Novel aspects of ethylene glycol catabolism.

Ethylene glycol (EG) is an industrially important two-carbon diol used as a solvent, antifreeze agent, and building block of polymers such as poly(ethylene terephthalate) (PET). Recently, the use of EG as a starting material for the production of bio-fuels or bio-chemicals is gaining attention as a sustainable process since EG can be derived from materials not competing with human food stocks including CO2, syngas, lignocellulolytic biomass, and PET waste. In order to design and construct microbial process for the conversion of EG to value-added chemicals, microbes capable of catabolizing EG such as Escherichia coli, Pseudomonas putida, Rhodococcus jostii, Ideonella sakaiensis, Paracoccus denitrificans, and Acetobacterium woodii are candidates of chassis for the construction of synthetic pathways. In this mini-review, we describe EG catabolic pathways and catabolic enzymes in these microbes, and further review recent advances in microbial conversion of EG to value-added chemicals by means of metabolic engineering. KEY POINTS: • Ethylene glycol is a potential next-generation feedstock for sustainable industry. • Microbial conversion of ethylene glycol to value-added chemicals is gaining attention. • Ethylene glycol-utilizing microbes are useful as chassis for synthetic pathways.

A mycofactocin-associated dehydrogenase is essential for ethylene glycol metabolism by Rhodococcus jostii RHA1.

Ethylene glycol is an industrially important diol in many manufacturing processes and a building block of polymers, such as poly(ethylene terephthalate). In this study, we found that a mycolic acid-containing bacterium Rhodococcus jostii RHA1 can grow with ethylene glycol as a sole source of carbon and energy. Deletion of a putative glycolate dehydrogenase gene (RHA1_ro03227) abolished growth with ethylene glycol, indicating that ethylene glycol is assimilated via glycolate in R. jostii RHA1. Transcriptome sequencing and gene deletion analyses revealed that a gene homologous to mycofactocin (MFT)-associated dehydrogenase (RHA1_ro06057), hereafter referred to as EgaA, is essential for ethylene glycol assimilation. Furthermore, egaA deletion also negatively affected the utilization of ethanol, 1-propanol, propylene glycol, and 1-butanol, suggesting that EgaA is involved in the utilization of various alcohols in R. jostii RHA1. Deletion of MFT biosynthetic genes abolished growth with ethylene glycol, indicating that MFT is the physiological electron acceptor of EgaA. Further genetic studies revealed that a putative aldehyde dehydrogenase (RHA1_ro06081) is a major aldehyde dehydrogenase in ethylene glycol metabolism by R. jostii RHA1. KEY POINTS: • Rhodococcus jostii RHA1 can assimilate ethylene glycol via glycolate • A mycofactocin-associated dehydrogenase is involved in the oxidation of ethylene glycol • An aldehyde dehydrogenase gene is important for the ethylene glycol assimilation.

Regulation of Ribonuclease J Expression in Corynebacterium glutamicum.

RNase J exerts both 5'-3' exoribonuclease and endoribonuclease activities and plays a major role in ribonucleotide metabolism in various bacteria; however, its gene regulation is not well understood. In this study, we investigated the regulation of rnj expression in Corynebacterium glutamicum. rnj mRNA expression was increased in a strain with an rnj mutation. Deletion of the genes encoding RNase E/G also resulted in increased rnj mRNA levels, although the effect was smaller than that of the rnj mutation. rnj mRNA was more stable in the rnj mutant strain than in wild-type cells. These results indicate that RNase J regulates its own gene by degrading its mRNA. The growth of rnj and pnp mutant cells was impaired at cold temperatures. The expression of rnj mRNA was transiently induced by cold shock; however, this induction was not observed in the rnj mutant strain, suggesting that autoregulation by self-degradation is responsible for inducing of rnj expression under cold-shock conditions. IMPORTANCE Corynebacterium glutamicum harbors one RNase E/G-type enzyme and one RNase J-type enzyme which are major ribonucleases in various bacteria. However, little is known about these gene regulations. Here, we show that RNase J autoregulates its own gene expression and RNase E/G is also involved in the rnj mRNA degradation. Furthermore, we show that transient induction of the rnj mRNA in the cold-shock condition is dependent on RNase J autoregulation. This study sheds light on the regulatory mechanism of RNase J in C. glutamicum.

