Acetyl-CoA Production via ATP-Independent ADA Enzyme
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Solution Overview
Problem
The existing methods for producing acetyl-CoA derived isoprenoids are energetically inefficient due to high ATP requirements and redox imbalances, limiting the yield and productivity of these compounds in genetically modified microorganisms.
Innovation Solution
The use of heterologous acylating acetaldehyde dehydrogenase (ADA) to produce acetyl-CoA without ATP expenditure, combined with modifications such as introducing NADH-using enzymes and acetoacetyl-CoA synthase to address redox imbalances and enhance thermodynamic driving forces, optimizes the mevalonate pathway for improved yield and efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If the PDH-bypass pathway is used to generate cytosolic acetyl-CoA, then NADPH is produced which benefits the MEV pathway, but six ATP equivalents are expended per mevalonate generated resulting in energetically inefficient production
Solution Approach 1:
The invention extracts and replaces the ATP-consuming steps (acetaldehyde dehydrogenase and acetyl-CoA synthetase) from the PDH-bypass pathway with a single ATP-independent acetaldehyde dehydrogenase enzyme from Clostridium kluyveri that directly converts acetaldehyde to acetyl-CoA, eliminating the energy expenditure while preserving NADPH production
Solution Approach 2:
The invention changes the biochemical parameters of the pathway by introducing a heterologous enzyme with different catalytic properties (ATP-independent acetyl-CoA formation) to fundamentally alter the energy metabolism of the pathway while maintaining its redox balance benefits
2Productivity
If three acetyl-CoA molecules are consumed in the MEV pathway to generate one mevalonate, then isoprenoid synthesis proceeds, but the high ATP requirements limit the yield and productivity of isoprenoids
Solution Approach 1:
The invention performs preliminary action by pre-establishing an ATP-independent acetyl-CoA supply pathway using the heterologous C. kluyveri acetaldehyde dehydrogenase, which removes the energy bottleneck before isoprenoid synthesis begins, thereby enabling higher productivity without additional ATP expenditure
3Ease of manufacture
If the native PDH-bypass is used for acetyl-CoA production, then the pathway is energetically costly, but it provides a functional mechanism for cytosolic acetyl-CoA generation
Solution Approach 1:
The invention creates a copied and improved version of the acetyl-CoA generation mechanism by introducing the C. kluyveri acetaldehyde dehydrogenase enzyme that replicates the function of the PDH-bypass but eliminates the ATP-consuming steps, providing an energetically superior alternative pathway
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 reduces ATP equivalents required for isoprenoid synthesis, alleviates redox imbalances, and increases the maximum theoretical yield of isoprenoids like farnesene, leading to more efficient oxygen use and reduced oxygen demand, thereby enhancing production efficiency and yield.
Implementation Method 1
the use of heterologous acylating acetaldehyde dehydrogenase (ADA) to produce acetyl-CoA without ATP expenditure
Implementation Method 2
modifications such as introducing NADH-using enzymes and acetoacetyl-CoA synthase to address redox imbalances and enhance thermodynamic driving forces
Data Source
AI summary
Provided herein are compositions and methods for the heterologous production of acetyl-CoA-derived isoprenoids in a host cell. In some embodiments, the host cell is genetically modified to comprise a heterologous nucleotide sequence encoding an acetaldehyde dehydrogenase, acetylating (ADA, E.C. 1.2.1.10) and an MEV pathway comprising an NADH-using HMG-CoA reductase. In some embodiments, the host cell is genetically modified to comprise a heterologous nucleotide sequence encoding an ADA and an MEV pathway comprising an acetoacetyl-CoA synthase. In some embodiments, the genetically modified host cell further comprises one or more heterologous nucleotide sequences encoding a phosphoketolase and a phosphotransacetylase. In some embodiments, the genetically modified host cell further comprises a functional disruption of the native PDH-bypass. The compositions and methods described herein provide an energy-efficient yet redox balanced route for the heterologous production of acetyl-CoA-derived isoprenoids.