Sigma Factor Modulation to Prolong Growth-Phase Biosynthesis
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Solution Overview
Problem
The production of heterologous products in bacterial cells is hindered by bottlenecks in enzymatic processes and cellular stress responses, particularly during aerobic fermentation, leading to inefficient mixing and premature entry into the stationary phase, which limits yield and biomass production.
Innovation Solution
Genetically engineered cells with a modulated RNA transcription pathway that reduces or abolishes the functionality of stationary phase sigma factors like RpoS or SigB, combined with promoters recognized by growth-phase factors like RpoD, to prolong the growth phase and enhance heterologous product production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If cells are cultured in large-scale bioreactors with aerobic fermentation, then productivity and biomass production increase, but inefficient mixing creates heterogeneity and glucose gradients that trigger overflow metabolism and cellular stress
Solution Approach 1:
The patent extracts and removes the harmful stationary phase response by deleting the rpoS gene, which encodes the sigma factor that triggers stationary phase entry under stress conditions. This eliminates the cellular stress response that would otherwise be activated by glucose gradients and heterogeneity in large-scale fermenters, allowing cells to maintain growth phase metabolism even when exposed to suboptimal mixing conditions.
Solution Approach 2:
The patent changes the physiological state parameter of the cells by modifying the rpoS gene functionality. This genetic modification alters the cells' response to environmental stress, shifting them from a stress-resistant stationary phase state to a sustained growth phase state, thereby changing how they metabolize glucose and respond to heterogeneity in large-scale bioreactors.
2Reliability
If cells enter stationary phase in response to stress or limited carbon, then cellular stress is reduced, but growth phase is terminated and product yield is limited
Solution Approach 1:
The patent removes the rpoS gene that mediates the transition to stationary phase, thereby extracting the stress response mechanism that would otherwise terminate growth. This allows cells to maintain productive growth phase metabolism without entering the non-productive stationary phase, even when exposed to stress conditions or carbon limitation.
Solution Approach 2:
The patent enables continuous useful action by maintaining cells in the growth phase throughout the fermentation process. By eliminating the rpoS-mediated stationary phase entry, the cells continuously perform growth-associated metabolism and heterologous product synthesis rather than transitioning to a non-productive stationary state.
3Productivity
If expression levels of enzymes are increased to overcome bottlenecks, then production efficiency improves, but balancing expression of multiple enzymes becomes more difficult
Solution Approach 1:
The patent employs a universal promoter system that is recognized by the growth-phase sigma factor (RpoD) rather than the stationary phase sigma factor (RpoS). This universal promoter can drive expression of multiple different enzymes in the heterologous pathway with coordinated efficiency, eliminating the need to individually balance each gene's expression level while maintaining high overall pathway flux.
Data Source
AI summary
The present invention relates to the biosynthetic production of heterologous products in a genetically engineered cell comprising heterologous nucleic acids encoding a heterologous product and/or a polypeptide required for the production of the heterologous products, wherein the expression of the growth phase and/or stationary phase RNA polymerase sigma factors in said cell is modulated.