Bacillus subtilis N-acetylneuraminic acid production
Solution Overview
Problem
Current methods for producing N-acetylneuraminic acid using Bacillus subtilis face challenges such as high costs due to the need for expensive substrates and difficulties in increasing production levels due to protein synthesis pressure from multiple enzyme proteins, limiting market application.
Innovation Solution
The use of recombinant Bacillus subtilis expressing glucosamine-6-phosphate-N-acetyltransferase, N-acetylglucosamine isomerase, and N-acetylneuraminic acid synthase, optimized with different promoters to enhance expression levels and reduce protein synthesis pressure, along with overexpression of glycerol kinase for dual-carbon source utilization, to improve N-acetylneuraminic acid production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If multiple foreign enzyme proteins are introduced to construct N-acetylneuraminic acid metabolic pathway, then the pathway can be established, but cell pressure increases which is not conducive to cell growth and product synthesis
Solution Approach 1:
The patent utilizes the host organism's endogenous enzymes (Gna1, Age, NeuB from Bacillus subtilis) to construct the N-acetylneuraminic acid metabolic pathway, eliminating the need to introduce multiple foreign enzyme proteins. This self-service approach allows the pathway to be established using the host's own enzymatic capabilities, thereby avoiding cell pressure from foreign protein expression while maintaining pathway functionality.
2Adaptability or versatility
If multiple enzyme proteins are introduced simultaneously, then the metabolic pathway can be constructed, but it is not conducive to cell growth and product synthesis
Solution Approach 1:
The patent employs endogenous Bacillus subtilis enzymes (Gna1, Age, NeuB) to construct the metabolic pathway, eliminating the need to introduce multiple foreign proteins simultaneously. This approach maintains pathway construction capability while avoiding the burden of multiple foreign protein expressions, thereby preserving cell growth and product synthesis productivity.
3Reliability
If expensive substrates such as acetylglucosamine and pyruvate are used, then N-acetylneuraminic acid can be obtained by whole cell transformation, but the production cost increases
Solution Approach 1:
The patent changes the substrate parameter from expensive acetylglucosamine and pyruvate to cheap glucose. By modifying the metabolic pathway to utilize glucose as the carbon source through endogenous enzyme action, the system maintains reliable N-acetylneuraminic acid production while dramatically reducing production costs.
4Reliability
If natural product extraction methods are used, then N-acetylneuraminic acid can be obtained, but other products are difficult to separate and the cost is high
Solution Approach 1:
The patent uses endogenous Bacillus subtilis enzymes to construct a metabolic pathway that produces N-acetylneuraminic acid directly from glucose. This biological synthesis approach eliminates the need for complex extraction and separation processes required by natural product methods, thereby simplifying the production process while maintaining product obtainability.
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 achieves significant increases in N-acetylneuraminic acid production up to 10.4 g/L, reducing production costs and enhancing metabolic engineering applications, while minimizing protein synthesis pressure on the cells.
Implementation Method 1
recombinant Bacillus subtilis expressing glucosamine-6-phosphate-N-acetyltransferase (Gna1), N-acetylglucosamine isomerase (Age) and N-acetylneuraminic acid synthase (NeuB)
Implementation Method 2
expression of the coding genes of the glucosamine-6-phosphate-N-acetyltransferase, the N-acetylglucosamine isomerase and the N-acetylneuraminic acid synthase in the recombinant Bacillus subtilis is enhanced through promoters
Implementation Method 3
the recombinant Bacillus subtilis also overexpresses glycerol kinase (GlpK)
Data Source
AI summary
The disclosure discloses Bacillus subtilis for producing N-acetylneuraminic acid and application thereof, and belongs to the field of genetic engineering. The disclosure optimizes the expression levels of key enzymes in N-acetylneuraminic acid synthesis pathways on genome through promoters of different strength, reduces the protein synthesis pressure caused by the expression of enzymes on cells, and further integrates the three N-acetylneuraminic acids in a same Bacillus subtilis engineering strain. Bacillus subtilis with improved N-acetylneuraminic acid production is obtained, and the production reaches 10.4 g/L at the shake flask level, laying a foundation for further improving the NeuAc production from Bacillus subtilis.