Recombinant expression of glpK and glpD genes improves the accumulation of shikimic acid in E. coli grown on glycerol

Shikimic acid (SA) is an industrially important chiral compound used in diverse commercial applications, and the insufficient supply by isolation from plants and expensive chemical synthesis of SA has increased the importance of developing strategies for SA synthesis. In our previous studies, glycerol was observed to be an effective carbon source for SA accumulation in E. coli DHPYAAS-T7, where the PTS operon (ptsHIcrr) and aroL and aroK genes were inactivated, and the tktA, glk, aroE, aroF ^fbr , and aroB genes were overexpressed. For further investigation of the effects of glycerol aerobic fermentation on SA accumulation in E. coli BL21(DE3), the glpD, glpK genes and tktA, glk, aroE, aroF ^fbr , aroB genes were overexpressed simultaneously. The results indicated that SA production was increased 5.6-fold, while the yield was increased 5.3-fold over that of parental strain in shake flasks. It is demonstrated that the aerobic fermentation of glycerol associated with glpD and glpK gene overexpression increased glycerol flux, resulting in higher SA accumulation in E. coli BL21(DE3)-P-DK.     

Synthetic biology platform of CoryneBrick vectors for gene expression in Corynebacterium glutamicum and its application to xylose utilization

Currently, the majority of tools in synthetic biology have been designed and constructed for model organisms such as Escherichia coli and Saccharomyces cerevisiae. In order to broaden the spectrum of organisms accessible to such tools, we established a synthetic biological platform, called CoryneBrick, for gene expression in Corynebacterium glutamicum as a set of E. coli-C. glutamicum shuttle vectors whose elements are interchangeable with BglBrick standard parts. C. glutamicum is an established industrial microorganism for the production of amino acids, proteins, and commercially promising chemicals. Using the CoryneBrick vectors, we showed various time-dependent expression profiles of a red fluorescent protein. This CoryneBrick platform was also applicable for two-plasmid expression systems with a conventional C. glutamicum expression vector. In order to demonstrate the practical application of the CoryneBrick vectors, we successfully reconstructed the xylose utilization pathway in the xylose-negative C. glutamicum wild type by fast BglBrick cloning methods using multiple genes encoding for xylose isomerase and xylulose kinase, resulting in a growth rate of 0.11?±?0.004 h^?1 and a xylose uptake rate of 3.35 mmol/gDW/h when 1 % xylose was used as sole carbon source. Thus, CoryneBrick vectors were shown to be useful engineering tools in order to exploit Corynebacterium as a synthetic platform for the production of chemicals by controllable expression of the genes of interest.     

Glycerol as a substrate for aerobic succinate production in minimal medium with Corynebacterium glutamicum

Corynebacterium glutamicum, an established microbial cell factory for the biotechnological production of amino acids, was recently genetically engineered for aerobic succinate production from glucose in minimal medium. In this work, the corresponding strains were transformed with plasmid pVWEx1-glpFKD coding for glycerol utilization genes from Escherichia coli. This plasmid had previously been shown to allow growth of C. glutamicum with glycerol as sole carbon source. The resulting strains were tested in minimal medium for aerobic succinate production from glycerol, which is a by-product in biodiesel synthesis. The best strain BL-1/pVWEx1-glpFKD formed 79 mM (9.3 g l⁻¹) succinate from 375 mM glycerol, representing 42% of the maximal theoretical yield under aerobic conditions. A specific succinate production rate of 1.55 mmol g⁻¹ (cdw) h⁻¹ and a volumetric productivity of 3.59 mM h⁻¹ were obtained, the latter value representing the highest one currently described in literature. The results demonstrate that metabolically engineered strains of C. glutamicum are well suited for aerobic succinate production from glycerol.     

Engineering a glycerol utilization pathway in Corynebacterium glutamicum for succinate production under O<Subscript>2</Subscript> deprivation

Objective

To explore the glycerol utilization pathway in Corynebacterium glutamicum for succinate production under O₂ deprivation.