Construction of a Rhodobacter sphaeroides Strain That Efficiently Produces Hydrogen Gas from Acetate without Poly(ß-Hydroxybutyrate) Accumulation: Insight into the Role of PhaR in Acetate Metabolism.

The purple nonsulfur phototrophic bacterium Rhodobacter sphaeroides produces hydrogen gas (H2) from acetate. An approach to improve the H2 production is preventing accumulation of an intracellular energy storage molecule known as poly(ß-hydroxybutyrate) (PHB), which competes with H2 production for reducing power. However, disruption of PHB biosynthesis has been reported to severely impair the acetate assimilation depending on the genetic backgrounds and/or culture conditions. To solve this problem, we analyzed the relationship between PHB accumulation and acetate metabolism in R. sphaeroides. Gene deletion analyses based on the wild-type strain revealed that among the two polyhydroxyalkanoate synthase genes in the genome, phaC1, but not phaC2, is essential for PHB accumulation, and the phaC1 deletion mutant exhibited slow growth with acetate. On the other hand, a strain with the deletion of phaC1 together with phaR, which encodes a transcriptional regulator capable of sensing PHB accumulation, exhibited growth comparable to that of the wild-type strain despite no accumulation of PHB. These results suggest that PHB accumulation is required for normal growth with acetate by altering the expression of genes under the control of phaR. This hypothesis was supported by a transcriptome sequencing (RNA-seq) analysis revealing that phaR is involved in the regulation of the ethylmalonyl coenzyme A pathway for acetate assimilation. Consistent with these findings, deletion of phaC1 in a genetically engineered H2-producing strain resulted in lower H2 production from acetate due to growth defects, whereas deletion of phaR together with phaC1 restored growth with acetate and increased H2 production from acetate without PHB accumulation. IMPORTANCE This study provides a novel approach for increasing the yield of photofermentative H2 production from acetate by purple nonsulfur phototrophic bacteria. This study further suggests that polyhydroxyalkanoate is not only a storage substance for carbon and energy in bacteria, but may also act as a signaling molecule that mediates bacterial metabolic adaptations to specific environments. This notion will be helpful for understanding the physiology of polyhydroxyalkanoate-producing bacteria, as well as for their metabolic engineering via synthetic biology.

Protocatechuate overproduction by Corynebacterium glutamicum via simultaneous engineering of native and heterologous biosynthetic pathways.

Protocatechuic acid (3, 4-dihydroxybenzoic acid, PCA) is a natural bioactive phenolic acid potentially valuable as a pharmaceutical raw material owing to its diverse pharmacological activities. Corynebacterium glutamicum forms PCA as a key intermediate in a native pathway to assimilate shikimate/quinate through direct conversion of the shikimate pathway intermediate 3-dehydroshikimate (DHS), which is catalyzed by qsuB-encoded DHS dehydratase (the DHS pathway). PCA can also be formed via an alternate pathway extending from chorismate by introducing heterologous chorismate pyruvate lyase that converts chorismate into 4-hydroxybenzoate (4-HBA), which is then converted into PCA catalyzed by endogenous 4-HBA 3-hydroxylase (the 4-HBA pathway). In this study, we generated three plasmid-free C. glutamicum strains overproducing PCA based on the markerless chromosomal recombination by engineering each or both of the above mentioned two PCA-biosynthetic pathways combined with engineering of the host metabolism to enhance the shikimate pathway flux and to block PCA consumption. Aerobic growth-arrested cell reactions were performed using the resulting engineered strains, which revealed that strains dependent on either the DHS or 4-HBA pathway as the sole PCA-biosynthetic route produced 43.8 and 26.2 g/L of PCA from glucose with a yield of 35.3% and 10.0% (mol/mol), respectively, indicating that PCA production through the DHS pathway is significantly efficient compared to that produced through the 4-HBA pathway. Remarkably, a strain simultaneously using both DHS and 4-HBA pathways achieved the highest reported PCA productivity of 82.7 g/L with a yield of 32.8% (mol/mol) from glucose in growth-arrested cell reaction. These results indicated that simultaneous engineering of both DHS and 4-HBA pathways is an efficient method for PCA production. The generated PCA-overproducing strain is plasmid-free and does not require supplementation of aromatic amino acids and vitamins due to the intact shikimate pathway, thereby representing a promising platform for the industrial bioproduction of PCA and derived chemicals from renewable sugars.

