Microbial Terpene Production via MEP Pathway Flux Control
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Solution Overview
Problem
Current methods for industrial-scale production of terpenes and terpenoids face challenges due to the high cost and low availability of plant-derived terpenes, inefficient extraction processes, and limitations in carbon flux through the MEP pathway in bacterial systems, which hinder the efficient conversion of renewable resources like glucose into terpene products.
Innovation Solution
A bacterial strain is engineered to overexpress 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (IspG) and 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (IspH) to enhance carbon flux through the MEP pathway, with a balanced activity favoring IspH over IspG to prevent HMBPP accumulation, and includes genetic modifications such as overexpression of pyruvate:flavodoxin oxidoreductase (PFOR) to improve electron supply, allowing for increased production of terpene and terpenoid products like Farnesene, Artemisinic acid, and Limonene.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If IspG and IspH are overexpressed to increase carbon flux through the MEP pathway, then terpene productivity is improved, but HMBPP accumulates and feeds back to reduce pathway flux
Solution Approach 1:
The patent applies feedback control by monitoring HMBPP accumulation levels and dynamically adjusting the expression or activity of IspH enzyme. When HMBPP accumulates to inhibitory levels, the system reduces IspH activity to prevent further accumulation, thereby maintaining stable pathway flux while still achieving high terpene productivity through controlled overexpression of IspG and IspH.
Solution Approach 2:
The patent changes the operational parameters of the MEP pathway by overexpressing IspG and IspH enzymes and adjusting their relative activities. Specifically, it optimizes the IspH/IspG activity ratio to ensure that HMBPP is converted to IPP at a rate that prevents accumulation while maintaining high carbon flux through the pathway, thereby resolving the contradiction between productivity and flux stability.
2Productivity
If IspH activity is increased to convert HMBPP to IPP, then carbon flux to terpene products is improved, but HMBPP accumulates and inhibits the pathway
Solution Approach 1:
The system implements feedback control where HMBPP accumulation serves as the feedback signal. When HMBPP levels rise to inhibitory concentrations, the system automatically reduces IspH enzyme activity or expression, preventing further HMBPP accumulation and the associated feedback inhibition, thus maintaining high carbon flux without pathway suppression.
Solution Approach 2:
The patent introduces an intermediary control mechanism that mediates between HMBPP production (by IspG) and consumption (by IspH). By carefully balancing the activities of these two enzymes and using HMBPP accumulation as a control signal, the system ensures that HMBPP serves as a transient intermediate rather than an accumulated inhibitor, enabling sustained high productivity.
3Quantity of substance
If traditional extraction methods are used to obtain terpenes from plants, then terpene products are obtained, but the process is tedious and inefficient for industrial-scale production
Solution Approach 1:
The patent replaces the mechanical extraction process (solvent extraction, distillation, etc.) with a biological production system. Instead of extracting terpenes from plant materials through tedious mechanical and chemical processes, the invention uses genetically engineered microorganisms that biosynthesize terpenes de novo from renewable resources like glucose, thereby eliminating the need for plant extraction and dramatically improving productivity and efficiency.
Solution Approach 2:
The engineered microbial system performs self-service by autonomously converting renewable carbon sources into terpene products through metabolic engineering. The microorganisms contain the complete biosynthetic pathway and automatically produce terpenes without requiring external extraction processes, making the system self-sufficient and highly efficient for industrial-scale 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 significantly increases the yield of terpene and terpenoid products by optimizing carbon flux and preventing feedback inhibition, enabling efficient conversion of glucose into desired terpenoids, thus overcoming the limitations of traditional extraction methods and improving industrial scalability.
Implementation Method 1
the strain comprising one or more genetic modifications that enhance supply and/or transfer of electrons through the MEP pathway and/or to terpene and terpenoid products, and culturing the bacterial strain to produce the terpene or terpenoid product, wherein the bacterial strain contains an overexpression of a pyruvate:flavodoxin oxidoreductase (PFOR)
Implementation Method 2
wherein 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (IspG) and 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (IspH) are overexpressed in the bacterial strain to provide increased carbon flux to 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate (HMBPP) intermediate
Data Source
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AI summary
The invention relates to methods and bacterial strains for making terpene and terpenoid products, the bacterial strains having improved carbon pull through the MEP pathway and to a downstream recombinant synthesis pathway.