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Glycerol dehydratase is the key and rate-limiting enzyme in the 1,3-propanediol synthesis pathway of Klebsiella pneumoniae , which determined the producing rate and yield of 1,3-propanediol. However, the expression regulation mechanism of glycerol dehydratase gene dhaB remains poorly unknown. In this study, a histone-like nucleoid-structuring (H-NS) protein was identified and characterized as the positive transcription regulator for dhaB expression in K. pneumoniae 2e, which exhibited high tolerance against crude glycerol in our previous study. Deletion of hns gene significantly decreased the transcription level of dhaB in K. pneumoniae 2e, which led to a remarkable defect on strain growth, glycerol dehydratase activity, and 3-hydroxypropanal production during glycerol fermentation. The transcription level of dhaB was significantly up-regulated in crude glycerol relative to pure glycerol, while the inactivation of H-NS resulted in more negative effect for transcription level of dhaB in the former. Though the H-NS expression level was almost comparable in both substrates, its multimer state was reduced in crude glycerol relative to pure glycerol, suggesting that the oligomerization state of H-NS might have contributed for positive regulation of dhaB expression. Furthermore, electrophoretic mobility shift and DNase I footprinting assays showed that H-NS could directly bind to the upstream promoter region of dhaB by recognizing the AT-rich region. These findings provided new insight into the transcriptional regulation mechanism of H-NS for glycerol dehydratase expression in K. pneumoniae , which might offer new target for engineering bacteria to industrially produce 1,3-propanediol.IMPORTANCEThe biological production of 1,3-propanediol from glycerol by microbial fermentation shows great promising prospect on industrial application. Glycerol dehydratase catalyzes the penultimate step in glycerol metabolism and is regarded as one of the key and rate-limiting enzymes for 1,3-propanediol production. H-NS was reported as a pleiotropic modulator with negative effects on gene expression in most studies. Here, we reported for the first time that the expression of glycerol dehydratase gene is positively regulated by the H-NS. The results provide insight into a novel molecular mechanism of H-NS for positive regulation of glycerol dehydratase gene expression in K. pneumoniae , which holds promising potential for facilitating construction of engineering highly efficient 1,3-propanediol-producing strains., Competing Interests: The authors declare no conflict of interest.

Background: The direct bioconversion of crude glycerol, a byproduct of biodiesel production, into 1,3-propanediol by microbial fermentation constitutes a remarkably promising value-added applications. However, the low activity of glycerol dehydratase, which is the key and rate-limiting enzyme in the 1,3-propanediol synthetic pathway, caused by crude glycerol impurities is one of the main factors affecting the 1,3-propanediol yield. Hence, the exploration of glycerol dehydratase resources suitable for crude glycerol bioconversion is required for the development of 1,3-propanediol-producing engineered strains., Results: In this study, the novel glycerol dehydratase 2eGDHt, which has a tolerance against crude glycerol impurities from Klebsiella pneumoniae 2e, was characterized. The 2eGDHt exhibited the highest activity toward glycerol, with K m and V m values of 3.42 mM and 58.15 nkat mg -1 , respectively. The optimum pH and temperature for 2eGDHt were 7.0 and 37 °C, respectively. 2eGDHt displayed broader pH stability than other reported glycerol dehydratases. Its enzymatic activity was increased by Fe 2+ and Tween-20, with 294% and 290% relative activities, respectively. The presence of various concentrations of the crude glycerol impurities, including NaCl, methanol, oleic acid, and linoleic acid, showed limited impact on the 2eGDHt activity. In addition, the enzyme activity was almost unaffected by the presence of an impurity mixture that mimicked the crude glycerol environment. Structural analyses revealed that 2eGDHt possesses more coil structures than reported glycerol dehydratases. Moreover, molecular dynamics simulations and site-directed mutagenesis analyses implied that the existence of unique Val744 from one of the increased coil regions played a key role in the tolerance characteristic by increasing the protein flexibility., Conclusions: This study provides insight into the mechanism for enzymatic action and the tolerance against crude glycerol impurities, of a novel glycerol dehydratase 2eGDHt, which is a promising glycerol dehydratase candidate for biotechnological conversion of crude glycerol into 1,3-PDO., (© 2023. The Author(s).)