Isobutanol production in Corynebacterium glutamicum: Suppressed succinate by-production by pckA inactivation and enhanced productivity via the Entner-Doudoroff pathway.

On the basis of our previous studies of microbial L-valine production under oxygen deprivation, we developed isobutanol-producing Corynebacterium glutamicum strains. The artificial isobutanol synthesis pathway was composed of the first three steps of the L-valine synthesis pathway; and the subsequent Ehrlich Pathway: pyruvate was converted to 2-ketoisovalerate in the former reactions; and the 2-keto acid was decarboxylated into isobutyraldehyde, and subsequently reduced into isobutanol in the latter reactions. Although there exists redox cofactor imbalance in the overall reactions, i.e., NADH is generated via glycolysis whereas NADPH is required to synthesize isobutanol, it was resolved by taking advantage of the NAD-preferring mutant acetohydroxy acid isomeroreductase encoded by ilvCTM and the NAD-specific alcohol dehydrogenase encoded by adhA. Each enzyme activity to synthesize isobutanol was finely tuned by using two kinds of lac promoter derivatives. Efficient suppression of succinate by-production and improvement of isobutanol yield resulted from inactivation of pckA, which encodes phosphoenolpyruvate carboxykinase, whereas glucose consumption and isobutanol production rates decreased because of the elevated intracellular NADH/NAD+ ratio. On the other hand, introduction of the exogenous Entner-Doudoroff pathway effectively enhanced glucose consumption and productivity. Overexpression of phosphoenolpyruvate:carbohydrate phosphotransferase system specific to glucose and deletion of ilvE, which encodes branched-chain amino acid transaminase, further suppressed by-products and improved isobutanol productivity. Finally, the produced isobutanol concentration reached 280 mM at a yield of 84% (mol/mol glucose) in 24 h.

Evaluating the Accuracy of the Endoscopic ABC Classification System in Diagnosing Helicobacter pylori-Infected Gastritis.

AIMS: Evaluating the accuracy of the modified Endoscopic ABC (Endo ABC) classification with an electronic endoscopy with narrow band imaging without magnification in diagnosing Helicobacter pylori (H. pylori)-infected gastritis. METHODS: A total of 576 patients were enrolled and they underwent modified Endo ABC. They were stratified into 5 groups (A to E) based on the grades of endoscopic findings. H. pylori-infected gastritis status was determined in the following ways: current H. pylori gastritis was defined as active gastritis and/or chronic atrophic gastritis (CAG) seen on endoscopy and positive H. pylori test, naïve H. pylori gastritis was defined as regular arrangement of collecting venules in the angle of the lesser curvature without CAG and negative H. pylori test, and previous H. pylori gastritis was defined as negative H. pylori tests regardless of the presence of CAG. RESULTS: Endo A has 97% accuracy and 100% positive predictive value in diagnosing naïve H. pylori gastritis. Endo E has 97% accuracy and 100% positive predictive value in diagnosing previous H. pylori gastritis. The accuracy of Endo B and Endo C in diagnosing current H. pylori gastritis was 89 and 82% respectively. Endo D has 87% accuracy in diagnosing previous H. pylori gastritis. CONCLUSION: This study showed that the modified Endo ABC classification enables to accurately determine the H. pylori-infected gastritis status.

Anaerobic glucose consumption is accelerated at non-proliferating elevated temperatures through upregulation of a glucose transporter gene in Corynebacterium glutamicum.

