Malonyl-CoA Synthesis via Aminotransferase and Reductase Pathway
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Solution Overview
Problem
The natural synthesis pathways for malonyl-CoA, such as the PDH-ACC pathway and MCS pathway, suffer from catalytic defects like low carbon utilization, energy consumption, greenhouse gas emission, and strict regulation of key enzymes, limiting the efficient synthesis of malonyl-CoA derivatives.
Innovation Solution
An artificial synthesis method for malonyl-CoA is developed through heterologous expression of an aminotransferase and a malonyl-CoA reductase, using β-alanine as a precursor. This method constructs an artificial synthesis pathway that avoids the defects of natural pathways by forming 3-oxopropanoate and subsequently malonyl-CoA, improving the yield of malonyl-CoA and its derivatives.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Loss of substance
If the PDH-ACC pathway is used for malonyl-CoA synthesis, then malonyl-CoA can be produced through natural metabolism, but carbon utilization is low and CO2 is released reducing atomic economy
Solution Approach 1:
The invention extracts and removes the decarboxylation step (PDH enzyme) from the natural pathway that generates CO2 loss. By eliminating this step and using an alternative reductive carboxylation pathway, the method prevents carbon loss and CO2 emission while maintaining malonyl-CoA production capability.
Solution Approach 2:
Instead of using the conventional oxidative decarboxylation pathway (PDH) followed by carboxylation (ACC), the invention inverts the logic by using direct reductive carboxylation of acetyl-CoA. This reverses the traditional metabolic flow to achieve better carbon efficiency and avoid greenhouse gas emission.
2Productivity
If the PDH complex is modified and overexpressed to enhance malonyl-CoA synthesis, then synthesis capacity increases, but the complexity of the enzyme complex makes modification and overexpression difficult and burdensome for cells
Solution Approach 1:
The invention segments the complex PDH-ACC pathway into simpler, separate enzymatic steps using individual enzymes (acetyl-CoA carboxylase and malonyl-CoA synthetase) that can be independently expressed and regulated. This avoids the need to modify and coordinate the expression of multiple subunits in the massive PDH complex.
Solution Approach 2:
The invention replaces the expensive, complex, and difficult-to-modify PDH complex with simpler, more readily available enzymes that can be easily overexpressed and manipulated. The alternative pathway uses enzymes that are more amenable to genetic engineering and cellular overexpression strategies.
3Productivity
If acetyl-CoA carboxylase (ACC) is used to synthesize malonyl-CoA, then malonyl-CoA production occurs, but the pathway consumes additional ATP energy
Solution Approach 1:
The invention changes the energetic parameters of the pathway by using malonyl-CoA synthetase coupled with phosphoenolpyruvate (PEP) as the energy source instead of ATP. This substitution of energy currency (ATP → PEP) reduces the direct ATP consumption burden on the cell while maintaining malonyl-CoA synthesis capability.
4Productivity
If PDH and ACC are strictly regulated by cells, then metabolic homeostasis is maintained, but the synthesis of malonyl-CoA is limited and cannot be enhanced without disrupting regulation
Solution Approach 1:
The invention introduces an intermediary pathway using malonyl-CoA synthetase that operates parallel to the regulated PDH-ACC pathway. This intermediary enzyme system allows malonyl-CoA production to be enhanced without directly interfering with the cell's existing regulatory mechanisms for PDH and ACC, thus maintaining metabolic homeostasis while increasing 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 artificial pathway enhances the synthesis of malonyl-CoA derivatives, such as flaviolin, octanoic acid, phloroglucinol, pentadecaheptaene, natamycin, and spinosad, while also improving cellular tolerance to stress conditions like organic acids, osmotic pressure, and cytotoxic substances.
Implementation Method 1
forming 3-oxopropanoate and a compound of formula (2) by β-alanine and a compound of formula (1) under catalysis of an aminotransferase
Implementation Method 2
forming malonyl-CoA and 2[H] by the 3-oxopropanoate, CoA, and an electron acceptor by transferring electrons of the 3-oxopropanoate to the electron acceptor under catalysis of an oxidoreductase
Implementation Method 3
by transferring electrons of the 3-oxopropanoate to the electron acceptor under catalysis of an oxidoreductase
Data Source
AI summary
An artificial synthesis method for malonyl-CoA and use thereof are provided. By means of heterologous expression of an aminotransferase and a malonyl-CoA reductase, an artificial synthesis pathway for synthesizing malonyl-CoA by using β-alanine (β-ala) as a precursor is constructed as follows: firstly, under catalysis of a transaminase, β-ala transfers amino groups to α-ketonic acid (such as pyruvic acid, oxaloacetic acid, or α-ketoglutaric acid, etc.), to form an intermediate product 3-oxopropanoate and a corresponding amino acid; the 3-oxopropanoate generates malonyl-CoA under the action of the malonyl-CoA reductase. This pathway addresses the defects of the natural malonyl-CoA synthesis pathway, such as low carbon utilization, consumption of energy substance ATP, release of greenhouse gas CO2, and strict regulation of pathway enzymes, a pyruvate dehydrogenase (PDH) and an acetyl-CoA carboxylase (ACC), thereby achieving high yielding of products using malonyl-CoA as a precursor, including flaviolin, octanoic acid, phloroglucinol, pentadecaheptaene, natamycin, and spinosad.