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Microbiology spectrum 10, e01756-22 (2022). doi:10.1128/spectrum.01756-22, Published by ASM, Birmingham, Ala.

(1) Background: Hyaluronic acid (HA) is a polyanionic mucopolysaccharide extensively used in biomedical and cosmetic industries due to its unique rheological properties. Recombinant HA production using other microbial platforms has received increasing interest to avoid potential toxin contamination associated with its production by streptococcal fermentation. In this study, the Gram-negative strains

Genome analysis of Corynebacterium lactis revealed a bacteriocin gene cluster encoding a putative bacteriocin of the linaridin family of ribosomally synthesized and posttranslationally modified peptides (RIPPs). The locus harbors typical linaridin modification enzymes but lacks genes for a decarboxylase and methyltransferase, which is unusual for type B linaridins. Supernatants of Corynebacterium lactis RW3-42 showed antimicrobial activity against Corynebacterium glutamicum. Deletion of the precursor gene crdA clearly linked the antimicrobial activity of the producer strain to the identified gene cluster. Following purification, we observed potent activity of the peptide against Actinobacteria, mainly other members of the genus Corynebacterium including the pathogenic species Corynebacterium striatum and Corynebacterium amycolatum. Also, low activity against some Firmicutes was observed, but no activity against Gram-negative species. The peptide is resilient towards heat but sensitive to proteolytic degradation by trypsin and proteinase K. Notably, time-kill kinetics and experiments using live biosensors to monitor membrane integrity suggest bactericidal activity that does not involve formation of pores in the cytoplasmic membrane. As Corynebacterium species are ubiquitous in nature and include important commensal and pathogens of mammalian organisms, secretion of bacteriocins by species of this genus could be a hitherto neglected trait with high relevance for intra- and interspecies competition and infection.ImportanceBacteriocins are antimicrobial peptides produced by bacteria to fend off competitors in ecological niches and are considered to be important factors influencing the composition of microbial communities. However, bacteriocin production by bacteria of the genus Corynebacterium has been a hitherto neglected trait, although its species are ubiquitous in nature and make up large parts of the microbiome of humans and animals. In this study, we describe and characterize a novel linaridin-family bacteriocin from Corynebacterium lactis and show its narrow spectrum activity, mainly against other actinobacteria. Moreover, we were able to extend the limited knowledge on linaridin bioactivity in general and for the first time describe the mode of action of such a bacteriocin. Interestingly, the peptide, which was named corynaridin, appears bactericidal, but without formation of pores in the bacterial membrane.

In der lichtabhängigen Reaktion der Photosynthese koppeln grüne Pflanzen photoinduzierte Kaskaden von Redoxreaktionen mit transmembranen Protonentransfers um Reduktionsäquivalente und chemische Energie in Form von NADPH (Nikotinamidadenindinukleotidphosphat) und ATP (Adenosintriphosphat) zu erzeugen. Wir imitieren diese grundlegenden Prozesse durch Kombination von molekularen Rutheniumpolypyridinkomplexen und invertierten Vesikeln, die aus Escherichia coli gewonnen werden. Bei Bestrahlung mit sichtbarem Licht führt das Zusammenspiel von photokatalytischer Nikotinamidreduktion und enzymatischer, membranlokalisierter Atmung zur gleichzeitigen Bildung der beiden biologisch aktiven Cofaktoren NADH (Nikotinamidadenindinukleotid) und ATP. Dieses anorganisch-biologische Hybridsystem ahmt daher die Cofaktor-bereitstellende Funktion eines aktiven Chloroplasten nach.

Aerobic carboxydotrophic bacteria are a group of microorganisms which possess the unique trait to oxidize carbon monoxide (CO) as sole energy source with molecular oxygen (O

The bacteriocin nisin is one of the best studied antimicrobial peptides. It is widely used as a food preservative due to its antimicrobial activity against various Gram-positive bacteria including human pathogens such asListeriamonocytogenesand others. The receptor of nisin is the universal cell wall precursor lipid II, which is present in all bacteria. Thus, nisin has a broad spectrum of target organisms. Consequently, heterologous production of nisin with biotechnological relevant organisms includingCorynebacterium glutamicumis difficult. Nevertheless, bacteria have evolved several mechanisms of resistance against nisin and other cationic antimicrobial peptides (CAMPs). Here, we transferred resistance mechanisms described in other organisms toC. glutamicumwith the aim to improve nisin resistance. The presented approaches included: expression of (i) nisin immunity genesnisIand/ornisFEGor (ii) nisin ABC-transporter genes ofStaphylococcus aureusand its homologues ofC. glutamicum, (iii) genes coding for enzymes for alanylation or lysinylation of the cell envelope to introduce positive charges, and/or (iv) deletion of genes for porins of the outer membrane. None of the attempts alone increased resistance ofC. glutamicummore than two-fold. To increase resistance ofC. glutamicumto levels that will allow heterologous production of active nisin at relevant titers, further studies are needed.

Uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) is an acetylated amino sugar nucleotide that naturally serves as precursor in bacterial cell wall synthesis and is involved in prokaryotic and eukaryotic glycosylation reactions. UDP-GlcNAc finds application in various fields including the production of oligosaccharides and glycoproteins with therapeutic benefits. At present, nucleotide sugars are produced either chemically or in vitro by enzyme cascades. However, chemical synthesis is complex and non-economical, and in vitro synthesis requires costly substrates and often purified enzymes. A promising alternative is the microbial production of nucleotide sugars from cheap substrates. In this study, we aimed to engineer the non-pathogenic, Gram-positive soil bacterium Corynebacterium glutamicum as a host for UDP-GlcNAc production. The native glmS, glmU, and glmM genes and glmM of Escherichia coli, encoding the enzymes for UDP-GlcNAc synthesis from fructose-6-phosphate, were over-expressed in different combinations and from different plasmids in C. glutamicum GRS43, which lacks the glucosamine-6-phosphate deaminase gene (nagB) for glucosamine degradation. Over-expression of glmS, glmU and glmM, encoding glucosamine-6-phosphate synthase, the bifunctional glucosamine-1-phosphate acetyltransferase/N-acetyl glucosamine-1-phosphate uridyltransferase and phosphoglucosamine mutase, respectively, was confirmed using activity assays or immunoblot analysis. While the reference strain C. glutamicum GlcNCg1 with an empty plasmid in the exponential growth phase contained intracellularly only about 0.25 mM UDP-GlcNAc, the best engineered strain GlcNCg4 accumulated about 14 mM UDP-GlcNAc. The extracellular UDP-GlcNAc concentrations in the exponential growth phase did not exceed 2 mg/L. In the stationary phase, about 60 mg UDP-GlcNAc/L was observed extracellularly with strain GlcNCg4, indicating the potential of C. glutamicum to produce and to release the activated sugar into the culture medium. To our knowledge, the observed UDP-GlcNAc levels are the highest obtained with microbial hosts, emphasizing the potential of C. glutamicum as a suitable platform for activated sugar production.

The recent discovery that phosphoenolpyruvate carboxylase (PEPCx) is dispensable for growth and lysine production in

A series of three Ru(II) polypyridine complexes was investigated for the selective photocatalytic oxidation of NAD(P)H to NAD(P)+ in water. A combination of (time-resolved) spectroscopic studies and photocatalysis experiments revealed that ligand design can be used to control the mechanism of the photooxidation: For prototypical Ru(II) complexes a 1O2 pathway was found. Rudppz ([(tbbpy)2Ru(dppz)]Cl2, tbbpy = 4,4´-di-tert-butyl-2,2´-bipyridine, dppz = dipyrido[3,2-a:2′,3′-c]phenazine), instead, initiated the cofactor oxidation by electron transfer from NAD(P)H enabled by supramolecular binding between substrate and catalyst. Expulsion of the photoproduct NAD(P)+ from the supramolecular binding site in Rudppz allowed very efficient turnover. Therefore, Rudppz permits repetitive selective assembly and oxidative conversion of reduced naturally occurring nicotinamides by recognizing the redox state of the cofactor under formation of H2O2 as additional product. This photocatalytic process can fuel discontinuous photobiocatalysis.

Bacteriocins are antimicrobial peptides produced by bacteria to inhibit competitors in their natural environments. Some of these peptides have emerged as commercial food preservatives and, due to the rapid increase in antibiotic resistant bacteria, are also discussed as interesting alternatives to antibiotics for therapeutic purposes. Currently, commercial bacteriocins are produced exclusively with natural producer organisms on complex substrates and are sold as semi-purified preparations or crude fermentates. To allow clinical application, efficacy of production and purity of the product need to be improved. This can be achieved by shifting production to recombinant microorganisms. Here, we identify Corynebacterium glutamicum as a suitable production host for the bacteriocin pediocin PA-1. C. glutamicum CR099 shows resistance to high concentrations of pediocin PA-1 and the bacteriocin was not inactivated when spiked into growing cultures of this bacterium. Recombinant C. glutamicum expressing a synthetic pedACDCgl operon releases a compound that has potent antimicrobial activity against Listeria monocytogenes and Listeria innocua and matches size and mass:charge ratio of commercial pediocin PA-1. Fermentations in shake flasks and bioreactors suggest that low levels of dissolved oxygen are favorable for production of pediocin. Under these conditions, however, reduced activity of the TCA cycle resulted in decreased availability of the important pediocin precursor l-asparagine suggesting options for further improvement. Overall, we demonstrate that C. glutamicum is a suitable host for recombinant production of bacteriocins of the pediocin family., Highlights • C. glutamicum CR099 is resistant to high concentrations of pediocin PA-1. • Recombinant C. glutamicum CR099/pEKEx-pedACDCg produces of a compound with antimicrobial activity against Listeria sp. • The purified compound matches size and mass:charge ratio of commercial pediocin PA-1. • Low oxygen levels are favorable for production of active pediocin by C. glutamicum in batch fermentations.

