Application of whole-cell biosensors for analysis and improvement of L- and D-lactic acid fermentation by Lactobacillus spp. from the waste of glucose syrup production.

BACKGROUND: Lactic acid is one of the most important organic acids, with various applications in the food, beverage, pharmaceutical, cosmetic, and chemical industries. Optically pure forms of L- and D-lactic acid produced via microbial fermentation play an important role in the synthesis of biodegradable polylactic acid. Alternative substrates, including by-products and residues from the agro-food industry, provide a cost-effective solution for lactic acid production and are a promising avenue for the circular economy. RESULTS: In this study, the transcription factor (TF)-based whole-cell biosensor strategy was developed for the L- and D-lactic acid determination. It was cross validated with commonly used high-performance liquid chromatography and enzymatic methods. The utility of biosensors as an efficient analytical tool was demonstrated by their application for the lactic acid determination and fermentation improvement. We explored the ability of Lacticaseibacillus paracasei subsp. paracasei, Lactobacillus delbrueckii subsp. lactis, and Lactobacillus amylovorus to biosynthesize optically pure L-lactic acid, D-lactic acid or mixture of both from organic-rich residual fraction (ORRF), a waste of glucose syrup production from wheat starch. The fermentation of this complex industrial waste allowed the production of lactic acid without additional pretreatment obtaining yields from 0.5 to 0.9 Cmol/Cmol glucose. CONCLUSIONS: This study highlights the utility of whole cell biosensors for the determination of L- and D-forms of lactic acid. The fermentation of L-lactic acid, D-lactic acid and mixture of both by L. paracasei, L. lactis, and L. amylovorus, respectively, was demonstrated using waste of glucose syrup production, the ORRF.

Transcription factor-based biosensors for detection of naturally occurring phenolic acids.

Phenolic acids including hydroxybenzoic and hydroxycinnamic acids are secondary plant and fungal metabolites involved in many physiological processes offering health and dietary benefits. They are often utilised as precursors for production of value-added compounds. The limited availability of synthetic biology tools, such as whole-cell biosensors suitable for monitoring the dynamics of phenolic acids intracellularly and extracellularly, hinders the capabilities to develop high-throughput screens to study their metabolism and forward engineering. Here, by applying a multi-genome approach, we have identified phenolic acid-inducible gene expression systems composed of transcription factor-inducible promoter pairs responding to eleven different phenolic acids. Subsequently, they were used for the development of whole-cell biosensors based on model bacterial hosts, such as Escherichia coli, Cupriavidus necator and Pseudomonas putida. The dynamics and range of the biosensors were evaluated by establishing their response and sensitivity landscapes. The specificity and previously uncharacterised interactions between transcription factor and its effector(s) were identified by a screen of twenty major phenolic acids. To exemplify applicability, we utilise a protocatechuic acid-biosensor to identify enzymes with enhanced activity for conversion of p-hydroxybenzoate to protocatechuate. Transcription factor-based biosensors developed in this study will advance the analytics of phenolic acids and expedite research into their metabolism.

Improving carbon monoxide tolerance of Cupriavidus necator H16 through adaptive laboratory evolution.