Result

Overexpression of a glycerol facilitator, glycerol dehydrogenase and dihydroxyacetone kinase from Escherichia coli K-12 in C. glutamicum led to recombinant strains NC-3G diverting glycerol utilization towards succinate production under O₂ deprivation. Under these conditions, strain NC-3G efficiently consumed glycerol and produced succinate without growth. The recombinant C. glutamicum utilizing glycerol as the sole carbon source showed higher intracellular NADH/NAD⁺ ratio compare with utilizing glucose. The mass conversion of succinate increased from 0.64 to 0.95. Using an anaerobic fed-batch fermentation process, the final strain produced 38.4 g succinate/l with an average yield of 1.02 g/g.

Conclusions

The metabolically-engineered strains showed an efficient succinate production using glycerol as sole carbon source under O₂ deprivation.

     

Microaerobic Conversion of Glycerol to Ethanol in Escherichia coli

Glycerol has become a desirable feedstock for the production of fuels and chemicals due to its availability and low price, but many barriers to commercialization remain. Previous investigators have made significant improvements in the yield of ethanol from glycerol. We have developed a fermentation process for the efficient microaerobic conversion of glycerol to ethanol by Escherichia coli that presents solutions to several other barriers to commercialization: rate, titer, specific productivity, use of inducers, use of antibiotics, and safety. To increase the rate, titer, and specific productivity to commercially relevant levels, we constructed a plasmid that overexpressed glycerol uptake genes dhaKLM, gldA, and glpK, as well as the ethanol pathway gene adhE. To eliminate the cost of inducers and antibiotics from the fermentation, we used the adhE and icd promoters from E. coli in our plasmid, and we implemented glycerol addiction to retain the plasmid. To address the safety issue of off-gas flammability, we optimized the fermentation process with reduced-oxygen sparge gas to ensure that the off-gas remained nonflammable. These advances represent significant progress toward the commercialization of an E. coli-based glycerol-to-ethanol process.     

A CRISPR/dCas9-assisted system to clone toxic genes in Escherichia coli

BackgroundThe cloning of toxic genes in E. coli requires strict regulation of the target genes' leaky expression. Many methods facilitating successful gene cloning of toxic genes are commonly exploited, but the applicability is severely limited.MethodsA CRISPR/dCas9-assisted system was used to clone toxic genes in E. coli. The plasmid-based and genome-integrated systems were designed in this study. And the green fluorescent protein characterization system was used to test the repression efficiency of the two systems.ResultsWe optimized the plasmid-based CRISPR/dCas9-assisted repression system via testing different sgRNAs targeting the Ptrc promoter and achieved inhibition efficiency up to 64.8%. The genome-integrated system represented 35.9% decreased GFP expression and was successfully employed to cloned four toxic genes from Corynebacterium glutamicum in E. coli.ConclusionsUsing this method, we successfully cloned four C. glutamicum-derived toxic genes that had been failed to clone in conventional ways. The CRISPR/dCas9-assisted gene cloning method was a promising tool to facilitate precise gene cloning of different origins in E. coli.General significanceThis system will be useful for cloning toxic genes from different origins in E. coli, and can accelerate the related research of gene characterization and heterologous expression in the metagenomic era.     

Supplementation of Substrate Uptake Gene Enhances the Expression of rhIFN-β in High Cell Density Fed-Batch Cultures of Escherichia coli

