Acetolactate Decarboxylase Locus Knock-In Enables Stable Gas Fermentation
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Solution Overview
Problem
Existing microorganisms face challenges in achieving stable fermentation and high production yields of nonnative products from gaseous substrates like CO, CO2, and H2, with existing genetic engineering methods being tedious and unpredictable in Clostridia.
Innovation Solution
A genetically engineered microorganism with a knock-in of DNA at the acetolactate decarboxylase gene locus, replacing or adding enzymes to redirect metabolic flux towards desired products such as acetone, isopropanol, and other chemicals, using promoters like PbudA and Pfer to control enzyme expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If traditional genetic engineering methods are used in Clostridia, then product diversity can be achieved, but the process is tedious and unpredictable with low stability
Solution Approach 1:
The patent performs preliminary actions by disrupting the acetolactate decarboxylase gene (budA) before introducing heterologous pathways, thereby pre-establishing a metabolic state that favors acetone production and prevents byproduct formation, leading to more predictable and stable fermentation outcomes
Solution Approach 2:
The genetic engineering process is segmented into distinct sequential steps: first disrupting the budA gene, then introducing the heterologous acetone pathway genes (thlA, ctfAB, adc) separately. This segmentation makes the complex engineering process more manageable and predictable
2Productivity
If heterologous pathways are introduced to produce nonnative products, then product yield increases, but fermentation stability decreases
Solution Approach 1:
The patent extracts or removes the problematic native pathway by disrupting the budA gene, which eliminates the competing reaction that produces 2,3-butanediol. This extraction of the interfering pathway allows the heterologous acetone pathway to operate more efficiently and stably
Solution Approach 2:
The patent applies preliminary anti-action by disrupting the budA gene in advance to prevent the formation of unwanted byproducts (2,3-butanediol) before the heterologous pathway is fully established, thereby preventing metabolic conflicts that would reduce fermentation stability
3Quantity of substance
If multiple genetic modifications are made to redirect metabolic flux, then product titer increases, but the engineering process becomes more complex
Solution Approach 1:
The disrupted budA locus serves multiple functions: it eliminates the competing pathway for 2,3-butanediol production, creates a genomic insertion site for heterologous genes, and provides regulatory control through the remaining promoter region. This multi-functionality reduces the need for separate engineering steps
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 stabilizes fermentation and enhances production of heterologous products, overcoming the limitations of traditional genetic engineering in Clostridia by achieving higher product titers and metabolic efficiency.
Implementation Method 1
acetolactate decarboxylase gene locus... enzymes to redirect metabolic flux towards desired products such as acetone
Data Source
AI summary
Provided herein is a genetically engineered microorganism comprising knock-in of DNA at an acetolactate decarboxylase gene locus. Replacement of the acetolactate decarboxylase gene with DNA encoding one or more native or nonnative enzymes confers certain advantages, including fermentation stability and increased production of native and nonnative products from gaseous substrates.