Heterogeneous Protein Production via Alanine Aminotransferase Metabolic Engineering
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Solution Overview
Problem
Current methods for producing recombinant proteins using animal cells are limited by low production efficiency, and there is a need for a method to enhance the yield of desired polypeptides, particularly alanine aminotransferase (ALT) expression in host cells like CHO cells.
Innovation Solution
The method involves culturing cells that strongly express alanine aminotransferase (ALT) and a taurine transporter, which promotes the biosynthesis of pyruvate and glutamate from alanine, improving cell metabolism and increasing the yield of the desired polypeptide, such as antibodies, by utilizing the TCA cycle and glycogenesis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If animal cells are used to produce recombinant proteins, then post-translational modification and folding capabilities are improved, but production efficiency remains low
Solution Approach 1:
The patent changes the metabolic parameters of animal cells by introducing and overexpressing alanine aminotransferase (ALT), which catalyzes the conversion of alanine to pyruvate. This metabolic parameter change enables cells to efficiently utilize alanine as an energy source, thereby improving production efficiency while maintaining the inherent post-translational modification capabilities of animal cells
Solution Approach 2:
The patent introduces alanine aminotransferase as an intermediary enzyme that mediates the conversion of alanine to pyruvate, which then enters the TCA cycle to generate energy. This intermediary mechanism allows cells to efficiently metabolize alanine and improve overall production efficiency without compromising protein quality
2Productivity
If alanine aminotransferase is strongly expressed in host cells, then metabolic efficiency is improved, but cell behavior changes need to be understood
Solution Approach 1:
The patent employs feedback mechanisms where alanine aminotransferase activity is regulated based on cellular metabolic needs. The enzyme converts alanine to pyruvate, which feeds into the TCA cycle, creating a feedback loop that maintains metabolic balance and ensures stable cell culture behavior while improving metabolic efficiency
3Quantity of substance
If alanine is accumulated in cells during culture, then energy substrate is available, but metabolic pathway optimization is needed
Solution Approach 1:
The patent changes the metabolic parameter by overexpressing alanine aminotransferase, which shifts the metabolic pathway to efficiently convert accumulated alanine into pyruvate. This parameter change enables cells to utilize alanine accumulation as an energy source through enhanced conversion to pyruvate and entry into the TCA cycle, thereby improving overall metabolic pathway efficiency
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 significantly increases the yield of the desired polypeptide, as demonstrated by higher antibody production and improved cell survival ratios in fed-batch cultures, indicating enhanced cell culture behavior and metabolic efficiency.
Implementation Method 1
Alanine aminotransferase (EC 2.6.1.2.) has been known. This enzyme transfers an amino group from alanine to 2-oxoglutarate to synthesize glutamate.
Implementation Method 2
the products are utilized in metabolism during a TCA cycle and glucose production by glycogenesis
Implementation Method 3
the products are utilized in metabolism during a TCA cycle and glucose production by glycogenesis
Data Source
AI summary
The present invention provides a method capable of producing a natural or recombinant protein in high yield. The present invention relates to a method of producing a polypeptide, comprising culturing a cell which strongly expresses alanine aminotransferase and has a transferred DNA encoding a desired polypeptide and thereby allowing the cell to produce the polypeptide.