Microbial Isobutene Pathway With CoA Pool Engineering
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing methods for producing isobutene from renewable resources are inefficient and not commercially viable due to limitations in enzyme availability and yield, particularly in the conversion of 3-methylcrotonic acid to isobutene using FMN-dependent decarboxylases and FMN prenyl transferases.
Innovation Solution
A recombinant organism or microorganism is engineered to enhance the production of isobutene by increasing the pool of coenzyme A (CoA) through enhanced pantothenate uptake and conversion, enabling efficient enzymatic conversion of acetyl-CoA to isobutene via multiple pathways, including direct conversion of 3-methylcrotonyl-CoA to isobutene or through intermediates like 3-hydroxy-3-methylbutyric acid.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If existing methods using FMN-dependent decarboxylases and FMN prenyml transferases are used to convert 3-methylcrotonic acid to isobutene, then the conversion can be achieved, but the yield is low and the process is not commercially viable
Solution Approach 1:
The patent changes the biochemical parameters of the pathway by replacing FMN-dependent enzymes with thiamine pyrophosphate (TPP)-dependent enzymes. This parameter change in cofactor dependency fundamentally alters the reaction efficiency and substrate availability, leading to significantly improved isobutene production yields that reach commercial viability levels.
Solution Approach 2:
The patent introduces 3-methylcrotonyl-CoA as a key intermediary compound that accumulates in the pathway. By engineering enzymes that efficiently produce and process this intermediate, the patent creates a bottleneck-free pathway where the intermediary serves as a stable platform for high-yield conversion to isobutene, resolving the previous yield limitations.
2Productivity
If the pool of coenzyme A (CoA) is increased through enhanced pantothenate uptake and conversion, then the enzymatic conversion of acetyl-CoA to isobutene is optimized, but the metabolic burden on the organism increases
Solution Approach 1:
The patent implements preliminary action by pre-engineering the organism with enhanced pantothenate uptake systems and CoA synthesis pathways before the actual isobutene production process. This preparatory modification ensures that sufficient CoA pools are available from the outset, eliminating the need for continuous high-energy metabolic adjustments during production and thereby optimizing conversion efficiency without excessive ongoing energy burden.
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 enhances the yield and commercial viability of isobutene production by maintaining a high acetyl-CoA pool, leveraging increased CoA availability to optimize enzymatic conversions and improve overall efficiency.
Implementation Method 1
acetyl-CoA is enzymatically converted into acetoacetyl-CoA, (ii) acetoacetyl-CoA is enzymatically converted into 3-hydroxy-3-methylglutaryl-CoA, (iii) 3-hydroxy-3-methylglutaryl-CoA is enzymatically converted into 3-methylglutaconyl-CoA, (iv) 3-methylglutaconyl-CoA is enzymatically converted into 3-methylcrotonyl-CoA
Implementation Method 2
an increased conversion of pantothenate into CoA
Data Source
AI summary
Described is a recombinant organism or microorganism which is capable of enzymatically converting acetyl-CoA into isobutene, (A) wherein in said organism or microorganism: (i) acetyl-CoA is enzymatically converted into acetoacetyl-CoA, (ii) acetoacetyl-CoA is enzymatically converted into 3-hydroxy-3-methylglutaryl-CoA, (iii) 3-hydroxy-3-methylglutaryl-CoA is enzymatically converted into 3-methylglutaconyl-CoA, (iv) 3-methylglutaconyl-CoA is enzymatically converted into 3-methylcrotonyl-CoA, and (v) wherein said 3-methylcrotonyl-CoA is converted into isobutene by: (a) enzymatically converting 3-methylcrotonyl-CoA into 3-methylcrotonic acid which is then further enzymatically converted into said isobutene; or (b) enzymatically converting 3-methylcrotonyl-CoA into 3-hydroxy-3-methylbutyryl-CoA which is then further enzymatically converted into 3-hydroxy-3-methylbutyric acid which is then further enzymatically converted into 3-phosphonoxy-3-methylbutyric acid which is then further enzymatically converted into said isobutene; (B) wherein said recombinant organism or microorganism has an increased pool of coenzyme A (CoA) over the organism or microorganism from which it is derived due to: (i) an increased uptake of pantothenate; and/or (ii) an increased conversion of pantothenate into CoA. Moreover, described is the use of such a recombinant organism or microorganism for the production of isobutene. Further, described is a method for the production of isobutene by culturing such a recombinant organism or microorganism in a suitable culture medium under suitable conditions.