Multicopy Yeast Integration for High-Titre Hydrocortisone Production
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Solution Overview
Problem
Current methods for producing hydrocortisone using genetically modified Saccharomyces cerevisiae strains are inefficient, with low productivity and high costs due to limitations in transformation processes and the ability to introduce multiple genes, making industrial-scale production challenging.
Innovation Solution
A process involving stable multicopy integration of at least four expression cassettes into yeast using cotransformation of integration plasmids, allowing for high-level expression of multiple genes and improved productivity, enabling the conversion of endogenous or exogenous substrates into hydrocortisone.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional transformation processes are used to introduce heterologous genes into yeast, then the transformation process is simple, but the productivity is low and the number of genes that can be introduced is limited
Solution Approach 1:
The patent combines multiple expression cassettes (at least four) into a single integrated construct that can be introduced into yeast cells through a single transformation event. This merging approach allows simultaneous introduction of multiple genes required for hydrocortisone production, thereby increasing productivity without requiring multiple sequential transformation steps.
Solution Approach 2:
The patent divides the complex metabolic pathway for hydrocortisone production into separate expression cassettes, each containing specific genes required for different steps of the biosynthesis pathway. This segmentation allows for modular design and systematic optimization of each pathway component while maintaining the ability to introduce all components through integrated transformation.
2Productivity
If multiple expression cassettes are introduced to increase productivity, then hydrocortisone production increases, but the transformation process becomes more complex and costly
Solution Approach 1:
Multiple expression cassettes are merged into a single integrated DNA construct that can be transformed into yeast cells in one step. This approach maintains high productivity by introducing all necessary genes simultaneously while simplifying the manufacturing process by eliminating the need for multiple sequential transformation and selection steps.
Solution Approach 2:
The integrated expression cassette system is designed to self-assemble and function within the yeast cell after a single transformation event. The system includes all necessary regulatory elements and gene sequences required for proper expression of the hydrocortisone biosynthesis pathway, eliminating the need for complex external manipulation or multiple intervention steps.
3Productivity
If conventional single-copy integration is used, then the genetic stability is maintained, but the expression level and productivity are low
Solution Approach 1:
The patent introduces multiple copies of expression cassettes (at least four) into the yeast genome through integrated transformation. This excessive action of introducing more gene copies than the minimum single copy provides higher expression levels and productivity while the integrated nature of the construct ensures stable maintenance of all copies together as a single genetic unit.
Solution Approach 2:
Multiple expression cassettes are merged into a single integrated genetic construct that is introduced and maintained as one stable unit in the yeast genome. This merging ensures that all gene copies remain together and are co-inherited, providing both high expression levels from multiple copies and genetic stability through integrated maintenance.
Data Source
AI summary
The subject of the present invention is a process for preparing a genetically modified yeast by multicopy integration of at least four expression cassettes, allowing the production of a molecule of interest at high titer. The subject of the present invention is also yeasts transformed according to said process, and the use thereof for producing hydrocortisone.