Pseudomonas putida OMV Engineering for Hydrophobic Product Secretion
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Solution Overview
Problem
Current methods for extracting hydrophobic products like carotenoids and curcuminoids from gram-negative bacteria require cell lysis, and there is a need for innovative methods to convert aromatic compounds from waste and renewable resources into commodity and specialized chemicals.
Innovation Solution
Genetically modified Pseudomonas sp. with deletions in endogenous genes such as oprF and oprI to increase outer membrane vesicle (OMV) production, and the incorporation of exogenous enzymes connected to outer membrane proteins through linkers or vesicle nucleating peptides for targeted enzymatic reactions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If cell lysis is used to extract hydrophobic products, then product extraction is achieved, but cell integrity is compromised and purification is complex
Solution Approach 1:
The patent extracts hydrophobic products from the cell membrane into outer membrane vesicles (OMVs) without compromising cell integrity. The OMVs serve as a selective extraction mechanism that separates products from cellular contents, enabling easy purification through simple filtration and centrifugation steps while maintaining cell viability.
Solution Approach 2:
Outer membrane vesicles act as an intermediary carrier between the cell membrane and the extracellular environment. These vesicles encapsulate hydrophobic products, facilitating their transfer outside the cell while protecting them from degradation and simplifying separation from cellular debris through physical methods.
2Reliability
If enzymes are targeted to outer membrane vesicles, then enzymatic reaction coordination is improved, but genetic engineering complexity increases
Solution Approach 1:
The patent merges the enzyme of interest with outer membrane proteins through fusion constructs. This combining strategy allows the enzyme to be naturally targeted to OMVs via the outer membrane protein's localization signals, achieving coordinated enzymatic reactions without complex genetic engineering approaches.
Solution Approach 2:
Outer membrane proteins serve as universal targeting platforms for various enzymes. By utilizing the natural targeting mechanisms of these proteins, multiple different enzymes can be directed to OMVs using a common approach, reducing the need for enzyme-specific genetic engineering strategies.
3Productivity
If outer membrane vesicle production is increased, then secretion mechanism efficiency is improved, but cell membrane integrity may be compromised
Solution Approach 1:
The patent applies local quality changes by selectively modifying specific regions of the cell membrane to enhance vesicle formation. Through targeted genetic modifications in genes regulating OMV biogenesis, the cell membrane's ability to form vesicles is enhanced in specific locations without compromising overall membrane integrity or cell viability.
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
Enhanced production and isolation of compounds of interest through increased OMV production and targeted enzymatic reactions, improving enzyme stability and detoxification efficiency while simplifying purification and enabling long-term storage.
Implementation Method 1
Outer membrane vesicles (OMVs) are produced by gram-negative bacteria and represent a currently untapped resource for bioprocess engineering. Hydrophobic products without designated secretion mechanisms, such as carotenoids, curcuminoids, and other natural products can accumulate in the cell membrane and require cell lysis to extract the products. Thus, engineering increased vesiculation has potential to act as a secretion mechanism for these specialty chemicals.
Implementation Method 2
genetic tools to target specific enzymes to OMVs, would enable OMVs to be utilized as biocatalysts with coordinated enzymatic reactions
Implementation Method 3
the expressed enzyme encoded by the at least one exogenous gene is tagged with a vesicle nucleating peptide
Data Source
AI summary
Disclosed herein are methods, compositions and systems useful for genetically engineering subcellular compartments such as OMVs for synthetic biology applications. In an embodiment, genetically engineered bacteria use OMVs to secrete compounds or proteins of interest extracellularly where the compounds or proteins of interest can be isolated from the growth media.