Fusion Protein Linker Stabilizes Unstable Intermediate for Quinolinic Acid Yield
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Solution Overview
Problem
Current biological methods for producing quinolinic acid face challenges such as low yield due to transcriptional suppression, feedback inhibition, and the instability of intermediates like α-iminosuccinate, leading to high by-product formation and inefficient conversion to quinolinic acid.
Innovation Solution
Expressing a fusion protein of L-aspartate oxidase and quinolinate synthase linked via an amino acid linker in a recombinant microorganism to enhance the conversion rate of quinolinic acid from α-iminosuccinate, thereby increasing productivity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If individual enzymes NadB and NadA are expressed separately, then the biosynthetic pathway can proceed, but the conversion efficiency is low due to instability of intermediate α-iminosuccinate and feedback inhibition
Solution Approach 1:
The patent combines NadB (L-aspartate oxidase) and NadA (quinolinate synthase) into a single fusion protein by linking their coding sequences with a linker peptide. This merging ensures that the unstable intermediate α-iminosuccinate is immediately converted by the adjacent NadA domain, preventing its decomposition and improving conversion efficiency to quinolinic acid.
2Productivity
If transcriptional suppression by NadR is not relieved, then gene expression is regulated, but quinolinic acid production remains low
Solution Approach 1:
The patent removes the transcriptional repression mechanism by deleting the nadR gene, which encodes the NAD-related transcriptional repressor. This extraction of the suppression element allows constitutive high-level expression of the fusion protein without regulatory interference, significantly improving quinolinic acid production.
3Productivity
If feedback inhibition by NAD on NadB is not eliminated, then metabolic balance is maintained, but enzyme activity is suppressed
Solution Approach 1:
The fusion protein design ensures continuous conversion of the intermediate by placing NadA immediately adjacent to NadB. This continuous action prevents intermediate accumulation and drives the reaction forward despite feedback inhibition, maintaining high flux through the pathway.
4Productivity
If the biosynthetic pathway from carbon sources to L-aspartic acid is weak, then metabolic resources are conserved, but substrate availability for quinolinic acid synthesis is limited
Solution Approach 1:
The patent introduces plasmids carrying genes for phosphoenolpyruvate carboxylase (PPC) and L-aspartate aminotransferase (AspC) to enhance the biosynthetic pathway. This parameter change in enzyme capacity increases L-aspartic acid supply to the quinolinic acid pathway without requiring complete metabolic reprogramming.
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 fusion protein approach significantly improves the yield and productivity of quinolinic acid production by minimizing by-product formation and stabilizing the conversion process, achieving higher enzymatic conversion rates compared to individual enzyme expressions.
Implementation Method 1
L-aspartate oxidase (NadB) L-aspartic acid + oxygen <=> hydrogen peroxide + α-iminosuccinate + H + quinolinic acid + phosphate + 2H2O
Implementation Method 2
quinolinate synthase (NadA) converts α-iminosuccinate to quinolinic acid
Implementation Method 3
α-iminosuccinate as an intermediate of the quinolinic acid biosynthetic pathway is an unstable substance, and is converted to oxaloacetate by natural deamination reaction in cells
Data Source
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AI summary
The present invention relates to a quinolinic acid-producing recombinant microorganism expressing a fusion protein of L-aspartate oxidase and quinolinate synthase linked via a linker, and a method for producing quinolinic acid using the same.