Background: The toxic gas carbon monoxide (CO) is abundantly present in synthesis gas (syngas) and certain industrial waste gases that can serve as feedstocks for the biological production of industrially significant chemicals and fuels. For efficient bacterial growth to occur, and to increase productivity and titres, a high resistance to the gas is required. The aerobic bacterium Cupriavidus necator H16 can grow on CO2 + H2, although it cannot utilise CO as a source of carbon and energy. This study aimed to increase its CO resistance through adaptive laboratory evolution. Results: To increase the tolerance of C. necator to CO, the organism was continually subcultured in the presence of CO both heterotrophically and autotrophically. Ten individual cultures were evolved heterotrophically with fructose in this manner and eventually displayed a clear growth advantage over the wild type strain. Next-generation sequencing revealed several mutations, including a single point mutation upstream of a cytochrome bd ubiquinol oxidase operon (cydA2B2), which was present in all evolved isolates. When a subset of these mutations was engineered into the parental H16 strain, only the cydA2B2 upstream mutation enabled faster growth in the presence of CO. Expression analysis, mutation, overexpression and complementation suggested that cydA2B2 transcription is upregulated in the evolved isolates, resulting in increased CO tolerance under heterotrophic but not autotrophic conditions. However, through subculturing on a syngas-like mixture with increasing CO concentrations, C. necator could also be evolved to tolerate high CO concentrations under autotrophic conditions. A mutation in the gene for the soluble [NiFe]-hydrogenase subunit hoxH was identified in the evolved isolates. When the resulting amino acid change was engineered into the parental strain, autotrophic CO resistance was conferred. A strain constitutively expressing cydA2B2 and the mutated hoxH gene exhibited high CO tolerance under both heterotrophic and autotrophic conditions. Conclusion: C. necator was evolved to tolerate high concentrations of CO, a phenomenon which was dependent on the terminal respiratory cytochrome bd ubiquinol oxidase when grown heterotrophically and the soluble [NiFe]-hydrogenase when grown autotrophically. A strain exhibiting high tolerance under both conditions was created and presents a promising chassis for syngas-based bioproduction processes.

Development and Application of Whole-Cell Biosensors for the Detection of Gallic Acid.

Gallic acid is a prevalent secondary plant metabolite distinguished as one of the most effective free-radical scavengers among phenolic acids. This compound is also known for its cytotoxic, anti-inflammatory, and antimicrobial activities. Bulk quantities of gallic acid are conventionally produced by acid hydrolysis of tannins, a costly and environmentally hazardous process. With the aim to develop more sustainable approaches, microbial bioproduction strategies have been attempted recently. To advance synthetic biology and metabolic engineering of microorganisms for gallic acid production, we characterize here a transcription factor-based inducible system PpGalR/PPP_RS13150 that responds to the extracellular gallic acid in a dose-dependent manner in Pseudomonas putida KT2440. Surprisingly, this compound does not mediate induction when PpGalR/PPP_RS13150 is used in non-native host background. We show that the activation of the inducible system requires gallate dioxygenase activity encoded by galA gene. The 4-oxalomesaconic acid, an intermediate of gallic acid-metabolism, is identified as the effector molecule that interacts with the transcription factor GalR mediating activation of gene expression. Introduction of galA gene along galR enables development of biosensors suitable for detection and monitoring of gallic acid extracellularly using non-native hosts such as E. coli and C. necator. Moreover, the P. putida-based biosensor's applicability is demonstrated by detecting and measuring gallic acid in extracts of Camellia sinensis leaves. This study reports the strategy, which can be applied for developing gallic acid biosensors using bacterial species outside Pseudomonas genus.

Advances in Production of Hydroxycinnamoyl-Quinic Acids: From Natural Sources to Biotechnology.

Hydroxycinnamoyl-quinic acids (HCQAs) are polyphenol esters formed of hydroxycinnamic acids and (-)-quinic acid. They are naturally synthesized by plants and some micro-organisms. The ester of caffeic acid and quinic acid, the chlorogenic acid, is an intermediate of lignin biosynthesis. HCQAs are biologically active dietary compounds exhibiting several important therapeutic properties, including antioxidant, antimicrobial, anti-inflammatory, neuroprotective, and other activities. They can also be used in the synthesis of nanoparticles or drugs. However, extraction of these compounds from biomass is a complex process and their synthesis requires costly precursors, limiting the industrial production and availability of a wider variety of HCQAs. The recently emerged production through the bioconversion is still in an early stage of development. In this paper, we discuss existing and potential future strategies for production of HCQAs.

Development and Characterization of Indole-Responsive Whole-Cell Biosensor Based on the Inducible Gene Expression System from Pseudomonas putida KT2440.

