Engineered Bacillus Strain for De Novo CDP-Choline Synthesis
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Solution Overview
Problem
Current methods for synthesizing CDP-choline, such as chemical synthesis and microbial fermentation, face challenges like low yield, high cost, and the use of toxic reagents, while microbial fermentation requires complex two-stage enzyme production and is not suitable for large-scale production, and the addition of precursors like choline chloride can inhibit cellular processes.
Innovation Solution
A recombinant engineered strain of Bacillus subtilis 168N is developed by integrating phosphatidylethanolamine N-methyltransferase genes PEM1 and PEM2 from Saccharomyces cerevisiae, along with CKI and CCT genes, to enable de novo synthesis of CDP-choline directly from glucose as a substrate, bypassing the need for expensive precursors and reducing production costs.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If chemical synthesis is used to produce CDP-choline, then the synthesis can be achieved, but the yield is low, purity is low, production cost is high, and toxic reagents are used
Solution Approach 1:
The patent replaces chemical synthesis methods with biological synthesis methods using engineered E. coli strains. The mechanical/chemical system of chemical synthesis with toxic reagents is substituted by a biological system that uses enzymatic pathways to convert glucose and choline chloride into CDP-choline, eliminating the need for toxic chemical reagents while improving yield and purity
Solution Approach 2:
The patent optimizes multiple parameters including genetic modifications (introducing heterologous genes ck, cmk, ndk), culture conditions (glucose concentration, choline chloride concentration, pH, temperature), and fermentation parameters to maximize CDP-choline production. These parameter changes enable the biological system to outperform chemical synthesis in terms of yield, purity, and environmental safety
2Productivity
If microbial fermentation is used to produce CDP-choline, then production can be scaled up, but complex two-stage enzyme production is required which is time-consuming
Solution Approach 1:
The patent merges multiple enzymatic functions into a single engineered E. coli strain that possesses all necessary enzymes (choline kinase from Streptococcus pneumoniae, cytidine kinase, cytidylate kinase, and nucleoside diphosphate kinase) to convert glucose and choline chloride directly into CDP-choline in one fermentation step, eliminating the need for separate two-stage enzyme production processes
Solution Approach 2:
The engineered E. coli strain is designed with multi-functionality, serving as both the production host and containing all necessary enzymatic pathways in a single organism. This universal strain can simultaneously perform glucose metabolism, choline phosphorylation, and CDP-choline synthesis, greatly simplifying the production process and reducing time
3Productivity
If precursors like choline chloride are added to fermentation medium, then CDP-choline synthesis can proceed, but high chloride ion concentration interferes with cell membrane formation and inhibits cellular processes
Solution Approach 1:
The patent optimizes the concentration of choline chloride and chloride ions in the fermentation medium to maintain levels that support CDP-choline synthesis while avoiding inhibitory concentrations. By carefully controlling this parameter and monitoring cellular response, the system achieves productive synthesis without triggering harmful effects on cell membrane formation or metabolic processes
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 engineered strain allows for direct synthesis of CDP-choline from glucose, avoiding the use of costly precursors and inhibitory substances, thereby reducing production costs and enabling efficient large-scale synthesis.
Implementation Method 1
integrating phosphatidylethanolamine N-methyltransferase genes PEM1 and PEM2 from Saccharomyces cerevisiae, along with CKI and CCT genes, to enable de novo synthesis of CDP-choline directly from glucose
Data Source
AI summary
A recombinant engineered strain for de novo synthesis of CDP-choline using glucose as a substrate and its preparation method and application are provided. Using BS168N as the starting strain, firstly, the phosphatidylethanolamine N-methyltransferase gene PEM1 and phosphatidylethanolamine/phosphatidyl-N-methylethanolamine N-methyltransferase gene PEM2 from S. cerevisiae are integrated into the genome of the BS168N for induced expression, thereby opening up the synthesis pathway from phosphatidylethanolamine to phosphatidylcholine; subsequently, the CKI and CCT genes of S. cerevisiae are further integrated into the BS168N genome expressing PEM1-PEM2, opening up the synthesis pathway of choline to CDPC, thereby obtaining the recombinant engineered strain. Further, the recombinant engineered strain is subjected to shake flask fermentation to achieve de novo synthesis of CDP-choline using glucose as a substrate. The method of the present disclosure provides a fundamental research and theoretical basis for the construction of efficient cell factories for de novo synthesis of CDP-choline through synthetic biology.