Exploring the potential of sigma factors for strain development in Corynebacterium glutamicum

Taniguchi H. Exploring the potential of sigma factors for strain development in Corynebacterium glutamicum. Bielefeld: Universität Bielefeld; 2016.A sigma factor is one of the components of RNA polymerase holoenzyme and responsible for promoter recognition and transcription initiation. Bacteria usually possess multiple genes encoding sigma factors and strictly control their activity. Activation of a specific sigma factor alters the promoter recognition of the RNA polymerase holoenzyme and enhances the transcription of a specific set of genes with similar promoter sequences. Therefore, sigma factors can be used for activating expression of a set of genes at the same time and could be useful for strain development. In this study, the potential of sigma factors for strain development was examined in Corynebacterium glutamicum. This bacterium was first identified as a natural glutamate producer and has been used in industry for production of amino acids such as L-glutamate and L-lysine. Because of its genetic amenability, various strains have been developed to produce industrial relevant compounds using metabolic engineering. C. glutamicum possesses seven sigma factor genes, sigA, sigB, sigC, sigD, sigE, sigH and sigM. In order to test the effect of sigma factors on the cell behavior, all sigma factor genes were overexpressed in an IPTG-inducible system with different IPTG concentrations. Overexpression of each sigma factor gene affected the growth rate differently, and the effect was dependent on the IPTG concentration, thus, on the strength of expression. The strongest effect was observed for overexpression of sigD and sigH. Overexpression of sigH led to the strong yellow supernatant. HPLC analysis identified that this color was caused by riboflavin. Transcriptome analysis revealed increased expression of genes of riboflavin biosynthesis, the pentose phosphate pathway and of enzymes requiring FMN (flavin mononucleotide), FAD (flavin adenine dinucleotide) or NADPH as cofactor. Since riboflavin is a precursor of FMN, sigH overexpression was applied for the production of FMN. Balanced expression o f sigH and the bifunctional riboflavin kinase/FMN adenyltransferase gene ribF improved accumulation of riboflavin (20 ± 1 μM) and allowed for its conversion to FMN (33 ± 2 μM). sigD overexpression in C. glutamicum WT reduced foaming possibly due to secretion of anti-foaming compounds. Transcriptome analysis revealed that expression of genes encoding for proteins which are involved in the cell wall integrity such as mycomembrane synthesis increased as a consequence of sigD overexpression. TLC analysis revealed that sigD overexpression increased the content of trehalose dicorynomycolate, which is one of the components of the mycomembrane. In order to explore the potential of sigma factor for strain development, overexpression of sigma factor genes was performed in the recombinant lycopene producing strain LYC5, aiming at an increase of lycopene production. Among seven sigma factor genes, sigA was selected as the most effective gene for this purpose. Overexpression of sigA in the WT strain also increased production of decaprenoxanthin, which is naturally produced by C. glutamicum. Accumulation of lycopene and decaprenoxanthin in the sigA overexpressing strain was observed especially in the stationary phase. Transcriptome analysis identified many genes which expression was upregulated under sigA overexpression. Based on this transcriptome information, the supplement of thiamine and protocatechuic acid was found to further increase decaprenoxanthin production under sigA overexpression. In addition, the effect of sigA overexpression was successfully transferred to other carotenoid producing strains. In this study, it was proven that overexpression of sigma factor genes can perturb the transcriptome profile of C. glutamicum by artificially activating the native metabolic pathway and change cell physiology under normal conditions. Artificial activation of sigma factors was helpful to understand regulatory networks in bacteria by connecting genes at different loci with similar promoter sequences. In addition, overexpression of sigma factor and the following screening of the potent sigma factor gene for a specific purpose was shown to be an effective approach for strain development. Therefore, overexpression of genes encoding sigma factors is not only relevant to study the gene regulatory networks, but also effective in strain development of C. glutamicum with respect to production of value-added chemicals

