Cupriavidus Metabolic Redirection for Extracellular Product Yield
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Solution Overview
Problem
Current bioprocesses for producing non-biomass and non-polyhydroxyalkanoate products in Cupriavidus and Ralstonia organisms face limitations due to excessive polyhydroxybutyrate synthesis, which hampers the productivity and yield of desired extracellular products, and traditional fermentation methods have inefficiencies such as altered cell membrane composition and productivity limitations.
Innovation Solution
Modifying organisms like Cupriavidus and Ralstonia to decrease polyhydroxyalkanoate synthesis and increase extracellular product synthesis by using specific nutrient limitation conditions and inducible or constitutive promoters, and culturing them in continuous fermentation systems with controlled nutrient concentrations to optimize product yield.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If nutrient limitation is applied to induce overflow metabolism, then extracellular product formation is improved, but polyhydroxyalkanoate synthesis increases which reduces product yield
Solution Approach 1:
The patent removes the harmful metabolic pathway (polyhydroxyalkanoate synthesis) by deleting or inactivating the phaC gene, which encodes the key enzyme polyhydroxyalkanoate synthase. This extraction of the problematic pathway allows carbon to be redirected exclusively toward extracellular product formation, resolving the contradiction between overflow metabolism induction and polyhydroxyalkanoate accumulation.
Solution Approach 2:
The patent modifies the organism's metabolic parameters by introducing genetic modifications (phaC gene deletion or promoter mutations) that fundamentally change the carbon flux distribution. This parameter change transforms the organism from one that stores excess carbon as polyhydroxyalkanoate to one that excretes excess carbon as desired extracellular products under nutrient limitation conditions.
2Productivity
If traditional batch or fed-batch fermentation is used, then process simplicity is maintained, but productivity and efficiency are limited
Solution Approach 1:
The patent employs continuous fermentation instead of batch or fed-batch processes, maintaining a steady state where modified organisms continuously produce extracellular products. This continuous operation eliminates downtime between batches and maintains optimal metabolic conditions throughout, significantly improving productivity while the genetic modification ensures consistent product formation without requiring complex process control.
3Productivity
If phosphate limitation is applied to control cell membrane composition, then product yield may improve, but cell membrane integrity and function are altered
Solution Approach 1:
The patent removes the need for phosphate limitation by eliminating the polyhydroxyalkanoate synthesis pathway. Since polyhydroxyalkanoate accumulation is the primary mechanism by which phosphate limitation improves product yield, removing this pathway allows the system to achieve high productivity without altering cell membrane composition through phosphate restriction.
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 and productivity of extracellular products by redirecting carbon flux and reducing polyhydroxyalkanoate synthesis, thereby enhancing the efficiency of product formation and overcoming limitations of traditional batch or fed-batch processes.
Implementation Method 1
under conditions of nutrient limitation a phenomenon known as overflow metabolism (also known as energy spilling uncoupling or spillage) occurs in many bacteria
Implementation Method 2
aerobic continuous culturing of the organism under one or more specific limitation conditions
Data Source
AI summary
Provided herein are methods for increasing the yield of an extracellular product synthesized by an organism cultured in a continuous aerobic fermentation system. The extracellular product yield is increased through the use of an organism modified to decreased production of polyhydroxyalkanoate, to increase production of the extracellular product, and to include promoters that can be inducible in response to nutrient limitation conditions. The extracellular product yield is also increased by operating the continuous fermentation system under particular nutrient limitation conditions. Also provided are non-naturally occurring organisms that have been modified for use with the provided methods, and extracellular products made using the provided methods.