Engineered Nitrile Hydratase Bacterium Amidase Knockout
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Solution Overview
Problem
The production of acrylamide through microbiological processes is hindered by the presence of amidase, which converts acrylamide into by-product acrylic acid, affecting quality, yield, and increasing purification difficulties and costs.
Innovation Solution
An engineered nitrile hydratase-producing bacterium with the amidase gene knocked out, specifically Rhodococcus ruber TH3 (amdA−), is constructed using a suicide plasmid vector for homologous recombination, reducing amidase activity and minimizing acrylic acid production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If wild-type nitrile hydratase-producing bacteria are used for acrylamide production, then acrylamide can be produced through microbiological process, but amidase converts acrylamide into by-product acrylic acid, affecting quality and yield
Solution Approach 1:
The patent applies the 'Taking out (Extraction)' principle by removing the amidase gene (amdA) from the bacterial genome through homologous recombination using a suicide plasmid vector. This extraction of the harmful gene eliminates the amidase enzyme that converts acrylamide to acrylic acid, thereby resolving the contradiction between maintaining acrylamide production capability and preventing acrylic acid by-product formation.
Solution Approach 2:
The patent applies the 'Parameter changes' principle by modifying the genetic parameters of the bacterial strain through gene knockout. By changing the genetic composition (removing amdA gene), the metabolic pathway parameters are altered to prevent the conversion of acrylamide to acrylic acid, thus improving product quality and yield without affecting the nitrile hydratase-mediated acrylamide synthesis.
2Ease of manufacture
If amidase is present in the bacterial strain, then the bacterial metabolism is complete, but purification difficulty and production costs increase due to acrylic acid by-product
Solution Approach 1:
By extracting/removing the amidase gene through targeted gene knockout, the patent eliminates the source of acrylic acid by-product. This directly addresses the purification difficulty and production cost issues, as the absence of acrylic acid simplifies the downstream separation and purification processes for acrylamide.
3Ease of manufacture
If gene knockout is performed using traditional methods, then amidase activity can be reduced, but the construction process is complex and time-consuming
Solution Approach 1:
The patent employs a suicide plasmid vector as an intermediary tool to facilitate the gene knockout process. The suicide plasmid carries homologous sequences that enable precise recombination with the bacterial chromosome at the amdA gene locus, mediating the gene deletion process in a controlled and efficient manner, thereby simplifying the overall strain construction procedure.
Solution Approach 2:
The patent applies 'Preliminary action' by first constructing the suicide plasmid vector with the necessary homologous sequences and antibiotic resistance markers before introducing it to the bacterial strain. This preliminary preparation of the gene knockout tool enables subsequent straightforward transformation and selection, reducing the overall complexity and time required for strain construction.
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 reduces by-product acrylic acid production by approximately 90%, enhancing acrylamide quality and reducing production costs, making it suitable for industrial use.
Implementation Method 1
The monomer of polyacrylamide is acrylamide (with a molecular formula of C3H5NO, and a structural formula of H2C═CHCONH2), which is generally produced through catalytic hydration using acrylonitrile as raw material
Implementation Method 2
amidase which can further convert acrylamide produced by nitrile hydratase catalysis into by-product acrylic acid
Data Source
AI summary
An engineered nitrile hydratase-producing bacterium and its construction method as well as its applications, wherein the engineered nitrile hydratase-producing bacterium is a mutant strain of an original nitrile hydratase-producing bacterium strain obtained by knocking-out or inhibiting the amidase gene in the original strain. The construction method of the engineered bacterium is to block the expression of the amidase gene by inserting the large fragment of a recombinant suicide plasmid carrying an amidase gene fragment into a wild-type strain through the homologous recombination between the recombinant suicide plasmid and the amidase gene of the wild-type strain. Compared to the corresponding wild-type bacterium strain, both the cell growth and the nitrile hydratase expression of the engineered nitrile hydratase-producing bacterium according to the invention are increased. In the process of catalyzing the hydration of acrylonitrile to produce acrylamide, the yield of the product, acrylamide, is significantly increased, while the yield of the by-product acrylic acid is significantly decreased. The engineered nitrile hydratase-producing bacterium of the present invention has wide application prospect in the production of acrylamide by microbiological process.