Cell proliferation is achieved through numerous enzyme reactions. Temperature governs the activity of each enzyme, ultimately determining the optimal growth temperature. The synthesis of useful chemicals and fuels utilizes a fraction of available metabolic pathways, primarily central metabolic pathways including glycolysis and the tricarboxylic acid cycle. However, it remains unclear whether the optimal temperature for these pathways is correlated with that for cell proliferation. Here, we found that wild-type Corynebacterium glutamicum displayed increased glycolytic activity under non-growing anaerobic conditions at 42.5 °C, at which cells do not proliferate under aerobic conditions. At this temperature, glucose consumption was not inhibited and increased by 28% compared with that at the optimal growth temperature of 30 °C. Transcriptional analysis revealed that a gene encoding glucose transporter (iolT2) was upregulated by 12.3-fold compared with that at 30 °C, with concomitant upregulation of NCgl2954 encoding the iolT2-regulating transcription factor. Deletion of iolT2 decreased glucose consumption rate at 42.5 °C by 28%. Complementation of iolT2 restored glucose consumption rate, highlighting the involvement of iolT2 in the accelerating glucose consumption at an elevated temperature. This study shows that the optimal temperature for glucose metabolism in C. glutamicum under anaerobic conditions differs greatly from that for cell growth under aerobic conditions, being beyond the upper limit of the growth temperature. This is beneficial for fuel and chemical production not only in terms of increasing productivity but also for saving cooling costs. KEY POINTS: • C. glutamicum accelerated anaerobic glucose consumption at elevated temperature. • The optimal temperature for glucose consumption was above the upper limit for growth. • Gene expression involved in glucose transport was upregulated at elevated temperature. Graphical abstract.

Extracytoplasmic function sigma factor σD confers resistance to environmental stress by enhancing mycolate synthesis and modifying peptidoglycan structures in Corynebacterium glutamicum.

Mycolates are α-branched, ß-hydroxylated, long-chain fatty acid specifically synthesized in bacteria in the suborder Corynebacterineae of the phylum Actinobacteria. They form an outer membrane, which functions as a permeability barrier and confers pathogenic mycobacteria to resistance to antibiotics. Although the mycolate biosynthetic pathway has been intensively studied, knowledge of transcriptional regulation of genes involved in this pathway is limited. Here, we report that the extracytoplasmic function sigma factor σD is a key regulator of the mycolate synthetic genes in Corynebacterium glutamicum in the suborder. Chromatin immunoprecipitation with microarray analysis detected σD -binding regions in the genome, establishing a consensus promoter sequence for σD recognition. The σD regulon comprised acyl-CoA carboxylase subunits, acyl-AMP ligase, polyketide synthase and mycolyltransferases; they were involved in mycolate synthesis. Indeed, deletion or overexpression of sigD encoding σD modified the extractable mycolate amount. Immediately downstream of sigD, rsdA encoded anti-σD and was under the control of a σD -dependent promoter. Another σD regulon member, l,d-transpeptidase, conferred lysozyme resistance. Thus, σD modifies peptidoglycan cross-linking and enhances mycolate synthesis to provide resistance to environmental stress.

Glutamine-rich toxic proteins GrtA, GrtB and GrtC together with the antisense RNA AsgR constitute a toxin-antitoxin-like system in Corynebacterium glutamicum.

The Corynebacterium glutamicum R grtA (cgR_2936), grtB (cgR_2934) and grtC (cgR_2933) genes were identified as paralogs encoding glutamine-rich toxic proteins. We also identified a new antisense small RNA AsgR (antisense sRNA for grtA) that overlaps the 3' end of the grtA gene. Single over-expressions of grtA, grtB and grtC resulted in complete inhibition of Escherichia coli cell growth. This growth was rescued by co-expression of AsgR. Similar effects were observed in C. glutamicum, although the toxicities of these proteins were moderate. Inhibition of AsgR transcription resulted in increased levels and prolonged half-lives of grtA, grtB and grtC mRNAs. We also found that the expression levels of grtA, grtB and grtC were increased in an RNase III deletion mutant. Primer extension analysis revealed the RNase III cleavage site to be in the 3' untranslated region (3'-UTR) of the grtA mRNA. The expression levels of grtA, grtB and grtC were increased after exposure to several stresses, including heat shock, treatment with penicillin G, lysozyme or H2 O2 . The deletions of grtABC and asgR genes resulted in decreased survival rate under several stresses. These results indicate that GrtABC and AsgR constitute a type I toxin-antitoxin-like system in C. glutamicum.

Introduction of Glyoxylate Bypass Increases Hydrogen Gas Yield from Acetate and l-Glutamate in Rhodobacter sphaeroides.