Indole is a biologically active compound naturally occurring in plants and some bacteria. It is an important specialty chemical that is used as a precursor by the pharmaceutical and chemical industries, as well as in agriculture. Recently, indole has been identified as an important signaling molecule for bacteria in the mammalian gut. The regulation of indole biosynthesis has been studied in several bacterial species. However, this has been limited by the lack of in vivo tools suitable for indole-producing species identification and monitoring. The genetically encoded biosensors have been shown to be useful for real-time quantitative metabolite analysis. This paper describes the identification and characterization of the indole-inducible system PpTrpI/PPP_RS00425 from Pseudomonas putida KT2440. Indole whole-cell biosensors based on Escherichia coli and Cupriavidus necator strains are developed and validated. The specificity and dynamics of biosensors in response to indole and its structurally similar derivatives are investigated. The gene expression system PpTrpI/PPP_RS00425 is shown to be specifically induced up to 639.6-fold by indole, exhibiting a linear response in the concentration range from approximately 0.4 to 5 mM. The results of this study form the basis for the use of whole-cell biosensors in indole metabolism-relevant bacterial species screening and characterization.

Biosensor-informed engineering of Cupriavidus necator H16 for autotrophic D-mannitol production.

Cupriavidus necator H16 is one of the most researched carbon dioxide (CO2)-fixing bacteria. It can store carbon in form of the polymer polyhydroxybutyrate and generate energy by aerobic hydrogen oxidation under lithoautotrophic conditions, making C. necator an ideal chassis for the biological production of value-added compounds from waste gases. Despite its immense potential, however, the experimental evidence of C. necator utilisation for autotrophic biosynthesis of chemicals is limited. Here, we genetically engineered C. necator for the high-level de novo biosynthesis of the industrially relevant sugar alcohol mannitol directly from Calvin-Benson-Bassham (CBB) cycle intermediates. To identify optimal mannitol production conditions in C. necator, a mannitol-responsive biosensor was applied for screening of mono- and bifunctional mannitol 1-phosphate dehydrogenases (MtlDs) and mannitol 1-phosphate phosphatases (M1Ps). We found that MtlD/M1P from brown alga Ectocarpus siliculosus performed overall the best under heterotrophic growth conditions and was selected to be chromosomally integrated. Consequently, autotrophic fermentation of recombinant C. necator yielded up to 3.9 g/L mannitol, representing a substantial improvement over mannitol biosynthesis using recombinant cyanobacteria. Importantly, we demonstrate that at the onset of stationary growth phase nearly 100% of carbon can be directed from the CBB cycle into mannitol through the glyceraldehyde 3-phosphate and fructose 6-phosphate intermediates. This study highlights for the first time the potential of C. necator to generate sugar alcohols from CO2 utilising precursors derived from the CBB cycle.

Identification and characterization of L- and D-lactate-inducible systems from Escherichia coli MG1655, Cupriavidus necator H16 and Pseudomonas species.

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.

Bioproduction of l- and d-lactic acids: advances and trends in microbial strain application and engineering.

Lactic acid is an important platform chemical used in the food, agriculture, cosmetic, pharmaceutical, and chemical industries. It serves as a building block for the production of polylactic acid (PLA), a biodegradable polymer, which can replace traditional petroleum-based plastics and help to reduce environmental pollution. Cost-effective production of optically pure l- and d-lactic acids is necessary to achieve a quality and thermostable PLA product. This paper evaluates research advances in the bioproduction of l- and d-lactic acids using microbial fermentation. Special emphasis is given to the development of metabolically engineered microbial strains and processes tailored to alternative and flexible feedstock concepts such as: lignocellulose, glycerol, C1-gases, and agricultural-food industry byproducts. Alternative fermentation concepts that can improve lactic acid production are discussed. The potential use of inducible gene expression systems for the development of biosensors to facilitate the screening and engineering of lactic acid-producing microorganisms is discussed.

The pMTL70000 modular, plasmid vector series for strain engineering in Cupriavidus necator H16.

Cupriavidus necator H16 can convert CO2 into industrial chemicals and fuels. To facilitate its engineering, we designed, built and tested the pMTL70000 modular plasmids comprising standardised Cupriavidus and E. coli replicons, selectable markers and application specific modules. Plasmids were characterised in terms of transmissibility, stability, copy number and compatibility.

Engineering Cupriavidus necator H16 for the autotrophic production of (R)-1,3-butanediol.

