Programmable Synthetic Lysis System for Macromolecule Release
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for releasing macromolecules from prokaryotic cells, such as E. coli, are inefficient and costly, requiring mechanical, chemical, or enzymatic cell disruption, and existing regulatory systems for programmed lysis suffer from low activity and poor specificity, making them unsuitable for industrial-scale applications.
Innovation Solution
A dual-promoter expression system is introduced, where a carbon starvation-induced promoter activates a quorum sensing system, which in turn controls the expression of lytic proteins for controlled cell lysis, allowing for efficient release of macromolecules at high cell density without additional inducers or costly reagents.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If mechanical, chemical or enzymatic cell disruption methods are used to release macromolecules, then macromolecule extraction is achieved, but additional expensive reagents and equipments are required
Solution Approach 1:
The E. coli cells are engineered to perform cell disruption themselves by expressing bacteriophage-encoded lytic proteins (holin and lysozyme). The holin forms pores in the cytoplasmic membrane allowing lysozyme to access and degrade the peptidoglycan layer, causing autonomous cell lysis and macromolecule release without external mechanical, chemical or enzymatic intervention
Solution Approach 2:
The patent replaces mechanical cell disruption methods (ultrasonication, homogenization) and chemical/enzymatic treatments with a biological system using bacteriophage-derived lytic proteins. The holin-lysozyme system provides a self-contained mechanism that substitutes expensive external equipment and reagents with internally produced proteins
2Ease of operation
If supplementary chemical inducers are used to regulate lytic protein expression, then programmed cell lysis is achieved, but additional costly reagents are required for industrial scale-up
Solution Approach 1:
The system uses an auto-inducible promoter (ptsGPL) that automatically responds to physiological conditions (glucose exhaustion) to trigger lytic protein expression. The cells themselves sense their metabolic state and autonomously initiate the lysis program without requiring external addition of chemical inducers like IPTG, eliminating this cost burden
Solution Approach 2:
The ptsGPL promoter creates a feedback system where glucose depletion during batch culture automatically triggers the lysis cascade. The metabolic state of the culture feeds back to the promoter activity, which in turn controls lytic protein expression, creating a self-regulating system that eliminates the need for external inducer addition
3Productivity
If ptsGPL promoter is used to enable cell lysis at high cell density, then additional cell disruption steps are eliminated, but promoter activity is low and specificity is poor due to regulation by factors other than glucose
Solution Approach 1:
The patent divides the promoter function into two separate components: ptsGPL promoter for glucose-responsive activation and lasI promoter for quorum sensing-based cell density control. This segmentation allows each promoter to specialize in one regulatory function, with lasI providing the missing cell density specificity that ptsGPL lacks when regulated by other factors like oxygen and oxidative stress
4Ease of operation
If quorum sensing system is used to control lytic protein expression, then cell density control is achieved, but it is difficult to assert control over the threshold cell density without trial and error
Solution Approach 1:
The patent performs preliminary characterization of the lasI promoter's response to AHL concentrations and cell density, establishing predictable relationships between inducer levels and expression thresholds. This pre-established knowledge allows direct design of the quorum sensing circuit with desired thresholds without requiring extensive trial and error optimization during implementation
Solution Approach 2:
The system enables control over threshold cell density by modifying parameters such as the concentration of AHL inducer, the strength of the lasI promoter, or the expression level of the AHL synthase. These parameter changes allow tuning of the quorum sensing threshold to achieve desired cell density control without trial and error
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 system enables controlled and efficient release of macromolecules at high cell density, reducing cell viability loss and eliminating the need for external inducers, thus simplifying downstream processing and lowering costs.
Implementation Method 1
a first promoter that is a carbon starvation-induced promoter and controls a gene involved in activating a quorum sensing system
Implementation Method 2
lytic proteins such as holin are responsible for forming a lesion in the host cell membrane
Implementation Method 3
The combined work of holin and lysozyme results in the degradation of the two cell membranes of gram-negative bacteria, thus causing cell lysis
Implementation Method 4
a first promoter that is a carbon starvation-induced promoter... activated by a carbon starvation signal
Data Source
AI summary
The present invention relates to an expression system or a recombinant cell comprising one or more nucleic acid constructs, wherein the one or more nucleic acid constructs comprise: (i) a first gene encoding for one or more protein(s) that activate a quorum sensing system; and (ii) a second gene encoding for one or more lytic protein(s) capable of forming a lesion in a host cell's membrane; wherein the first gene is under control of a first promoter and the second gene is under control of a second promoter, wherein the first promoter controlling the first gene is a carbon starvation-induced promoter and the second promoter is a quorum sensing system promoter induced by the quorum sensing system activated by one or more protein(s) encoded by the first gene as well as recombinant cells hosting such an expression system. Further encompassed is the use of the expression systems and cells of the invention for the expression of gene products of interest and the respective methods of use.