Engineered Lactic Acid Bacteria for Enhanced DHAD Activity
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Solution Overview
Problem
There is a need to increase dihydroxy-acid dehydratase (DHAD) activity in lactic acid bacteria to enhance the production of compounds such as isobutanol, valine, isoleucine, leucine, and pantothenic acid, as existing methods do not effectively achieve high enough levels of DHAD activity for optimal production.
Innovation Solution
Recombinant lactic acid bacterial cells are engineered to express a heterologous polypeptide with DHAD activity and are made substantially free of lactate dehydrogenase activity, resulting in increased DHAD specific activity, allowing for improved production of these compounds.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If lactic acid bacteria are engineered to express heterologous DHAD polypeptide, then DHAD activity is increased, but the presence of endogenous lactate dehydrogenase activity reduces the specific activity of DHAD
Solution Approach 1:
The patent removes endogenous lactate dehydrogenase activity from lactic acid bacteria through genetic modification (disruption of ldh genes). This extraction of the interfering enzyme allows the heterologous DHAD polypeptide to function with higher specific activity, as there is no competing endogenous activity to reduce the measured DHAD specific activity.
Solution Approach 2:
The patent changes the physiological parameters of the lactic acid bacteria by eliminating lactate dehydrogenase activity. This parameter change (from having LDH activity to being substantially free of LDH activity) creates an optimized environment for DHAD function, allowing the heterologous enzyme to achieve higher specific activity levels.
2Productivity
If endogenous lactate dehydrogenase activity is present in lactic acid bacteria, then the bacteria can perform normal lactate metabolism, but it interferes with measuring and achieving high DHAD specific activity
Solution Approach 1:
By removing endogenous lactate dehydrogenase activity through genetic modification, the patent eliminates the interference that makes DHAD specific activity difficult to measure. Without competing LDH activity, DHAD specific activity can be accurately detected and measured, enabling optimization of compound production pathways.
3Quantity of substance
If heterologous DHAD polypeptide is expressed in wild-type lactic acid bacteria, then DHAD activity is introduced, but the specific activity remains limited due to endogenous LDH interference
Solution Approach 1:
The patent extracts endogenous lactate dehydrogenase activity from the lactic acid bacteria through disruption of ldh genes. This removal eliminates the source of interference, allowing the heterologous DHAD polypeptide to achieve higher and more precise specific activity levels, improving the precision of DHAD-catalyzed reactions in compound production pathways.
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 engineered cells exhibit enhanced DHAD activity, leading to increased production of isobutanol and other relevant compounds, reducing the reliance on petrochemical fuels and improving biochemical pathway efficiency.
Implementation Method 1
Dihydroxy-acid dehydratase (DHAD), also called acetohydroxy acid dehydratase, catalyzes the conversion of 2,3-dihydroxyisovalerate to α-ketoisovalerate
Implementation Method 2
The DHAD enzyme requires binding of an iron-sulfur (Fe—S) cluster for activity
Data Source
AI summary
Lactic acid bacterial (LAB) cells were modified such that they have a specific activity of dihydroxy-acid dehydratase enzyme activity that is increased to about 0.1 μmol min−1 mg−1. LAB cells with even higher activities of 0.2 to 0.6 μmol min−1 mg−1 of DHAD activity were obtained.These modified cells may be used to produce isobutanol when additional isobutanol biosynthetic pathway enzymes are expressed.