Modified Benzalacetone Synthase Enzyme T281A Mutation
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
The existing BAS enzymes from Rheum palmatum are inefficient in converting coumaroyl-CoA into 4-hydroxybenzalacetone and ferulic acid into 4-(4-hydroxy-3-methoxyphenyl)but-3-en-2-one, limiting the production of raspberry ketone and zingerone in biotechnological applications due to low catalytic constants, which slows down metabolic flux and yields.
Innovation Solution
A modified benzalacetone synthase enzyme with a T281A substitution is developed, derived from wild-type BAS enzymes, which enhances catalytic activity, allowing for more efficient conversion of coumaroyl-CoA into 4-hydroxybenzalacetone and ferulic acid into 4-(4-hydroxy-3-methoxyphenyl)but-3-en-2-one, using genetic engineering techniques to introduce non-natural mutations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If wild-type BAS enzyme is used, then the enzyme structure is simple and easy to manufacture, but the catalytic activity is low and productivity is insufficient
Solution Approach 1:
The patent applies parameter changes by modifying the amino acid sequence of the BAS enzyme through site-directed mutagenesis. Specifically, threonine at position 281 is substituted with alanine (T281A mutation), which changes the local chemical environment and sterics of the active site, thereby enhancing catalytic activity while maintaining the overall enzyme structure.
Solution Approach 2:
The invention applies local quality by making a targeted mutation at position 281 of the BAS enzyme. This local modification alters the properties of a specific region (the active site vicinity) without changing the entire enzyme structure, thus improving catalytic activity while keeping the rest of the enzyme simple and manageable.
2Productivity
If wild-type BAS enzyme is used, then the manufacturing process is simple, but the metabolic flux is slow and yield is low
Solution Approach 1:
The patent changes the kinetic parameters of the BAS enzyme by introducing the T281A mutation, which increases the turnover number (kcat) and/or decreases the Michaelis constant (Km) for the substrates. This parameter change accelerates the enzymatic reaction rate, thereby increasing metabolic flux and product yield without complicating the manufacturing process.
3Productivity
If BAS enzyme with higher catalytic activity is developed, then productivity increases, but the risk of altering enzyme stability and specificity increases
Solution Approach 1:
The invention applies local quality by making a targeted mutation at position 281 of the BAS enzyme. This local modification alters the properties of a specific region (the active site vicinity) without changing the entire enzyme structure, thus improving catalytic activity while keeping the rest of the enzyme simple and manageable.
Solution Approach 2:
The patent creates a copy of the wild-type BAS enzyme with a specific mutation (T281A). This mutant enzyme retains the overall structure and function of the wild-type enzyme but with improved catalytic properties, allowing for reliable performance while achieving higher 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 modified BAS enzyme significantly increases the production of 4-hydroxybenzalacetone and raspberry ketone, with Pseudomonas putida strains expressing this enzyme achieving up to 6 times higher yields compared to strains with wild-type BAS, making the process more economically viable and efficient.
Implementation Method 1
Benzalacetone Synthases ("BAS" or "BAS enzyme") catalyze the transformation of coumaroyl-coA into 4-hydroxybenzalacetone
Implementation Method 2
the modified BAS has the capability to produce 4-(4-hydroxy-3-methoxyphenyl)but-3-en-2-one from ferulic acid
Data Source
Figure 1
Figure 2
Figure 3
AI summary
The present invention is in the field of genetically modified enzyme and microorganism comprising such a modified enzyme for the production of raspberry ketone or zingerone. The modified enzyme is a recombinant benzalacetone synthase (BAS) issued or derived from a wild-type BAS. The modified BAS has the capability to produce 4-hydroxybenzalacetone from 4-coumaroyl-CoA in a more effective way as compared to wild-type BAS.