Rhodobacter sphaeroides produces hydrogen gas (H2) from organic compounds via nitrogenase under anaerobic-light conditions in the presence of poor nitrogen sources, such as l-glutamate. R. sphaeroides utilizes the ethylmalonyl-coenzyme A (EMC) pathway for acetate assimilation, but its H2 yield from acetate in the presence of l-glutamate has been reported to be low. In this study, the deletion of ccr encoding crotonyl-coenzyme A (crotonyl-CoA) carboxylase/reductase, a key enzyme for the EMC pathway in R. sphaeroides, revealed that the EMC pathway is essential for H2 production from acetate and l-glutamate but not for growth and acetate consumption in the presence of l-glutamate. We introduced a plasmid expressing aceBA from Rhodobacter capsulatus encoding two key enzymes for the glyoxylate bypass into R. sphaeroides, which resulted in a 64% increase in H2 production. However, compared with the wild-type strain expressing heterologous aceBA genes, the strain with aceBA introduced in the genetic background of an EMC pathway-disrupted mutant showed a lower H2 yield. These results indicate that a combination of the endogenous EMC pathway and a heterologously expressed glyoxylate bypass is beneficial for H2 production. In addition, introduction of the glyoxylate bypass into a polyhydroxybutyrate (PHB) biosynthesis-disrupted mutant resulted in a delay in growth along with H2 production, although its H2 yield was comparable to that of the wild-type strain expressing heterologous aceBA genes. These results suggest that PHB production is important for fitness to the culture during H2 production from acetate and l-glutamate when both acetate-assimilating pathways are present.IMPORTANCE As an alternative to fossil fuel, H2 is a promising renewable energy source. Although photofermentative H2 production from acetate is key to developing an efficient process of biohydrogen production from biomass-derived sugars, H2 yields from acetate and l-glutamate by R. sphaeroides have been reported to be low. In this study, we observed that in addition to the endogenous EMC pathway, heterologous expression of the glyoxylate bypass in R. sphaeroides markedly increased H2 yields from acetate and l-glutamate. Therefore, this study provides a novel strategy for improving H2 yields from acetate in the presence of l-glutamate and contributes to a clear understanding of acetate metabolism in R. sphaeroides during photofermentative H2 production.

Engineering the transcriptional activator NifA for the construction of Rhodobacter sphaeroides strains that produce hydrogen gas constitutively.

Purple non-sulfur photosynthetic bacteria such as Rhodobacter sphaeroides and Rhodopseudomonas palustris produce hydrogen gas (H2) via proton reduction, which is catalyzed by nitrogenase. Although the expression of nitrogenase is usually repressed under nitrogen-sufficient conditions, a partial deletion of nifA, which encodes a transcriptional activator of nitrogen-fixation genes, has been reported to enable the constitutive expression of nitrogenase in R. palustris. In this study, we evaluated the effects of a similar mutation (nifA* mutation) on H2 production during the photoheterotrophic growth of R. sphaeroides, based on the notion that H2 production by nitrogenase compensates for the loss of CO2 fixation via the Calvin cycle, thereby restoring the redox balance. The chromosomal nifA* mutation resulted in the slight restoration of the photoheterotrophic growth of a mutant strain lacking ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), the key enzyme of the Calvin cycle, when the strain was cultured in van Niel's yeast medium. In addition, the strain with the nifA* mutation produced detectable levels of H2 during photoheterotrophic growth with acetate and ammonium; however, the H2 production was considerably lower than that observed during the photoheterotrophic growth of the strain with acetate and L-glutamate, where L-glutamate serves as a poor nitrogen source, thereby causing nitrogenase derepression. On the other hand, introduction of a multicopy plasmid harboring nifA* markedly restored the photoheterotrophic growth of the RubisCO-deletion mutant in van Niel's yeast medium and resulted in efficient H2 production during the photoheterotrophic growth with acetate and ammonium.

Metabolic engineering of Corynebacterium glutamicum for hyperproduction of polymer-grade L- and D-lactic acid.

