Construction Of Lactate Biosensors Based On The Transcriptional Regulatory Factor LldR

As an important platform compound,lactic acid is widely applied in pharmaceutical,cosmetic,food and chemical industries.Nowadays,lactic acid is majorly produced through microbiol fermentation.In addition,lactic acid is the final product of anaerobic glycolysis of the human cells.It also palys a key role in human energy metabolism.Lactic acid in blood is an important evaluation index reflecting the function of body organs.Rapid and sensitive lactic acid detection method is of great significance for the monitoring of lactic acid during microbial fermentation and the clinical diagnosis of lactic acidemia.Lactic acid has two optical isomers:D-lactic acid and L-lactic acid.However,all of the reported lactic acid biosensor can only detect one optical isomer.The lactic acid utilization operon of Pseudomonas aeruginosa PAO1 contains both the genes encoding NAD-independent D-lactate dehydrogenase(D-iLDH)and NAD-independent L-lactate dehydrogenase(L-iLDH).The transcriptional regulatory factor LldR can simultaneously respond to D-lactic acid and L-lactic acid.Alpha(Amplified luminescent proximity homogeneous assay)technology is a high-sensitivity homogeneous detection technology based on microbeads with the characteristics of fast,sensitive and simple.In the second chapter of this dissertation,LldR from P.aeruginosa PAO1 was coupled with Alpha technology to obtained a biosensor that can simultaneously detect D-lactic acid and L-lactic acid.The sensitivity of the sensor was optimized through site-directed mutagenesis of the binding sites of LldR.The detection limit of the biosensor was reduced from 32.78 mM to 2.33 mM.This biosensor can be used in rapid and accurate detection of D-lactate,L-lactate and DL-lactate in microbiol fermentation samples.The lactic acid utilization operon of P.fluorescens A506 only contains the coding gene of D-iLDH,and its LldR was reported to show specificity for D-lactic acid.The lactic acid utilization operon of Escherichia coli K-12 MG1655 only contains the gene encoding L-iLDH,and its LldR was reported to show specificity for L-lactic acid.In the third chapter of this dissertation,LldR from P.fluorescens A506 and LldR from E.coli K-12 MG 1655 was coupled with Alpha technology.The two developed biosensors can respond to lactate,but they can not make specific response to D-lactic acid and L-lactic acid.Thus,this study confirmed that both LldR from P.fluorescens A506 and LldR from E.coli K-12 MG1655 can respond to D-lactic acid and L-lactic acid without specificity.In vitro transcription(IVT)refers to the use of DNA as a template to transcribe RNA in an in vitro cell-free system.It uses transcription regulators to generate RNA in response to the analyte.Quantitative detection of transcripted RNA can be used to indicate the concentration of the analyte.In the fourth chapter of this dissertation,LldR from P.aeruginosa PAO1 was combined with IVT technology to develop another biosensor that can simultaneously detect D-lactic acid and L-lactic acid.However,the optimized lactate biosensor could not respond to exogenous lactic acid.In order to verify the applicability of IVT technology,a D-2-hydroxyglutaric acid biosensor was constructed by combining the transcription factor DhdR of D-2-hydroxyglutaric acid metabolism from Achromobacter denitrificans NBRC 15123 with IVT technology.The biosensor can respond to high concentration of D-2-hydroxyglutaric acid,its sensitivity can not meet the needs of clinical detection of D-2-hydroxyglutaric acid.