E. coli Gene Combination for Terrequinone A Biosynthesis
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Solution Overview
Problem
The high production cost and low synthesis efficiency of terrequinone A in vitro due to complex chemical synthesis and spatial constraints in enzyme-catalyzed systems limit the effective production of this bis-indolequinone compound, which has potential anticancer properties.
Innovation Solution
A gene combination for expressing and producing terrequinone A in E. coli, involving specific genes (tdiAS, tdiBS, tdiCS, tdiDS, sfpS, ScCKS, and AtIPKS) optimized for E. coli expression, integrated into recombinant plasmids pC02 and pU03, which are then transformed into E. coli to facilitate efficient biosynthesis using L-tryptophan and prenol substrates.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If chemical synthesis method is used to produce terrequinone A, then the compound can be obtained, but the production cost is high and synthesis efficiency is low due to complex multi-step synthesis
Solution Approach 1:
The patent replaces the traditional chemical synthesis system with a biological system (E. coli expression system). By introducing the tdiABCDE gene cluster from Aspergillus nidulans into E. coli, the mechanical/chemical synthesis process is substituted with a biological biosynthesis process, achieving simpler operation and higher efficiency
Solution Approach 2:
The engineered E. coli system achieves self-service by autonomously producing terrequinone A through the introduced biosynthetic pathway. The bacteria utilize available substrates (tryptophan, dimethylallyl diphosphate) and endogenous enzymes to automatically complete the multi-step synthesis without requiring external chemical reagents or complex synthetic procedures
2Productivity
If enzyme-catalyzed in vitro synthesis is used to produce terrequinone A, then the synthesis can be performed, but the production cost increases due to expensive cofactors and cofactor regeneration enzyme systems
Solution Approach 1:
The E. coli expression system provides self-service by utilizing the bacterium's endogenous cofactor pools and metabolic pathways. The introduced tdiABCDE genes work within the native E. coli cellular environment, automatically accessing available NADPH, ATP, and other cofactors without requiring external addition of expensive cofactors or separate regeneration systems
Solution Approach 2:
The E. coli host cell provides universal support for the biosynthetic pathway by contributing its entire metabolic machinery. The bacterial cell simultaneously provides cofactors, energy, protein synthesis capabilities, and structural components, making the system universally applicable without needing specialized in vitro components for each reaction step
3Productivity
If enzyme-catalyzed in vitro synthesis is used to produce terrequinone A, then the reaction can proceed, but the synthesis efficiency is reduced due to spatial constraints limiting substrate and product delivery between enzymes
Solution Approach 1:
The patent replaces the in vitro enzyme system with spatial constraints with an in vivo cellular system. Within the E. coli cell, the introduced enzymes are distributed throughout the cytoplasm where substrates and products can freely diffuse, eliminating the spatial constraints and delivery limitations that plague in vitro multi-enzyme systems
Solution Approach 2:
The patent merges the five separate enzymatic reactions of the terrequinone A biosynthetic pathway into a single cellular environment. By co-expressing all five Tdi proteins (TdiA, TdiB, TdiC, TdiD, TdiE) in E. coli, the system combines multiple enzymatic functions into one integrated biological system, allowing seamless substrate channeling and product formation without external intervention
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 improves the yield and efficiency of terrequinone A production, achieving a concentration of 106.3 mg/L in the fermentation broth, surpassing natural strain A. terreus production levels, with potential applications in the biopharmaceutical field.
Implementation Method 1
The tdiAS gene encoding nonribosomal peptide synthetase (NRPS), the tdiBS gene encoding methylpropanoyl-L-tryptophan synthetase
Implementation Method 2
the ScCKS gene encoding choline kinase, and the AtIPKS gene encoding isopentenyl phosphate kinase
Implementation Method 3
the ScCKS gene encoding choline kinase, and the AtIPKS gene encoding isopentenyl phosphate kinase
Implementation Method 4
inoculating the recombinant E. coli strain in M9 liquid medium supplemented with 100 μg/mL ampicillin and 50 μg/mL kanamycin to culture at 37° C. until a bacterial suspension reaches an OD600 of 0.6, adding 0.2% arabinose, continuing culturing at 25° C. for 14-18 h to obtain a fermentation broth
Data Source
AI summary
The present disclosure provides a gene combination for expressing and producing terrequinone A in Escherichia coli and use thereof. The gene combination includes a tdiAS gene, a tdiBS gene, a tdiCS gene, a tdiDS gene, a tdiES gene, an sfpS gene, an ScCKS gene, and an AtIPKS gene with nucleotide sequences set forth in SEQ ID NOS:1 to 8. In the present disclosure, a recombinant engineered strain capable of producing terrequinone A having anti-cancer activity is obtained by separately constructing recombinant plasmids pC02 and pU03 through the eight genes and transforming the two recombinant plasmids into E. coli. The content of terrequinone A in a fermentation broth thereof is 106.3 mg/L, which has potential application value in the biopharmaceutical field.