Strain development is critical for microbial production of bio-based chemicals. The stereo-complex form of polylactic acid, a complex of poly-L- and poly-D-lactic acid, is a promising polymer candidate due to its high thermotolerance. Here, we developed Corynebacterium glutamicum strains producing high amounts of L- and D-lactic acid through intensive metabolic engineering. Chromosomal overexpression of genes encoding the glycolytic enzymes, glucokinase, glyceraldehyde-3-phosphate dehydrogenase, phosphofructokinase, triosephosphate isomerase, and enolase, increased L- and D-lactic acid concentration by 146% and 56%, respectively. Chromosomal integration of two genes involved in the Entner-Doudoroff pathway (6-phosphogluconate dehydratase and 2-dehydro-3-deoxyphosphogluconate aldolase), together with a gene encoding glucose-6-phosphate dehydrogenase from Zymomonas mobilis, to bypass the carbon flow from glucose, further increased L- and D-lactic acid concentration by 11% and 44%, respectively. Finally, additional chromosomal overexpression of a gene encoding NADH dehydrogenase to modulate the redox balance resulted in the production of 212 g/L L-lactic acid with a 97.9% yield and 264 g/L D-lactic acid with a 95.0% yield. The optical purity of both L- and D-lactic acid was 99.9%. Because the constructed metabolically engineered strains were devoid of plasmids and antibiotic resistance genes and were cultivated in mineral salts medium, these strains could contribute to the cost-effective production of the stereo-complex form of polylactic acid in practical scale.

Production of 4-Hydroxybenzoic Acid by an Aerobic Growth-Arrested Bioprocess Using Metabolically Engineered Corynebacterium glutamicum.

Corynebacterium glutamicum was metabolically engineered to produce 4-hydroxybenzoic acid (4-HBA), a valuable aromatic compound used as a raw material for the production of liquid crystal polymers and paraben. C. glutamicum was found to have a higher tolerance to 4-HBA toxicity than previously reported hosts used for the production of genetically engineered 4-HBA. To obtain higher titers of 4-HBA, we employed a stepwise overexpression of all seven target genes in the shikimate pathway in C. glutamicum Specifically, multiple chromosomal integrations of a mutated aroG gene from Escherichia coli, encoding a 3-deoxy-d-arabinoheptulosonic acid 7-phosphate (DAHP) synthase, and wild-type aroCKB from C. glutamicum, encoding chorismate synthase, shikimate kinase, and 3-dehydroquinate synthase, were effective in increasing product titers. The last step of the 4-HBA biosynthesis pathway was recreated in C. glutamicum by expressing a highly 4-HBA-resistant chorismate pyruvate-lyase (UbiC) from the intestinal bacterium Providencia rustigianii To enhance the yield of 4-HBA, we reduced the formation of by-products, such as 1,3-dihydroxyacetone and pyruvate, by deleting hdpA, a gene coding for a haloacid dehalogenase superfamily phosphatase, and pyk, a gene coding for a pyruvate kinase, from the bacterial chromosome. The maximum concentration of 4-HBA produced by the resultant strain was 36.6 g/liter, with a yield of 41% (mol/mol) glucose after incubation for 24 h in minimal medium in an aerobic growth-arrested bioprocess using a jar fermentor. To our knowledge, this is the highest concentration of 4-HBA produced by a metabolically engineered microorganism ever reported.IMPORTANCE Since aromatic compound 4-HBA has been chemically produced from petroleum-derived phenol for a long time, eco-friendly bioproduction of 4-HBA from biomass resources is desired in order to address environmental issues. In microbial chemical production, product toxicity often causes problems, but we confirmed that wild-type C. glutamicum has high tolerance to the target 4-HBA. A growth-arrested bioprocess using this microorganism has been successfully used for the production of various compounds, such as biofuels, organic acids, and amino acids. However, no production method has been applied for aromatic compounds to date. In this study, we screened for a novel final reaction enzyme possessing characteristics superior to those in previously employed microbial 4-HBA production. We demonstrated that the use of the highly 4-HBA-resistant UbiC from the intestinal bacterium P. rustigianii is very effective in increasing 4-HBA production.

Recent advances in metabolic engineering of Corynebacterium glutamicum for bioproduction of value-added aromatic chemicals and natural products.

