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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 significant ly 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 significant ly 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. [ABSTRACT FROM AUTHOR]

1,3-Propanediol (1,3-PDO) is one of the important organic chemical materials and is widely used in polyester synthesis, and it also shows great potential in medicine, cosmetics, resins, and biodegradable plastics. So far, 1,3-PDO mainly comes from chemical synthesis. However, the by-products and the side effects during chemical synthesis of 1,3-PDO bring about serious damage to the environment. In recent years, the biosynthetic pathway of 1,3-PDO has been elucidated in microorganisms. Under the action of glycerol dehydratase (GDHt) and propanediol oxidoreductase (PDOR), glycerol can be catalyzed to form 1,3-PDO through the reduction pathway. Compared to the chemical synthesis, the biosynthesis of 1,3-PDO is environmentally friendly but would face the problem of low production. To improve the yield, the native 1,3-PDO producing strains have been modified by genetic engineering, and the biosynthetic pathway has been reconstructed in the model microorganism, Escherichia coli. In this review, we summarize the research progress of the 1,3-PDO biosynthesis in microorganisms, and hopefully, it will provide reference for the renewable production of 1,3-PDO in industry.

Abstract 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 Fe2+ 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.

The redox imbalance is the major obstacle in the microbial production of 3-hydroxypropionic acid (3-HP). Co-production of propanediol (PDO) and 3-HP is an effective strategy to overcome the redox imbalance issue. The biocatalyst Klebsiellapneumoniae naturally co-produces PDO and 3-HP. Its poor growth characteristics and excessive production of lipopolysaccharide hurdle its usage in industries. The objective of this study is to circumvent these constraints by isolating a novel strain of K.quasipneumoniae MSN12 (KpMSN12) from soil samples. From a numerous isolate, the new isolate KpMSN12 has produced 58 mM PDO and 4 mM 3-HP, when it was grown in shake flask under microaerobic conditions, which is higher than the control Kp109. The effect of overexpression of an aldH (KGSADH) on co-production of 3-HP and PDO from glycerol was investigated in recombinant KpMSN12 (KpMSN12K). KpMSN12K produced 45 mM of 3-HP and 30 mM PDO which was observed higher (3-HP 25 mM, PDO 28 mM) than the control strain. While over expressing dhaB in another recombinant (KpMSN12D), 3-HP was reduced to 9 mM and 11 mM PDO due to the accumulation of 3-HPA and the growth was severely hampered. In addition, combined overexpression of dhaB and aldH (KpMSN12DK) produced 33 mM 3-HP and 25 mM PDO. The lower lipopolysaccharide content in the KpMSN12 strain was confirmed by comparing 2-keto3-deoxyoctonate content (KpMSN12—3.8 µg/mL; Kp109—5.14 µg/mL). In addition, this new strain exhibited enhanced sedimentation characteristics and higher susceptibility to a variety of antibiotics when compared to Kp109 strain. These potential characteristics of KpMSN12 would shape it as a desirable host for the production of industrial chemicals. [ABSTRACT FROM AUTHOR]

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 Km and Vm 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 Fe2+ 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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.

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.

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. • Diol dehydratase and glycerol dehydratase are simultaneously responsible for glycerol catabolism. • Large amounts of diol dehydratase were presented in the cytoplasm. • Pdu microcompartments reduce the accumulation of 3-hydroxypropionaldehyde. • PduG can cross reactivate glycerol dehydratase instead of DhaF. [ABSTRACT FROM AUTHOR]

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.)