Mutant Sucrose Permease Polypeptides for Industrial Fermentation
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Solution Overview
Problem
Industrial microbial strains struggle to efficiently utilize sucrose as a carbon source due to low sucrose utilization rates and productivity, limiting the production of glycosylated products in a cost-effective and time-efficient manner.
Innovation Solution
Development of novel sucrose permease polypeptides with specific mutations, integrated into metabolically engineered cells, enabling enhanced sucrose uptake and utilization, and a method for producing and purifying glycosylated products such as monosaccharides, phosphorylated monosaccharides, disaccharides, oligosaccharides, glycoproteins, or glycolipids.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional sucrose permease (CscB) is used in industrial microbial strains, then the strain can utilize sucrose as a carbon source, but the sucrose utilization rate and productivity remain low
Solution Approach 1:
The patent applies parameter changes by introducing specific amino acid mutations (E106K, E106R, D107K, D107R, E106K/D107K, E106R/D107R) at positions 106 and 107 of the CscB sucrose permease sequence. These parameter changes in the protein structure directly improve the sucrose utilization rate and productivity while maintaining reliable sucrose utilization ability in industrial microbial strains.
2Ease of manufacture
If sucrose is used as a carbon source instead of glucose, then cost and environmental benefits are improved, but the ability to utilize sucrose efficiently is limited in most industrial strains
Solution Approach 1:
The patent modifies the sucrose permease protein parameters through specific amino acid substitutions at positions 106 and 107, enabling industrial strains to efficiently utilize sucrose as a carbon source. This resolves the contradiction by maintaining the cost-effectiveness of sucrose while improving the utilization rate through engineered parameter changes in the transport protein.
3Productivity
If existing sucrose utilization systems (cscBKA or cscBKAR) are used, then sucrose utilization is enabled, but growth rates on sucrose and product productivity remain low
Solution Approach 1:
The patent introduces parameter changes in the sucrose permease protein structure through amino acid mutations at positions 106 and 107. These changes enhance both the growth rate on sucrose and product productivity by improving the efficiency of sucrose transport into the cell, thereby resolving the contradiction between speed and productivity.
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 novel sucrose permease polypeptides improve sucrose utilization rates and productivity in metabolically engineered cells, facilitating the efficient production and purification of glycosylated products, thereby addressing the limitations of existing technologies in using sucrose as a carbon source.
Implementation Method 1
extracellular sucrose is taken up in the cell by a sucrose permease
Implementation Method 2
sucrose:H+ symporter (sucrose permease)
Implementation Method 3
Sucrose-6-P is then hydrolyzed in the cell by a sucrose-6-P hydrolase (invertase) generating fructose and glucose-6-phosphate
Implementation Method 4
Fructose is then catalysed by an ATP-dependent fructokinase to generate fructose-6-P
Implementation Method 5
glucose is catalysed by an ATP-dependent glucokinase to generate glucose-6-P
Implementation Method 6
Glucose-1-P can be converted to glucose-6-P by a phosphoglucomutase
Data Source
AI summary
The present invention is in the technical field of synthetic biology and metabolic engineering. More particularly, the present invention is in the technical field of fermentation of metabolically engineered cells. The present invention describes new sucrose permease polypeptides, and their applications. The present invention also describes a metabolically engineered cell for the production of a glycosylated product using the novel sucrose permease polypeptides. Furthermore, the present invention provides a method for the production of a glycosylated product by a cell using the novel sucrose permease polypeptides as well as the purification of said glycosylated product from the cultivation.