Recombinant Polypeptide for Adipyl-CoA Reduction
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Solution Overview
Problem
Current fermentation processes for producing aliphatic compounds, such as adipic acid, face challenges due to low enzyme activity for acyl-CoA compounds, particularly adipyl-CoA, which limits the production of target compounds like 5-formylpentanoic acid and 1,6-diaminohexane.
Innovation Solution
A recombinant polypeptide with enhanced acyl-CoA compound reducing activity is developed by introducing specific mutations into succinic semialdehyde dehydrogenase, improving the enzyme's activity for converting adipyl-CoA into 5-formylpentanoic acid while reducing activity for succinyl-CoA, thereby increasing the production of target aliphatic compounds.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If succinic semialdehyde dehydrogenase is used for converting adipyl-CoA into 5-formylpentanoic acid, then the enzyme can catalyze the reaction, but the enzyme activity for adipyl-CoA is low and insufficient product amount is obtained
Solution Approach 1:
The patent applies parameter changes by modifying the amino acid sequence of succinic semialdehyde dehydrogenase through site-directed mutagenesis. Specifically, mutations are introduced at positions corresponding to the substrate-binding site (e.g., F287L, F287W, F386L, F386W, F484L, F484W) to alter the enzyme's catalytic properties. This changes the enzyme's parameters to improve its activity toward adipyl-CoA while reducing activity toward succinyl-CoA, thereby resolving the contradiction between low productivity and insufficient enzyme activity.
2Productivity
If succinic semialdehyde dehydrogenase is used for synthesizing adipic semialdehyde from adipyl-CoA, then the reaction can proceed, but the enzyme has high activity for succinyl-CoA which is an intermediate of the adipic acid pathway, diverting substrate away from the desired product
Solution Approach 1:
The patent applies local quality by making specific local modifications to the enzyme's substrate-binding site through targeted amino acid substitutions. The mutations (e.g., F287L, F386L, F484L) are localized to the substrate-binding region, creating a binding pocket that selectively accommodates adipyl-CoA over succinyl-CoA. This local modification changes the enzyme's specificity, reducing harmful activity toward succinyl-CoA while maintaining or enhancing activity toward the desired substrate adipyl-CoA.
3Ease of manufacture
If existing enzymes are used for the adipic acid fermentation pathway, then the pathway can be constructed, but the availability and actual catalytic activity of the enzymes are not sufficient for industrial production
Solution Approach 1:
The patent applies parameter changes by optimizing the key enzymatic step in the adipic acid fermentation pathway. Instead of using wild-type succinic semialdehyde dehydrogenase with insufficient activity, the patent introduces specific amino acid mutations to change the enzyme's catalytic parameters. This creates a hyper-active variant that can efficiently convert adipyl-CoA to 5-formylpentanoic acid, thereby enabling industrial-scale production while maintaining pathway constructibility.
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 polypeptide efficiently converts adipyl-CoA into 5-formylpentanoic acid, enhancing the production of aliphatic compounds like 1,6-diaminohexane and 6-aminocaproic acid, addressing the limitations of existing enzyme activities and improving fermentation yields.
Implementation Method 1
a recombinant polypeptide having (a) an amino acid sequence A having a sequence identity of 60% or higher with an amino acid sequence set forth in SEQ ID NO: 1; (b) a substitution of at least one amino acid at a position corresponding to a substrate-binding site of a polypeptide having an amino acid sequence set forth in SEQ ID NO: 1 in the amino acid sequence A; and (c) a reducing activity R of converting CoA thioester of an acyl-CoA compound into an aldehyde group
Data Source
AI summary
Provided are a recombinant polypeptide having an excellent acyl-CoA compound reducing activity, and a production method of an aliphatic compound using the recombinant polypeptide.A recombinant polypeptide has (a) an amino acid sequence A having a sequence identity of 60% or higher, 65% or higher, 70% or higher, 75% or higher, 80% or higher, 85% or higher, 88% or higher, 90% or higher, 93% or higher, 95% or higher, 97% or higher, 98% or higher, or 99% or higher with an amino acid sequence set forth in SEQ ID NO: 1; (b) a substitution of at least one amino acid at a position corresponding to a substrate-binding site of a polypeptide having an amino acid sequence set forth in SEQ ID NO: 1 in the amino acid sequence A; and (c) a reducing activity R of converting CoA thioester of an acyl-CoA compound into an aldehyde group, in which the reducing activity R includes (c-1) a reducing activity R1 of converting adipyl-CoA into 5-formylpentanoic acid in one step; and (c-2) a reducing activity R2 of converting succinyl-CoA into succinic semialdehyde.