Recent progress in synthetic and systems metabolic engineering technologies has explored the potential of microbial cell factories for the production of industrially relevant bulk and fine chemicals from renewable biomass resources in an eco-friendly manner. Corynebacterium glutamicum, a workhorse for industrial amino acid production, has currently evolved into a promising microbial platform for bioproduction of various natural and non-natural chemicals from renewable feedstocks. Notably, it has been recently demonstrated that metabolically engineered C. glutamicum can overproduce several commercially valuable aromatic and related chemicals such as shikimate, 4-hydroxybenzoate, and 4-aminobenzoate from sugars at remarkably high titer suitable to commercial application. On the other hand, overexpression and/or extension of its endogenous metabolic pathways by integrating heterologous metabolic pathways enabled production of structurally intricate and valuable natural chemicals like plant polyphenols, carotenoids, and fatty acids. In this review, we summarize recent advances in metabolic engineering of C. glutamicum for production of those value-added aromatics and other natural products, which highlights high potential and the versatility of this microbe for bioproduction of diverse chemicals.

Polynucleotide Phosphorylase, RNase E/G, and YbeY Are Involved in the Maturation of 4.5S RNA in Corynebacterium glutamicum.

Corynebacterium glutamicum has been applied for the industrial production of various metabolites, such as amino acids. To understand the biosynthesis of the membrane protein in this bacterium, we investigated the process of signal recognition particle (SRP) assembly. SRP is found in all three domains of life and plays an important role in the membrane insertion of proteins. SRP RNA is initially transcribed as precursor molecules; however, relatively little is known about its maturation. In C. glutamicum, SRP consists of the Ffh protein and 4.5S RNA lacking an Alu domain. In this study, we found that 3'-to-5' exoribonuclease, polynucleotide phosphorylase (PNPase), and two endo-type RNases, RNase E/G and YbeY, are involved in the 3' maturation of 4.5S RNA in C. glutamicum The mature form of 4.5S RNA was inefficiently formed in ΔrneG Δpnp mutant cells, suggesting the existence of an alternative pathway for the 3' maturation of 4.5S RNA. Primer extension analysis also revealed that the 5' mature end of 4.5S RNA corresponds to that of the transcriptional start site. Immunoprecipitated Ffh protein contained immature 4.5S RNA in Δpnp, ΔrneG, and ΔybeY mutants, suggesting that 4.5S RNA precursors can interact with Ffh. These results imply that the maturation of 4.5S RNA can be performed in the 4.5S RNA-Ffh complex.IMPORTANCE Overproduction of a membrane protein, such as a transporter, is useful for engineering of strains of Corynebacterium glutamicum, which is a workhorse of amino acid production. To understand membrane protein biogenesis in this bacterium, we investigated the process of signal recognition particle (SRP) assembly. SRP contains the Ffh protein and SRP RNA and plays an important role in the membrane insertion of proteins. Although SRP RNA is highly conserved among the three domains of life, relatively little is known about its maturation. We show that PNPase, RNase E/G, and YbeY are involved in the 3' maturation of the SRP RNA (4.5S RNA) in this bacterium. This indicates that 3' end processing in this organism is different from that in other bacteria, such as Escherichia coli.

The extracytoplasmic function σ factor σ(C) regulates expression of a branched quinol oxidation pathway in Corynebacterium glutamicum.

Bacteria modify their expression of different terminal oxidases in response to oxygen availability. Corynebacterium glutamicum, a facultative anaerobic bacterium of the phylum Actinobacteria, possesses aa3 -type cytochrome c oxidase and cytochrome bd-type quinol oxidase, the latter of which is induced by oxygen limitation. We report that an extracytoplasmic function σ factor, σ(C) , is responsible for the regulation of this process. Chromatin immunoprecipitation with microarray analysis detected eight σ(C) -binding regions in the genome, facilitating the identification of a consensus promoter sequence for σ(C) recognition. The promoter sequences were found upstream of genes for cytochrome bd, heme a synthesis enzymes and uncharacterized membrane proteins, all of which were upregulated by sigC overexpression. However, one consensus promoter sequence found on the antisense strand upstream of an operon encoding the cytochrome bc1 complex conferred a σ(C) -dependent negative effect on expression of the operon. The σ(C) regulon was induced by cytochrome aa3 deficiency without modifying sigC expression, but not by bc1 complex deficiency. These findings suggest that σ(C) is activated in response to impaired electron transfer via cytochrome aa3 and not directly to a shift in oxygen levels. Our results reveal a new paradigm for transcriptional regulation of the aerobic respiratory system in bacteria.

