Utilization of Glycerol as a Hydrogen Acceptor by Lactobacillus reuteri: Purification of 1,3-Propanediol:NAD+ Oxidoreductase

Lactobacillus reuteri utilizes exogenously added glycerol as a hydrogen acceptor during carbohydrate fermentations, resulting in higher growth rates and cell yields than those obtained during growth on carbohydrates alone. Glycerol is first converted to 3-hydroxypropionaldehyde by a coenzyme B₁₂-dependent glycerol dehydratase and then reduced to 1,3-propanediol by an NAD⁺ -dependent oxidoreductase. The latter enzyme was purified and determined to have a molecular weight of 180,000; it is predicted to exist as a tetramer of identical 42,000-molecular-weight subunits.     

Glycerol fermentation in Klebsiella pneumoniae: functions of the coenzyme B12-dependent glycerol and diol dehydratases.

Glycerol and diol dehydratases are inducible, coenzyme B12-dependent enzymes found together in Klebsiella pneumoniae ATCC 25955 during anaerobic growth on glycerol. Mutants of this strain isolated by a novel procedure were separately constitutive for either dehydratase, showing the structural genes for the two enzymes to be under independent control in vivo. Glycerol dehydratase and a trimethylene glycol dehydrogenase were implicated as members of a pleiotropic control system that includes glycerol dehydrogenase and dihydroxyacetone kinase for the anaerobic dissimilation of glycerol (the "dha system"). The dehydratase and dehydrogenases were induced by dihydroxyacetone and were jointly constitutive in mutants isolated as constitutive for either the dha system or glycerol dehydratase. These data and the stimulation of growth by Co2+ suggested that glycerol dehydratase and trimethylene glycol dehydrogenase are obligatory enzymes for anaerobic growth on glycerol as the sole carbon source.     

Characterization of a glycerol kinase mutant of Aspergillus niger

A glycerol-kinase-deficient mutant of Aspergillus niger was isolated. Genetic analysis revealed that the mutation is located on linkage group VI. The phenotype of this mutant differed from that of a glycerol kinase mutant of Aspergillus nidulans in its ability to utilize dihydroxyacetone (DHA). The weak growth on glycerol of the A. niger glycerol kinase mutant showed that glycerol phosphorylation is an important step in glycerol catabolism. The mutant could still grow normally on DHA because of the presence of a DHA kinase. This enzyme, probably in combination with an NAD(+)-dependent glycerol dehydrogenase, present only in the mutant, is responsible for the weak growth of the mutant on glycerol. Enzymic analysis of both the mutant and the parental strain showed that at least three different glycerol dehydrogenases were formed under different physiological conditions: the NAD(+)-dependent enzyme described above, a constitutive NADP(+)-dependent enzyme and a D-glyceraldehyde-specific enzyme induced on D-galacturonate. The glycerol kinase mutant showed impaired growth on D-galacturonate.     

Insights into the genome of the enteric bacterium Escherichia blattae: cobalamin (B12) biosynthesis, B12-dependent reactions, and inactivation of the gene region encoding B12-dependent glycerol dehydratase by a new mu-like prophage

The enteric bacterium Escherichia blattae has been analyzed for the presence of cobalamin (B12) biosynthesis and B12-dependent pathways. Biochemical studies revealed that E. blattae synthesizes B12 de novo aerobically and anaerobically. Genes exhibiting high similarity to all genes of Salmonella enterica serovar Typhimurium, which are involved in the oxygen-independent route of B12 biosynthesis, were present in the genome of E. blattae DSM 4481. The dha regulon encodes the key enzymes for the anaerobic conversion of glycerol to 1,3-propanediol, including coenzyme B12-dependent glycerol dehydratase. E. blattae DSM 4481 lacked glycerol dehydratase activity and showed no anaerobic growth with glycerol, but the genome of E. blattae DSM 4481 contained a dha regulon. The E. blattaedha regulon is unusual, since it harbors genes for two types of dihydroxyacetone kinases. The major difference to dha regulons of other enteric bacteria is the inactivation of the dehydratase-encoding gene region by insertion of a 33,339-bp prophage (MuEb). Sequence analysis revealed that MuEb belongs to the Mu family of bacteriophages. The E. blattae strains ATCC 33429 and ATCC 33430 did not contain MuEb. Accordingly, both strains harbored an intact dehydratase-encoding gene region and fermented glycerol. The properties of the glycerol dehydratases and the correlating genes (dhaBCE) of both strains were similar to other B12-dependent glycerol and diol dehydratases, but both dehydratases exhibited the highest affinity for glycerol of all B12-dependent dehydratases characterized so far. In addition to the non-functional genes encoding B12-dependent glycerol dehydratase, the genome of E. blattae DSM 4481 contained the genes for only one other B12-dependent enzyme, the methylcobalamin-dependent methionine synthase.     