Over-expression of recombinant proteins in Escherichia coli triggers a metabolic stress response which causes a sharp decline in both growth and product formation rates post induction. We identified a key down-regulated substrate utilization gene, glycerol kinase (glpK), whose up-regulation could help alleviate this stress response. In a proof of principal study conducted in shake flask cultures, the glpK gene under the “ara” promoter in a pPROLar.A122 vector was co-transformed along with the recombinant interferon-β (rhIFN-β) gene in a pET22b vector into E. coli BL-21(DE3) cells. Co-expression of glpK improved the expression levels of rhIFN-β in glycerol containing medium, while no such gain was observed in medium without glycerol. This study was extended to high cell density fed-batch cultures where exponential feeding of complex substrates was done to increase biomass and hence product titers. For this we first constructed a modified E. coli strain BL-21(glpK ⁺) where the glpK gene was inserted downstream of the ibpA promoter in the host chromosome. There was a significant improvement in growth as well as expression levels of rhIFN-β in this modified strain when the feed medium contained high glycerol. A final product concentration of 4.8 g/l of rhIFN-β was obtained with the modified strain which was 35 % higher than the control.     

Phosphate Starvation-Inducible Gene ushA Encodes a 5′ Nucleotidase Required for Growth of Corynebacterium glutamicum on Media with Nucleotides as the Phosphorus Source

Phosphorus is an essential component of macromolecules, like DNA, and central metabolic intermediates, such as sugar phosphates, and bacteria possess enzymes and control mechanisms that provide an optimal supply of phosphorus from the environment. UDP-sugar hydrolases and 5′ nucleotidases may play roles in signal transduction, as they do in mammals, in nucleotide salvage, as demonstrated for UshA of Escherichia coli, or in phosphorus metabolism. The Corynebacterium glutamicum gene ushA was found to encode a secreted enzyme which is active as a 5′ nucleotidase and a UDP-sugar hydrolase. This enzyme was synthesized and secreted into the medium when C. glutamicum was starved for inorganic phosphate. UshA was required for growth of C. glutamicum on AMP and UDP-glucose as sole sources of phosphorus. Thus, in contrast to UshA from E. coli, C. glutamicum UshA is an important component of the phosphate starvation response of this species and is necessary to access nucleotides and related compounds as sources of phosphorus.     

Genetic Characterization of the Resorcinol Catabolic Pathway in Corynebacterium glutamicum

Corynebacterium glutamicum grew on resorcinol as a sole source of carbon and energy. By genome-wide data mining, two gene clusters, designated NCgl1110-NCgl1113 and NCgl2950-NCgl2953, were proposed to encode putative proteins involved in resorcinol catabolism. Deletion of the NCgl2950-NCgl2953 gene cluster did not result in any observable phenotype changes. Disruption and complementation of each gene at NCgl1110-NCgl1113, NCgl2951, and NCgl2952 indicated that these genes were involved in resorcinol degradation. Expression of NCgl1112, NCgl1113, and NCgl2951 in Escherichia coli revealed that NCgl1113 and NCgl2951 both coded for hydroxyquinol 1,2-dioxygenases and NCgl1112 coded for maleylacetate reductases. NCgl1111 encoded a putative monooxygenase, but this putative hydroxylase was very different from previously functionally identified hydroxylases. Cloning and expression of NCgl1111 in E. coli revealed that NCgl1111 encoded a resorcinol hydroxylase that needs NADPH as a cofactor. E. coli cells containing Ncgl1111 and Ncgl1113 sequentially converted resorcinol into maleylacetate. NCgl1110 and NCgl2950 both encoded putative TetR family repressors, but only NCgl1110 was transcribed and functional. NCgl2953 encoded a putative transporter, but disruption of this gene did not affect resorcinol degradation by C. glutamicum. The function of NCgl2953 remains unclear.     

Design of a recombinant Escherichia coli for producing l-phenylalanine from glycerol