Biotechnological processes at biorefineries are considered one of the most attractive alternatives for valorizing biomasses by converting them into bioproducts, biofuels, and bioenergy. For example, biodiesel can be obtained from oils and grease but generates glycerol as a byproduct. Glycerol recycling has been studied in several bioprocesses, with one of them being its conversion to 1,3-propanediol (1,3-PDO) by Clostridium . Clostridium beijerinckii is particularly interesting because it can produce a range of industrially relevant chemicals, including solvents and organic acids, and it is non-pathogenic. However, while Clostridium species have many potential advantages as chassis for synthetic biology applications, there are significant limitations when considering their use, such as their limited genetic tools, slow growth rate, and oxygen sensitivity. In this work, we carried out the overexpression of the genes involved in the synthesis of 1,3-PDO in C. beijerinckii Br21, which allowed us to increase the 1,3-PDO productivity in this strain. Thus, this study contributed to a better understanding of the metabolic pathways of glycerol conversion to 1,3-PDO by a C. beijerinckii isolate. Also, it made it possible to establish a transformation method of a modular vector in this strain, therefore expanding the limited genetic tools available for this bacterium, which is highly relevant in biotechnological applications.

Bio-based propionate attracts increasing attention owing to its green nature and specific food additive market. To date, the time-consuming and costly fermentation process by strict anaerobes makes propionate production not ideal. In this study, we designed a new route for propionate production, in which 1,2-propanediol was first dehydrated to propionaldehyde and then to propionate by taking advantage of the robust oxidization capacity of the Pseudomonas putida KT2440 strain. The high atom economy (0.97 g/g) in this proposed pathway is more advantageous than the previous l-threonine-derived route (0.62 g/g). The molecular mechanism of the extraordinary oxidation capacity of P. putida KT2440 was first deciphered. The propionate production was realized in P. putida KT2440 by screening suitable glycerol dehydratases and optimizing the expression to eliminate the formation of 1-propanol and the accumulation of the intermediate propionaldehyde. The engineered strain produced propionate with a molar conversion rate of >99% from 1,2-propanediol. A high titer of 46.5 g/L pure propionic acid with a productivity of 1.55 g/L/h and a mass yield of 0.96 g/g was achieved in fed-batch biotransformation. Thus, this study provides another idea for the production of high-purity bio-based propionate from renewable materials with high atom economy.

