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L-phenylglycine (L-Phg) is a rare non-proteinogenic amino acid, which only occurs in some natural compounds, such as the streptogramin antibiotics pristinamycin I and virginiamycin S or the bicyclic peptide antibiotic dityromycin. Industrially, more interesting than L-Phg is the enantiomeric D-Phg as it plays an important role in the fine chemical industry, where it is used as a precursor for the production of semisynthetic β-lactam antibiotics. Based on the natural L-Phg operon from Streptomyces pristinaespiralis and the stereo-inverting aminotransferase gene hpgAT from Pseudomonas putida, an artificial D-Phg operon was constructed. The natural L-Phg operon, as well as the artificial D-Phg operon, was heterologously expressed in different actinomycetal host strains, which led to the successful production of Phg. By rational genetic engineering of the optimal producer strains S. pristinaespiralis and Streptomyces lividans, Phg production could be improved significantly. Here, we report on the development of a synthetic biology-derived D-Phg pathway and the optimization of fermentative Phg production in actinomycetes by genetic engineering approaches. Our data illustrate a promising alternative for the production of Phgs., Deutsche Forschungsgemeinschaft, Baden-Württemberg-Stiftung, Deutsches Zentrum für Infektionsforschung, Projekt DEAL

Mandelic acids are valuable products which are used in a broad field of applications. The enzyme hydroxymandelate synthase (HMS) is a non-heme iron dioxygenase which converts para-hydroxyphenylpyruvate and other 3-aryl pyruvates by decarboxylation to the corresponding mandelates. In the present work, the gene hms encoding the hydroxymandelate synthase from Amycolatopsis mediterranei was cloned and overexpressed in Escherichia coli BL21(DE3) for in vivo cascade catalysis taking advantage of resident aromatic amino acid transaminases. The resulting recombinant cells were used for whole cell biotransformations. We successfully accomplished the production of para-hydroxymandelate exclusively by using the aromatic amino acid l -tyrosine in biotransformation. Furthermore, by utilizing different phenylalanine derivatives (including chloro-, fluoro- and hydroxylated amino acids), the corresponding S-mandelic acids were obtained with high conversion (21–87 %) and high ee (38–97%). This process is an alternative and attractive way to get access to a variety of mandelic acids.

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Aromatic amines are an important class of chemicals which are used as building blocks for the synthesis of polymers and pharmaceuticals. In this study we establish a

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.

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.

In bacteria, the uptake of dicarboxylates, such as the tricarboxylic acid (TCA) cycle intermediates succinate, fumarate, and l-malate, is mediated by transporters of different protein families. Whereas Dcu-type transporters facilitate dicarboxylate uptake under anaerobic conditions, the most common aerobic dicarboxylate transporters are members of the dicarboxylate amino acid-cation symporter (DAACS), divalent anion sodium symporter (DASS), tripartite ATP-independent periplasmic (TRAP), and CitMHS transporter families. DAACS transporters are responsible for C4-dicarboxylate uptake under aerobic conditions in various bacteria, e.g., DctA from Escherichia coli, Bacillus subtilis, or Rhizobium leguminosarum, and are involved in different physiological functions (2, 4, 27, 41). The first described member of the TRAP family is the C4-dicarboxylate transporter DctPQM from Rhodobacter capsulatus, which facilitates substrate uptake by the use of an extracytoplasmic solute receptor (8). An example of the DASS family, members of which occur in bacteria, as well in eukaryotes, is the well-characterized transporter SdcS from Staphylococcus aureus (13). Members of the CitHMS family import citrate in symport with the cation Mg2+ or Ca2+. Whereas E. coli possesses one DctA and four different Dcu carriers, no Dcu transporter-encoding genes were found in Corynebacterium glutamicum (16, 19), which is used for the industrial production of amino acids, such as glutamate (33) or l-lysine (39), and is capable of succinate and l-lactate production under oxygen deprivation conditions. A dctA gene was annotated (19); however, C. glutamicum is not able to utilize succinate, malate, or fumarate as a sole carbon source. The uptake systems CitH and TctCBA have been characterized recently as citrate uptake systems (3, 26). Interestingly, we and others have shown that C. glutamicum possesses a DASS family transporter (DccT) for uptake of the C4-dicarboxylates succinate, fumarate, and l-malate (36, 40). Spontaneous mutants showing fast growth in succinate or fumarate minimal medium were isolated and shown to possess promoter-up mutations in the dccT gene (40). In l-malate minimal medium, these spontaneous mutants showed relatively slow growth, and the affinity of DccT for succinate and fumarate was found to be 5- and 12-fold higher than for l-malate, respectively (40). These findings prompted us to search for other uptake systems for l-malate in C. glutamicum. Here, we describe the identification and characterization of the DAACS family protein DctA from C. glutamicum as a proton motive force-driven uptake system for C4-dicarboxylate intermediates of the TCA cycle. Additionally, we compare both uptake systems, DccT and DctA, from C. glutamicum.

