Modified Gram Positive Bacteria Trehalose Accumulation
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Solution Overview
Problem
Gram-positive bacteria, such as Lactococcus lactis, face challenges in maintaining viability during storage and processing, especially under stress conditions like acidity and bile salts, due to limited methods for intracellular trehalose accumulation, which is crucial for protection against detrimental agents and environmental stresses.
Innovation Solution
A novel approach involving the partial or complete deletion of the trehalose 6-phosphate phosphorylase (TrePP) gene to inhibit TrePP activity, combined with overexpression of trehalose transporters, allows for intracellular trehalose accumulation without relying on external trehalose or heterologous enzymes like otsB, enhancing resistance to stress and storage conditions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If externally added trehalose is used as cryopreservant, then protective effect during freeze drying is improved, but internal trehalose accumulation in microorganisms is not achieved because it is not taken up or is metabolized rapidly
Solution Approach 1:
The patent uses heterologous enzymes (OtsA and OtsB from Saccharomyces cerevisiae) as intermediaries to convert externally added trehalose into intracellular trehalose-6-phosphate and then free trehalose. These enzymatic mediators enable the conversion pathway that naturally occurs in S. cerevisiae but is absent in L. lactis, allowing internal accumulation of trehalose from external sources.
Solution Approach 2:
The patent copies the trehalose metabolism pathway from Saccharomyces cerevisiae into Lactococcus lactis by introducing heterologous genes otsA and otsB. This genetic copying enables L. lactis to acquire the capability to synthesize and accumulate trehalose intracellularly, a function native to yeast but not to lactic acid bacteria.
2Adaptability or versatility
If L. lactis is used for food fermentations and medicaments, then viability during storage and processing is improved, but viability drops pronounced during storage, processing, and under stress conditions like high acidity and bile salts
Solution Approach 1:
The patent applies preliminary protective action by accumulating high concentrations of intracellular trehalose in L. lactis before exposure to stress conditions. This pre-accumulated trehalose forms a protective glass matrix during freeze drying and provides osmoprotection against acidity and bile salts, ensuring high viability is maintained during subsequent storage and processing operations.
Solution Approach 2:
The patent changes the physiological parameters of L. lactis by modifying its metabolic capabilities through genetic engineering. By introducing otsA and otsB genes, the bacteria's ability to accumulate trehalose is fundamentally altered, transforming it from a bacterium that cannot store trehalose to one that can accumulate high internal concentrations, thereby changing its stress resistance profile.
3Reliability
If de-novo synthesized trehalose through plasmid driven overexpression of otsA and otsB is used, then intracellular trehalose accumulation is achieved and protection from bile lysis and cell death is improved, but device complexity increases
Solution Approach 1:
The patent merges multiple functions into a single genetic construct by combining the otsA and otsB genes from Saccharomyces cerevisiae into a plasmid system that can be introduced into L. lactis. This combined approach simultaneously provides both trehalose-6-phosphate synthase activity (from otsA) and trehalose-6-phosphate phosphatase activity (from otsB), enabling complete de-novo trehalose synthesis in one integrated system.
Data Source
AI summary
The present invention relates to a gram positive bacterium, preferably a lactic acid bacterium (LAB) or Bifidobacterium, with increased stress resistance and/or improved manufacturing, processing and/or storage characteristics. In particular, the invention relates to a gram positive bacterium which accumulate intracellular trehalose. The gram positive bacterium according to the invention lack trehalose 6-phosphate phosphorylase (TrePP) activity. The gram positive bacterium may further lack cellobiose-specific PTS system IIC component (ptcC) activity. The gram positive bacterium may further overexpress trehalose transporters. The invention further relates to compositions comprising such gram positive bacterium as well as methods and uses thereof.