Anaerobic growth of Escherichia coli on glycerol by importing genes of the dha regulon from Klebsiella pneumoniae

The dha regulon of Klebsiella pneumoniae specifying fermentative dissimilation of glycerol was mobilized by the broad-host-range plasmid RP4:mini Mu and introduced conjugatively into Escherichia coli. The recipient E. coli was enabled to grow anaerobically on glycerol without added hydrogen acceptors, although its cell yield was less than that of K. pneumoniae. The reduced cell yield was probably due to the lack of the coenzyme-B12-dependent glycerol dehydratase of the dha system. This enzyme initiates the first step in an auxiliary pathway for disposal of the extra reducing equivalents from glycerol. The lack of this enzyme would also account for the absence of 1,3-propanediol (a hallmark fermentation product of glycerol) in the spent culture medium. In a control experiment, a large quantity of this compound was detected in a similar culture medium following the growth of K. pneumoniae. The other three known enzymes of the dha system, glycerol dehydrogenase, dihydroxyacetone kinase and 1,3-propanediol oxidoreductase, however, were synthesized at levels comparable to those found in K. pneumoniae. Regulation of the dha system in E. coli appeared to follow the same pattern as in K. pneumoniae: the three acquired enzymes were induced by glycerol, catabolite repressed by glucose, and glycerol dehydrogenase was post-translationally inactivated during the shift from anaerobic to aerobic growth. The means by which the E. coli recipient can achieve redox balance without formation of 1,3-propanediol during anaerobic growth on glycerol remains to be discovered.     

Immunochemical evidence for the difference between coenzyme-B12-dependent diol dehydratase and glycerol dehydratase.

Klebsiella pneumoniae ATCC 25955 (formerly named Aerobacter aerogenes PZH 572, Warsaw), which is known to produce coenzyme-B12-dependent glycerol dehydratase when grown anaerobically in a glycerol medium, formed coenzyme-B12-dependent diol dehydratase in a 1,2-propanediol-containing medium. Both the diol dehydratase and the glycerol dehydratase produced by the organism catalyzed the conversion of glycerol, 1,2-propanediol and 1,2-ethanediol to the corresponding aldehydes and underwent concomitant inactivation during the catalysis of glycerol dehydration, as does the diol dehydratase of K. pneumoniae (A. aerogenes) ATCC 8724. However, the two enzymes were distinguishable from each other by the monovalent-cation-selectivity pattern and by substrate specificity; that is, glycerol dehydratase preferred glycerol to 1,2-propanediol as a substrate, whereas diol dehydratase preferred 1,2-propanediol to glycerol, as judged from initial velocity studies. Ouchterlony double-diffusion analysis and immunochemical titration with rabbit antiserum against diol dehydratase of K. pneumoniae ATCC 8724 established clearly that the diol dehydratase of K. pneumoniae ATCC 25955 is immunologically similar to that of K. pneumoniae ATCC 8724, while the glycerol dehydratase of the former is different from the diol dehydratase of both strains. Both the enzymes were found to be distributed in several bacteria of the family Enterobacteriaceae.     