Due to climate change and worldwide pollution, development of highly sustainable routes for industrial production of basic and specialty chemicals is critical nowadays. One possible approach is the use of CO

Summary The reduction of CO 2 emissions is a global effort which is not only supported by the society and politicians but also by the industry. Chemical producers worldwide follow the strategic goal to reduce CO 2 emissions by replacing existing fossil‐based production routes with sustainable alternatives. The smart use of CO and CO 2/H2 mixtures even allows to produce important chemical building blocks consuming the said gases as substrates in carboxydotrophic fermentations with acetogenic bacteria. However, existing industrial infrastructure and market demands impose constraints on microbes, bioprocesses and products that require careful consideration to ensure technical and economic success. The mini review provides scientific and industrial facets finally to enable the successful implementation of gas fermentation technologies in the industrial scale.

A heterobinuclear dyad containing a ruthenium polypyridyl moiety bound via an aromatic bridging ligand to an organometallic catalytic center has been used for the light driven reduction of BNA+, NAD+ and NADP+ yielding the two electron reduced analog. Direct coupling with enzymatic conversion could be proven by means of UV-vis spectroscopy as well as liquid chromatography, showing cofactor recycling and enzymatic conversion with a turnover number of 350 per photocatalyst. First insights into the complex behavior of the catalytic system under irradiation points towards multiple prerequisites on the molecular as well as on the macroscopic level in order to generate highly efficient semiartificial photobiocatalytic systems for future energy storage applications.

The development of CRISPR interference (CRISPRi) technology has dramatically increased the pace and the precision of target identification during platform strain development. In order to develop a simple, reliable, and dual-inducible CRISPRi system for the industrially relevant Corynebacterium glutamicum, we combined two different inducible repressor systems in a single plasmid to separately regulate the expression of dCas9 (anhydro-tetracycline-inducible) and a given single guide RNA (IPTG-inducible). The functionality of the resulting vector was demonstrated by targeting the l-arginine biosynthesis pathway in C. glutamicum. By co-expressing dCas9 and a specific single guide RNA targeting the 5'-region of the argininosuccinate lyase gene argH, the specific activity of the target enzyme was down-regulated and in a l-arginine production strain, l-arginine formation was shifted towards citrulline formation. The system was also employed for down-regulation of multiple genes by concatenating sgRNA sequences encoded on one plasmid. Simultaneous down-regulated expression of both argH and the phosphoglucose isomerase gene pgi proved the potential of the system for multiplex targeting. The system can be a promising tool for further pathway engineering in C. glutamicum. Cumulative effects on targeted genes can be rapidly evaluated avoiding tedious and time-consuming traditional gene knockout approaches.

Gasification is a suitable technology to generate energy-rich synthesis gas (syngas) from biomass or waste streams, which can be utilized in bacterial fermentation processes for the production of chemicals and fuels. Established microbial processes currently rely on acetogenic bacteria which perform an energetically inefficient anaerobic CO oxidation and acetogenesis potentially hampering the biosynthesis of complex and ATP-intensive products. Since aerobic oxidation of CO is energetically more favorable, we exploit in this study the Gram-negative beta-proteobacterium Hydrogenophaga pseudoflava DSM1084 as novel host for the production of chemicals from syngas. We sequenced and annotated the genome of H.pseudoflava and established a genetic engineering toolbox, which allows markerless chromosomal modification via the pk19mobsacB system and heterologous gene expression on pBBRMCS2-based plasmids. The toolbox was extended by identifying strong endogenous promotors such as PgapA2 which proved to yield high expression under heterotrophic and autotrophic conditions. H.pseudoflava showed relatively fast heterotrophic growth in complex and minimal medium with sugars and organic acids which allows convenient handling in lab routines. In autotrophic bioreactor cultivations with syngas, H.pseudoflava exhibited a growth rate of 0.06 h-1 and biomass specific uptakes rates of 14.2 ± 0.3 mmol H2 gCDW-1 h-1, 73.9 ± 1.8 mmol CO gCDW-1 h-1, and 31.4 ± 0.3 mmol O2 gCDW-1 h-1. As proof of concept, we engineered the carboxydotrophic bacterium for the aerobic production of the C15 sesquiterpene (E)-alpha-bisabolene from the C1 carbon source syngas by heterologous expression of the (E)-alpha-bisabolene synthase gene agBIS. The resulting strain H.pseudoflava (pOCEx1:agBIS) produced 59 ± 8 mug (E)-alpha-bisabolene L-1 with a volumetric productivity Qp of 1.2 ± 0.2 mug L-1 h-1 and a biomass-specific productivity qp of 13.1 ± 0.6 mug gCDW-1 h-1. The intrinsic properties and the genetic repertoire of H.pseudoflava make this carboxydotrophic bacterium a promising candidate for future aerobic production processes to synthesize more complex or ATP-intensive chemicals from syngas. Copyright © 2019 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

