Microbial Fermentation Using Oxygen-Sensitive Promoters
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Solution Overview
Problem
The commercial viability of synthetic biology for producing biofuels and chemicals is hindered by strain stability issues, particularly in non-catabolic fermentation processes where acetyl-CoA derived compounds require ATP, NADPH, and oxygen, leading to strain degeneration due to evolutionary mutations and selection pressures.
Innovation Solution
Implementing a fermentation process with a genetically modified host cell that utilizes oxygen-sensitive and maltose-responsive promoters to control the production of non-catabolic compounds, allowing for a 'build' stage with reduced production under aerobic conditions and an 'on' stage under microaerobic conditions or in the absence of maltose, thereby alleviating selective pressure and enhancing stability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If non-catabolic fermentation is used to produce acetyl-CoA derived compounds, then product yield can be achieved, but strain stability deteriorates due to evolutionary mutations and selection pressures
Solution Approach 1:
The fermentation process is divided into distinct phases: a growth phase where biomass is accumulated without product formation, and a production phase where product is synthesized. This temporal segmentation allows the strain to maintain stability during growth while achieving high product yield during production, resolving the contradiction between yield and stability.
Solution Approach 2:
The system dynamically adjusts metabolic flux through regulated enzyme expression. Key enzymes in the non-catabolic pathway are controlled to be active only during the production phase, allowing the strain to adapt its metabolic state between growth and production modes, thereby maintaining stability while achieving high yield.
2Productivity
If continuous production of non-catabolic compounds is maintained, then productivity is sustained, but strain degeneration increases due to metabolic burden
Solution Approach 1:
The system employs periodic induction of product formation through controlled addition of inducers or switching of cultivation conditions. This periodic activation of the non-catabolic pathway prevents continuous metabolic burden while maintaining high productivity during active production periods, thereby reducing strain degeneration.
Solution Approach 2:
The system changes key parameters such as dissolved oxygen concentration, pH, or nutrient composition to trigger and control product formation. By modulating these parameters, the system can switch between growth and production modes, maintaining productivity while minimizing the metabolic burden that leads to genetic instability.
Data Source
AI summary
The present disclosure relates to the use of a switch for the production of heterologous non-catabolic compounds in microbial host cells. In one aspect, provided herein are genetically modified microorganisms that produce non-catabolic compounds more stably when serially cultured under aerobic conditions followed by microaerobic conditions, and methods of producing non-catabolic compounds by culturing the genetically modified microbes under such culture conditions. In another aspect, provided herein are genetically modified microorganisms that produce non-catabolic compounds more stably when serially cultured in the presence of maltose followed by the reduction or absence of maltose, and methods of producing non-catabolic compounds by culturing the genetically modified microbes under such culture conditions.