Metabolic Engineering for Phase Separation in Bioprocessing
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Solution Overview
Problem
Current biotechnological processes face challenges in efficiently separating hydrophobic organic solutions from aqueous culture media, particularly in large-scale production of industrially demanded chemical compounds like 12-aminolauric acid, due to slow phase separation and potential toxicity of hydrophobic solvents to metabolically active cells, which hampers resource efficiency and product yield.
Innovation Solution
Employing metabolically active prokaryotic cells with reduced activity of enzymes involved in β-oxidation of fatty acids, such as FadE and FadL, and incorporating recombinant alkane hydroxylases to enhance the separation of hydrophobic organic solutions from aqueous culture media, thereby reducing solvent stress and oxygen consumption while maintaining production performance.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Speed
If conventional biotechnological processes are used to produce hydrophobic compounds in aqueous culture medium, then product formation is achieved, but phase separation between hydrophobic organic solution and aqueous culture medium is slow
Solution Approach 1:
The invention modifies the metabolic parameters of the cells by reducing the activity of enzymes involved in beta-oxidation of fatty acids (FadE and FadL). This parameter change in cell metabolism leads to reduced formation of hydrophobic substances by the cells, thereby accelerating phase separation between the hydrophobic organic solution and aqueous culture medium, while maintaining product formation capability.
2Quantity of substance
If hydrophobic organic solvents are used to extract products from aqueous culture medium, then product concentration is improved, but solvent toxicity to metabolically active cells increases
Solution Approach 1:
By changing the metabolic state of the cells through reduced enzyme activity in beta-oxidation pathways, the invention reduces the cells' susceptibility to solvent toxicity. This parameter change allows for more effective product extraction into hydrophobic phase while maintaining cell viability and metabolic activity.
Solution Approach 2:
The invention enables rapid phase separation, allowing the process to quickly transition from the toxic two-phase contact period to separation. This rushing through the harmful contact period minimizes solvent exposure time to cells, reducing toxic effects while maintaining extraction efficiency.
3Reliability
If cells with normal beta-oxidation enzyme activity are used, then metabolic activity is maintained, but phase separation is slow and oxygen consumption is high
Solution Approach 1:
The invention optimizes the metabolic parameter of beta-oxidation enzyme activity to a reduced but non-zero level. This parameter change achieves a balance where cells maintain sufficient metabolic activity for product formation while consuming less oxygen and enabling faster phase separation, unlike complete knockout which would stop metabolism.
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 approach significantly accelerates phase separation, reduces oxygen consumption, and improves the yield of hydrophobic compounds, making the process more resource-efficient and environmentally friendly by minimizing contact between cells and toxic solvents.
Implementation Method 1
A fundamental problem in processes for the production of fine chemicals based on renewable raw materials instead of fossil fuels is to convert the product once obtained, which is typically initially in a large-volume aqueous phase, into a hydrophobic organic phase.
Data Source
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AI summary
The invention relates to a method for the improved separation of a hydrophobic organic solution from an aqueous culture medium, comprising the steps of providing an aqueous culture medium comprising a metabolically active cell, contacting the aqueous culture medium with a hydrophobic organic solution, and separating the hydrophobic organic solution from the aqueous culture medium, wherein the cell has an activity of at least one enzyme that is reduced compared to the wild type of the cell, which enzyme catalyzes one of the reactions of the ß oxidation of fatty acids. The invention further relates to the use of a metabolically active cell having an activity of an enzyme that is reduced compared to the wild type of said cell, which enzyme catalyzes one of the reactions of the ß oxidation of fatty acids, preferably an enzyme selected from the group comprising FadA, FadB, FadD, FadL, and FadE and variants thereof, preferably FadE or a variant thereof, for the improved separation of a hydrophobic organic solution from an aqueous culture medium comprising the metabolically active cell.