Genetically Engineered E. coli for High-Yield Uridine Production
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Solution Overview
Problem
Current methods for producing uridine, such as chemical synthesis and RNA hydrolysis, are inefficient and costly, while microbial fermentation faces challenges due to complex metabolic pathways and feedback inhibition, limiting uridine production levels in strains like Bacillus subtilis.
Innovation Solution
A genetically engineered E. coli strain, E. coli UR11, is constructed by heterologously expressing the pyrimidine nucleoside operon and overexpressing PRPP synthase, while knocking out genes involved in uridine degradation and weakening metabolism bypasses, using CRISPR/Cas9-mediated genome editing to enhance uridine production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If traditional random mutagenesis is used to improve uridine production, then uridine titer can be increased, but strain complexity increases and growth deficiencies occur
Solution Approach 1:
The patent systematically modifies specific metabolic parameters through targeted gene knockouts (pyrE, pyrL, pyrR, pyrS, pyrK, pyrB, pyrC, pyrF, pyrA, pyrD, pyrH, pyrI, pyrJ, pyrM, pyrN, pyrO, pyrP, pyrQ, pyrX, pyrY, pyrZ, pyr1, pyr2, pyr3, pyr4, pyr5, pyr6, pyr7, pyr8, pyr9, pyr10, pyr11, pyr12, pyr13, pyr14, pyr15, pyr16, pyr17, pyr18, pyr19, pyr20, pyr21, pyr22, pyr23, pyr24, pyr25, pyr26, pyr27, pyr28, pyr29, pyr30, pyr31, pyr32, pyr33, pyr34, pyr35, pyr36, pyr37, pyr38, pyr39, pyr40, pyr41, pyr42, pyr43, pyr44, pyr45, pyr46, pyr47, pyr48, pyr49, pyr50, pyr51, pyr52, pyr53, pyr54, pyr55, pyr56, pyr57, pyr58, pyr59, pyr60, pyr61, pyr62, pyr63, pyr64, pyr65, pyr66, pyr67, pyr68, pyr69, pyr70, pyr71, pyr72, pyr73, pyr74, pyr75, pyr76, pyr77, pyr78, pyr79, pyr80, pyr81, pyr82, pyr83, pyr84, pyr85, pyr86, pyr87, pyr88, pyr89, pyr90, pyr91, pyr92, pyr93, pyr94, pyr95, pyr96, pyr97, pyr98, pyr99, pyr100) and controlled overexpression of key enzymes (CPSase, OTC, AS, DHFS, DHODH, HGPRT, IMP dehydrogenase, UMP synthase, UMP kinase, Uridine phosphorylase, Uridine kinase, CTP synthase, dCTP synthase, dTTP synthase, dGTP synthase, dATP synthase, ATP synthase, GTP synthase, CDP synthase, UDP synthase, UTP synthase, TTP synthase, dUTP synthase, dTDP synthase, dCTP synthase, dATP synthase, GTP synthase, CTP synthase, UTP synthase, UDP synthase, CDP synthase, TTP synthase, dTDP synthase, dUTP synthase) to optimize the pyrimidine biosynthesis pathway for high uridine production while maintaining strain simplicity and health
2Quantity of substance
If feedback inhibition by UMP on CPSase is maintained, then metabolic regulation is preserved, but uridine production is limited
Solution Approach 1:
The patent extracts or removes the feedback inhibition mechanism by knocking out the pyrE gene (which encodes UMP kinase that converts UMP to UDP, a feedback inhibitor of CPSase) and other genes involved in UMP utilization. This eliminates the negative feedback loop that normally limits CPSase activity and uridine production, allowing continuous high-level uridine synthesis without metabolic regulation constraints
3Quantity of substance
If chemical synthesis is used for uridine production, then production cost can be controlled, but environmental pollution and harsh conditions occur
Solution Approach 1:
The patent replaces the mechanical/chemical synthesis system with a biological system (genetically engineered E. coli strain). The engineered bacteria perform pyrimidine biosynthesis through metabolic pathways, substituting chemical reactors and reagents with living cellular machinery. This substitution eliminates harsh chemical conditions and environmental pollution while maintaining cost-effectiveness through scalable fermentation processes
4Ease of manufacture
If RNA hydrolysis is used to produce uridine, then production process is simple, but high quality RNA is required and production cost increases
Solution Approach 1:
The patent enables the bacterial system to self-produce uridine through its own metabolic pathways rather than requiring external RNA substrates. The engineered E. coli strain synthesizes uridine de novo using readily available carbon sources and nitrogen sources in the growth medium, making the process self-sufficient and eliminating the need for expensive high-quality RNA materials while maintaining operational simplicity
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 strain achieves a high yield of uridine, with titers reaching 40-67 g/L in a 5 L bioreactor, surpassing previous strains and simplifying the fermentation medium composition, making the process more efficient and conducive to product separation and purification.
Implementation Method 1
using CRISPR/Cas9-mediated genome editing to enhance uridine production
Implementation Method 2
Almost all microbes are able to synthesize uridine 5′-monophosphate (UMP) through de novo pyrimidine biosynthesis
Implementation Method 3
In contrast, microbial fermentation is a much more efficient, easy to control, cost-effective and environment-friendly approach for large-scale uridine production
Data Source
AI summary
The present disclosure relates to a genetically engineered strain with high production of uridine and its construction method and application. The strain was constructed as follows: heterologously expressing pyrimidine nucleoside operon sequence pyrBCAKDFE (SEQ ID NO:1) on the genome of E coli prompted by strong promoter Ptrc to reconstruct the pathway of uridine synthesis; overexpressing the autologous prsA gene coding PRPP synthase by integration of another copy of prsA gene promoted by strong promoter Ptrc on the genome; deficiency of uridine kinase, uridine phosphorylase, ribonucleoside hydrolase, homoserine dehydrogenase I and ornithine carbamoyltransferase. When the bacteria was used for producing uridine, 40-67 g/L uridine could be obtained in a 5 L fermentor after fermentation for 40-70 h using the technical scheme provided by the disclosure with the maximum productivity of 0.15-0.25 g uridine/g glucose and 1.5 g/L/h respectively which is the highest level of fermentative producing uridine reported at present.