A recombinant Escherichia coli was engineered to produce the commercially important amino acid L: -phenylalanine (L: -Phe) using glycerol as the carbon source. Compared to the conventionally used glucose and sucrose, glycerol is a less expensive carbon source. As phenylalanine dehydrogenase (PheDH) activity is involved in the last step of L: -Phe synthesis in E. coli, a phenylalanine dehydrogenase gene (phedh) from the thermotolerant Bacillus lentus was cloned into pRSFDuet-1 (pPheDH) and expressed in E. coli BL21(DE3). The resulting clone had a limited ability to produce L: -Phe from glycerol, possibly because of a poor glycerol uptake by the cell, or an inability to excrete L: -Phe, or both. Therefore, yddG gene encoding an aromatic amino acid exporter and glpF gene encoding a glycerol transport facilitator were coexpressed with the phedh in a reengineered E. coli. In a glycerol medium, the maximum L: -Phe production rates of the clones pPY (phedh and yddG genes) and pPYF (phedh, yddG and glpF genes) were 1.4- and 1.8-fold higher than the maximum production rate of the pPheDH clone. The better producing pPYF clone was further evaluated in a 5?l stirred-tank fermenter (37?°C, an aeration rate of 1 vvm, an agitation speed of 400?rpm). In the fermenter, the maximum concentration of L: -Phe (366?mg/l) was achieved in a much shorter period compared to in the shake flasks. In the latter, the highest titer of L: -Phe was only 76?% of the maximum value attained in the fermenter.     

Impact of Heterologous Expression of Escherichia coli UDP-Glucose Pyrophosphorylase on Trehalose and Glycogen Synthesis in Corynebacterium glutamicum

Trehalose is a disaccharide with a wide range of applications in the food industry. We recently proposed a strategy for trehalose production based on improved strains of the gram-positive bacterium Corynebacterium glutamicum. This microorganism synthesizes trehalose through two major pathways, OtsBA and TreYZ, by using UDP-glucose and ADP-glucose, respectively, as the glucosyl donors. In this paper we describe improvement of the UDP-glucose supply through heterologous expression in C. glutamicum of the UDP-glucose pyrophosphorylase gene from Escherichia coli, either expressed alone or coexpressed with the E. coli ots genes (galU otsBA synthetic operon). The impact of such expression on trehalose accumulation and excretion, glycogen accumulation, and the growth pattern of new recombinant strains is described. Expression of the galU otsBA synthetic operon resulted in a sixfold increase in the accumulated and excreted trehalose relative to that in a wild-type strain. Surprisingly, single expression of galU also resulted in an increase in the accumulated trehalose. This increase in trehalose synthesis was abolished upon deletion of the TreYZ pathway. These results proved that UDP-glucose has an important role not only in the OtsBA pathway but also in the TreYZ pathway.     

Putrescine production by engineered Corynebacterium glutamicum

Here, we report the engineering of the industrially relevant Corynebacterium glutamicum for putrescine production. C. glutamicum grew well in the presence of up to 500 mM of putrescine. A reduction of the growth rate by 34% and of biomass formation by 39% was observed at 750 mM of putrescine. C. glutamicum was enabled to produce putrescine by heterologous expression of genes encoding enzymes of the arginine- and ornithine decarboxylase pathways from Escherichia coli. The results showed that the putrescine yield by recombinant C. glutamicum strains provided with the arginine-decarboxylase pathway was 40 times lower than the yield by strains provided with the ornithine decarboxylase pathway. The highest production efficiency was reached by overexpression of speC, encoding the ornithine decarboxylase from E. coli, in combination with chromosomal deletion of genes encoding the arginine repressor ArgR and the ornithine carbamoyltransferase ArgF. In shake-flask batch cultures this strain produced putrescine up to 6 g/L with a space time yield of 0.1 g/L/h. The overall product yield was about 24 mol% (0.12 g/g of glucose).     

Enhanced production of l-phenylalanine in Corynebacterium glutamicum due to the introduction of Escherichia coli wild-type gene aroH