The Gram-positive bacterium Corynebacterium glutamicum represents a promising platform for the production of amino acids, organic acids, and other bio-products. However, the availability of only few expression vectors limits its use for production purposes, using metabolic engineering approaches when co-expression of several target genes is desired. To widen the scope for co-expression, the pCG1/p15A and pBL1/colE1 replicons were employed to construct the two differentially-inducible and compatible expression vectors pRG_Duet1 and pRG_Duet2. To functionally validate these newly constructed expression vectors, target genes for easily measurable enzymes were cloned and over-expression of these genes was investigated using respective enzyme assays. Furthermore, functionality and co-existence of the pCG1-based C. glutamicum - E. coli shuttle vector pRG_Duet1 were confirmed with pBL1-based expression vectors pRG_Duet2 and pEKEx2, using co-transformation and enzyme assays. The novel shuttle expression vectors pRG_Duet1 and pRG_Duet2 are attractive additions to the existing set of vectors for co-expression studies and metabolic engineering of C. glutamicum.

Corynebacterium glutamicum is an industrial workhorse used for the production of amino acids and a variety of other chemicals and fuels. Within its regulatory repertoire, C. glutamicum possesses RamA which was initially identified as essential transcriptional regulator of acetate metabolism. Further studies revealed its relevance for ethanol and propionate catabolism and also identified RamA to function as global regulator in the metabolism of C. glutamicum. Thereby, RamA acts as transcriptional activator or repressor of genes encoding enzymes which are involved in carbon uptake, central carbon metabolism, and cell wall synthesis. RamA controls the expression of target genes either directly and/or indirectly by constituting feed-forward loop type of transcriptional motifs with other regulators such as GlxR, SugR, RamB, and GntR1. In this review, we summarize the current knowledge on RamA, its regulon, and its regulatory interplay with other transcriptional regulators coordinating the metabolism of C. glutamicum.

Pyruvate as a key metabolite of the central metabolism in bacteria is also a precursor for several biotechnologically relevant compounds. Therefore, efforts have been made to tailor the metabolism of the industrially relevant Corynebacterium glutamicum for optimal pyruvate availability and an optimal flux towards pyruvate-derived products. Here we summarize the recent achievements on metabolic engineering of C. glutamicum for production of pyruvate, L-valine, 2-ketoisovalerate and isobutanol.

The branched chain amino acid L-valine is an essential nutrient for higher organisms, such as animals and humans. Besides the pharmaceutical application in parenteral nutrition and as synthon for the chemical synthesis of e.g. herbicides or anti-viral drugs, L-valine is now emerging into the feed market, and significant increase of sales and world production is expected. In accordance, well-known microbial production bacteria, such as Escherichia coli and Corynebacterium glutamicum strains, have recently been metabolically engineered for efficient L-valine production under aerobic or anaerobic conditions, and the respective cultivation and production conditions have been optimized. This review summarizes the state of the art in L-valine biosynthesis and its regulation in E. coli and C. glutamicum with respect to optimal metabolic network for microbial L-valine production, genetic strain engineering and bioprocess development for L-valine production, and finally, it will shed light on emerging technologies that have the potential to accelerate strain and bioprocess engineering in the near future.

Exchange of the native Corynebacterium glutamicum promoter of the aceE gene, encoding the E1p subunit of the pyruvate dehydrogenase complex (PDHC), with mutated dapA promoter variants led to a series of C. glutamicum strains with gradually reduced growth rates and PDHC activities. Upon overexpression of the l -valine biosynthetic genes ilvBNCE , all strains produced l -valine. Among these strains, C. glutamicum aceE A16 (pJC4 ilvBNCE ) showed the highest biomass and product yields, and thus it was further improved by additional deletion of the pqo and ppc genes, encoding pyruvate:quinone oxidoreductase and phosphoenolpyruvate carboxylase, respectively. In fed-batch fermentations at high cell densities, C. glutamicum aceE A16 Δ pqo Δppc (pJC4 ilvBNCE ) produced up to 738 mM (i.e., 86.5 g/liter) l -valine with an overall yield ( Y P/S ) of 0.36 mol per mol of glucose and a volumetric productivity ( Q P ) of 13.6 mM per h [1.6 g/(liter × h)]. Additional inactivation of the transaminase B gene ( ilvE ) and overexpression of ilvBNCD instead of ilvBNCE transformed the l -valine-producing strain into a 2-ketoisovalerate producer, excreting up to 303 mM (35 g/liter) 2-ketoisovalerate with a Y P/S of 0.24 mol per mol of glucose and a Q P of 6.9 mM per h [0.8 g/(liter × h)]. The replacement of the aceE promoter by the dapA -A16 promoter in the two C. glutamicum l -lysine producers DM1800 and DM1933 improved the production by 100% and 44%, respectively. These results demonstrate that C. glutamicum strains with reduced PDHC activity are an excellent platform for the production of pyruvate-derived products.

