Abstract
Lactic acid is an important platform chemical used for the production of various compounds including polylactic acid (PLA). Optically pure L- and D-lactic acids are required to obtain high quality PLA. To advance the development and selection of microbial strains for improved production of lactic acid enantiomers, a high-throughput screening, dynamic pathway control, or real-time monitoring are often applied. Inducible gene expression systems and their application in the genetically encoded biosensors contribute to the development of these techniques and are important devices for the advancement of lactic acid biotechnology. Here, we identify and characterize eleven lactate-inducible systems from Escherichia coli, Cupriavidus necator, and Pseudomonas spp. The specificity and dynamics of these systems in response to L- and D-lactate, or structurally similar compounds are investigated. We demonstrate that the inducible systems EcLldR/PlldP and CnGntR/PH16_RS19190 respond only to the L-lactate, exhibiting approximately 19- and 24-fold induction, respectively. Despite neither of the examined bacteria possess the D-lactate-specific inducible system, the PaPdhR/PlldP and PfPdhR/PlldP are induced approximately 37- and 366-fold, respectively, by D-lactate and can be used for developing biosensor with improved specificity. The findings of this study provide an insight into understanding of L- and D-lactate-inducible systems that can be employed as sensing and tuneable devices in synthetic biology.
Highlights
Pure L- and D-lactic acids are used for synthesis of bioplastic PLA1 that has shown potential to replace traditional petroleum-based plastics
Bacteria can produce lactic acid and utilise it as a carbon source, typically involving two types of lactate dehydrogenases, NAD-dependent lactate dehydrogenases and NAD-independent lactate dehydrogenases. nLDH catalyses the oxidation of lactate to pyruvate by using NADH as electron acceptor, whereas iLDH uses a flavin-dependent m echanism5. iLDHs are classified into NAD-independent L-lactate dehydrogenase (L-iLDH) and NAD-independent D-lactate dehydrogenase (D-iLDH) according to the substrate specificity
Based on the homology of functional genes encoding LDHs and lactate permeases, we identified potential operons that contained at least two genes related to the lactate catabolism and the adjacent transcriptional regulators (TRs) gene in E. coli MG1655, C. necator H16, and Pseudomonas spp
Summary
Pure L- and D-lactic acids are used for synthesis of bioplastic PLA1 that has shown potential to replace traditional petroleum-based plastics. Most of the L-iLDHs belong to α-hydroxy acid oxidizing flavoproteins family and use flavin mononucleotide (FMN) as a cofactor These dehydrogenases can be divided into three groups by the electron acceptor, which can utilise quinones, oxygen or cytochrome c5. Genes encoding for L-iLDH, D-iLDH and lactate permease often form lactate utilization operon, the expression of which is regulated by a transcription factor Such lactate catabolism-related genes and their corresponding transcriptional regulators (TRs) have been identified previously in several classes of bacteria including Gammaproteobacteria[7,8,9], Actinobacteria[10], Bacilli[11,12], Deltaproteobacteria[13,14], and Clostridia[15]. We report the identification, characterization, and specificity of L- and D-lactate inducible-gene expression systems from gammaproteobacteria E. coli MG1655 and Pseudomonas spp., and betaproteobacterium C. necator H16. To expedite synthetic biology and metabolic engineering efforts, the dynamics of inducible systems is parameterized and their suitability for controlling orthogonal gene expression is demonstrated
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