Metabolic engineering is a powerful tool which has been widely used for producing valuable products. For improving l-phenylalanine (l-Phe) accumulation in Corynebacterium glutamicum, we have investigated the target genes involved in the biosynthetic pathways. The genes involved in the biosynthesis of l-Phe were found to be strictly regulated genes by feedback inhibition. As a result, overexpression of the native wild-type genes aroF, aroG or pheA resulted in a slight increase of l-Phe. In contrast, overexpression of aroF ^wt or pheA ^fbr from E. coli significantly increased l-Phe production. Co-overexpression of aroF ^wt and pheA ^fbr improved the titer of l-Phe to 4.46 ± 0.06 g l^?1. To further analyze the target enzymes in the aromatic amino acid synthesis pathway between C. glutamicum and E. coli, the wild-type gene aroH from E. coli was overexpressed and evaluated in C. glutamicum. As predicted, upregulation of the wild-type gene aroH resulted in a remarkable increase of l-Phe production. Co-overexpression of the mutated pheA ^fbr and the wild-type gene aroH resulted in the production of l-Phe up to 4.64 ± 0.09 g l^?1. Based on these results we conclude that the wild-type gene aroH from E. coli is an appropriate target gene for pathway engineering in C. glutamicum for the production of aromatic amino acids.     

Enhancement of glycerol utilization ability of Ralstonia eutropha H16 for production of polyhydroxyalkanoates

Ralstonia eutropha H16 is a well-studied bacterium with respect to biosynthesis of polyhydroxyalkanoates (PHAs), which has attracted attentions as biodegradable bio-based plastics. However, this strain shows quite poor growth on glycerol of which bulk supply has been increasing as a major by-product of biodiesel industries. This study examined enhancement of glycerol assimilation ability of R. eutropha H16 by introduction of the genes of aquaglyceroporin (glpF) and glycerol kinase (glpK) from Escherichia coli. Although introduction of glpFK _Ec into the strain H16 using a multi-copy vector was not successful, a recombinant strain possessing glpFK _Ec within the chromosome showed much faster growth on glycerol than H16. Further analyses clarified that weak expression of glpK _Ec alone allowed to establish efficient glycerol assimilation pathway, indicating that the poor growth of H16 on glycerol was caused by insufficient kination activity to glycerol, as well as this strain had a potential ability for uptake of extracellular glycerol. The engineered strains expressing glpFK _Ec or glpK _Ec produced large amounts of poly[(R)-3-hydroxybutyrate] [P(3HB)] from glycerol with much higher productivity than H16. Unlike other glycerol-utilizable wild strains of R. eutropha, the H16-derived engineered strains accumulated P(3HB) with no significant decrease in molecular weights on glycerol, and the polydispersity index of the glycerol-based P(3HB) synthesized by the strains expressing glpFK _Ec was lower than those by the parent strains. The present study demonstrated possibility of R. eutropha H16-based platform for production of useful compounds from inexpensive glycerol.     

Heterologous Carotenoid-Biosynthetic Enzymes: Functional Complementation and Effects on Carotenoid Profiles in Escherichia coli

A limited number of carotenoid pathway genes from microbial sources have been studied for analyzing the pathway complementation in the heterologous host Escherichia coli. In order to systematically investigate the functionality of carotenoid pathway enzymes in E. coli, the pathway genes of carotenogenic microorganisms (Brevibacterium linens, Corynebacterium glutamicum, Rhodobacter sphaeroides, Rhodobacter capsulatus, Rhodopirellula baltica, and Pantoea ananatis) were modified to form synthetic expression modules and then were complemented with Pantoea agglomerans pathway enzymes (CrtE, CrtB, CrtI, CrtY, and CrtZ). The carotenogenic pathway enzymes in the synthetic modules showed unusual activities when complemented with E. coli. For example, the expression of heterologous CrtEs of B. linens, C. glutamicum, and R. baltica influenced P. agglomerans CrtI to convert its substrate phytoene into a rare product—3,4,3′,4′-tetradehydrolycopene—along with lycopene, which was an expected product, indicating that CrtE, the first enzyme in the carotenoid biosynthesis pathway, can influence carotenoid profiles. In addition, CrtIs of R. sphaeroides and R. capsulatus converted phytoene into an unusual lycopene as well as into neurosporene. Thus, this study shows that the functional complementation of pathway enzymes from different sources is a useful methodology for diversifying biosynthesis as nature does.     