The dha operon of Klebsiella pneumoniae is responsible for glycerol catabolism and 1,3-propanediol formation. Subunits of glycerol dehydratase and the large subunit of glycerol dehydratase reactivating factor are encoded by dhaBCE and dhaF, respectively. Proteins of pdu operon form a microcompartment (bacteria organelle) and responsible for 1,2-propanediol catabolism. In this operon, pduCDE and pduG encode subunits of diol dehydratase and its reactivating factor. Diol dehydratase is an isofunctional enzyme of glycerol dehydratase, but its role in glycerol catabolism was not entirely clear. In this study, dhaBCE, pduCDE, dhaF, and pduG in K. pneumoniae were knocked out individually or combinedly. These strains were cultured with glycerol as a substrate, and dehydratase activities in the cytoplasm and microcompartment were detected. Results showed that glycerol dehydratase and diol dehydratase were simultaneously responsible for glycerol catabolism in K. pneumoniae. Besides being packaged in microcompartment, large amounts of diol dehydratase was also presented in the cytoplasm. However, the Pdu microcompartment reduced the accumulation of 3-hydroxypropionaldehyde in the fermentation broth. PduG can cross reactivate glycerol dehydratase instead of DhaF. However, DhaF is not involved in reactivation of diol dehydratase. In conclusion, diol dehydratase and Pdu microcompartment play important roles in glycerol catabolism in K. pneumoniae., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Adenosylcobalamin (AdoCbl) or coenzyme B 12 -dependent enzymes tend to undergo mechanism-based inactivation during catalysis or inactivation in the absence of substrate. Such inactivation may be inevitable because they use a highly reactive radical for catalysis, and side reactions of radical intermediates result in the damage of the coenzyme. How do living organisms address such inactivation when enzymes are inactivated by undesirable side reactions? We discovered reactivating factors for radical B 12 eliminases. They function as releasing factors for damaged cofactor(s) from enzymes and thus mediate their exchange for intact AdoCbl. Since multiple turnovers and chaperone functions were demonstrated, they were renamed "reactivases" or "reactivating chaperones." They play an essential role in coenzyme recycling as part of the activity-maintaining systems for B 12 enzymes. In this chapter, we describe our investigations on reactivating chaperones, including their discovery, gene cloning, preparation, characterization, activity assays, and mechanistic studies, that have been conducted using a wide range of biochemical and structural methods that we have developed., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Adenosylcobalamin (AdoCbl) or coenzyme B 12 -dependent enzymes catalyze intramolecular group-transfer reactions and ribonucleotide reduction in a wide variety of organisms from bacteria to animals. They use a super-reactive primary-carbon radical formed by the homolysis of the coenzyme's Co-C bond for catalysis and thus belong to the larger class of "radical enzymes." For understanding the general mechanisms of radical enzymes, it is of great importance to establish the general mechanism of AdoCbl-dependent catalysis using enzymes that catalyze the simplest reactions-such as diol dehydratase, glycerol dehydratase and ethanolamine ammonia-lyase. These enzymes are often called "eliminases." We have studied AdoCbl and eliminases for more than a half century. Progress has always been driven by the development of new experimental methodologies. In this chapter, we describe our investigations on these enzymes, including their metabolic roles, gene cloning, preparation, characterization, activity assays, and mechanistic studies, that have been conducted using a wide range of biochemical and structural methodologies we have developed., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Pseudomonas asiatica C1, which could grow on glucose and aerobically synthesize coenzyme B 12 , was isolated and developed as a microbial cell factory for the production of 3-hydroxypropionic acid (3-HP) from glycerol. Three heterologous enzymes, glycerol dehydratase (GDHt), GDHt reactivase (GdrAB) and aldehyde dehydrogenase (ALDH), constituting the 3-HP synthesis pathway, were introduced, and three putative dehydrogenases, responsible for 3-HP degradation, were disrupted. In addition, the transcriptional repressor glpR and the glycerol kinase glpK were removed to increase glycerol import while eliminating the catabolic use of glycerol. Furthermore, the global regulatory protein encoded by crc and several putative oxidoreductases (PDORs) were disrupted. One resulting strain, when grown on glucose, could produce 3-HP at ~ 700 mM in 48 h in a fed-batch bioreactor experiment, with the molar yield > 0.99 on glycerol without much by-products. This study demonstrates that P. asiatica C1 is a promising host for production of 3-HP from glycerol., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

3-Hydroxypropionic acid (3-HP) and 1,3-propanediol (1,3-PDO) have tremendous potential markets in many industries. This study evaluated the simultaneous biosynthesis of the 2 compounds using the new psychrophile-based simple biocatalyst (PSCat) reaction system. The PSCat method is based on the expression of glycerol dehydratase, 1,3-propanediol dehydrogenase, and aldehyde dehydrogenase from Klebsiella pneumoniae in Shewanella livingstonensis Ac10 and Shewanella frigidimarina DSM 12253, individually. Heat treatment at 45 °C for 15 min deactivated the intracellular metabolic flux, and the production process was started after adding substrate, cofactor, and coenzyme. In the solo production process after 1 h, the maximum production of 3-HP was 62.0 m m. For 1,3-PDO, the maximum production was 25.0 m m. In the simultaneous production process, productivity was boosted, and the production of 3-HP and 1,3-PDO increased by 13.5 and 4.9 m m, respectively. Hence, the feasibility of the individual production and the simultaneous biosynthesis system were verified in the new PSCat approach., (© The Author(s) 2020. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

The necessity of costly co-enzyme B 12 for the activity of glycerol dehydratase (GDHt) is considered as a major bottleneck in sustainable bioproduction of 1,3-propanediol (1,3-PD) from glycerol. Here, an E. coil Rosetta-dhaB1-dhaB2 strain was constructed by overexpressing a B 12 -independent GDHt (dhaB1) and its activating factor (dhaB2) from Clostridium butyricum. Subsequently, it was used in designing a co-culture with E. coli BL21-dhaT that overexpressed 1,3-PD oxidoreductase (dhaT), to produce 1,3-PD during co-fermentation of glycerol and glucose. The optimum initial ratio of BL21-dhaT to Rosetta-dhaB1-dhaB2 strains in the co-culture was 1.5. Compared to the fermentation of glycerol alone, co-fermentation approach provided 1.3-folds higher 1,3-PD. Finally, co-fermentation was done in a 10 L bioreactor that produced 41.65 g/L 1,3-PD, which corresponded to 0.69 g/L/h productivity and 0.67 mol/mol yield of 1,3-PD. Hence, the developed co-culture could produce 1,3-PD cost-effectively without requiring vitamin B 12 ., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Coenzyme B 12 -dependent glycerol dehydratase (GDHt) catalyzes the dehydration reaction of glycerol in the presence of adenosylcobalamin to yield 3-hydroxypropanal (3-HPA), which can be converted biologically to versatile platform chemicals such as 1,3-propanediol and 3-hydroxypropionic acid. Owing to the increased demand for biofuels, developing biological processes based on glycerol, which is a byproduct of biodiesel production, has attracted considerable attention recently. In this review, we will provide updates on the current understanding of the catalytic mechanism and structure of coenzyme B 12 -dependent GDHt, and then summarize the results of engineering attempts, with perspectives on future directions in its engineering., (Copyright © 2020 Nasir, Ashok, Shim, Park and Yoo.)

