Nitrogen-Fixing Bacteria with Stable nifA Cassette Integration
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Solution Overview
Problem
Existing nitrogen-fixing bacteria exhibit unstable ammonia accumulation and gene cassette shedding when cultured without selection agents, limiting their industrial application for ammonia production and protein production.
Innovation Solution
Introduce a nifA gene expression cassette by homologous recombination into a gene locus other than the nifL-nifA gene locus, using the double crossover method to ensure stable ammonia accumulation and prevent gene cassette shedding, thereby enhancing nitrogen-fixing ability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If a nifA gene expression cassette is introduced into the nifL-nifA gene locus, then nitrogen-fixing ability is enhanced, but gene cassette shedding occurs when cultured without selection agents
Solution Approach 1:
The patent uses a selectable marker gene (antibiotic resistance gene) as an intermediary element linked to the nifA gene expression cassette. This marker gene serves as a mediator that enables selection and maintenance of the cassette during culture, preventing shedding while allowing the nitrogen-fixing ability to be expressed. The marker gene acts as a bridge that connects the beneficial nifA cassette to a selection mechanism.
Solution Approach 2:
The patent combines the nifA gene expression cassette with a selectable marker gene into a single integrated genetic construct. By merging these two functional elements, the system ensures that the nitrogen-fixing enhancement (from nifA) and the stability mechanism (from selectable marker) work together as a unified genetic unit, preventing cassette shedding while maintaining enhanced nitrogen fixation.
2Stability of the object's composition
If nitrogen-fixing bacteria are cultured without selection agents, then gene cassette shedding is prevented, but ammonia accumulation becomes unstable
Solution Approach 1:
The selectable marker gene serves as an intermediary that enables stable maintenance of the nifA cassette without requiring continuous presence of selection agents during production. The marker gene creates a genetic linkage that ensures cassette retention, while the system is designed to allow ammonia accumulation stability to be maintained through this genetic stability rather than continuous selection pressure.
Solution Approach 2:
The patent establishes gene cassette stability through preliminary genetic engineering (introducing the cassette with linked marker gene) before the production phase. This preliminary action of creating a stable genetic construct allows the bacteria to maintain both cassette integrity and ammonia accumulation capability without requiring selection agents during the actual production process.
3Productivity
If conventional nitrogen-fixing bacteria are used, then industrial ammonia production is limited, but energy consumption and cost remain high
Solution Approach 1:
The patent changes the genetic parameters of nitrogen-fixing bacteria by introducing an enhanced nifA gene expression cassette. This parameter change in gene expression leads to increased nitrogen fixation rate and higher ammonia production capacity. The system achieves improved productivity through genetic parameter modification rather than through energy-intensive industrial processes.
Solution Approach 2:
The genetically modified nitrogen-fixing bacteria perform ammonia production autonomously using their enhanced nitrogen-fixing ability. The bacteria self-service by converting atmospheric nitrogen to ammonia through their metabolic processes, eliminating the need for energy-intensive external industrial processing. The system uses the bacteria's own biological machinery to produce ammonia efficiently.
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
Stable production of ammonia at high concentrations (up to 510 mg/L) is achieved, enabling efficient production of ammonia-utilizing microorganism biomass and microbial preparations, with the bacteria themselves serving as protein sources for foods and feeds.
Implementation Method 1
The ability to convert nitrogen molecules in the atmosphere to ammonia is referred to as 'nitrogen-fixing ability'
Implementation Method 2
The enzymes involved in nitrogen-fixing ability are nitrogenases, which catalyze the following reaction under ideal reaction conditions in vitro
Data Source
Figure 1(A)~1(B)
Figure 2(A)~2(C)
Figure 3
AI summary
Nitrogen-fixing bacteria having a gene cassette that produces high expression of the known nifA gene transferred into the regulatory region of the nifA gene, have been found to shed the gene cassette after repeated subculturing in the absence of a selection agent such as kanamycin. A nifA gene expression cassette comprising the nifA gene, and tac or trc promoter, Lac operator and the ribosome binding site (RBS), disposed in a manner allowing expression of the nifA gene, has been transferred into a gene locus other than the nifL-nifA gene locus, to obtain nitrogen-fixing bacteria without such shedding of the gene cassette, even after subculturing. A method has been devised for producing amino acid- or protein-containing culture product or a meat alternative, using the nitrogen-fixing bacteria.