The shortage of oil resources, the steadily rising oil prices and the impact of its use on the environment evokes an increasing political, industrial and technical interest for development of safe and efficient processes for the production of chemicals from renewable biomass. Thus, microbial fermentation of renewable feedstocks found its way in white biotechnology, complementing more and more traditional crude oil-based chemical processes. Rational strain design of appropriate microorganisms has become possible due to steadily increasing knowledge on metabolism and pathway regulation of industrially relevant organisms and, aside from process engineering and optimization, has an outstanding impact on improving the performance of such hosts. Corynebacterium glutamicum is well known as workhorse for the industrial production of numerous amino acids. However, recent studies also explored the usefulness of this organism for the production of several organic acids and great efforts have been made for improvement of the performance. This review summarizes the current knowledge and recent achievements on metabolic engineering approaches to tailor C. glutamicum for the bio-based production of organic acids. We focus here on the fermentative production of pyruvate, L- and D-lactate, 2-ketoisovalerate, 2-ketoglutarate, and succinate. These organic acids represent a class of compounds with manifold application ranges, e.g. in pharmaceutical and cosmetics industry, as food additives, and economically very interesting, as precursors for a variety of bulk chemicals and commercially important polymers.

The stereochemistry of 2,3-butanediol (2,3-BD) synthesis in microbial fermentations is important for many applications. In this work, we showed that Corynebacterium glutamicum endowed with the Lactococcus lactis genes encoding α-acetolactate synthase and decarboxylase activities produced meso-2,3-BD as the major end product, meaning that (R)-acetoin is a substrate for endogenous 2,3-butanediol dehydrogenase (BDH) activity. This is curious in view of the reported absolute stereospecificity of C. glutamicum BDH for (S)-acetoin (Takusagawa et al. Biosc Biotechnol Biochem 65:1876–1878, 2001). To resolve this discrepancy, the enzyme encoded by butACg was produced in Escherichia coli and purified, and the stereospecific properties of the pure protein were examined. Activity assays monitored online by 1H-NMR using racemic acetoin and an excess of NADH showed an initial, fast production of (2S,3S)-2,3-BD, followed by a slow (∼20-fold lower apparent rate) formation of meso-2,3-BD. Kinetic parameters for (S)-acetoin, (R)-acetoin, meso-2,3-BD and (2S,3S)-BD were determined by spectrophotometric assays. Vmax values for (S)-acetoin and (R)-acetoin were 119 ± 15 and 5.23 ± 0.06 μmol min−1 mg protein−1, and Km values were 0.23 ± 0.02 and 1.49 ± 0.07 mM, respectively. We conclude that C. glutamicum BDH is not absolutely specific for (S)-acetoin, though this is the preferred substrate. Importantly, the low activity of BDH with (R)-acetoin was sufficient to support high yields of meso-2,3-BD in the engineered strain C. glutamicum ΔaceEΔpqoΔldhA(pEKEx2-als,aldB,butACg). Additionally, we found that the BDH activity was nearly abolished upon inactivation of butACg (from 0.30 ± 0.03 to 0.004 ± 0.001 μmol min−1 mg protein−1), indicating that C. glutamicum expresses a single BDH under the experimental conditions examined.

Corynebacterium glutamicum is a Gram-positive nonpathogenic bacterium that is used for the biotechnological production of amino acids. Here, we investigated the transcriptional control of the malE gene encoding malic enzyme (MalE) in C. glutamicum ATCC 13032, which is known to involve the nitrogen regulator AmtR. Gel shift experiments using purified regulators RamA and RamB revealed binding of these regulators to the malE promoter. In DNA-affinity purification experiments a hitherto uncharacterized transcriptional regulator belonging to the MarR family was found to bind to malE promoter DNA and was designated as MalR. C. glutamicum cells overexpressing malR showed reduced MalE activities in LB medium or in minimal media with acetate, glucose, pyruvate or citrate. Deletion of malR positively affected MalE activities during growth in LB medium and minimal media with pyruvate, glucose or the TCA cycle dicarboxylates l-malate, succinate and fumarate. Transcriptional fusion analysis revealed elevated malE promoter activity in the malR deletion mutant during growth in pyruvate minimal medium suggesting that MalR acts as a repressor of malE. Purified MalR bound malE promoter DNA in gel shift experiments. Two MalR binding sites were identified in the malE promoter by mutational analysis. Thus, MalR contributes to the complex transcriptional control of malE which also involves RamA, RamB and AmtR.