Many bacteria can utilize C 4 -carboxylates as carbon and energy sources. However, Corynebacterium glutamicum ATCC 13032 is not able to use tricarboxylic acid cycle intermediates such as succinate, fumarate, and l -malate as sole carbon sources. Upon prolonged incubation, spontaneous mutants which had gained the ability to grow on succinate, fumarate, and l -malate could be isolated. DNA microarray analysis showed higher mRNA levels of cg0277, which subsequently was named dccT , in the mutants than in the wild type, and transcriptional fusion analysis revealed that a point mutation in the promoter region of dccT was responsible for increased expression. The overexpression of dccT was sufficient to enable the C. glutamicum wild type to grow on succinate, fumarate, and l -malate as the sole carbon sources. Biochemical analyses revealed that DccT, which is a member of the divalent anion/Na + symporter family, catalyzes the effective uptake of dicarboxylates like succinate, fumarate, l -malate, and likely also oxaloacetate in a sodium-dependent manner.

Sustainable supply of feedstock has become a key issue in process development in microbial biotechnology. The workhorse of industrial amino acid production Corynebacterium glutamicum has been engineered towards utilization of alternative carbon sources. Utilization of the chitin-derived aminosugar N-acetyl-glucosamine (GlcNAc) for both cultivation and production with C. glutamicum has hitherto not been investigated. Albeit this organism harbors the enzymes N-acetylglucosamine-6-phosphatedeacetylase and glucosamine-6P deaminase of GlcNAc metabolism (encoded by nagA and nagB, respectively) growth of C. glutamicum with GlcNAc as substrate was not observed. This was attributed to the lack of a functional system for GlcNAc uptake. Of the 17 type strains of the genus Corynebacterium tested here for their ability to grow with GlcNAc, only Corynebacterium glycinophilum DSM45794 was able to utilize this substrate. Complementation studies with a GlcNAc-uptake deficient Escherichia coli strain revealed that C. glycinophilum possesses a nagE-encoded EII permease for GlcNAc uptake. Heterologous expression of the C. glycinophilum nagE in C. glutamicum indeed enabled uptake of GlcNAc. For efficient GlcNac utilization in C. glutamicum, improved expression of nagE with concurrent overexpression of the endogenous nagA and nagB genes was found to be necessary. Based on this strategy, C. glutamicum strains for the efficient production of the amino acid l-lysine as well as the carotenoid lycopene from GlcNAc as sole substrate were constructed.

Crop production may benefit from plant growth-promoting bacteria. The knowledge on bacterial communities is indispensable in agricultural systems that intend to apply beneficial bacteria to improve plant health and production of crops such as canola. In this work, the diversity of root bacterial communities associated to two different developmental phases of canola (Brassica napus L.) plants was assessed through the application of new generation sequencing technology. Total bacterial DNA was extracted from root samples from two different growth states of canola (rosette and flowering). It could be shown how bacterial communities inside the roots changed with the growing stage of the canola plants. There were differences in the abundance of the genera, family, and even the phyla identified for each sample. While in both root samples Proteobacteria was the most common phylum, at the rosette stage, the most common bacteria belonged to the family Pseudomonadaceae and the genus Pseudomonas, and in the flowering stage, the Xanthomonadaceae family and the genus Xanthomonas dominated the community. This implies in a switch in the predominant bacteria in the different developmental stages of the plant, suggesting that the plant itself interferes with the associated microbial community.

Corynebacterium glutamicum grows with a variety of carbohydrates and carbohydrate derivatives as sole carbon sources; however, growth with glucosamine has not yet been reported. We isolated a spontaneous mutant (M4) which is able to grow as fast with glucosamine as with glucose as sole carbon source. Glucosamine also served as a combined source of carbon, energy and nitrogen for the mutant strain. Characterisation of the M4 mutant revealed a significantly increased expression of the nagB gene encoding the glucosamine-6P deaminase NagB involved in degradation of glucosamine, as a consequence of a single mutation in the promoter region of the nagAB-scrB operon. Ectopic nagB overexpression verified that the activity of the NagB enzyme is in fact the growth limiting factor under these conditions. In addition, glucosamine uptake was studied, which proved to be unchanged in the wild-type and M4 mutant strains. Using specific deletion strains, we identified the PTS(Glc) transport system to be responsible for glucosamine uptake in C. glutamicum. The affinity of this uptake system for glucosamine was about 40-fold lower than that for its major substrate glucose. Because of this difference in affinity, glucosamine is efficiently taken up only if external glucose is absent or present at low concentrations. C. glutamicum was also examined for its suitability to use glucosamine as substrate for biotechnological purposes. Upon overexpression of the nagB gene in suitable C. glutamicum producer strains, efficient production of both the amino acid L-lysine and the diamine putrescine from glucosamine was demonstrated.

