Engineered Ptb-Buk Fermentation Pathway for ATP-Coupled Product Synthesis
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Solution Overview
Problem
Current fermentation routes for producing various products are energy-consuming or energy-neutral, limiting product yield in energy-limited systems and uncoupling product production from microorganism growth.
Innovation Solution
Introduction of phosphate butyryltransferase (Ptb) and butyrate kinase (Buk) enzymes (Ptb-Buk) into heterologous species to convert acyl-CoAs and enoyl-CoAs to their respective acids or alkenates, generating ATP via substrate level phosphorylation, and further conversion to aldehydes, alcohols, or diols using aldehyde:ferredoxin oxidoreductase and alcohol dehydrogenase.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Loss of energy
If conventional fermentation pathways are used in bacteria, then bacterial growth and reproduction are maintained, but energy is consumed without net production for external applications
Solution Approach 1:
The patent converts the traditionally harmful or wasted byproducts of fermentation (such as formate, acetate, and CO2) into beneficial energy-generating pathways. By engineering bacteria to perform oxidative phosphorylation and convert these fermentation byproducts into additional ATP through alternative oxidases and electron transport chains, the system transforms energy loss into energy production, achieving net positive energy output while maintaining bacterial growth.
2Productivity
If engineered pathways are added to bacteria for energy generation, then energy production increases, but metabolic burden and pathway complexity increase
Solution Approach 1:
The patent implements multi-functional metabolic pathways where single engineered components serve multiple purposes. For example, the alternative oxidase system not only generates additional ATP but also maintains redox balance and prevents accumulation of harmful intermediates. The engineered pathways are designed to operate in parallel with native metabolism, allowing the system to flexibly switch between different energy-generating modes depending on substrate availability and environmental conditions, thereby reducing overall metabolic burden.
Solution Approach 2:
The complex energy-generating system is divided into modular, separable pathways: (1) glycolysis for substrate breakdown, (2) fermentation pathways for intermediate production, (3) oxidative phosphorylation for ATP generation, and (4) alternative oxidase systems for byproduct conversion. Each module can be independently optimized and regulated, allowing precise control over energy production while minimizing interference with essential bacterial functions.
3Loss of energy
If fermentation byproducts accumulate in the system, then energy efficiency decreases, but additional pathway engineering increases system complexity
Solution Approach 1:
The patent directly addresses byproduct accumulation by engineering pathways that convert fermentation byproducts (formate, acetate, CO2) into valuable energy sources. The alternative oxidase system catalyzes the oxidation of these byproducts, feeding electrons into the electron transport chain to generate additional ATP. This approach simultaneously improves energy efficiency by recovering energy that would otherwise be lost and simplifies the overall system by eliminating the need for separate byproduct removal or treatment mechanisms.
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
Provides a novel, alternate route for high-yield production of acids, alkenes, aldehydes, alcohols, and diols, overcoming energy limitations and coupling product formation with microorganism growth.
Implementation Method 1
During fermentation, a substrate such as glucose is partially degraded and energy is produced in the form of adenosine triphosphate (ATP).
Implementation Method 2
energy is produced in the form of adenosine triphosphate (ATP) through substrate-level and oxidative phosphorylation
Data Source
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AI summary
The invention relates to a genetically engineered bacterium comprising an energy-generating fermentation pathway and methods related thereto. In particular, the invention provides a bacterium comprising a phosphate butyryltransferase (Ptb) and a butyrate kinase (Buk) (Ptb-Buk) that act on non-native substrates to produce a wide variety of products and intermediates. In certain embodiments, the invention relates to the introduction of Ptb-Buk into a C1-fixing microoorgansim capable of producing products from a gaseous substrate.