Regulation of hydrogenase in Rhizobium japonicum: analysis of mutants altered in regulation by carbon substrates and oxygen

The synthesis of the H2 uptake system in free-living Rhizobium japonicum SR is repressed both by oxygen and by carbon substrates. Mutants selected for the ability to express hydrogenase in 10.0% partial pressure O2 were also less sensitive than the wild type to repression by carbon substrates such as arabinose, glycerol, gluconate, and succinate. The H2 uptake system in another class of mutants, previously shown to be hypersensitive to repression by O2, is also more sensitive to repression by carbon substrates. The oxygen- and carbon-insensitive mutants express the hydrogen uptake system during heterotrophic growth in the absence of hydrogen and thus can be considered constitutive (Hupc). The amount of cytochromes in the Hupc mutants is similar to that in the wild-type strain; however, the Hupc mutants contain greater methylene blue-dependent and O2-dependent hydrogenase activity, both as free-living cells and as bacteroids. Two-dimensional polyacrylamide gel electrophoresis revealed that during heterotrophic growth the Hupc mutant strain SR470 synthesized at least six peptides not found in the wild-type strain. The concentrations of cyclic AMP and guanosine tetraphosphate were similar in strain SR and the Hupc mutants during heterotrophic growth.     

Physiology and Bioenergetics of [NiFe]-Hydrogenase 2-Catalyzed H2-Consuming and H2-Producing Reactions in Escherichia coli

Escherichia coli uptake hydrogenase 2 (Hyd-2) catalyzes the reversible oxidation of H₂ to protons and electrons. Hyd-2 synthesis is strongly upregulated during growth on glycerol or on glycerol-fumarate. Membrane-associated Hyd-2 is an unusual heterotetrameric [NiFe]-hydrogenase that lacks a typical cytochrome b membrane anchor subunit, which transfers electrons to the quinone pool. Instead, Hyd-2 has an additional electron transfer subunit, termed HybA, with four predicted iron-sulfur clusters. Here, we examined the physiological role of the HybA subunit. During respiratory growth with glycerol and fumarate, Hyd-2 used menaquinone/demethylmenaquinone (MQ/DMQ) to couple hydrogen oxidation to fumarate reduction. HybA was essential for electron transfer from Hyd-2 to MQ/DMQ. H₂ evolution catalyzed by Hyd-2 during fermentation of glycerol in the presence of Casamino Acids or in a fumarate reductase-negative strain growing with glycerol-fumarate was also shown to be dependent on both HybA and MQ/DMQ. The uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) inhibited Hyd-2-dependent H₂ evolution from glycerol, indicating the requirement for a proton gradient. In contrast, CCCP failed to inhibit H₂-coupled fumarate reduction. Although a Hyd-2 enzyme lacking HybA could not catalyze Hyd-2-dependent H₂ oxidation or H₂ evolution in whole cells, reversible H₂-dependent reduction of viologen dyes still occurred. Finally, hydrogen-dependent dye reduction by Hyd-2 was reversibly inhibited in extracts derived from cells grown in H₂ evolution mode. Our findings suggest that Hyd-2 switches between H₂-consuming and H₂-producing modes in response to the redox status of the quinone pool. Hyd-2-dependent H₂ evolution from glycerol requires reverse electron transport.     

Cloning and characterization of a plastidic glycerol 3-phosphate dehydrogenase cDNA from Dunaliella salina

A cDNA encoding a nicotinamide adenine dinucleotide (NAD+) -dependent glycerol 3-phosphate dehydrogenase (GPDH) has been cloned by rapid amplification of cDNA ends from Dunaliella salina. The cDNA is 3032 base pairs long with an open reading frame encoding a polypeptide of 701 amino acids. The polypeptide shows high homology with published NAD+ -dependent GPDHs and has at its N-terminal a chloroplast targeting sequence. RNA gel blot analysis was performed to study GPDH gene expression under different conditions, and changes of the glycerol content were monitored. The results indicate that the cDNA may encode an osmoregulated isoform primarily involved in glycerol synthesis. The 701-amino-acid polypeptide is about 300 amino acids longer than previously reported plant NAD+ -dependent GPDHs. This 300-amino-acid fragment has a phosphoserine phosphatase domain. We suggest that the phosphoserine phosphatase domain functions as glycerol 3-phosphatase and that, consequently, NAD+ -dependent GPDH from D. salina can catalyze the step from dihydroxyacetone phosphate to glycerol directly. This is unique and a possible explanation for the fast glycerol synthesis found in D. salina.     

