SAM-Dependent Methyltransferase Activity via In Vivo Growth Selection
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Solution Overview
Problem
There is a need for improved SAM-dependent methyltransferase activity for biosynthetic applications, as existing methods do not effectively enhance the activity of these enzymes in cells.
Innovation Solution
An in vivo growth selection system is used to improve SAM-dependent methyltransferase activity by genetically engineering host cells to rely solely on the SAM cycle for homocysteine production, with homocysteine serving as a precursor for cysteine, 2-oxobutanoate, or isoleucine production. This is achieved by introducing a heterologous SAM-dependent methyltransferase and disrupting endogenous pathways for homocysteine biosynthesis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If existing methods are used to enhance methyltransferase activity, then biosynthetic applications can proceed, but the methyltransferase activity is not effectively enhanced
Solution Approach 1:
The invention employs a feedback mechanism where the methyltransferase activity is coupled to cell growth through the SAM cycle. The enzyme converts SAM to SAH, and the subsequent conversion of SAH back to SAM provides a feedback loop that selects for improved enzyme activity, as higher activity leads to more efficient methylation and better growth under selection pressure
Solution Approach 2:
The invention changes the physiological parameters of the host cell by deleting specific genes (CHO2, OPI3, MET17) to create a dependency on the SAM cycle for homocysteine production. This parameter change forces the system to rely on methyltransferase activity for essential metabolite synthesis, thereby selecting for improved enzyme activity
2Reliability
If the SAM cycle is made the sole source of homocysteine, then methyltransferase activity can be selected for, but the cell requires multiple gene disruptions
Solution Approach 1:
The invention extracts and removes alternative homocysteine biosynthesis pathways by deleting genes encoding enzymes in those pathways (CHO2, OPI3, MET17). This extraction of competing pathways isolates the SAM cycle as the sole source of homocysteine, enabling selection for methyltransferase activity despite the complexity of multiple gene disruptions
3Reliability
If endogenous pathways for homocysteine biosynthesis are disrupted, then SAM cycle becomes the only source, but the cell becomes auxotrophic requiring supplementation
Solution Approach 1:
The invention implements self-service by enabling the cell to produce its own homocysteine and essential metabolites (cysteine, 2-oxobutanoate, isoleucine) through the SAM cycle and associated pathways. The methyltransferase enzyme itself becomes part of the essential biosynthetic machinery, converting SAM to SAH and enabling the cycle to continue, thereby eliminating the need for external supplementation of these metabolites
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 system effectively enhances SAM-dependent methyltransferase activity, leading to improved growth rates and increased production of desired metabolites, such as melatonin, in genetically modified bacterial and yeast cells.
Implementation Method 1
Methyltransferases are an important group of enzymes which facilitate methylation events between methyl donors and acceptors. They can generally be divided into three classes, of which the most common one is that of S-adenosyl methionine- (SAM-) dependent methyltransferases, i.e., methyltransferases requiring the SAM molecule as a methyl donor, resulting in its conversion to S-adenosyl-L-homocysteine (SAH).
Implementation Method 2
The SAH can then be converted back to SAM via a SAM cycle, an enzymatic pathway existing in all known organisms.
Implementation Method 3
an in vivo growth selection system is used to improve SAM-dependent methyltransferase activity by genetically engineering host cells to rely solely on the SAM cycle for homocysteine production, with homocysteine serving as a precursor for cysteine, 2-oxobutanoate, or isoleucine production
Data Source
Figure 1
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Figure 3A~3B
AI summary
Methods for evolving cells or strains towards improved methyltransferase activity, particularly SAM-dependent methyltransferase activity, as well as to cells and strains useful in such methods and methods of using the evolved cells in the production of methylated products.