Acetyl-CoA Fermentation Switching for Stable Microbial Production
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Solution Overview
Problem
The production of acetyl-CoA derived compounds, such as isoprenoids, fatty acids, and polyketides, is non-catabolic in nature, requiring ATP, NADPH, and oxygen, leading to strain degeneration due to evolutionary mutations and reduced product yields, as these compounds are not produced during phases where biomass generation is needed, increasing operational and capital expenditures.
Innovation Solution
A fermentation process that includes an 'off' and 'on' stage for non-catabolic compound production, controlled by oxygen levels and oxygen-sensitive promoters, utilizing microaerobic conditions to switch on production when optimal cell densities are reached, and by maltose-sensitive promoters to regulate gene expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If non-catabolic compound production is maintained continuously, then product yield is improved, but strain stability deteriorates due to evolutionary mutations and selection
Solution Approach 1:
The patent implements periodic action by cycling between aerobic and microaerobic phases. During aerobic phases, biomass accumulates while compound production is suppressed. During microaerobic phases, compound production is activated. This periodic switching prevents continuous metabolic burden while maintaining productivity over extended cultivation periods, thereby resolving the contradiction between product yield and strain stability.
Solution Approach 2:
The patent applies dynamics by making the production system adjustable through oxygen level control. The system transitions from a static continuous production mode to a dynamic phased mode where aerobic and microaerobic conditions are alternated. This dynamic control allows optimization of both biomass growth and compound production at different times, preventing strain degeneration while maintaining high productivity.
2Productivity
If non-catabolic compound production is activated, then product output is improved, but growth rate deteriorates due to metabolic burden
Solution Approach 1:
The patent segments the fermentation process into distinct phases: aerobic growth phase and microaerobic production phase. During the aerobic phase, metabolic resources are dedicated to biomass accumulation with compound production suppressed. During the microaerobic phase, resources are directed toward compound production. This segmentation eliminates the metabolic burden during growth phases while ensuring high output during production phases.
Solution Approach 2:
By periodically switching between aerobic and microaerobic conditions, the system ensures that high growth rates occur during aerobic phases when production genes are repressed, and high product output occurs during microaerobic phases when production genes are activated. This periodic action resolves the contradiction by decoupling growth and production in time.
3Loss of time
If continuous fermentation is extended, then operational efficiency is improved, but production characteristics deteriorate due to strain degeneration
Solution Approach 1:
The phased aerobic-microaerobic cycling prevents strain degeneration during extended fermentations by periodically relieving the metabolic burden. This allows continuous fermentation to be extended over multiple cycles while maintaining stable production characteristics, as the population is repeatedly reset to a healthy state during aerobic phases.
Solution Approach 2:
The patent achieves continuity of useful action by maintaining a cycle of growth and production phases. Rather than interrupting fermentation, the system continuously alternates between biomass accumulation and compound production, extending operational efficiency while preventing strain degeneration through periodic metabolic relief.
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 improves strain stability and productivity by reducing metabolic burden, enhancing growth rates, and maintaining favorable production characteristics, thus extending the duration of non-catabolic compound production and reducing costs.
Implementation Method 1
controlled by oxygen levels and oxygen-sensitive promoters
Implementation Method 2
utilizing microaerobic conditions to switch on production when optimal cell densities are reached
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.