Optimal Detection for Diffusion-Based Molecular Timing Channels

This work studies optimal detection for communication over diffusion-based molecular timing (DBMT) channels. The transmitter simultaneously releases multiple information particles, where the information is encoded in the time of release. The receiver decodes the transmitted information based on the random time of arrival of the information particles, which is modeled as an additive noise channel. For a DBMT channel without flow, this noise follows the L\'evy distribution. Under this channel model, the maximum-likelihood (ML) detector is derived and shown to have high computational complexity. It is also shown that under ML detection, releasing multiple particles improves performance, while for any additive channel with $\alpha$-stable noise where $\alpha<1$ (such as the DBMT channel), under linear processing at the receiver, releasing multiple particles degrades performance relative to releasing a single particle. Hence, a new low-complexity detector, which is based on the first arrival (FA) among all the transmitted particles, is proposed. It is shown that for a small number of released particles, the performance of the FA detector is very close to that of the ML detector. On the other hand, error exponent analysis shows that the performance of the two detectors differ when the number of released particles is large.Comment: 16 pages, 9 figures. Submitted for publicatio

Exploring the role of sigma factor gene expression on production by Corynebacterium glutamicum: sigma factor H and FMN as example

Taniguchi H, Wendisch VF. Exploring the role of sigma factor gene expression on production by Corynebacterium glutamicum: sigma factor H and FMN as example. Frontiers in Microbiology. 2015;6: 740.Bacteria are known to cope with environmental changes by using alternative sigma factors binding to RNA polymerase core enzyme. Sigma factor is one of the targets to modify transcription regulation in bacteria and to influence production capacities. In this study, the effect of overexpressing each annotated sigma factor gene in Corynebacterium glutamicum WT was assayed using an IPTG inducible plasmid system and different IPTG concentrations. It was revealed that growth was severely decreased when sigD or sigH were overexpressed with IPTG concentrations higher than 50 μM. Overexpression of sigH led to an obvious phenotypic change, a yellow-colored supernatant. High performance liquid chromatography analysis revealed that riboflavin was excreted to the medium when sigH was overexpressed and DNA microarray analysis confirmed increased expression of riboflavin biosynthesis genes. In addition, genes for enzymes related to the pentose phosphate pathway and for enzymes dependent on flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), or NADPH as cofactor were upregulated when sigH was overexpressed. To test if sigH overexpression can be exploited for production of riboflavin-derived FMN or FAD, the endogenous gene for bifunctional riboflavin kinase/FMN adenyltransferase was co-expressed with sigH from a plasmid. Balanced expression of sigH and ribF improved accumulation of riboflavin (19.8 ± 0.3 μM) and allowed for its conversion to FMN (33.1 ± 1.8 μM) in the supernatant. While a proof-of-concept was reached, conversion was not complete and titers were not high. This study revealed that inducible and gradable overexpression of sigma factor genes is an interesting approach to switch gene expression profiles and to discover untapped potential of bacteria for chemical production

交通行動と健康診断データ・心的傾向の関連分析 : 神奈川県大和市職員を対象として

本研究では，交通行動と健康状態，ならびにクルマの運転動機，交通手段への態度，五大性格特性，主観的幸福感等の心的傾向を計測する指標との関係性を把握することを目的に，神奈川県大和市職員を対象としたアンケート調査(n=479)を行った．このうち健康診断データの提供に同意した職員は180名であった．分析の結果，クルマ・バイク通勤者はメタボ該当者がそれ以外の通勤者の倍程度であった．心的傾向については，組織との関係が良好で地域のボランティアに参加しているほど，メタボや高BMIの傾向が示された．この理由としては，勤務先やボランティア等で飲食を伴う交流が盛んであった可能性が考えられる．メタボは危険因子が集積した状態であり，自覚症状が希薄であるため，健康指標として心的傾向と組み合わせた分析を行う際には留意が必要である