Formation of glycerol as by-product of amino acid production by Corynebacterium glutamicum has been observed under certain conditions, but the enzyme(s) involved in its synthesis from glycerol-3-phosphate were not known. It was shown here that cg1700 encodes an enzyme active as a glycerol-3-phosphatase (GPP) hydrolyzing glycerol-3-phosphate to inorganic phosphate and glycerol. GPP was found to be active as a homodimer. The enzyme preferred conditions of neutral pH and requires Mg²⁺ or Mn²⁺ for its activity. GPP dephosphorylated both L- and D-glycerol-3-phosphate with a preference for the D-enantiomer. The maximal activity of GPP was estimated to be 31.1 and 1.7 U mg⁻¹ with K(M) values of 3.8 and 2.9 mM for DL- and L-glycerol-3-phosphate, respectively. For physiological analysis a gpp deletion mutant was constructed and shown to lack the ability to produce detectable glycerol concentrations. Vice versa, gpp overexpression increased glycerol accumulation during growth in fructose minimal medium. It has been demonstrated previously that intracellular accumulation of glycerol-3-phosphate is growth inhibitory as shown for a recombinant C. glutamicum strain overproducing glycerokinase and glycerol facilitator genes from E. coli in media containing glycerol. In this strain, overexpression of gpp restored growth in the presence of glycerol as intracellular glycerol-3-phosphate concentrations were reduced to wild-type levels. In C. glutamicum wild type, GPP was shown to be involved in utilization of DL-glycerol-3-phosphate as source of phosphorus, since growth with DL-glycerol-3-phosphate as sole phosphorus source was reduced in the gpp deletion strain whereas it was accelerated upon gpp overexpression. As GPP homologues were found to be encoded in the genomes of many other bacteria, the gpp homologues of Escherichia coli (b2293) and Bacillus subtilis (BSU09240, BSU34970) as well as gpp1 from the plant Arabidosis thaliana were overexpressed in E. coli MG1655 and shown to significantly increase GPP activity.

Corynebacterium glutamicum call utilize acetic acid and propionic acid for growth and amino acid production. Growth on acetate as sole carbon source requires acetate activation by acetate kinase (AK) and phosphotransacetylase (PTA), encoded in the pta-ack operon. Genetic and enzymatic Studies, showed that these enzymes also catalyze propionate activation and were required for growth oil propionate as sole carbon source. However, when glucose was present as a co-substrate Strain lacking the AK-PTA pathway was still able to utilize acetate or propionate for growth indicating that an alternative activation pathway exists. As shown by C-13-labelling experiments, the carbon skeleton of acetate is conserved during activation to acetyl-CoA in this pathway. Metabolic flux analysis during growth on an acetate-glucose Mixture revealed that in the absence of the AK-PTA pathway carbon fluxes in glycolysis, the tricarboxylic acid (TCA) cycle and anaplerosis via PEP carboxylase and/or pyruvate carboxylase were increased, while the glyoxylate cycle flux was decreased. DNA microarray experiments identified cg2840 as a constitutively and highly expressed gene putatively encoding a CoA transferase. Purified His-tagged Cg2840 Protein was active as CoA transferase interconverting acetyl-, propionyl- and succinyl-moieties as CoA acceptors and donors. Strains lacking both the CoA transferase and the AK-PTA pathway could neither activate acetate nor propionate in the presence or absence of glucose. Thus, when these short-chain fatty acids are co-metabolized with other carbon Sources, CoA transferase and the AK-PTA pathway constitute a redundant system for activation of acetate and propionate. (C) 2008 Elsevier B.V. All rights reserved.

In this study, we demonstrate that sigma(E), an alternative sigma factor of Corynebacterium glutamicum, is involved in cell surface stresses. Cells in which the sigE gene was deleted evidenced increased sensitivity to magnesium deficiency, as well as to SDS, lysozymes, EDTA and heat. We utilized physiological analyses to show that the downstream gene, designated cseE, encodes an anti-sigma factor. The retarded growth of the cseE mutant cells under ordinary growth conditions could be recovered by an additional deletion of sigE encoding sigma(E). Under stress conditions, the phenotype of the cseE-overexpressing cells mimicked that of the sigE mutant. The sigE and cseE genes were transcribed into a single transcript, and gene transcription was stimulated by heat. The SigE and CseE proteins interacted physically in vitro, in the form of glutathione S-transferase (GST) and maltose binding protein (MBP) fusion proteins, respectively. 2D-PAGE analysis of the wild-type and mutant crude extracts showed that the sigE mutant failed to synthesize a 34 kDa polypeptide that was normally induced in wild-type cells grown under heat (or SDS)-stressed conditions. The protein turned out to be expressed from ORF NCgl1070 and showed similarity to methyltransferases which may confer resistance to antibiotics. Accordingly, the sigE mutant evidenced extreme sensitivity to antibiotics, including nalidixic acid, penicillin and vancomycin. Finally, we present a discussion of the possible role of the sigE and cseE genes in the acclimation of C. glutamicum to cell surface stress conditions.