Corynebacterium glutamicum transiently accumulates glycogen as carbon capacitor during the early exponential growth phase in media containing carbohydrates. In some bacteria glycogen is synthesized by the consecutive action of ADP-glucose pyrophosphorylase (GlgC), glycogen synthase (GlgA) and glycogen branching enzyme (GlgB). GlgC and GlgA of C. glutamicum have been shown to be necessary for glycogen accumulation in this organism. However, although cg1381 has been annotated as the putative C. glutamicum glgB gene, cg1381 and its gene product have not been characterized and their role in transient glycogen accumulation has not yet been investigated. We show here that the cg1381 gene product of C. glutamicum catalyses the formation of α-1,6-glycosidic bonds in polysaccharides and thus represents a glycogen branching enzyme. RT-PCR experiments revealed glgB to be co-transcribed with glgE, probably encoding a maltosyltransferase. Promoter activity assays with the glgE promoter region revealed carbon-source-dependent expression of the glgEB operon. Characterization of the growth and glycogen content of glgB-deficient and glgB-overexpressing strains showed that the glycogen branching enzyme GlgB is essential for glycogen formation in C. glutamicum. Taken together these results suggest that an interplay of the enzymes GlgC, GlgA and GlgB is not essential for growth, but is required for synthesis of the transient carbon capacitor glycogen in C. glutamicum.

Expression profiling of Corynebacterium glutamicum in comparison to a derivative deficient in the transcriptional regulator AtlR (previously known as SucR or MtlR) revealed eight genes showing more than 4-fold higher mRNA levels in the mutant. Four of these genes are located in the direct vicinity of the atlR gene, i.e., xylB, rbtT, mtlD, and sixA, annotated as encoding xylulokinase, the ribitol transporter, mannitol 2-dehydrogenase, and phosphohistidine phosphatase, respectively. Transcriptional analysis indicated that atlR and the four genes are organized as atlR-xylB and rbtT-mtlD-sixA operons. Growth experiments with C. glutamicum and C. glutamicum ΔatlR, ΔxylB, ΔrbtT, ΔmtlD, and ΔsixA derivatives with sugar alcohols revealed that (i) wild-type C. glutamicum grows on D-arabitol but not on other sugar alcohols, (ii) growth in the presence of D-arabitol allows subsequent growth on D-mannitol, (iii) D-arabitol is cometabolized with glucose and preferentially utilized over D-mannitol, (iv) RbtT and XylB are involved in D-arabitol but not in D-mannitol metabolism, (v) MtlD is required for D-arabitol and D-mannitol metabolism, and (vi) SixA is not required for growth on any of the substrates tested. Furthermore, we show that MtlD confers D-arabitol and D-mannitol dehydrogenase activities, that the levels of these and also xylulokinase activities are generally high in the C. glutamicum ΔatlR mutant, whereas in the parental strain, they were high when cells were grown in the presence of D-arabitol and very low when cells were grown in its absence. Our results show that the XylB, RbtT, and MtlD proteins allow the growth of C. glutamicum on D-arabitol and that D-arabitol metabolism is subject to arabitol-dependent derepression by AtlR.

Citrate synthase (CS) is located at a major branch point in the metabolism and is required for both tricarboxylic acid and glyoxylic acid cycle activity. Here we show that the CS gene gltA of Corynebacterium glutamicum is monocistronic, but that two transcripts are formed with their transcript initiation sites located 121 bp and 357 bp upstream of the translational start codon, respectively. Northern blot analyses revealed that during growth on acetate the short transcript prevails, whereas during growth on glucose the long transcript is dominant. Further Northern blots, reporter gene fusions, and CS activity measurements in mutants devoid of the transcriptional regulators RamA and RamB or with the global regulator GlxR overexpressed revealed a complex involvement of these regulators in gltA transcription. This was confirmed by demonstrating the direct interaction of isolated RamA, RamB and GlxR proteins with specific gltA promoter regions in vitro. The combined analyses point to an elaborate control of gltA transcript formation, which is possibly required as a dedicated mechanism to balance the total CS activity according to the physiological requirements.

