ACC BCCP Mutations for Malonyl-CoA Titer
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Solution Overview
Problem
There is a need for alternative routes to produce fuels and chemicals currently derived from petroleum, with a requirement for improved genetically modified enzymes, recombinant host cells, and methods to achieve robust and cost-effective production of malonyl-derived compounds through fermentation.
Innovation Solution
Development of ACC variants with specific mutations in their amino acid sequences, particularly in the biotin carboxyl carrier protein (BCCP), which are expressed in recombinant host cells to increase the production of malonyl-CoA-derived compounds such as fatty acid derivatives and non-fatty acid compounds.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional ACC enzymes are used in recombinant host cells, then the production process is simpler, but the yield and titer of malonyl-CoA-derived compounds are insufficient
Solution Approach 1:
The patent applies parameter changes by introducing specific amino acid mutations in the ACC enzyme structure, particularly in the BCCP subunit. These mutations (such as D2N, D2H, D2I substitutions at position 2) modify the enzymatic parameters to enhance activity and stability, thereby increasing the yield and titer of malonyl-CoA-derived compounds without requiring complex production systems
Solution Approach 2:
The patent creates improved copies of the natural ACC enzyme by generating variant versions with specific mutations. These copied and modified enzyme versions retain the core function of catalyzing acetyl-CoA carboxylation while exhibiting enhanced performance characteristics, allowing simple recombinant expression systems to achieve high productivity
2Productivity
If ACC variants with specific mutations are developed, then the production of malonyl-CoA-derived compounds is enhanced, but the enzyme structure becomes more complex
Solution Approach 1:
The patent applies local quality by introducing mutations at specific local positions within the ACC enzyme structure, particularly focusing on the BCCP subunit. Rather than redesigning the entire enzyme, targeted mutations at specific amino acid positions (such as position 2) locally modify the enzyme's properties to enhance overall productivity while maintaining the rest of the structure
Solution Approach 2:
The patent segments the ACC enzyme into its four subunits (BC, BCCP, CTα, CTβ) and focuses mutations specifically on the BCCP component. This segmentation allows independent optimization of the carrier protein portion without affecting the catalytic domains, enabling enhanced production capability with minimal structural complexity increase
3Ease of manufacture
If fermentation methods are used for production, then the process is more cost-effective, but achieving robust production requires improved enzymes and cell lines
Solution Approach 1:
The patent applies preliminary action by pre-engineering the ACC enzyme variants with specific mutations before introducing them into recombinant host cells. This preliminary optimization of the enzyme structure ensures that when the fermentation process is executed, the cells already possess the enhanced enzymatic capability needed for robust and reliable production, eliminating the need for complex process adjustments during fermentation
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
The ACC variants significantly enhance the yield and titer of malonyl-CoA-derived compounds, improving acetyl-CoA carboxylase activity and facilitating the production of fatty acid derivatives and other compounds in host cells, such as fatty esters, fatty alcohols, and polyketides.
Implementation Method 1
Acetyl CoA carboxylase (ACC) plays an important role in regulating fatty acid synthesis and degradation. It is a biotin-dependent enzyme complex that catalyzes the first committed step of fatty acid biosynthesis, i.e., the irreversible carboxylation of acetyl-CoA to malonyl-CoA.
Data Source
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AI summary
The disclosure relates to acetyl-CoA carboxylase (ACC) variants and host cells expressing them for the production of malonyl-CoA derived compounds including fatty acid derivatives. Further contemplated are methods of producing increased amounts of malonyl-CoA derived compounds and related cell cultures.