Push-Pull Metabolic Engineering for Lipid Synthesis
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Solution Overview
Problem
Existing methods for engineering microbes for oil production focus on amplifying rate-controlling steps in fatty acid synthesis pathways, leading to increased saturated fatty acids and negative feedback loops, limiting carbohydrate to oil conversion yields.
Innovation Solution
A push-pull metabolic engineering strategy that combines amplification of upstream metabolite-forming pathways with downstream product-sequestering pathways to increase carbon flux into lipid synthesis without significant deviations in intermediate metabolite concentrations, using genetic modifications such as overexpression of diacylglycerol acyltransferase (DGA1) and acetyl-CoA carboxylase (ACC1) in oleaginous yeast like Yarrowia lipolytica.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If upstream metabolite-forming pathways are amplified to increase carbon flux into lipid synthesis, then lipid production increases, but intermediate metabolite concentrations deviate from homeostatic levels causing feedback inhibition
Solution Approach 1:
The patent segments the lipid synthesis pathway into distinct push (upstream metabolite-forming) and pull (downstream product-sequestering) components. By independently engineering these segments with specific enzyme overexpressions, the system achieves balanced flux amplification without disrupting metabolite homeostasis, resolving the contradiction between increased productivity and maintained reliability.
Solution Approach 2:
The patent employs parameter changes by selectively overexpressing specific enzymes (ACC1, DGA1, LRO1) at controlled levels to modulate pathway flux. This targeted parameter adjustment increases lipid production while maintaining intermediate metabolite concentrations within homeostatic ranges, avoiding feedback inhibition and resolving the technical contradiction.
2Productivity
If downstream product-sequestering pathways are amplified to remove feedback inhibition, then lipid synthesis increases, but carbon flux into the pathway is insufficient without upstream amplification
Solution Approach 1:
The patent merges push and pull pathway amplifications into a unified metabolic engineering strategy. By simultaneously overexpressing upstream enzymes (ACC1 for metabolite formation) and downstream enzymes (DGA1 for product sequestration), the system achieves both sufficient carbon flux and effective feedback inhibition relief, resolving the contradiction between synthesis rate and carbon availability.
3Productivity
If single-branch modifications are made to lipid biosynthesis pathways, then some improvement in conversion efficiency is achieved, but compensatory regulation limits further gains
Solution Approach 1:
The patent creates a composite metabolic engineering strategy by combining multiple genetic modifications (ACC1 overexpression, DGA1 overexpression, LRO1 overexpression) into an integrated system. This composite approach overrides compensatory regulatory mechanisms through coordinated pathway amplification, achieving synergistic effects that surpass single-branch modifications and resolve the contradiction between productivity improvement and regulatory adaptability.
Data Source
AI summary
Some aspects of this invention provide engineered microbes for oil production. Methods for microbe engineering and for use of engineered microbes are also provided herein. In some embodiments, microbes are provided that are engineered to modulate a combination of rate-controlling steps of lipid synthesis, for example, a combination of a step generating metabolites, acetyl-CoA, ATP or NADPH for lipid synthesis (a push step), and a step sequestering a product or an intermediate of a lipid synthesis pathway that mediates feedback inhibition of lipid synthesis (a pull step). Such push-and-pull engineered microbes exhibit greatly enhanced conversion yields and TAG synthesis and storage properties.