RNase III mediated cleavage of the coding region of mraZ mRNA is required for efficient cell division in Corynebacterium glutamicum.

The Corynebacterium glutamicum R cgR_1959 gene encodes an endoribonuclease of the RNase III family. Deletion mutant of cgR_1959 (Δrnc mutant) showed an elongated cell shape, and presence of several lines on the cell surface, indicating a required of RNase III for maintaining normal cell morphology in C. glutamicum. The level of mraZ mRNA was increased, whereas cgR_1596 mRNA encoding a putative cell wall hydrolase and ftsEX mRNA were decreased in the Δrnc mutant. The half-life of mraZ mRNA was significantly prolonged in the Δrnc and the Δpnp mutant strains. This indicated that the degradation of mraZ mRNA was performed by RNase III and the 3'-to-5' exoribonuclease, PNPase. Northern hybridization and primer extension analysis revealed that the cleavage site for mraZ mRNA by RNase III is in the coding region. Overproduction of MraZ resulted in an elongated cell shape. The expression of ftsEX decreased while that of cgR_1596 unchanged in an MraZ-overexpressing strain. An electrophoretic mobility shift assay and a transcriptional reporter assay indicate that MraZ is a transcriptional repressor of ftsEX in C. glutamicum. These results indicate that RNase III is required for efficient expression of MraZ-dependent ftsEX and MraZ-independent cgR_1596.

Enhanced Glucose Consumption and Organic Acid Production by Engineered Corynebacterium glutamicum Based on Analysis of a pfkB1 Deletion Mutant.

In the analysis of a carbohydrate metabolite pathway, we found interesting phenotypes in a mutant strain of Corynebacterium glutamicum deficient in pfkB1, which encodes fructose-1-phosphate kinase. After being aerobically cultivated with fructose as a carbon source, this mutant consumed glucose and produced organic acid, predominantly l-lactate, at a level more than 2-fold higher than that of the wild-type grown with glucose under conditions of oxygen deprivation. This considerably higher fermentation capacity was unique for the combination of pfkB1 deletion and cultivation with fructose. In the metabolome and transcriptome analyses of this strain, marked intracellular accumulation of fructose-1-phosphate and significant upregulation of several genes related to the phosphoenolpyruvate:carbohydrate phosphotransferase system, glycolysis, and organic acid synthesis were identified. We then examined strains overexpressing several of the identified genes and demonstrated enhanced glucose consumption and organic acid production by these engineered strains, whose values were found to be comparable to those of the model pfkB1 deletion mutant grown with fructose. l-Lactate production by the ppc deletion mutant of the engineered strain was 2,390 mM (i.e., 215 g/liter) after 48 h under oxygen deprivation, which was a 2.7-fold increase over that of the wild-type strain with a deletion of ppc IMPORTANCE: Enhancement of glycolytic flux is important for improving microbiological production of chemicals, but overexpression of glycolytic enzymes has often resulted in little positive effect. That is presumably because the central carbon metabolism is under the complex and strict regulation not only transcriptionally but also posttranscriptionally, for example, by the ATP/ADP ratio. In contrast, we studied a mutant strain of Corynebacterium glutamicum that showed markedly enhanced glucose consumption and organic acid production and, based on the findings, identified several genes whose overexpression was effective in enhancing glycolytic flux under conditions of oxygen deprivation. These results will further understanding of the regulatory mechanisms of glycolytic flux and can be widely applied to the improvement of the microbial production of useful chemicals.

Postgenomic approaches to using corynebacteria as biocatalysts.

Corynebacterium glutamicum exhibits numerous ideal intrinsic attributes as a factory of primary and secondary metabolites. The versatile capabilities of this organism have long been implemented at the industrial scale to produce an array of amino acids at high yields and conversion rates, thereby enabling the development of an entire industry. The postgenomic era provides a new technological platform not only to further optimize the intrinsic attributes of C. glutamicum whole cells as biocatalysts, but also to dramatically expand the product portfolio that can be manufactured by this organism, from amino acids to commodity chemicals. This review addresses the methods and strain optimization strategies enabled by genomic information and associated techniques. Their implementation has provided important additional incremental improvements to the economics of industry-scale manufacturing in which C. glutamicum and its episomal elements are used as a performing host-vector system.