Fps1, a yeast member of the MIP family of channel proteins, is a facilitator for glycerol uptake and efflux and is inactive under osmotic stress.

The Saccharomyces cerevisiae FPS1 gene, which encodes a channel protein belonging to the MIP family, has been isolated previously as a multicopy suppressor of the growth defect of the fdp1 mutant (allelic to GGS1/TPS1) on fermentable sugars. Here we show that overexpression of FPS1 enhances glycerol production. Enhanced glycerol production caused by overexpression of GPD1 encoding glycerol-3-phosphate dehydrogenase also suppressed the growth defect of ggs1/tps1 delta mutants, suggesting a novel role for glycerol production in the control of glycolysis. The suppression of ggs1/tps1 delta mutants by GPD1 depends on the presence of Fps1. Mutants lacking Fps1 accumulate a greater part of the glycerol intracellularly, indicating that Fps1 is involved in glycerol efflux. Glycerol-uptake experiments showed that the permeability of the yeast plasma membrane for glycerol consists of an Fps1-independent component probably due to simple diffusion and of an Fps1-dependent component representing facilitated diffusion. The Escherichia coli glycerol facilitator expressed in a yeast fps1 delta mutant can restore the characteristics of glycerol uptake, production and distribution fully, but restores only partially growth of a ggs1/tps1 delta fps1 delta double mutant on glucose. Fps1 appears to be closed under hyperosmotic stress when survival depends on intracellular accumulation of glycerol and apparently opens rapidly when osmostress is lifted. The osmostress-induced High Osmolarity Glycerol (HOG) response pathway is not required for inactivation of Fps1. We conclude that Fps1 is a regulated yeast glycerol facilitator controlling glycerol production and cytosolic concentration, and might have additional functions.     

Analysis of glycerol dehydrogenase activities present in <Emphasis Type="Italic">Mucor circinelloides</Emphasis> YR-1

Two inducible NADP⁺-dependent glycerol dehydrogenase (GlcDH) activities were identified in Mucor circinelloides strain YR-1. One of these, denoted iGlcDH2, was specifically induced by n-decanol when it was used as sole carbon source in the culture medium, and the second, denoted iGlcDH1, was induced by alcohols and aliphatic or aromatic hydrocarbons when glycerol was used as the only substrate. iGlcDH2 was found to have a much broader substrate specificity than iGlcDH1, with a low activity as an ethanol dehydrogenase with NAD⁺ or NADP⁺ as cofactor. Both isozymes showed an optimum pH for activity of 9.0 in Tris-HCl buffer and are subject to carbon catabolite repression. In contrast, the constitutive NADP⁺-dependent glycerol dehydrogenases (GlcDHI, II, and III) were only present in cell extracts when the fungus was grown in glycolytic carbon sources or glycerol under oxygenation, and their optimum pH was 7.0 in Tris-HCl buffer. In addition to these five NADP⁺-dependent glycerol dehydrogenases, a NAD⁺-dependent alcohol dehydrogenase is also present in glycerol or n-decanol medium; this enzyme was found to have weak activity as a glycerol dehydrogenase.     

