Phosphoketolase Pathway Engineering for Acetyl-CoA Yield
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Solution Overview
Problem
Current methods for producing isoprene and isoprenoids are limited by the loss of carbon atoms during the conversion of glucose to acetyl-CoA, resulting in decreased yields, and existing pathways rely on enzymes like those in the Wood-Ljungdahl pathway, which are not universally applicable.
Innovation Solution
Engineering recombinant cells with a heterologous phosphoketolase pathway that converts xylulose 5-phosphate or fructose 6-phosphate to acetyl phosphate, bypassing the typical carbon loss, thereby potentially producing three molecules of acetyl-CoA from one glucose molecule without relying on the Wood-Ljungdahl pathway enzymes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional glycolysis pathway is used to convert glucose to acetyl-CoA, then the process is simple and well-established, but carbon atoms are lost during conversion resulting in decreased yields
Solution Approach 1:
The patent divides the conventional glycolysis pathway into segments and replaces specific enzymatic steps with alternative pathways. The phosphoketolase pathway segments the conversion process to bypass decarboxylation steps that cause carbon loss, allowing recovery of carbon atoms that would otherwise be lost as CO2.
Solution Approach 2:
Instead of following the conventional glycolytic route that loses carbon, the patent inverts the approach by using phosphoketolase to cleave ketose phosphates into glyceraldehyde-3-phosphate and acetyl phosphate, then converting acetyl phosphate to acetyl-CoA without carbon loss. This reverse engineering of the metabolic route eliminates the harmful decarboxylation step.
2Productivity
If Wood-Ljungdahl pathway enzymes are used for acetyl-CoA production, then carbon efficiency may be improved, but the pathway is not universally applicable to all host organisms
Solution Approach 1:
The patent employs phosphoketolase, an enzyme with broad substrate specificity that can process multiple ketose phosphates (xylulose-5-phosphate, fructose-6-phosphate, sedoheptulose-7-phosphate). This universal enzyme can be implemented across diverse host organisms including bacteria, yeast, and plant cells, making the pathway broadly applicable while maintaining carbon efficiency.
Solution Approach 2:
The patent changes the biochemical parameters of the metabolic pathway by introducing phosphoketolase activity, which alters the stoichiometry of the conversion from 2:1 (conventional) to potentially 3:1 acetyl-CoA to glucose ratio. This parameter change enables carbon-efficient production without requiring the complex anaerobic conditions needed for Wood-Ljungdahl pathway.
3Productivity
If phosphoketolase pathway is engineered into recombinant cells, then carbon flux to acetyl-CoA is maximized, but the device complexity and pathway engineering requirements increase
Solution Approach 1:
The patent merges the phosphoketolase pathway with existing central metabolism by utilizing native enzymes for downstream processing. Acetyl phosphate produced by phosphoketolase is converted to acetyl-CoA by existing acetyl-CoA synthetase, and glyceraldehyde-3-phosphate feeds into glycolysis. This merging reduces the number of heterologous components needed compared to implementing entirely separate pathways.
Solution Approach 2:
The engineered pathway utilizes the host organism's own metabolic infrastructure to complete the conversion process. Native enzymes handle substrate phosphorylation, acetyl phosphate activation, and product utilization, allowing the system to be self-sufficient and reducing the burden of introducing multiple external components.
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 enhances the yield of isoprene, isoprenoid precursors, and acetyl-CoA-derived metabolites by maximizing carbon flux through the phosphoketolase pathway, increasing productivity and reducing carbon loss, thus improving industrial applicability.
Implementation Method 1
Engineering recombinant cells with a heterologous phosphoketolase pathway that converts xylulose 5-phosphate or fructose 6-phosphate to acetyl phosphate
Data Source
AI summary
This present invention relates to cultured recombinant cells comprising heterologous phosphoketolase (PKL) polypeptides that are capable of increased production of acetyl coenzyme A-derived metabolites, as well as methods for producing and using the same. In some embodiments, the recombinant cells further comprise one or more mevalonate (MVA) pathway polypeptides for the production of isoprenoid precursors, isoprene and isoprenoids.