Recombinant Cell Gene Knockout for Ester Yield
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Solution Overview
Problem
Biotechnological processes for reacting carboxylic acid esters face challenges such as low yield, carbon and nitrogen imbalance, and purity issues due to uncontrolled secondary product formation and enzyme competition, particularly when using recombinant cells with wild-type enzyme activities.
Innovation Solution
A process involving a recombinant cell with reduced activity of specific polypeptides, such as BioH, and the use of alkane hydroxylases, transaminases, and other enzymes to control reactions and improve the efficiency of carboxylic acid ester conversion, including the knockout of genes coding for these polypeptides to enhance yield and balance.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If wild-type cells with full enzyme activity are used for biotechnological processes, then the cell possesses complete metabolic capabilities, but unwanted secondary products are formed and yield is reduced due to enzyme competition
Solution Approach 1:
The patent applies gene knockout technology to remove specific genes (such as bioH, acs, acnA) from the wild-type cell genome, thereby eliminating the production of unwanted secondary products. This extraction of problematic genetic elements resolves the contradiction by maintaining the cell's overall metabolic versatility while removing specific enzymatic activities that compete for substrates and reduce yield.
Solution Approach 2:
The patent modifies the genetic parameters of the cell by creating recombinant strains with specific gene deletions. This changes the enzymatic profile of the cell, reducing the activity of polypeptides that cause unwanted side reactions. By altering these genetic parameters, the cell maintains its metabolic capabilities while improving productivity through reduced enzyme competition.
2Adaptability or versatility
If multiple enzymes are present in the cell to catalyze various reactions, then metabolic versatility is maintained, but carbon and nitrogen balance is disrupted due to competing pathways
Solution Approach 1:
The patent removes specific genes responsible for unwanted metabolic pathways through gene knockout. This extraction eliminates competing reactions that disrupt carbon and nitrogen balance, while the cell retains other essential metabolic pathways through its recombinant enzyme expression system.
Solution Approach 2:
The patent segments the metabolic pathways by separating the desired reactions (catalyzed by recombinant enzymes) from unwanted side reactions (eliminated through gene knockout). This segmentation allows independent optimization of the productive pathway while removing competing pathways that cause substance loss.
3Manufacturing precision
If recombinant cells are used to improve selectivity, then secondary product formation is reduced, but process complexity increases due to genetic modification requirements
Solution Approach 1:
The patent employs whole-cell catalysts that perform the desired chemical transformations autonomously through their endogenous enzymatic systems. The recombinant cells self-regulate the reaction process, reducing the need for external process control and simplifying overall process complexity despite the genetic modifications required to achieve selectivity.
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
The process achieves higher yield, carbon and nitrogen balance, and purity of products by reducing unwanted secondary product formation and improving the re-usability of solvents and cation exchangers, thereby optimizing the reaction conditions for carboxylic acid esters.
Implementation Method 1
employing various organisms that possess interesting synthesizing capabilities
Implementation Method 2
the cell is a recombinant cell which has reduced activity of a polypeptide having SEQ ID NO: 2 or a variant thereof
Implementation Method 3
A process involving a recombinant cell with reduced activity of specific polypeptides, such as BioH, and the use of alkane hydroxylases, transaminases, and other enzymes
Implementation Method 4
improving the re-usability of solvents and cation exchangers
Data Source
AI summary
The invention provides a process for reacting a carboxylic acid ester of the formula (I)R1-A-COOR2 (I),wherein R1 is hydrogen, —CH2OH, —CHO, —COOR3, —CH2SH, —CH2OR3 or —CH2NH2, R2 is an alkyl group, R3 is hydrogen or an alkyl group, and A is a substituted, unsubstituted, linear, branched and/or cyclic alkylene, alkenylene, arylene or aralkylene radical having at least 4 carbons, in the presence of a cell. The process comprises a) contacting the cell with said carboxylic acid ester in an aqueous solution, wherein the cell is a recombinant cell which has reduced activity of a polypeptide comprising SEQ ID NO: 2 or a variant thereof over the wild-type cell.