We recently engineered Corynebacterium glutamicum for aerobic production of 2-ketoisovalerate by inactivation of the pyruvate dehydrogenase complex, pyruvate:quinone oxidoreductase, transaminase B, and additional overexpression of the ilvBNCD genes, encoding acetohydroxyacid synthase, acetohydroxyacid isomeroreductase, and dihydroxyacid dehydratase. Based on this strain, we engineered C. glutamicum for the production of isobutanol from glucose under oxygen deprivation conditions by inactivation of l -lactate and malate dehydrogenases, implementation of ketoacid decarboxylase from Lactococcus lactis , alcohol dehydrogenase 2 (ADH2) from Saccharomyces cerevisiae , and expression of the pntAB transhydrogenase genes from Escherichia coli . The resulting strain produced isobutanol with a substrate-specific yield (Y P/S ) of 0.60 ± 0.02 mol per mol of glucose. Interestingly, a chromosomally encoded alcohol dehydrogenase rather than the plasmid-encoded ADH2 from S. cerevisiae was involved in isobutanol formation with C. glutamicum , and overexpression of the corresponding adhA gene increased the Y P/S to 0.77 ± 0.01 mol of isobutanol per mol of glucose. Inactivation of the malic enzyme significantly reduced the Y P/S , indicating that the metabolic cycle consisting of pyruvate and/or phosphoenolpyruvate carboxylase, malate dehydrogenase, and malic enzyme is responsible for the conversion of NADH+H + to NADPH+H + . In fed-batch fermentations with an aerobic growth phase and an oxygen-depleted production phase, the most promising strain, C. glutamicum Δ aceE Δ pqo Δ ilvE Δ ldhA Δ mdh (pJC4 ilvBNCD-pntAB )(pBB1 kivd-adhA ), produced about 175 mM isobutanol, with a volumetric productivity of 4.4 mM h −1 , and showed an overall Y P/S of about 0.48 mol per mol of glucose in the production phase.

When grown in glucose-, fructose- or sucrose-containing medium, the amino acid producer Corynebacterium glutamicum transiently accumulates large amounts of glycogen (up to 10 % of its dry weight), whereas only a marginal amount of glycogen is formed during growth with acetate. This carbon-source-dependent regulation is at least partially due to transcriptional control of glgC, encoding ADP-glucose pyrophosphorylase, the first enzyme of glycogen synthesis from glucose-1-phosphate. Here, we have analysed a possible regulatory role for the transcriptional regulators RamA and RamB on glycogen content of the cells and on control of expression of glgC and of glgA, which encodes the second enzyme of glycogen synthesis, glycogen synthase. Determination of the glycogen content of RamA- and RamB-deficient C. glutamicum indicated that RamA and RamB influence glycogen synthesis positively and negatively, respectively. In accordance with the identification of putative RamA and RamB binding sites upstream of glgC and glgA, both regulators were found to bind specifically to the glgC–glgA intergenic promoter region. Promoter activity assays in wild-type and RamA- and RamB-deficient strains of C. glutamicum revealed that (i) RamA is a positive regulator of glgC and glgA, (ii) RamB is a negative regulator of glgA and (iii) neither RamA nor RamB alone is responsible for the carbon-source-dependent regulation of glycogen synthesis in C. glutamicum.

Corynebacterium glutamicum transiently accumulates large amounts of glycogen, when cultivated on glucose and other sugars as a source of carbon and energy. Apart from the debranching enzyme GlgX, which is required for the formation of maltodextrins from glycogen, α-glucan phosphorylases were assumed to be involved in glycogen degradation, forming α-glucose 1-phosphate from glycogen and from maltodextrins. We show here that C. glutamicum in fact possesses two α-glucan phosphorylases, which act as a glycogen phosphorylase (GlgP) and as a maltodextrin phosphorylase (MalP). By chromosomal inactivation and subsequent analysis of the mutant, cg1479 was identified as the malP gene. The deletion mutant C. glutamicum ΔmalP completely lacked MalP activity and showed reduced intracellular glycogen degradation, confirming the proposed pathway for glycogen degradation in C. glutamicum via GlgP, GlgX and MalP. Surprisingly, the ΔmalP mutant showed impaired growth, reduced viability and altered cell morphology on maltose and accumulated much higher concentrations of glycogen and maltodextrins than the wild-type during growth on this substrate, suggesting an additional role of MalP in maltose metabolism of C. glutamicum. Further assessment of enzyme activities revealed the presence of 4-α-glucanotransferase (MalQ), glucokinase (Glk) and α-phosphoglucomutase (α-Pgm), and the absence of maltose hydrolase, maltose phosphorylase and β-Pgm, all three known to be involved in maltose utilization by Gram-positive bacteria. Based on these findings, we conclude that C. glutamicum metabolizes maltose via a pathway involving maltodextrin and glucose formation by MalQ, glucose phosphorylation by Glk and maltodextrin degradation via the reactions of MalP and α-Pgm, a pathway hitherto known to be present in Gram-negative rather than in Gram-positive bacteria.

