Engineered L-Alanine Strain for High-Temperature Fermentation
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Solution Overview
Problem
Existing methods for producing L-alanine, such as chemical synthesis and enzymatic catalysis, are inefficient and costly, while genetically engineered Escherichia coli strains are not suitable for high-temperature fermentation.
Innovation Solution
A genetically engineered strain is constructed by introducing a pyruvate synthesis pathway, overexpressing 6-Phosphofructokinase and pyruvate kinase, incorporating thermostable alanine dehydrogenase, and inactivating or deleting alanine racemase genes, enabling high-temperature fermentation for L-alanine production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If Escherichia coli strains are used for L-alanine fermentation, then production cost is reduced and yield is improved, but the strain cannot withstand high-temperature fermentation conditions
Solution Approach 1:
The patent changes the temperature parameter from conventional 37°C to 42-55°C high-temperature fermentation, and modifies the strain's genetic parameters by introducing thermostable enzymes (alanine dehydrogenase from Bacillus subtilis, pyruvate kinase from Bacillus licheniformis) to enable the strain to function reliably at elevated temperatures while maintaining high productivity
2Object-affected harmful factors
If high-temperature fermentation is implemented, then contamination risk is reduced and heat exchange cost is reduced, but conventional strains cannot grow effectively
Solution Approach 1:
The patent raises the fermentation temperature parameter to 42-55°C, which inherently reduces contamination risk from common mesophilic contaminants. Simultaneously, it introduces thermostable metabolic enzymes to ensure the strain maintains effective growth and production capability at this elevated temperature, resolving the contradiction between reduced contamination and maintained productivity
3Productivity
If chemical synthesis is used for L-alanine production, then production capacity is achieved, but the process becomes costly and inefficient
Solution Approach 1:
The patent replaces the chemical synthesis mechanism (propionic acid chlorination process) with a biological fermentation mechanism using genetically engineered strains. This substitution maintains production capacity while dramatically reducing costs by using inexpensive substrates like glucose and glycerol, eliminating the need for expensive chemical reagents and complex multi-step synthesis procedures
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 strain achieves high-yield, optically pure L-alanine production at temperatures of 42° C. to 55° C., reducing contamination risks and production costs, with yields up to 100 g/L.
Implementation Method 1
L-alanine can be produced by fermentation with a strain constructed by genetic engineering. This approach has attracted widespread attention thanks to the advantages of the inexpensive and easily available raw material, high product yield, low cost and so forth.
Implementation Method 2
enhancing the glycolysis pathway or/and introducing thermostable alanine dehydrogenase, a significantly increased yield of alanine can be obtained
Data Source
AI summary
The present invention discloses an L-alanine-producing genetically engineered strain, as well as a method of construction and use thereof, and pertains to the field of bioengineering. According to the present invention, through enhancing the glycolysis pathway or/and introducing a gene for thermostable alanine dehydrogenase, a genetically engineered strain capable of high-yield production of alanine under a high temperature condition of 42° C. to 55° C. can be constructed. Moreover, through knocking out alanine racemase genes, optical purity of L-alanine can be significantly increased. When the original strain possesses a lactate synthesis pathway, blocking this lactate synthesis pathway can augment the proportion of a pyruvate synthesis pathway, resulting in an additionally increased yield of L-alanine. The present invention overcomes the problems of fermentation at a low temperature, high cost and the like, which arise from the use of conventional L-alanine production techniques, enables production of L-alanine by fermentation at a high temperature of 42° C. to 55° C. with a yield of 95 g/L or higher, and is of high value to industrial application.