Cell-Free UPO Production via Eukaryotic Extracts
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Solution Overview
Problem
Current methods for producing unspecific peroxygenases (UPOs) are limited by low yields, long production times, and the inability to express and isolate enzymes in sufficient diversity and yield, particularly in eukaryotic cell systems, hindering their industrial application.
Innovation Solution
A cell-free production process using eukaryotic cell extracts from fungi, which includes preparing these extracts, adding a nucleic acid template, and providing necessary components for protein synthesis, enabling rapid and efficient production of native and genetically modified UPOs without the need for living cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If cell-based systems are used to produce UPOs, then the enzymes can be produced with post-translational modifications, but the production time is long (several days to weeks) and yields are low (10-450 mg protein/L)
Solution Approach 1:
The patent extracts the essential protein synthesis machinery from living cells to create a cell-free system. By removing the need for intact cellular processes and using purified cell extracts containing ribosomes, tRNAs, and other translation components, the system achieves rapid UPO production without the time-consuming cellular growth and differentiation phases required in cell-based systems.
Solution Approach 2:
The patent performs preliminary extraction and purification of cell components (ribosomes, tRNAs, enzymes) to create a pre-prepared translation system. This preliminary action allows the actual protein synthesis to occur rapidly without needing to wait for cellular processes to unfold naturally, thus reducing production time while maintaining protein functionality.
2Reliability
If homologous production is used, then UPOs can be produced in their native organism, but the diversity of UPOs that can be produced is limited and yields are insufficient
Solution Approach 1:
The patent creates a universal cell-free translation system that can synthesize any UPO protein sequence by simply changing the mRNA template. This system is not limited to producing only the native UPO from a specific organism, but can produce diverse UPOs from different fungal species and engineered variants using the same basic translation machinery, thereby achieving both reliability and versatility.
Solution Approach 2:
The patent uses mRNA templates that copy the genetic information of desired UPOs into the cell-free system. By using synthetic or purified mRNA rather than relying on the organism's own genome expression, the system can accurately reproduce diverse UPO sequences without being constrained by the host organism's genetic limitations, enabling production of many different UPO variants.
3Adaptability or versatility
If genetic engineering methods are applied to wild types, then genetically modified UPO variants with desired properties can be produced, but many wild types do not have available genetic engineering methods
Solution Approach 1:
The patent bypasses the need for genetic engineering in the host organism by using mRNA templates that directly encode the desired UPO variants. This allows production of genetically modified UPOs with desired properties (improved stability, substrate specificity, etc.) without needing to genetically modify the wild-type fungus, thus maintaining ease of manufacture while achieving versatility.
Solution Approach 2:
The patent introduces mRNA as an intermediary carrier that bridges the gap between the desired UPO sequence and the cell-free translation system. This intermediary allows the system to produce genetically modified variants without requiring the host organism to have compatible genetic engineering tools, as the mRNA template can be synthesized independently and then translated using the universal cell-free system.
4Productivity
If cell-free production is used, then production time is reduced to a few hours, but the system requires complex preparation of eukaryotic cell extracts
Solution Approach 1:
The patent performs cell extract preparation as a preliminary action that can be done in advance. By pre-extracting and purifying the necessary cellular components (ribosomes, tRNAs, enzymes) before the actual protein production, the system achieves rapid UPO synthesis in a few hours. The complexity of extract preparation is separated from the production step, allowing for optimized preparation protocols that enable fast downstream synthesis.
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 process allows for the rapid production of UPOs in a few hours to less than two days, enabling high throughput and variability, facilitating the production of diverse UPOs and their variants, and overcoming the limitations of traditional cell-based systems.
Implementation Method 1
cell-free production of unspecific peroxygenases using eukaryotic cell extracts, preferably from fungi, in particular filamentous fungi
Data Source
AI summary
The present invention relates to a process for the cell-free production of unspecific peroxygenases, preferably from fungi and/or genetically modified variants thereof, using eukaryotic cell extracts, preferably from fungi, in particular filamentous fungi, and its use.