Recombinant Cell Isoprene Production via MVA Pathway
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for producing isoprene and terpene face challenges in achieving stable and high-yield mass production, as they often rely on petrochemical processes or extraction from natural sources, and biotechnological approaches using recombinant microorganisms struggle with cytotoxicity and instability in precursor synthesis pathways.
Innovation Solution
A recombinant cell is developed that synthesizes isopentenyl diphosphate exclusively through the mevalonate pathway by deleting key enzymes of the non-mevalonate pathway and introducing genes for isoprene or terpene synthases, allowing for stable production of isoprene or terpene with 10, 15, 20, or 40 carbon atoms using C1 compounds like carbon monoxide or methane as carbon sources.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If the non-mevalonate pathway is deleted to improve isopentenyl diphosphate synthesis stability, then pathway function stability is improved, but productivity may be affected due to loss of precursor synthesis capability
Solution Approach 1:
The patent extracts and removes the non-mevalonate pathway from the host cell to eliminate pathway instability and cytotoxicity issues. By deleting genes encoding key enzymes in the non-mevalonate pathway, the invention isolates the mevalonate pathway as the sole functional route for isopentenyl diphosphate synthesis, thereby improving stability while maintaining productivity through optimized mevalonate pathway expression
Solution Approach 2:
The patent changes the metabolic pathway parameters by completely eliminating the non-mevalonate pathway and optimizing the mevalonate pathway expression levels. This parameter change transforms the system from a dual-pathway state with instability to a single-pathway state with controlled and stable high-level expression of isopentenyl diphosphate synthesis enzymes
2Productivity
If the mevalonate pathway is introduced to improve precursor synthesis ability, then isopentenyl diphosphate synthesis ability is improved, but cytotoxicity occurs due to pathway imbalance
Solution Approach 1:
The patent converts the potential harm of pathway imbalance into benefit by completely eliminating the non-mevalonate pathway. This prevents the formation of toxic intermediates that would arise from competing pathways, while the introduced mevalonate pathway genes are expressed at optimized levels to provide sufficient precursor supply without causing cytotoxicity
Solution Approach 2:
The patent changes the expression parameters of mevalonate pathway genes to achieve optimal balance between productivity and cytotoxicity avoidance. By controlling the expression levels of introduced genes, the system maintains high isopentenyl diphosphate synthesis ability while preventing toxic intermediate accumulation that would occur with unbalanced pathway expression
3Productivity
If exogenous mevalonate pathway genes are introduced to improve mass production capability, then isopentenyl diphosphate synthesis is improved, but pathway function is lost due to mutation over time
Solution Approach 1:
The patent performs preliminary deletion of the non-mevalonate pathway before introducing and expressing the mevalonate pathway genes. This preliminary action prevents future pathway conflicts and mutations by eliminating competing routes, ensuring that the introduced mevalonate pathway genes maintain their function stably over time without being subject to mutational loss
Solution Approach 2:
The patent optimizes the expression parameters of introduced mevalonate pathway genes to achieve stable mass production. By carefully controlling gene expression levels and ensuring proper integration into the host genome, the system maintains high productivity while preventing the pathway function loss that occurs with unoptimized expression systems
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 enables stable and efficient production of isoprene or terpene, overcoming previous limitations in yield and stability, and allows for continuous production without loss of pathway function over multiple subcultures.
Implementation Method 1
Enzymes acting in the mevalonate pathway include, in the order from the upstream, acetyl CoA acetyl transferase, HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, 5-phosphomevalonate kinase, and diphosphomevalonate decarboxylase
Implementation Method 2
Enzymes acting in the non-mevalonate pathway include, in the order from the upstream, DOXP synthase, DOXP reductoisomerase, 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase, 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase, 2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase, HMB-PP synthase and HMB-PP reductase
Data Source
AI summary
Provided is a recombinant cell that produces isoprene or terpene, wherein the recombinant cell includes an ability to synthesize isopentenyl diphosphate through a mevalonate pathway (MVA pathway), wherein the recombinant cell lacks an ability to synthesize isopentenyl diphosphate through an endogenous non-mevalonate pathway (MEP pathway), wherein the recombinant cell includes an isoprene synthase gene or a terpene synthase gene as a foreign gene, and wherein the recombinant cell produces, with the expression of the foreign gene, isoprene or terpene having 10, 15, 20, 30, or 40 carbon atoms. The mevalonate pathway is preferably an exogenous mevalonate pathway.