Functional Identification of Novel Genes Involved in the Glutathione-Independent Gentisate Pathway in Corynebacterium glutamicum

Corynebacterium glutamicum used gentisate and 3-hydroxybenzoate as its sole carbon and energy source for growth. By genome-wide data mining, a gene cluster designated ncg12918-ncg12923 was proposed to encode putative proteins involved in gentisate/3-hydroxybenzoate pathway. Genes encoding gentisate 1,2-dioxygenase (ncg12920) and fumarylpyruvate hydrolase (ncg12919) were identified by cloning and expression of each gene in Escherichia coli. The gene of ncg12918 encoding a hypothetical protein (Ncg12918) was proved to be essential for gentisate-3-hydroxybenzoate assimilation. Mutant strain RES167Δncg12918 lost the ability to grow on gentisate or 3-hydroxybenzoate, but this ability could be restored in C. glutamicum upon the complementation with pXMJ19-ncg12918. Cloning and expression of this ncg12918 gene in E. coli showed that Ncg12918 is a glutathione-independent maleylpyruvate isomerase. Upstream of ncg12920, the genes ncg12921-ncg12923 were located, which were essential for gentisate and/or 3-hydroxybenzoate catabolism. The Ncg12921 was able to up-regulate gentisate 1,2-dioxygenase, maleylpyruvate isomerase, and fumarylpyruvate hydrolase activities. The genes ncg12922 and ncg12923 were deduced to encode a gentisate transporter protein and a 3-hydroxybenzoate hydroxylase, respectively, and were essential for gentisate or 3-hydroxybenzoate assimilation. Based on the results obtained in this study, a GSH-independent gentisate pathway was proposed, and genes involved in this pathway were identified.     

Construction and Screening of Metagenomic Libraries Derived from Enrichment Cultures: Generation of a Gene Bank for Genes Conferring Alcohol Oxidoreductase Activity on Escherichia coli

Enrichment of microorganisms with special traits and the construction of metagenomic libraries by direct cloning of environmental DNA have great potential for identifying genes and gene products for biotechnological purposes. We have combined these techniques to isolate novel genes conferring oxidation of short-chain (C₂ to C₄) polyols or reduction of the corresponding carbonyls. In order to favor the growth of microorganisms containing the targeted genes, samples collected from four different environments were incubated in the presence of glycerol and 1,2-propanediol. Subsequently, the DNA was extracted from the four samples and used to construct complex plasmid libraries. Approximately 100,000 Escherichia coli strains of each library per test substrate were screened for the production of carbonyls from polyols on indicator agar. Twenty-four positive E. coli clones were obtained during the initial screen. Sixteen of them contained a plasmid (pAK101 to pAK116) which conferred a stable carbonyl-forming phenotype. Eight of the positive clones exhibited NAD(H)-dependent alcohol oxidoreductase activity with polyols or carbonyls as the substrates in crude extracts. Sequencing revealed that the inserts of pAK101 to pAK116 encoded 36 complete and 17 incomplete presumptive protein-encoding genes. Fifty of these genes showed similarity to sequenced genes from a broad collection of different microorganisms. The genes responsible for the carbonyl formation of E. coli were identified for nine of the plasmids (pAK101, pAK102, pAK105, pAK107 to pAK110, pAK115, and pAK116). Analyses of the amino acid sequences deduced from these genes revealed that three (orf12, orf14, and orf22) encoded novel alcohol dehydrogenases of different types, four (orf5, sucB, fdhD, and yabF) encoded novel putative oxidoreductases belonging to groups distinct from alcohol dehydrogenases, one (glpK) encoded a putative glycerol kinase, and one (orf1) encoded a protein which showed no similarity to any other known gene product.     

