Modified Isoprene Synthase Enzyme Productivity
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for producing isoprene monomer face challenges in achieving high productivity due to limitations in enzymatic properties, particularly with the decreasing supply of isoprene derived from oil resources and the need for a cost-effective, stable production system.
Innovation Solution
A modified isoprene synthase enzyme is developed by mutating specific amino acid residues in the isoprene synthase derived from Mucuna, enhancing its productivity and stability, which is then used in a host cell to produce isoprene through fermentation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If isoprene synthase from natural sources (kudzu, poplar, Mucuna) is used for fermentation production, then isoprene monomer can be produced, but productivity is insufficient due to limitations in enzymatic properties
Solution Approach 1:
The patent applies parameter changes by mutating specific amino acid residues in the isoprene synthase enzyme sequence. Multiple mutant variants were created with substitutions at different positions (e.g., F31E, L35E, L67E, I90E, V97E, L114E, Q123C, D124E, V125E, K127D, K127E, K127N, K127R, R128K, K130G, D131C, G134P, C137E, C137I, C137L, C137M, C137S, V144E, V187E, R202D, R202N, L224E, L247D, L247E, L247Q, S248E, S248K, S248R, L249E, L249K, L249R, L249T, T257L, K259D, K259E, K259N, K259Q, K259R, R264F, R264M, R264T, D265E, D265N, D265Q, D265R, R266N, R266Q, E269D, E269I, E269Y, C286V, C286N, K292L, T298I, I300N, D301R, Y304F, D305R, E312I, E312R, F316E, E321D, V325K, I328E, C338V, C338L, L340K, C370E, C370N, C370K, C370V, E371D, C373V, C373Y, E379T, S383I, K386R, I388C, A390C, Y394F, S401T, S402L, G404A, G404M, V405C, L414C, C416E, C440S, R444T, N447L, N447R, R458Q, T461R, T462M, T462S, T466F, T466C, T466W, T466M, T466Y, T466H, T466P, T466Q, T466N, T466A, E471C, C480N, C480Y, C480V, K481E, R484K, K492E, V499I, P505H, F508C, F508Q, F508R, I518C, I518S, S519N, H520N, C521N, C521S, C521V, C521E, C521I, Y523I, Y523L, and G530K). These parameter changes in the enzyme structure resulted in improved productivity while maintaining enzymatic stability.
2Quantity of substance
If isoprene is extracted from C5 fraction obtained by cracking of naphtha, then isoprene monomer is available, but production amount is decreasing due to lightening of cracker field
Solution Approach 1:
The patent replaces the mechanical/industrial process of extracting isoprene from naphtha cracking with a biological fermentation system. Instead of relying on oil refining processes, the invention uses genetically modified host cells expressing mutated isoprene synthase to produce isoprene monomer through fermentation. This substitution of production methodology ensures stable supply independent of oil price fluctuations and cracker production changes.
3Quantity of substance
If natural rubber plantation is expanded to meet increasing demand, then raw material supply increases, but farm plantation increase is difficult due to deforestation regulation and competition with palms
Solution Approach 1:
The patent replaces agricultural production of natural rubber with industrial fermentation using engineered microorganisms. Instead of expanding physical plantations which face regulatory and land competition constraints, the invention uses host cells (bacteria, yeast, or other microorganisms) that can be cultivated in fermentation reactors. This substitution transforms a land-intensive agricultural process into a space-efficient biotechnological process, enabling increased production without deforestation or land competition issues.
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 modified isoprene synthase significantly improves isoprene monomer production, establishing an efficient and cost-effective production system that overcomes the limitations of traditional methods by increasing productivity and stability.
Implementation Method 1
a modified isoprene synthase, wherein a host cell that expresses the modified isoprene synthase produces a greater amount of isoprene compared to a host cell that expresses an isoprene synthase according to SEQ ID NO: 4
Implementation Method 2
a method of producing the isoprene monomer using a transformant obtained by integrating an isolated isoprene synthase gene derived from kudzu or poplar and its mutant into a bacterium for fermentation production
Data Source
Figure 1~2
Figure 3~4
AI summary
The present invention provides an enzyme and the like useful for establishing an excellent isoprene monomer production system. Specifically, the present invention provides a modified isoprene synthase that has a mutation(s) of a given amino acid residue(s) in any amino acid sequence of (a) the amino acid sequence of SEQ ID NO:4, (b) an amino acid sequence having one or several amino acid substitutions, deletions, insertions or additions in the amino acid sequence of SEQ ID NO:4, or (c) an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO:4, and has an isoprene synthetic activity; a polynucleotide encoding the modified isoprene synthase; a vector comprising the polynucleotide; a host cell comprising an expression unit comprising the polynucleotide; and a method for producing an isoprene monomer comprising producing the isoprene monomer from dimethylallyl diphosphate in the presence of the modified isoprene synthase.