Butanediols are widely used in the synthesis of polymers, specialty chemicals and important chemical intermediates. Optically pure R-form of 1,3-butanediol (1,3-BDO) is required for the synthesis of several industrial compounds and as a key intermediate of ß-lactam antibiotic production. The (R)-1,3-BDO can only be produced by application of a biocatalytic process. Cupriavidus necator H16 is an established production host for biosynthesis of biodegradable polymer poly-3-hydroxybutryate (PHB) via acetyl-CoA intermediate. Therefore, the utilisation of acetyl-CoA or its upstream precursors offers a promising strategy for engineering biosynthesis of value-added products such as (R)-1,3-BDO in this bacterium. Notably, C. necator H16 is known for its natural capacity to fix carbon dioxide (CO2) using hydrogen as an electron donor. Here, we report engineering of this facultative lithoautotrophic bacterium for heterotrophic and autotrophic production of (R)-1,3-BDO. Implementation of (R)-3-hydroxybutyraldehyde-CoA- and pyruvate-dependent biosynthetic pathways in combination with abolishing PHB biosynthesis and reducing flux through the tricarboxylic acid cycle enabled to engineer strain, which produced 2.97 g/L of (R)-1,3-BDO and achieved production rate of nearly 0.4 Cmol Cmol-1 h-1 autotrophically. This is first report of (R)-1,3-BDO production from CO2.

Valorization of Bilberry (Vaccinium myrtillus L.) Pomace by Enzyme-Assisted Extraction: Process Optimization and Comparison with Conventional Solid-Liquid Extraction.

Bilberry (Vaccinium myrtillus L.) pomace contains a significant amount of polyphenols and can serve as a basis for food additives, nutraceuticals, and functional foods. Although various techniques can be employed to recover bioactive fractions from berry pomaces, data on enzyme-assisted extraction (EAE) of bilberry pomace are rather scarce. This study aimed to optimize critical EAE parameters using Viscozyme L to obtain a high-yield extract with enhanced antioxidant capacity. Central composite design and response surface methodology evaluating the effect of four independent variables, namely, pH, temperature, extraction time, and enzyme concentration on three responses, were employed to define optimal EAE conditions. Under the optimal conditions (pH: 4.5, temperature 46 °C, 1 h of extraction, and 2 active units (AU) of Viscozyme L/g of pomace), EAE yielded 56.15 g/100 g DW of the water-soluble fraction. Comparison with conventional maceration indicated that EAE, besides the yield, significantly increased the in vitro antioxidant capacity measured by the total phenolic content, ABTS, ORAC, and CUPRAC assays. Moreover, an increase was observed for the measured mono- and disaccharide as well as anthocyanin content. Overall, this study demonstrates the improved efficiency of EAE over conventional solid-liquid extraction to recover fractions with a higher yield and enhanced functional properties in a fast and sustainable manner.

Advances and Prospects of Phenolic Acids Production, Biorefinery and Analysis.

Biotechnological production of phenolic acids is attracting increased interest due to their superior antioxidant activity, as well as other antimicrobial, dietary, and health benefits. As secondary metabolites, primarily found in plants and fungi, they are effective free radical scavengers due to the phenolic group available in their structure. Therefore, phenolic acids are widely utilised by pharmaceutical, food, cosmetic, and chemical industries. A demand for phenolic acids is mostly satisfied by utilising chemically synthesised compounds, with only a low quantity obtained from natural sources. As an alternative to chemical synthesis, environmentally friendly bio-based technologies are necessary for development in large-scale production. One of the most promising sustainable technologies is the utilisation of microbial cell factories for biosynthesis of phenolic acids. In this paper, we perform a systematic comparison of the best known natural sources of phenolic acids. The advances and prospects in the development of microbial cell factories for biosynthesis of these bioactive compounds are discussed in more detail. A special consideration is given to the modern production methods and analytics of phenolic acids.

A genome-wide approach for identification and characterisation of metabolite-inducible systems.

Inducible gene expression systems are vital tools for the advancement of synthetic biology. Their application as genetically encoded biosensors has the potential to contribute to diagnostics and to revolutionise the field of microbial cell factory development. Currently, the number of compounds of biological interest by far exceeds the number of available biosensors. Here, we address this limitation by developing a generic genome-wide approach to identify transcription factor-based inducible gene expression systems. We construct and validate 15 functional biosensors, provide a characterisation workflow to facilitate forward engineering efforts, exemplify their broad-host-range applicability, and demonstrate their utility in enzyme screening. Previously uncharacterised interactions between sensors and compounds of biological relevance are identified by employing the largest reported library of metabolite-responsive biosensors in an automated high-throughput screen. With the rapidly growing genomic data these innovative capabilities offer a platform to vastly increase the number of biologically detectable molecules.

