Lysine Production from Sucrose via ptsF Mutation and Glucose Isomerase
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Solution Overview
Problem
Microorganisms like Corynebacterium glutamicum, optimized for glucose, exhibit reduced efficiency in producing amino acids like L-lysine when using sucrose as a carbon source due to inefficient metabolic pathways, leading to suboptimal growth on sucrose and potential loss of flexibility in using either glucose or sucrose as a carbon source.
Innovation Solution
Development of novel strains with attenuated or blocked fructose transport mechanisms and expression of enzymes like glucose isomerase and glucokinase to drive fructose toward the pentose phosphate pathway, enhancing NADPH production and lysine synthesis by converting sucrose into glucose-6-phosphate for efficient utilization.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If Corynebacterium glutamicum is optimized for glucose utilization, then growth rate and amino acid production efficiency on glucose is improved, but efficiency of amino acid production using sucrose as carbon source deteriorates
Solution Approach 1:
The patent applies parameter changes by modifying the metabolic parameters of C. glutamicum through genetic engineering. Specifically, it overexpresses fructose-1,6-bisphosphatase (FBP) to alter the metabolic flux distribution, enabling the bacterium to efficiently utilize sucrose by redirecting carbon flow through the pentose phosphate pathway, thus improving both sucrose utilization efficiency and maintaining glucose productivity.
Solution Approach 2:
The patent introduces fructose-1,6-bisphosphatase as an intermediary enzyme to facilitate the conversion of fructose-1,6-bisphosphate to fructose-6-phosphate. This intermediary enzyme acts as a metabolic bridge, enabling efficient carbon flow from sucrose through the pentose phosphate pathway to produce NADPH and precursor molecules for amino acid synthesis, thereby resolving the incompatibility between sucrose utilization and amino acid production.
2Ease of operation
If multiple sucrose uptake systems are present in C. glutamicum, then sucrose can be transported into the cell, but metabolic efficiency and lysine production are reduced
Solution Approach 1:
The patent extracts or removes the problematic fructose export mechanism from the cellular metabolism. By eliminating fructose export, the system prevents the loss of carbon that would otherwise be wasted, and redirects all fructose derived from sucrose hydrolysis into the pentose phosphate pathway through FBP overexpression, thereby improving lysine production efficiency while maintaining sucrose uptake capability.
3Reliability
If fructose is exported from the cell after sucrose hydrolysis, then cellular fructose concentration is reduced, but carbon is wasted and lysine production efficiency decreases
Solution Approach 1:
The patent converts the previously harmful fructose export mechanism into a beneficial process. By overexpressing fructose-1,6-bisphosphatase, the system transforms fructose that would have been exported and wasted into a valuable metabolic intermediate (fructose-6-phosphate) that enters the pentose phosphate pathway. This converts the harmful carbon loss into a beneficial source of NADPH and precursor molecules for amino acid synthesis.
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 modified strains significantly increase lysine production by optimizing carbon flux through the pentose phosphate pathway, improving NADPH availability and reducing fructose export, thus enhancing the economic viability of amino acid production from both sucrose and glucose.
Implementation Method 1
glucose isomerase and/or invertase have been added
Implementation Method 2
production of L-lysine from Corynebacterium glutamicum using sucrose as a carbon source
Implementation Method 3
a phosphotransferase system (PTS), in which sucrose is phosphorylated at the glucose ring and subsequently hydrolyzed in the cell to glucose-6-phosphate and fructose
Data Source
AI summary
Methods and compositions for increased production of amino acids from C. glutamicum using sucrose as a carbon source are described. In one aspect, increased production of L-lysine from C. glutamicum is accomplished by using a strain having a mutation in the ptsF gene encoding fructose-PTS enzyme that attenuates or blocks fructose import into the cell when such strain is grown on media containing sucrose as a carbon source and production is increased by providing glucose isomerase in the fermentation media. The glucose isomerase may be exogenously added or expressed in the strain and exported into the media. In certain embodiments the media also contain an invertase. In another aspect increased production of L-lysine is accomplished by making a C. glutamicum strain having the ptsF mutation and a second mutation in a fructose exporter function. The dual mutation retains imported fructose in the cell. In certain embodiments, the strain also overexpresses at least one of a glucose isomerase and glucokinase activity in the cell to drive imported fructose toward the pentose phosphate pathway to increase L-lysine production.