Background: In their quest for sustainable development and effective management of greenhouse gas emissions, our societies pursue a shift away from fossil-based resources towards renewable resources. With 95% of our current transportation energy being petroleum based, the application of alternative, carbon-neutral products-among them biodiesel-is inevitable. In order to enhance the cost structure of biodiesel biorefineries, the valorization of the crude glycerol waste stream into high-value platform chemicals is of major importance., Results: The purpose of this study is the production of 3-hydroxypropionaldehyde (3-HPA) from biodiesel-derived crude glycerol by Lactobacillus diolivorans . Particular focus is given on overcoming potential limitations of glycerol transport into the cell, in order to use the cells' total glycerol dehydratase capability towards the formation of 3-HPA as the main product. Recombinant overexpression of the endogenous glycerol uptake facilitating protein PduF results in a significant increase of glycerol conversion by a factor of 1.3. Concomitantly, glycerol dehydratase activity increased from initially 1.70 ± 0.03 U/mg protein to 2.23 ± 0.11 U/mg protein. With this approach, an average productivity of 4.8 g 3-HPA /(g CDM  h) yielding up to 35.9 g/L 3-HPA and 0.91 mol 3-HPA /mol Glycerol have been obtained., Conclusion: Lactobacillus diolivorans proves to be a valuable cell factory for the utilization of crude glycerol delivering high-value C3 chemicals like 3-HPA, 1,3-propanediol (1,3-PDO) and 3-hydroxypropionic acid (3-HP). Enhancing the glycerol influx into the cell by genetic engineering was successful paving the way towards the commercial production of 3-HPA.

3-Hydroxypropionic acid (3-HP) is an important compound and precursor for a series of chemicals and polymeric materials. In this study, the 3-HP producing bacteria were constructed and studied for efficient synthesis of 3-HP. The results indicated that the instability of glycerol dehydratase (GDHt) affected the 3-HP production significantly, which was successfully solved by the expression of glycerol dehydratase reactivase (GdrB), with fivefold increase in 3-HP yield. Meanwhile, NAD + -regenerating enzymes GPD1 (glycerol-3-phosphate dehydrogenase) was expressed; however, the results showed 3-HP was significantly decreased from 56.73-4 mM, and malic acid was obviously increased. Analysis of the C flux distribution showed that the main reason for the results was the lack of NAD + . The addition of NAD + further increased the 3-HP production to 23.87 mM, demonstrating that the "regeneration of NAD + " was the major factor for enhancing 3-HP production.

