Recombinant Gram-Negative Bacteria LPS Engineering
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Solution Overview
Problem
Current technologies face challenges in addressing the strong immune response and pathogenic effects of lipopolysaccharides (LPS) from Gram-negative bacteria, particularly in humans, which are highly sensitive and can lead to severe clinical manifestations such as septic shock.
Innovation Solution
Development of recombinant Gram-negative bacteria lacking genes involved in LPS biosynthesis, specifically those required for core oligosaccharide biosynthesis, and introduction of exogenous KDO transferases and heptosyltransferases to generate novel LPS glycoforms, which can be used in therapeutic, preventative, and research applications.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If LPS is used as an immunogen or adjuvant, then immune response is enhanced, but severe immune reactions and endotoxin-related disorders occur
Solution Approach 1:
The patent applies this principle by taking the harmful LPS molecule and modifying it through genetic engineering to create a version that retains immunostimulatory benefits while eliminating toxic effects. Specifically, LPS from bacteria lacking core oligosaccharide biosynthesis genes (waa mutants) is used to generate Lipid A variants that are less immunotoxic but still effective as adjuvants, converting the harmful endotoxin into a beneficial therapeutic agent
Solution Approach 2:
The patent changes the chemical parameters of LPS by modifying the bacterial genes responsible for LPS biosynthesis. By deleting or mutating specific genes (waaF, waaC, waaL, etc.) involved in core oligosaccharide synthesis, the patent creates LPS variants with altered structures that have reduced toxicity but maintained immunogenicity, thereby changing the parameters of the LPS molecule to resolve the contradiction
2Object-affected harmful factors
If LPS structure is modified to reduce toxicity, then safety is improved, but immune response effectiveness may be reduced
Solution Approach 1:
The patent applies local quality by making specific localized modifications to the LPS molecule rather than altering the entire structure. By targeting specific genes responsible for core oligosaccharide biosynthesis while leaving other LPS components intact, the patent creates variants with localized structural changes that reduce toxicity at specific sites while preserving the immunostimulatory properties of other regions of the LPS molecule
Solution Approach 2:
The patent uses partial action by selectively modifying only certain aspects of LPS biosynthesis rather than completely eliminating LPS production. By deleting specific genes involved in core oligosaccharide synthesis while maintaining production of other LPS components, the patent achieves partial modification that reduces toxicity while preserving sufficient immunogenicity to maintain effectiveness
3Object-generated harmful factors
If genes for core oligosaccharide biosynthesis are deleted, then LPS toxicity is reduced, but bacterial structural integrity may be compromised
Solution Approach 1:
The patent applies this principle by extracting or removing the harmful core oligosaccharide component from LPS through genetic deletion of biosynthesis genes. By taking out the specific toxic portion (core oligosaccharide) while leaving the essential Lipid A portion intact, the patent reduces endotoxin production while maintaining the structural integrity of the bacterial outer membrane through the remaining LPS components
Data Source
AI summary
The present invention provides novel, recombinant Gram-negative bacteria. In particular, the invention provides recombinant Gram-negative bacteria (e.g., E. coli) lacking genes involved in lipopolysaccharide (LPS, endotoxin) biosynthesis (e.g., lacking genes required for core oligosaccharide biosynthesis) and also provides recombinant Gram-negative bacteria lacking genes involved in LPS biosynthesis that contain one or more exogenous KDO transferases and/or one or more exogenous heptosyltransferases (e.g., from one or more types and/or strains of bacteria). The invention further provides methods of generating and utilizing (e.g., as or in an immunogenic composition (e.g., as or in an adjuvant and/or vaccine)) the recombinant Gram-negative bacteria therapeutic, preventative, and/or research applications.