Bacillus subtilis Strain Engineering for Butyric Acid Yield
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for butyric acid biosynthesis through microbial fermentation face challenges such as low yield and high costs due to end product inhibition and the need for large amounts of glucose and mannitol as carbon sources, limiting their economic viability and industrial application.
Innovation Solution
A butyric acid-producing Bacillus subtilis strain is developed by knocking out autolysis-related and acetic acid metabolism genes while introducing the butyric acid synthesis-related gene BCoAT, using a CRISPR/Cas9 gene editing system to optimize the metabolic pathway for enhanced butyric acid production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If microbial fermentation is used to produce butyric acid, then environmental benefits are improved and chemical synthesis can be replaced, but the yield is low due to end product inhibition and economic benefit is poor
Solution Approach 1:
The patent extracts and removes the end product inhibition effect by knocking out the butyryl-CoA dehydrogenase gene (bcd), which catalyzes the rate-limiting step that causes accumulation of butyric acid and subsequent inhibition. This allows the fermentation system to continue producing butyric acid without the harmful feedback inhibition, thereby resolving the contradiction between environmental benefits and productivity
Solution Approach 2:
The patent changes the metabolic parameters of the Bacillus subtilis strain by introducing the butyryl-CoA:acetate CoA-transferase gene (BCoAT) from Clostridium tyrobutyricum. This gene encoding BCoAT enzyme redirects the metabolic flux from the inhibited pathway to an alternative pathway that produces butyric acid without end product inhibition, thereby improving both yield and economic viability while maintaining environmental sustainability
2Productivity
If glucose and mannitol are used as carbon sources for fermentation, then butyric acid production is achieved, but the cost increases due to large amounts required
Solution Approach 1:
The patent changes the metabolic efficiency parameters of the strain by introducing the BCoAT gene, which enables more efficient conversion of carbon sources to butyric acid. This increases the yield per unit of carbon source consumed, thereby reducing the total amount of glucose and mannitol needed and lowering fermentation costs while maintaining high productivity
Solution Approach 2:
The engineered strain gains enhanced versatility in utilizing different carbon sources more efficiently. The BCoAT gene enables the strain to redirect metabolic flux from multiple carbon sources through a unified alternative pathway, improving overall carbon conversion efficiency and reducing the total carbon source requirement regardless of whether glucose, mannitol, or other substrates are used
3Ease of manufacture
If waste is used as a carbon source to reduce cost, then fermentation cost decreases, but toxicity inhibitors are present which complicates the process
Solution Approach 1:
The patent converts the harmful effect of toxicity inhibitors present in waste substrates into a beneficial outcome by engineering the strain with enhanced tolerance and efficiency. The BCoAT gene introduction creates a metabolic pathway that is less sensitive to inhibition, allowing the strain to efficiently convert waste carbon sources into butyric acid while tolerating the presence of toxic inhibitors, thereby turning a disadvantage into an advantage
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 optimized strain achieves a significant increase in butyric acid yield, up to 1.53 g/L, which is 6.95 times higher than the original bacterium, improving the economic feasibility of microbial fermentation.
Implementation Method 1
butyric acid biosynthesis is attracting more and more attention... Microbiological fermentation leads to a lower yield of butyric acid... CN109355318A discloses a method for producing butyric acid through fermentation
Data Source
Figure 1
Figure 2~3
Figure 4~5
AI summary
The present disclosure describes a butyric acid-producing Bacillus subtilis strain, a method of construction and application thereof. Autolysis-related genes and acetic acid metabolism-related genes are deleted on the genome of the chassis cell B. Subtilis. Meanwhile, the genome further contains a butyric acid synthesis-related gene BCoAT. In the present disclosure, autolysis-related genes and acetic acid metabolism-related genes in a chassis B. subtilis strain are knocked out, the butyric acid synthesis-related gene BCoAT is inserted, the butyric acid metabolic pathway of the B. subtilis is optimized, a bypass gene is knocked out and a heterogenous metabolic pathway is introduced. Finally, the butyric acid yield of the optimized B. subtilis strain increases by 6.95 times compared with that of the original strain.