The metabolism of monocarboxylic acids is of central importance for bacteria in their natural habitat as well as during biotechnological production. Although biosynthesis and degradation are well understood, the transport of such compounds is still a matter of discussion. Here we present the identification and characterization of a new transport system in Corynebacterium glutamicum with high affinity for acetate and propionate and with lower affinity for pyruvate. Biochemical analysis of this monocarboxylic acid transporter (MctC) revealed for the first time a quantitative discrimination of passive diffusion and active transport of acetate by bacterial cells. MctC is a secondary transporter and belongs to the class of sodium solute symporters, but it is driven by the electrochemical proton potential. The mctC gene is preceded by and cotranscribed with cg0952, a locus encoding a small membrane protein, and the transcription of the cg0952- mctC operon is under the control of the transcriptional regulators RamA and RamB. Both of these proteins directly bind to the promoter region of the operon; RamA is essential for expression and RamB exerts a slightly negative control on expression of the cg0952- mctC operon. mctC expression is induced in the presence of pyruvate and beneficial under substrate-limiting conditions for C. glutamicum .

The transcriptional regulators RamA, RamB and GlxR were detected to bind to the promoter region of the resuscitation promoting factor 2 (rpf2) gene involved in growth and culturability of Corynebacterium glutamicum. DNA-binding sites were identified by bioinformatic analysis and verified by electrophoretic mobility shift assays with purified hexahistidyl-tagged proteins. Carbon source-dependent deregulation of rpf2 expression was demonstrated in vivo in ramA and ramB mutants and in a C. glutamicum strain overexpressing glxR. The deduced network of regulatory interactions provided insights into the complex regulation pattern of rpf2 expression in C. glutamicum.

Corynebacterium glutamicum has recently been shown to grow on ethanol as a carbon and energy source and to possess high alcohol dehydrogenase (ADH) activity when growing on this substrate and low ADH activity when growing on ethanol plus glucose or glucose alone. Here we identify the C. glutamicum ADH gene ( adhA ), analyze its transcriptional organization, and investigate the relevance of the transcriptional regulators of acetate metabolism RamA and RamB for adhA expression. Sequence analysis of adhA predicts a polypeptide of 345 amino acids showing up to 57% identity with zinc-dependent ADH enzymes of group I. Inactivation of the chromosomal adhA gene led to the inability to grow on ethanol and to the absence of ADH activity, indicating that only a single ethanol-oxidizing ADH enzyme is present in C. glutamicum . Transcriptional analysis revealed that the C. glutamicum adhA gene is monocistronic and that its expression is repressed in the presence of glucose and of acetate in the growth medium, i.e., that adhA expression is subject to catabolite repression. Further analyses revealed that RamA and RamB directly bind to the adhA promoter region, that RamA is essential for the expression of adhA , and that RamB exerts a negative control on adhA expression in the presence of glucose or acetate in the growth medium. However, since the glucose- and acetate-dependent down-regulation of adhA expression was only partially released in a RamB-deficient mutant, there might be an additional regulator involved in the catabolite repression of adhA .

In Corynebacterium glutamicum, the transcriptional regulator RamB negatively controls the expression of genes involved in acetate metabolism. Here we show that during growth in media containing glucose and in complex medium without glucose RamB activates expression of the aceE gene, encoding the E1p subunit of the pyruvate dehydrogenase complex. Thus, RamB functions both as repressor and as activator in C. glutamicum.

Bifidobacteria constitute up to 3% of the total microbiota and represent one of the most important health-promoting bacterial groups of the human intestinal microflora. The presence ofBifidobacteriumin the human gastrointestinal tract has been directly related to several health-promoting activities; however, to date, no information about the specific mechanisms of interaction with the host is available. In order to provide some insight into the molecular mechanisms involved in the interaction with the host, we investigated whetherBifidobacteriumwas able to capture human plasminogen on the cell surface. By using flow cytometry, we demonstrated a dose-dependent human plasminogen-binding activity for four strains belonging to three bifidobacterial species:Bifidobacterium lactis,B. bifidum, andB. longum. The binding of human plasminogen toBifidobacteriumwas dependent on lysine residues of surface protein receptors. By using a proteomic approach, we identified five putative plasminogen-binding proteins in the cell wall fraction of the model strainB. lactisBI07. The data suggest that plasminogen binding toB. lactisis due to the concerted action of a number of proteins located on the bacterial cell surface, some of which are highly conserved cytoplasmic proteins which have other essential cellular functions. Our findings represent a step forward in understanding the mechanisms involved in theBifidobacterium-host interaction.