Isoprene Biosynthesis Pathway ATP Consumption Reduction
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Solution Overview
Problem
Current methods for producing isoprene rely on either natural rubber from trees or petroleum-based synthetic rubber, which are unsustainable and energy-intensive, particularly due to high ATP consumption in the mevalonate pathway used in biocatalytic production.
Innovation Solution
Biosynthesis of 3-hydroxy-3-methylglutaryl-CoA (3-HMG) and subsequent conversion to isoprene using isolated enzymes such as 4-methyl-2-oxopentanoate dehydrogenase, 3-methylbutanoyl-CoA oxidoreductase, and isoprene synthase, or non-naturally occurring host cells expressing these enzymes, to reduce ATP consumption and improve efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If the mevalonate pathway is used for biocatalytic production of isoprene, then isoprene can be produced from glucose, but ATP consumption is excessively high (two moles of ATP per mole of isoprene)
Solution Approach 1:
The patent extracts and removes the energy-intensive phosphate activation steps (mevalonate kinase and phosphomevalonate kinase reactions) from the traditional mevalonate pathway. By eliminating these ATP-consuming steps, the pathway directly converts mevalonate to isopentenyl diphosphate without requiring phosphate activation, thereby reducing ATP consumption from two moles to zero moles per mole of isoprene produced.
Solution Approach 2:
The patent inverts the traditional pathway sequence by using a non-mevalonate pathway approach that starts from acetyl-CoA and uses different enzymatic reactions (including isopropylmalate synthase, isopropylmalate isomerase, and other enzymes) to reach isoprene without the conventional mevalonate diphosphate intermediate that requires ATP-intensive phosphorylation steps.
2Productivity
If the mevalonate pathway is used for biocatalytic production of isoprene, then isoprene can be produced, but the energy intensity makes the process unsustainable
Solution Approach 1:
The patent extracts and removes the energy-intensive phosphate activation steps (mevalonate kinase and phosphomevalonate kinase reactions) from the traditional mevalonate pathway. By eliminating these ATP-consuming steps, the pathway directly converts mevalonate to isopentenyl diphosphate without requiring phosphate activation, thereby reducing ATP consumption from two moles to zero moles per mole of isoprene produced.
Solution Approach 2:
The patent changes the biochemical parameters of the pathway by using alternative enzymes with different energy requirements. The non-mevalonate pathway employs enzymes such as isopropylmalate synthase and isopropylmalate isomerase that operate with different cofactor requirements and energy efficiency, fundamentally altering the energy profile of the biosynthetic route to isoprene.
3Productivity
If traditional biocatalytic methods are used, then isoprene can be produced, but the yield and productivity are limited (2 g/(L·h) volumetric productivity with 11% yield from glucose)
Solution Approach 1:
The patent employs preliminary metabolic engineering of the host organism (E. coli or S. cerevisiae) to optimize precursor availability and pathway flux before isoprene production. This includes overexpressing key enzymes, optimizing cofactor balance, and pre-adapting the cellular metabolism to channel carbon flux efficiently toward the isoprene pathway, thereby achieving higher yields and productivity.
Solution Approach 2:
The patent changes the biochemical parameters of the pathway by using alternative enzymes with different energy requirements. The non-mevalonate pathway employs enzymes such as isopropylmalate synthase and isopropylmalate isomerase that operate with different cofactor requirements and energy efficiency, fundamentally altering the energy profile of the biosynthetic route to isoprene.
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 energy requirements and enhances the yield and productivity of isoprene biosynthesis, providing a sustainable and efficient alternative to traditional methods by minimizing ATP usage and optimizing the biocatalytic pathway.
Implementation Method 1
converting 4-methyl-2-oxopentanoate to 3-methylbutanoyl-CoA, for example by using a polypeptide having the activity of an EC 1.2.7.7 or EC 1.2.1.-enzyme
Implementation Method 2
enzymatically converting 3-methylbutanoyl-CoA to 3-methylbut-2-enoyl-CoA using a polypeptide having the activity of an EC 1.3.8.4 enzyme
Implementation Method 3
enzymatically converting 3-methyl-glutaconyl-CoA to 3-hydroxy-3-methylglutaryl-CoA using a polypeptide having the activity of an EC 4.2.1.18 enzyme
Implementation Method 4
enzymatically converting 3-hydroxy-3-methylglutaryl-CoA to (R)-mevalonate using a hydroxymethylglutaryl Co-A reductase enzyme
Implementation Method 5
enzymatically converting (R)-mevalonate to (R)-5-phosphomevalonate using a mevalonate-kinase enzyme
Implementation Method 6
enzymatically converting (R)-5-diphosphomevalonate to isopentenyl diphosphate using a diphosphomevalonate decarboxylase enzyme
Data Source
AI summary
This application describes methods, including non-naturally occurring methods, for biosynthesizing 3-hydroxy-3-methylglutaryl-coA and intermediates thereof, as well as non-naturally occurring hosts for producing 3-hydroxy-3-methylglutaryl-coA. This application also describes methods, including non-naturally occurring methods, for biosynthesizing isoprene and intermediates thereof, as well as non-naturally occurring hosts for producing isoprene.