Design, cloning and characterization of transcription factor-based inducible gene expression systems.

Cellular functions are often controlled by small molecular weight molecules such as metabolites. Microorganisms, mainly prokaryotes, have evolved sensing and regulatory mechanisms based on transcriptional regulators (TRs) that are able to activate gene expression in response to changes in intra- and extracellular metabolite (ligand) concentrations. To understanding control mechanisms and cell factory development in synthetic biology applications, high throughput analytical procedures are required. In this chapter, we outline a methodological pipeline to design and build reporter constructs enabling the characterization of metabolite-responsive inducible gene expression systems. As an example, we present the design, cloning and characterization of the itaconate-inducible system which is composed of the LysR-type transcriptional regulator ItcR and the promoter Pccl from Yersinia pseudotuberculosis. Fluorescence-based plate reader and flow cytometry assays are described and the steps for performing data analysis are provided.

Functional Genetic Elements for Controlling Gene Expression in Cupriavidus necator H16.

A robust and predictable control of gene expression plays an important role in synthetic biology and biotechnology applications. Development and quantitative evaluation of functional genetic elements, such as constitutive and inducible promoters as well as ribosome binding sites (RBSs), are required. In this study, we designed, built, and tested promoters and RBSs for controlling gene expression in the model lithoautotroph Cupriavidus necator H16. A series of variable-strength, insulated, constitutive promoters exhibiting predictable activity within a >700-fold dynamic range was compared to the native P phaC , with the majority of promoters displaying up to a 9-fold higher activity. Positively (AraC/P araBAD -l-arabinose and RhaRS/P rhaBAD -l-rhamnose) and negatively (AcuR/P acuRI -acrylate and CymR/P cmt -cumate) regulated inducible systems were evaluated. By supplying different concentrations of inducers, a >1,000-fold range of gene expression levels was achieved. Application of inducible systems for controlling expression of the isoprene synthase gene ispS led to isoprene yields that exhibited a significant correlation to the reporter protein synthesis levels. The impact of designed RBSs and other genetic elements, such as mRNA stem-loop structure and A/U-rich sequence, on gene expression was also evaluated. A second-order polynomial relationship was observed between the RBS activities and isoprene yields. This report presents quantitative data on regulatory genetic elements and expands the genetic toolbox of C. necatorIMPORTANCE This report provides tools for robust and predictable control of gene expression in the model lithoautotroph C. necator H16. To address a current need, we designed, built, and tested promoters and RBSs for controlling gene expression in C. necator H16. To answer a question on how existing and newly developed inducible systems compare, two positively (AraC/P araBAD -l-arabinose and RhaRS/P rhaBAD -l-rhamnose) and two negatively (AcuR/P acuRI -acrylate and CymR/P cmt -cumate) regulated inducible systems were quantitatively evaluated and their induction kinetics analyzed. To establish if gene expression can be further improved, the effect of genetic elements, such as mRNA stem-loop structure and A/U-rich sequence, on gene expression was evaluated. Using isoprene production as an example, the study investigated if and to what extent chemical compound yield correlates to the level of gene expression of product-synthesizing enzyme.

A Transcription Factor-Based Biosensor for Detection of Itaconic Acid.