Metabolic engineering of Corynebacterium glutamicum to produce GDP-l-fucose from glucose and mannose

Wild-type Corynebacterium glutamicum was metabolically engineered to convert glucose and mannose into guanosine 5′-diphosphate (GDP)-l-fucose, a precursor of fucosyl-oligosaccharides, which are involved in various biological and pathological functions. This was done by introducing the gmd and wcaG genes of Escherichia coli encoding GDP-d-mannose-4,6-dehydratase and GDP-4-keto-6-deoxy-d-mannose-3,5-epimerase-4-reductase, respectively, which are known as key enzymes in the production of GDP-l-fucose from GDP-d-mannose. Coexpression of the genes allowed the recombinant C. glutamicum cells to produce GDP-l-fucose in a minimal medium containing glucose and mannose as carbon sources. The specific product formation rate was much higher during growth on mannose than on glucose. In addition, the specific product formation rate was further increased by coexpressing the endogenous phosphomanno-mutase gene (manB) and GTP-mannose-1-phosphate guanylyl-transferase gene (manC), which are involved in the conversion of mannose-6-phosphate into GDP-d-mannose. However, the overexpression of manA encoding mannose-6-phosphate isomerase, catalyzing interconversion of mannose-6-phosphate and fructose-6-phosphate showed a negative effect on formation of the target product. Overall, coexpression of gmd, wcaG, manB and manC in C. glutamicum enabled production of GDP-l-fucose at the specific rate of 0.11 mg g cell^?1 h^?1. The specific GDP-l-fucose content reached 5.5 mg g cell^?1, which is a 2.4-fold higher than that of the recombinant E. coli overexpressing gmd, wcaG, manB and manC under comparable conditions. Well-established metabolic engineering tools may permit optimization of the carbon and cofactor metabolisms of C. glutamicum to further improve their production capacity.     