Biosynthetic Pathways of Glycerol Accumulation under Salt Stress in Aspergillus nidulans

Redkar, R. J., Locy. R. D., and Singh, N. K. 1995. Biosynthetic pathways of glycerol accumulation under salt stress in Aspergillus nidulans. Experimental Mycology 19, 241-246. A culture of Aspergillus nidulans (FGSC 359) was gradually adapted for growth in media containing up to 2 M NaCl or was exposed to a salt shock with 2 M NaCl. The intracellular glycerol level increased by about 7.9-fold in salt-adapted and 2.4-fold in salt-shocked cultures when compared to the unadapted culture. The biosynthetic pathway involved in the accumulation of glycerol was investigated under long-term salt adaptation and short-term salt shock. Glycerol 3-phosphate dehydrogenase (EC 1.1.1.8) was induced 1.4-fold in salt-shocked but not in salt-adapted cultures. An alternate enzymatic pathway involving glycerol dehydrogenase (NADP⁺-dependent) utilizing dihydroxyacetone (DHA) and/or DL-glyceraldehyde (DL-GAD) was induced by NaCl. DHA-dependent glycerol dehydrogenase activity was induced about 6.3-fold in salt adapted and 1.35-fold in salt-shocked cultures, while DL-GAD-dependent activity was induced about 6.1-fold in salt-adapted and 1.2-fold in salt shocked cultures. However, the level of glycerol dehydrogenase activity with DL-GAD as substrate was 7% of the DHA-dependent activity. We conclude that a salt-inducible NADP⁺-dependent glycerol dehydrogenase activity electrophoretically indistinguishable from previously described glycerol dehydrogenase I results in glycerol accumulation in salt-stressed A. nidulans.     

Towards the exploitation of glycerol's high reducing power in Saccharomyces cerevisiae-based bioprocesses

One advantage of using glycerol as a carbon source for industrial bioprocesses is its higher degree of reduction compared to glucose. In order to exploit this reducing power for the production of reduced compounds thereby significantly increasing maximum theoretical yields, the electrons derived from glycerol oxidation must first be saved in the form of cytosolic NAD(P)H. However, the industrial platform organism Saccharomyces cerevisiae naturally uses an FAD-dependent pathway for glycerol catabolism transferring the electrons to the respiratory chain. Here, we developed a pathway replacement strategy forcing glycerol catabolism through a synthetic, NAD⁺-dependent route. The required expression cassettes were integrated via CRISPR-Cas9 targeting the endogenous GUT1 locus, thereby abolishing the native FAD-dependent pathway. Interestingly, this pathway replacement even established growth in synthetic glycerol medium of strains naturally unable to grow on glycerol and an engineered derivative of CEN.PK even showed the highest ever reported maximum specific growth rate on glycerol (0.26 h⁻¹).     

Minimization of glycerol synthesis in industrial ethanol yeast without influencing its fermentation performance

To synthesize glycerol, a major by-product during anaerobic production of ethanol, the yeast Saccharomyces cerevisiae would consume up to 4% of the sugar feedstock in typical industrial ethanol processes. The present study was dedicated to decreasing the glycerol production mostly in industrial ethanol producing yeast without affecting its desirable fermentation properties including high osmotic and ethanol tolerance, natural robustness in industrial processes. In the present study, the GPD1 gene, encoding NAD+-dependent glycerol-3-phosphate dehydrogenase in an industrial ethanol producing strain of S. cerevisiae, was deleted. Simultaneously, a non-phosphorylating NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPN) from Bacillus cereus was expressed in the mutant deletion of GPD1. Although the resultant strain AG1A (gpd1△ P(PGK)-gapN) exhibited a 48.7±0.3% (relative to the amount of substrate consumed) lower glycerol yield and a 7.6±0.1% (relative to the amount of substrate consumed) higher ethanol yield compared to the wild-type strain, it was sensitive to osmotic stress and failed to ferment on 25% glucose. However, when trehalose synthesis genes TPS1 and TPS2 were over-expressed in the above recombinant strain AG1A, its high osmotic stress tolerance was not only restored but also improved. In addition, this new recombinant yeast strain displayed further reduced glycerol yield, indistinguishable maximum specific growth rate (μ(max)) and fermentation ability compared to the wild type in anaerobic batch fermentations. This study provides a promising strategy to improve ethanol yields by minimization of glycerol production.     

