Enzyme Relocation to Periplasmic Space for Metabolic Flux Control
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Solution Overview
Problem
The production of chemicals via synthetic enzymatic pathways in microbial hosts is limited by deleterious effects on the engineered cell's metabolism, such as unregulated consumption of cellular resources and accumulation of toxic pathway intermediates, due to complex interactions within the cell that are difficult to predict.
Innovation Solution
The solution involves manipulating metabolic pathway flux by relocating key enzymes to non-naturally occurring compartments, such as the periplasmic space, and controlling their concentrations during growth and production phases using genetically modified cells and cell-free systems, allowing for high-level production of desired products without disrupting cellular health.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If key pathway enzymes are overexpressed in intact cells during growth phase, then product yield increases, but cellular metabolism is disrupted and toxic intermediates accumulate
Solution Approach 1:
The patent divides the metabolic pathway into two spatial segments: enzymes are relocated to the periplasmic compartment while substrates and products remain in the cytoplasm. This spatial segmentation prevents toxic intermediates from accumulating in the cytoplasm while maintaining high product yield through periplasmic enzyme activity.
Solution Approach 2:
The periplasmic space acts as an intermediary compartment that receives substrates from the cytoplasm, processes them through relocated enzymes, and releases products back to the cytoplasm. This intermediary system allows high-level product production while preventing direct accumulation of toxic intermediates in the main cellular metabolism.
2Productivity
If enzyme concentrations are increased to enhance pathway flux, then production efficiency improves, but metabolic burden on the cell increases
Solution Approach 1:
The patent moves enzyme expression from the traditional cytoplasmic dimension to the periplasmic dimension, creating a separate production space. This allows high enzyme concentrations to be achieved in the periplasm without imposing proportional metabolic burden on the cytoplasmic housekeeping functions, as the periplasm has distinct resource pools and regulatory mechanisms.
3Productivity
If enzymes are relocated to non-natural compartments, then pathway flux control improves, but cellular regulatory mechanisms are disrupted
Solution Approach 1:
The patent applies local quality control by relocating specific pathway enzymes to the periplasm while leaving other cellular enzymes and regulatory mechanisms in their native cytoplasmic locations. This selective relocation allows precise control of pathway flux through the relocated enzymes without disrupting the overall cellular regulatory network, as each compartment maintains its own quality and regulatory characteristics.
Data Source
AI summary
Genetically manipulated cells, lysates of such cells, systems, and methods of use thereof are provided, where one or more enzymes in a pathway of interest are genetically modified to incorporate a peptide sequence that provides for relocation of the protein, e.g., to the periplasm, so as to sequester the enzyme, and where the enzyme controls flux in the pathway of interest.