Recombinant Plasmid Biosensor for D-Lactic Acid Strain Mining
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Solution Overview
Problem
The challenge of achieving high-yield production of D-lactic acid in Zymomonas mobilis requires the identification and integration of suitable target genes and genetic engineering elements to enhance the biosynthesis process.
Innovation Solution
A method involving the use of a recombinant plasmid carrying an operon for catabolizing D-lactic acid and a gene for reporting, coupled with a CRISPRi library and CRISPR-Cas12a system, to screen and regulate D-lactic acid biosynthesis in Zymomonas mobilis strains, enabling high-throughput mining of strains and genes for enhanced production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional methods are used to mine D-lactic acid producing strains, then the process is simple, but the productivity and yield of D-LA are insufficient
Solution Approach 1:
The patent implements a feedback mechanism by constructing a biosensor system where the lldR-Plldp-eGFP connector responds to D-LA concentration changes. The eGFP fluorescence intensity provides real-time feedback on D-LA production levels, enabling automated screening and selection of high-yield strains through flow cytometry, thus dramatically improving productivity while managing complexity through systematic design
Solution Approach 2:
The patent introduces an intermediary biosensor system comprising the lldR-Plldp-eGFP connector as a mediator between the target strain and the screening process. This intermediary converts D-LA production into measurable fluorescence signals, enabling indirect but highly efficient strain mining without requiring complex direct measurement methods
2Measurement precision
If comprehensive gene screening is performed to identify D-LA biosynthesis genes, then the precision of strain selection improves, but the time and resource consumption increase
Solution Approach 1:
The patent replaces traditional mechanical/chemical detection methods with a biological sensing system. The lldR-Plldp-eGFP connector acts as a biological transducer that automatically converts D-LA concentration into fluorescence signals, eliminating the need for time-consuming manual assays and enabling high-throughput automated screening with high precision
Solution Approach 2:
The patent changes the measurement parameter from direct chemical analysis of D-LA to optical detection of eGFP fluorescence. This parameter transformation enables rapid, non-destructive, high-throughput screening while maintaining high measurement precision, significantly reducing screening time and resource consumption
3Productivity
If multiple genes are introduced to enhance D-LA production, then the yield increases, but the stability of the strain may decrease
Solution Approach 1:
The patent employs a multi-functional biosensor system that simultaneously serves as a detection tool, a selection marker, and a regulatory element. The lldR-Plldp-eGFP connector integrates promoter regulation, reporter expression, and flow cytometry compatibility into a single universal system, enabling precise strain evaluation and selection to maintain stability while enhancing productivity
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 allows for the effective mining of strains and genes that increase D-lactic acid production, demonstrated by a 24.5-fold increase in fluorescence intensity and linear correlation with D-lactic acid concentration, leading to improved yield and efficiency in biosynthesis.
Implementation Method 1
screening positive colonies according to the fluorescence intensity of the strain to be mined
Data Source
AI summary
Method for mining strains and genes, and use are provided. The method includes using a recombinant plasmid, and transferring the plasmid into a strain to be mined. The recombinant plasmid carries an operon for catabolizing lactic acid and a gene for report. A use of ZMO1323 mining by the method in biosynthesis of D-LA is provided.