bypass ergosterol synthesis
Implementation Method 2
using a carbon source like waste vegetable oils
Data Source
AI summary
The present invention relates to genetically-modified oleaginous yeasts for producing non-native sterols at commercially useful levels, especially for example in providing sterols individually or as a mixture in an artificial dietary composition for honeybees or other insects or animals. For this purpose, an oleaginous yeast, e.g. Yarrowia lipolytica, may be employed wherein the yeast has reduced production of ergosterol compared with a wild-type oleaginous yeast or is incapable of producing ergosterol and is provided with a sterol surrogate to aid growth. From such yeast, however, other yeast may be engineered which retain useful sterol production without need for a sterol surrogate, e.g. production of sterol mixtures in which 24-methylenecholesterol or campesterol is the dominant sterol.