Itaconic acid is an important platform chemical that can easily be incorporated into polymers and has the potential to replace petrochemical-based acrylic or methacrylic acid. A number of microorganisms have been developed for the biosynthesis of itaconate including Aspergillus terreus, Escherichia coli, and Saccharomyces cerevisiae. However, the number of strains and conditions that can be tested for increased itaconate titers are currently limited because of the lack of high-throughput screening methods. Here we identified itaconate-inducible promoters and their corresponding LysR-type transcriptional regulators from Yersinia pseudotuberculosis and Pseudomonas aeruginosa. We show that the YpItcR/P ccl inducible system is highly inducible by itaconic acid in the model gammaproteobacterium E. coli and the betaproteobacterium Cupriavidus necator (215- and 105-fold, respectively). The kinetics and dynamics of the YpItcR/P ccl inducible system are investigated, and we demonstrate, that in addition to itaconate, the genetically encoded biosensor is capable of detecting mesaconate, cis-, and trans-aconitate in a dose-dependent manner. Moreover, the fluorescence-based biosensor is applied in E. coli to identify the optimum expression level of cadA, the product of which catalyzes the conversion of cis-aconitate into itaconate. The fluorescence output is shown to correlate well with itaconate concentrations quantified using high-performance liquid chromatography coupled with ultraviolet spectroscopy. This work highlights the potential of the YpItcR/P ccl inducible system to be applied as a biosensor for high-throughput microbial strain development to facilitate improved itaconate biosynthesis.

13C-assisted metabolic flux analysis to investigate heterotrophic and mixotrophic metabolism in Cupriavidus necator H16.

INTRODUCTION: Cupriavidus necator H16 is a gram-negative bacterium, capable of lithoautotrophic growth by utilizing hydrogen as an energy source and fixing carbon dioxide (CO2) through Calvin-Benson-Bassham (CBB) cycle. The potential to utilize synthesis gas (Syngas) and the prospects of rerouting carbon from polyhydroxybutyrate synthesis to value-added compounds makes C. necator an excellent chassis for industrial application. OBJECTIVES: In the context of lack of sufficient quantitative information of the metabolic pathways and to advance in rational metabolic engineering for optimized product synthesis in C. necator H16, we carried out a metabolic flux analysis based on steady-state 13C-labelling. METHODS: In this study, steady-state carbon labelling experiments, using either d-[1-13C]fructose or [1,2-13C]glycerol, were undertaken to investigate the carbon flux through the central carbon metabolism in C. necator H16 under heterotrophic and mixotrophic growth conditions, respectively. RESULTS: We found that the CBB cycle is active even under heterotrophic condition, and growth is indeed mixotrophic. While Entner-Doudoroff (ED) pathway is shown to be the major route for sugar degradation, tricarboxylic acid (TCA) cycle is highly active in mixotrophic condition. Enhanced flux is observed in reductive pentose phosphate pathway (redPPP) under the mixotrophic condition to supplement the precursor requirement for CBB cycle. The flux distribution was compared to the mRNA abundance of genes encoding enzymes involved in key enzymatic reactions of the central carbon metabolism. CONCLUSION: This study leads the way to establishing 13C-based quantitative fluxomics for rational pathway engineering in C. necator H16.

Characterisation of a 3-hydroxypropionic acid-inducible system from Pseudomonas putida for orthogonal gene expression control in Escherichia coli and Cupriavidus necator.

3-hydroxypropionic acid (3-HP) is an important platform chemical used as a precursor for production of added-value compounds such as acrylic acid. Metabolically engineered yeast, Escherichia coli, cyanobacteria and other microorganisms have been developed for the biosynthesis of 3-HP. Attempts to overproduce this compound in recombinant Pseudomonas denitrificans revealed that 3-HP is consumed by this microorganism using the catabolic enzymes encoded by genes hpdH, hbdH and mmsA. 3-HP-inducible systems controlling the expression of these genes have been predicted in proteobacteria and actinobacteria. In this study, we identify and characterise 3-HP-inducible promoters and their corresponding LysR-type transcriptional regulators from Pseudomonas putida KT2440. A newly-developed modular reporter system proved possible to demonstrate that PpMmsR/P mmsA and PpHpdR/P hpdH are orthogonal and highly inducible by 3-HP in E. coli (12.3- and 23.3-fold, respectively) and Cupriavidus necator (51.5- and 516.6-fold, respectively). Bioinformatics and mutagenesis analyses revealed a conserved 40-nucleotide sequence in the hpdH promoter, which plays a key role in HpdR-mediated transcription activation. We investigate the kinetics and dynamics of the PpHpdR/P hpdH switchable system in response to 3-HP and show that it is also induced by both enantiomers of 3-hydroxybutyrate. These findings pave the way for use of the 3-HP-inducible system in synthetic biology and biotechnology applications.