Yeast Acetyl-CoA Pathways Without ATP-Consuming PDH Bypass
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Solution Overview
Problem
Yeast cells require an efficient metabolic route to produce cytosolic acetyl-CoA without relying on the energy-inefficient pyruvate dehydrogenase (PDH) by-pass, which limits the yield of acetyl-CoA-derived products like butanol due to ATP imbalance.
Innovation Solution
Introduction of heterologous enzymes such as acetylating acetaldehyde dehydrogenase (ACDH) or pyruvate:NADP oxidoreductase (PNO) to convert pyruvate, acetaldehyde, or acetate into acetyl-CoA, bypassing the PDH by-pass, ensuring ATP balance and increased production of acetyl-CoA in the cytosol.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If the PDH by-pass is used to produce cytosolic acetyl-CoA in yeast, then acetyl-CoA is supplied for product synthesis, but ATP is consumed leading to energy imbalance and reduced productivity
Solution Approach 1:
The patent changes the biochemical parameters of the metabolic pathway by replacing the ATP-consuming PDH by-pass enzymes with alternative enzymes that do not require ATP hydrolysis. Specifically, it uses pyruvate decarboxylase followed by acetaldehyde dehydrogenase (acetylating type) to convert pyruvate to acetyl-CoA without ATP consumption, thereby maintaining energy balance while ensuring sufficient acetyl-CoA supply for butanol production.
2Quantity of substance
If the PDH by-pass is used to produce cytosolic acetyl-CoA, then acetyl-CoA is available for butanol synthesis, but carbon source must be diverted for ATP generation lowering overall product yield
Solution Approach 1:
The patent modifies the metabolic pathway parameters by eliminating the ATP-hydrolyzing step in acetyl-CoA synthesis. By using the alternative pathway (pyruvate → acetaldehyde → acetyl-CoA) that does not consume ATP, more carbon from the glucose substrate can be directed toward acetyl-CoA and subsequently butanol production, rather than being consumed for ATP regeneration, thereby increasing the theoretical and actual yield of butanol on carbon substrate.
3Ease of manufacture
If yeast is used instead of bacteria for acetyl-CoA derived product production, then sterile process requirements are eliminated, but existing metabolic pathways are energy-inefficient
Solution Approach 1:
The patent accepts yeast as the production host (which eliminates sterile processing requirements compared to bacteria) but addresses the energy inefficiency by changing the metabolic pathway parameters. It introduces heterologous genes encoding alternative enzymes for acetyl-CoA synthesis that bypass the ATP-consuming step, thereby combining the manufacturing advantages of yeast with improved energy efficiency suitable for industrial production.
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 allows for the production of acetyl-CoA without ATP consumption, enabling higher yields of desirable fermentation products like butanol, overcoming the limitations of the PDH by-pass and improving the energy balance in yeast cells.
Implementation Method 1
acetaldehyde dehydrogenase (acdh; E.C. 1.2.1.10) also referred to as acetaldehyde:NAD+ oxidoreductase (CoA-acetylating), catalyzing the following reaction: acetaldehyde+NAD++CoA⇌acetyl-CoA+NADH+H+
Implementation Method 2
The heterologous enzyme may produce the acetyl-CoA from acetaldehyde. An enzyme capable of catalyzing said reaction is acetylating acetaldehyde dehydrogenase
Implementation Method 3
An enzyme capable of converting pyruvate directly into acetyl-CoA is a pyruvate:NADP oxidoreductase (pno; E.C. 1.2.1.51), catalyzing the following reaction: pyruvate+NADP++CoA⇌acetyl-CoA+CO2+NADPH+H+
Implementation Method 4
pyruvate:NADP oxidoreductase (pno; E.C. 1.2.1.51), catalyzing the following reaction: pyruvate+NADP++CoA⇌acetyl-CoA+CO2+NADPH+H+
Implementation Method 5
The heterologous enzyme may also produce the acetyl-CoA from acetate. An enzyme capable of catalyzing said reaction is acetate:CoA ligase (ACL; E.C. 6.2.1.1), catalyzing the following reaction: acetate+ATP+CoA⇌acetyl-CoA+AMP+PPi
Implementation Method 6
Another enzyme capable of catalyzing said reaction is ATP:acetate phosphotransferase (AckA; E.C. 2.7.2.1) in combination with acetyl-CoA:Pi acetyltransferase (Pta; E.C. 2.3.1.8)
Implementation Method 7
In cytosol, acetyl-CoA is synthesized via the pyruvate dehydrogenase (PDH) by-pass, involving the enzymes pyruvate decarboxylase (PDC), acetaldehyde dehydrogenase (ALD), and acetyl-CoA synthetase (ACS), with the following overall reaction stoichiometry: Pyr+CoA+ATP+NAD(P)+=acetyl-CoA+CO2+NAD(P)H+AMP+Ppi+H+
Data Source
AI summary
A method of identifying a heterologous polypeptide having enzymatic activity for converting pyruvate, acetaldehyde or acetate into acetyl-CoA in cytosol of a yeast cell comprising: a) providing a mutated yeast cell comprising a deletion of at least one gene of the by-pass, selected from the genes encoding the enzymes pyruvate decarboxylase, acetaldehyde dehydrogenase, and acetylCoA synthetase; b) transforming said mutated yeast cell with an expression vector comprising a heterologous nucleotide sequence encoding a candidate polypeptide having potential enzymatic activity for converting pyruvate, acetaldehyde or acetate into acetyl-CoA; c) testing said recombinant mutated yeast cell for its ability to grown on minimal medium containing glucose as sole carbon source, and d) identifying said candidate polypeptide as a heterologous polypeptide having enzymatic activity for converting pyruvate, acetaldehyde or acetate into acetyl-CoA in the cytosol of said yeast cell when growth of said cell is observed.