Glycerol dehydratase (GDHt) is a pivotal enzyme for fermentative utilization of glycerol by catalyzing radical-mediated conversion of glycerol into 3-hydroxypropionaldehyde (3-HPA). Precise and sensitive monitoring of cellular GDHt activity during the fermentation process is a prerequisite for reliable metabolic analysis to afford efficient cellular engineering and process optimization. Here we report a new spectrophotometric assay for the sensitive measurement of the GDHt activity with a sub-nanomolar limit of detection (LOD). The assay method employs aldehyde dehydrogenase (ALDH) as a reporter enzyme, so the readout of the GDHt activity is recorded at 340 nm as an increase in UV absorbance which results from NADH generation accompanied by oxidation of 3-HPA to 3-hydroxypropionic acid (3-HP). The GDHt assay was performed under the reaction conditions where the ALDH activity overwhelms the GDHt activity (i.e., 50-fold higher activity of ALDH relative to GDHt activity), affording sensitive detection of GDHt with 360 pM LOD. The ALDH-coupled assay was used to determine kinetic parameters of GDHt for glycerol, leading to K M  = 0.73 ± 0.09 mM and k cat  = 400 ± 20 s -1 which are in reasonable agreements with the previous reports. Our assay method allowed measurement of even a 10 4 -fold decrease in the cellular GDHt activity during fermentative production of 3-HP, which demonstrates the detection sensitivity much higher than the previous methods., (Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Glycyl radical enzymes (GREs) represent a diverse superfamily of enzymes that utilize a radical mechanism to catalyze difficult, but often essential, chemical reactions. In this work we present the first biochemical and structural data for a GRE-type diol dehydratase from the organism Roseburia inulinivorans (RiDD). Despite high sequence (48% identity) and structural similarity to the GRE-type glycerol dehydratase from Clostridium butyricum, we demonstrate that the RiDD is in fact a diol dehydratase. In addition, the RiDD will utilize both (S)-1,2-propanediol and (R)-1,2-propanediol as a substrate, with an observed preference for the S enantiomer. Based on the new structural information we developed and successfully tested a hypothesis that explains the functional differences we observe., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The rapid accumulation of glycerol during biodiesel synthesis has attracted much interest on the potential usage. One of the promising applications is the conversion of glycerol to 1,3-propanediol (PDO), as well 2,3-butanediol (BDO), by using Klebsiella pneumonia. The results of this study indicate that the dissolved oxygen level (DO) is a determining factor in the distribution of PDO and BDO. The batch with a low DO could achieve a much higher PDO/BDO ratio than the high DO batch, with results of 9.9 and 0.2, respectively. The enzyme activity of glycerol dehydratase (GDHt) in the low DO batch was about three times that of the high DO batch, and this might be the cause of the enhanced PDO production. In conclusion, the results of this work show that high DO was beneficial to the production of BDO when using glycerol as the carbon source, while low DO could enhance PDO production., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Biosynthesis of 3-hydroxypropionic acid (3-HP) typically involves two sequential reactions catalyzed by glycerol dehydratase (DhaB) and aldehyde dehydrogenase (AldH). Although plasmid-dependent over-expression of the two enzymes is common, systematic investigation of gene arrangement in vector has not been reported. Here we show that gene arrangements have a noticeable influence on 3-HP production. Using Klebsiella pneumoniae as a host, three AldH-coding genes: ald4 from Saccharomyces cerevisiae, aldh from Escherichia coli, and puuC from host K. pneumoniae, were respectively ligated to dhaB. The recombinant Kp/pET-pk-ald4-dhaB (Kp refers to as K. pneumoniae, pk is a native promoter) produced the highest yield of 3-HP in comparison to both Kp/pET-pk-dhaB-ald4 and Kp/pET-pk-dhaB-pk-ald4, suggesting that the preferential expression of AldH can increase 3-HP production. Additionally, when different AldH-coding genes were respectively ligated downstream of dhaB, the recombinant Kp/pET-pk-dhaB-puuC produced more 3-HP than that by Kp/pET-pk-dhaB-aldh or Kp/pET-pk-dhaB-ald4, implying the intrinsic compatibility of native gene puuC with its host. These findings indicate the applicability of native AldH-coding gene and provide insights into strategies for metabolic engineering of multiple genes.

3-Hydroxypropionic acid (3-HP) is a valuable platform chemical that can be produced biologically from glucose or glycerol. This review article provides an overview and the current status of microbial 3-HP production. The constraints of microbial 3-HP production and possible solutions are also described. Finally, future prospects of biological 3-HP production are discussed., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Glycerol dehydratase (GD) catalyses glycerol reductive conversion to 3-hydroxypropanaldehyde (3-HPA), this being the first step required for the microbial conversion of glycerol to 1, 3 -propanodiol. GD has been functionally characterised to date and two main groups have been determined, one of them being vitamin B(12)-dependent and the other B(12)-independent. GD evolutionary history has been described and an exhaustive analysis made for detecting the functional residues responsible for type I divergence. GD phylogenetic tree topology was seen to be statistically robust and the data indicated strong purifying selection operating on the GD proteins within it. Two clades were indentified, one for vitamin B(12)-dependent and the other for B(12)- independent classes. The ancient hot-pot residues responsible for protein divergency for each clade were also identified. The basic evolutionary biology for GD proteins has been described, thereby opening the way forward for developing rational mutagenesis studies.