Mapping and cloning of gldA, the structural gene of the Escherichia coli glycerol dehydrogenase.

gldA, the structural gene for the NAD(+)-dependent glycerol dehydrogenase, was mapped at 89.2 min on the Escherichia coli linkage map, cotransducible with, but not adjacent to, the glpFKX operon encoding the proteins for the uptake and phosphorylation of glycerol. gldA was cloned, and its position on the physical map of E. coli was determined. The expression of gldA was induced by hydroxyacetone under stationary-phase growth conditions.     

Regulation of glycerol metabolism in Enterococcus faecalis by phosphoenolpyruvate-dependent phosphorylation of glycerol kinase catalyzed by enzyme I and HPr of the phosphotransferase system.

Using a polyclonal antibody against glycerol kinase from Enterococcus faecalis, we could demonstrate that glycerol kinase is inducible by growth on glycerol-containing medium and that during growth on glycerol the enzyme is mainly phosphorylated. Glucose and other sugars metabolized via the Embden-Meyerhof pathway strongly repressed the synthesis of glycerol kinase, while if glycerol was also present during growth, low activity, reflecting partial induction and the presence of mainly unphosphorylated, less active enzyme, was found. With gluconate, which is also a substrate of the phosphotransferase system, repression of glycerol kinase was less severe, but the enzyme was mainly present in the less active, unphosphorylated form. Effects of growth on different carbon sources on glycerol uptake are also reported.     

Utilization of exogenous glycerophosphodiesters and glycerol 3-phosphate by inositol-starved yeast, Saccharomyces uvarum

Inositol-starved Saccharomyces uvarum cells hydrolyse exogenous glycerophosphodiesters to glycerol 3-phosphate and the corresponding alcohol. Glycerophosphodiesterase activity is highest with glycerophosphoinositol as the substrate, followed by glycerophosphoethanolamine and glycerophosphocholine; the artificial substrate for phosphodiesterases, bis-p-nitrophenylphosphate,is hydrolysed at a similar rate as compared with glycerophosphoinositol. Competition experiments suggest that distinct phosphodiesterases are involved in the hydrolysis of the respective substrates. An Mg2+-dependent glycerophosphate phosphohydrolase with a pH-optimum around neutral cleaves glycerol 3-phosphate to glycerol and orthophosphate. The latter is taken up into cells without first entering the pool of orthophosphate present in the growth medium. Accessibility to substrates with whole cells, adhesion of enzymes to spheroplasts, and solubilization of enzymes by treatment of whole cells with Triton X-100 under mild conditions suggest that phosphodiesterases and glycerol-3-phosphate phosphohydrolase are loosely associated with the outer side of the yeast plasma membrane. Enzyme activities are only marginal in inositol-supplemented cells, but are derepressed not only by inositol deficiency, but also by starvation of orthophosphate.     

Genetic and biochemical interactions involving tricarboxylic acid cycle (TCA) function using a collection of mutants defective in all TCA cycle genes

The eight enzymes of the tricarboxylic acid (TCA) cycle are encoded by at least 15 different nuclear genes in Saccharomyces cerevisiae. We have constructed a set of yeast strains defective in these genes as part of a comprehensive analysis of the interactions among the TCA cycle proteins. The 15 major TCA cycle genes can be sorted into five phenotypic categories on the basis of their growth on nonfermentable carbon sources. We have previously reported a novel phenotype associated with mutants defective in the IDH2 gene encoding the Idh2p subunit of the NAD+-dependent isocitrate dehydrogenase (NAD-IDH). Null and nonsense idh2 mutants grow poorly on glycerol, but growth can be enhanced by extragenic mutations, termed glycerol suppressors, in the CIT1 gene encoding the TCA cycle citrate synthase and in other genes of oxidative metabolism. The TCA cycle mutant collection was utilized to search for other genes that can suppress idh2 mutants and to identify TCA cycle genes that display a similar suppressible growth phenotype on glycerol. Mutations in 7 TCA cycle genes were capable of functioning as suppressors for growth of idh2 mutants on glycerol. The only other TCA cycle gene to display the glycerol-suppressor-accumulation phenotype was IDH1, which encodes the companion Idh1p subunit of NAD-IDH. These results provide genetic evidence that NAD-IDH plays a unique role in TCA cycle function.