Maltose-Responsive Degron Control for Stable Fermentation Production
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Solution Overview
Problem
The production of non-catabolic compounds in microbial fermentation is hindered by strain degeneration due to evolutionary mutations and selection, leading to reduced productivity and yield, as these compounds require ATP, NADPH, and oxygen, creating a favorable environment for lower biomass-yielding genotypes.
Innovation Solution
Utilizing maltose-dependent degrons and maltose-responsive promoters to control protein stability and gene expression, allowing for a two-phase fermentation process where non-catabolic compound production is turned off during biomass accumulation and on during production, stabilizing high-yielding strains and reducing strain degeneration.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If non-catabolic compound production is maintained continuously, then productivity is improved, but strain stability deteriorates due to evolutionary mutations and selection
Solution Approach 1:
The patent implements periodic action by using a maltose-responsive promoter to alternately activate and deactivate non-catabolic compound production pathways. During biomass accumulation phase, the promoter is repressed; during production phase, it is activated. This periodic switching prevents continuous production that causes strain degeneration while maintaining high productivity during active production phases.
Solution Approach 2:
The patent applies parameter changes by utilizing maltose concentration as a controllable parameter to regulate promoter activity. By adjusting maltose levels in the culture medium, the system transitions between different production states (off during biomass accumulation, on during production), thereby resolving the contradiction between continuous productivity and strain stability.
2Productivity
If non-catabolic compound production is activated, then productivity is improved, but energy consumption increases due to ATP, NADPH, and oxygen requirements
Solution Approach 1:
The system uses periodic action to activate energy-intensive non-catabolic compound production only during specific production phases rather than continuously. The maltose-responsive promoter ensures that high ATP, NADPH, and oxygen consumption occurs only when maltose is present, aligning energy usage with productive phases and reducing overall energy waste.
3Reliability
If biomass accumulation is prioritized, then strain stability is improved, but non-catabolic compound production decreases
Solution Approach 1:
The patent employs periodic action to sequentially prioritize biomass accumulation followed by non-catabolic compound production. During the biomass accumulation phase, the maltose-responsive promoter is repressed, allowing strains to stabilize and grow. When maltose is added, the promoter activates and switches the system to production mode, thereby achieving both strain stability and high productivity at different time points.
Data Source
AI summary
The present disclosure relates to the use of a maltose dependent degron to control stability of a protein of interest fused thereto at the post-translational level. The present disclosure also relates to the use of a maltose dependent degron in combination with a maltose-responsive promoter to control gene expression at the transcriptional level and to control protein stability at the post-translational level. The present disclosure also relates to the use of a stabilization construct that couples expression of a cell-growth-affecting protein with the production of non-catabolic compounds. The present disclosure further relates to the use of a synthetic maltose-responsive promoter. The present disclosure further provides compositions and methods for using a maltose dependent degron, a maltose-responsive promoter, and a stabilization construct, either alone or in various combinations, for the production of non-catabolic compounds in genetically modified host cells.