Glycerol Metabolism Is Important for Cytotoxicity of Mycoplasma pneumoniae

Glycerol is one of the few carbon sources that can be utilized by Mycoplasma pneumoniae. Glycerol metabolism involves uptake by facilitated diffusion, phosphorylation, and the oxidation of glycerol 3-phosphate to dihydroxyacetone phosphate, a glycolytic intermediate. We have analyzed the expression of the genes involved in glycerol metabolism and observed constitutive expression irrespective of the presence of glycerol or preferred carbon sources. Similarly, the enzymatic activity of glycerol kinase is not modulated by HPr-dependent phosphorylation. This lack of regulation is unique among the bacteria for which glycerol metabolism has been studied so far. Two types of enzymes catalyze the oxidation of glycerol 3-phosphate: oxidases and dehydrogenases. Here, we demonstrate that the enzyme encoded by the M. pneumoniae glpD gene is a glycerol 3-phosphate oxidase that forms hydrogen peroxide rather than NADH₂. The formation of hydrogen peroxide by GlpD is crucial for cytotoxic effects of M. pneumoniae. A glpD mutant exhibited a significantly reduced formation of hydrogen peroxide and a severely reduced cytotoxicity. Attempts to isolate mutants affected in the genes of glycerol metabolism revealed that only the glpD gene, encoding the glycerol 3-phosphate oxidase, is dispensable. In contrast, the glpF and glpK genes, encoding the glycerol facilitator and the glycerol kinase, respectively, are essential in M. pneumoniae. Thus, the enzymes of glycerol metabolism are crucial for the pathogenicity of M. pneumoniae but also for other essential, yet-to-be-identified functions in the M. pneumoniae cell.Mycoplasma pneumoniae causes infections of the upper and lower respiratory tracts. These bacteria are responsible for a large fraction of community-acquired pneumonias. Although usually harmless for adult patients, M. pneumoniae may cause severe disease in children or elderly people. In addition, M. pneumoniae is involved in extrapulmonary complications such as pediatric encephalitis and erythema multiforme (for reviews, see references 15, 21, and 34).M. pneumoniae and its relatives, the Mollicutes, are all characterized by the lack of a cell wall and a very close adaptation to a life within a eukaryotic host. This close adaptation is reflected by degenerative genome evolution that resulted in an extreme genome reduction. As a result, the Mollicutes are the organisms that are capable of independent life with the smallest known genome. M. pneumoniae has a genome of 816 kb and encodes only 688 proteins (18). This genome reduction is taken even further in the close relative Mycoplasma genitalium, which has only 482 protein-coding genes (18). Thus, the analysis of the Mollicutes allows us to study a minimal form of natural life. This question has recently attracted much interest and resulted in the determination of the essential gene sets of M. pneumoniae, M. genitalium, and, more recently, Mycoplasma pulmonis (6, 20). In M. genitalium, with the most reduced genomes, only 100 out of the 482 protein-coding genes are dispensable, suggesting that the remaining 382 genes form the essential gene set (7).Reductive genome evolution in M. pneumoniae is still under way: the genes for the utilization of mannitol as a carbon source seem to be present in M. pneumoniae; however, this substrate cannot be used by the bacteria. M. genitalium, which is further advanced in genome reduction, has lost the genes for mannitol transport and oxidation. It was therefore suggested that the genes for mannitol utilization in M. pneumoniae either are not expressed or encode inactive proteins (12).In M. pneumoniae as well as in other Mollicutes, pathogenicity is closely linked to carbon metabolism (13). M. pneumoniae can use glucose, fructose, and glycerol as the only carbon sources (12). Studies with Mycoplasma mycoides revealed that glycerol metabolism has a major impact on the pathogenicity of these bacteria. Oxidation of glycerol involves the glycerol 3-phosphate oxidase, which produces hydrogen peroxide rather than NADH₂, which is generated by the glycerol 3-phosphate dehydrogenase in most other bacteria (28). In addition to the induction of autoimmune responses, the formation of hydrogen peroxide is the only established mechanism by which mycoplasmas cause damage to their hosts (31, 34). Pathogenic strains of M. mycoides possess a highly active ABC transport system for glycerol in addition to the ubiquitous glycerol facilitator (33). The efficient formation of hydrogen peroxide by the membrane-bound glycerol 3-phosphate oxidase is the major virulence factor of the highly pathogenic strains of M. mycoides (28).M. pneumoniae possesses the complete set of genes for glycerol utilization, and the bacteria do indeed use this carbon source (12). The first component in glycerol metabolism is the glycerol facilitator encoded by the glpF gene. The transported glycerol is then phosphorylated by the glycerol kinase (product of glpK), and glycerol 3-phosphate is subsequently oxidized to dihydroxyacetone phosphate, a glycolytic intermediate. The relevant enzyme is annotated as glycerol 3-phosphate dehydrogenase (encoded by the gene glpD) in M. pneumoniae (17).In all organisms studied so far, glycerol metabolism is under dual control: the genes involved in glycerol utilization are expressed only if glycerol or glycerol 3-phosphate is present in the medium, and they are not expressed in the presence of glucose, the preferred carbon source (3, 4). This second mode of regulation, carbon catabolite repression, involves two distinct mechanisms in the Firmicutes, from which the Mollicutes evolved. In the presence of preferred sugars, the CcpA repressor protein binds in the promoter regions of glycerol utilization genes and prevents their expression. Moreover, the molecular inducer of the system, glycerol 3-phosphate, is formed only in the absence of glucose. This results from the low activity of the glycerol kinase. This enzyme is activated upon phosphorylation by HPr, a protein of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). HPr can phosphorylate other proteins only in the absence of glucose, thus providing a link between glucose availability, the activity of the glycerol kinase, and the induction of the glycerol utilization genes (3). Nothing is known about the regulation of glycerol utilization in any member of the Mollicutes; however, regulatory events seem to be rare in these organisms due to the lack of regulatory proteins, among them CcpA.In this work, we studied the mechanisms of glycerol utilization in M. pneumoniae, its regulation, and its contribution to cytotoxicity. We demonstrate constitutive expression of the genes for glycerol utilization in M. pneumoniae. As observed in M. mycoides, glycerol 3-phosphate oxidation involves the formation of hydrogen peroxide and is important for damaging the host cells.