In vitro production of L-cysteine using thermophilic enzymes

L-Cysteine (L-Cys) is a commercially important amino acid and widely used in food, pharmaceutical, and cosmetic industries. Commercial production of L-Cys has long been done by an acid-hydrolysis of human hair and animal feather, leading to the generation of a large quantity of hazardous wastes. Although several biotechnology companies have recently launched a fermentative production of L-Cys using engineered bacteria, these processes suffer from the low product titer mainly due to the cytotoxic effect of L-Cys. To provide an alternative approach for the commercial production of L-Cys, we aimed at the development of a non-fermentative, in vitro manufacturing system using thermophilic enzymes. In this system, enzymes from (hyper)thermophilic bacteria and archaea were assembled to construct an in vitro synthetic pathway for the one-pot conversion of glucose to L-Cys (Figure 1). By using experimentally optimized concentrations of enzymes, L-Cys could be produced at a rate of 0.9 g/L/h with a molar conversion yield of 25%. Please click Additional Files below to see the full abstract

Novel Charge Ordering in the Trimer Iridium Oxide BaIrO3

We have prepared polycrystalline samples of the trimer Ir oxide BaIrO3 with face-shared Ir3O12 trimers, and have investigated the origin of the phase transition at 182 K by measuring resistivity, thermopower, magnetization and synchrotron x-ray diffraction. We propose a possible electronic model and transition mechanism, starting from a localized electron picture on the basis of the Rietveld refinement. Within this model, BaIrO3 can be basically regarded as a Mott insulator, when the Ir3O12 trimer is identified to one pseudo-atom or one lattice site. The transition can be viewed as a transition from the Mott insulator phase to a kind of charge ordered insulator phase.Comment: 8 pages 5 figures, Crystals (in press

Differences of Functional Connectivity Brain Network in Emotional Judgment

Using combined emotional stimuli, combining photos of faces and recording of voices, we investigated the neural dynamics of emotional judgment using scalp EEG recordings. Stimuli could be either combioned in a congruent, or a non-congruent way.. As many evidences show the major role of alpha in emotional processing, the alpha band was subjected to be analyzed. Analysis was performed by computing the synchronization of the EEGs and the conditions congruent vs. non-congruent were compared using statistical tools. The obtained results demonstrate that scalp EEG ccould be used as a tool to investigate the neural dynamics of emotional valence and discriminate various emotions (angry, happy and neutral stimuli)

Regulation of the pstSCAB operon in Corynebacterium glutamicum by the regulator of acetate metabolism RamB

Sorger-Herrmann U, Taniguchi H, Wendisch VF. Regulation of the pstSCAB operon in Corynebacterium glutamicum by the regulator of acetate metabolism RamB. BMC Microbiology. 2015;15(1): 113.Background The pstSCAB operon of Corynebacterium glutamicum, which encodes an ABC transport system for uptake of phosphate (Pi), is induced during the Pi starvation response. The two-component regulatory system PhoRS is involved in this response, but partial Pi starvation induction of pstSCAB in a ΔphoRS mutant indicated the involvement of additional regulator(s). Regulation of pstSCAB also involves the global transcriptional regulator GlxR. Results DNA affinity chromatography identified the regulator of acetate metabolism RamB as a protein binding to pstS promoter DNA in vitro. Gel mobility shift assays and mutational analysis of the pstS promoter region revealed that RamB binds to two sites localized at positions −74 to −88 and −9 to +2 with respect to the transcriptional start site of pstSCAB. Reporter gene studies supported the in vivo relevance of both binding sites for activation of pstSCAB by RamB. DNA microarray analysis revealed that expression of many Pi starvation genes reached higher levels during the Pi starvation response on minimal medium with glucose as sole carbon source than in Pi starved acetate-grown C. glutamicum cells. Conclusions In C. glutamicum, RamB is involved in expression control of pstSCAB operon. Thus, transcriptional regulation of pstSCAB is complex involving activation by the phosphate-responsive two-component regulatory system PhoSR and the regulators of carbon metabolism GlxR and RamB

Overexpression of the primary sigma factor gene sigA improved carotenoid production by Corynebacterium glutamicum: application to production of beta-carotene and the non-native linear C50 carotenoid bisanhydrobacterioruberin

Taniguchi H, Henke NA, Heider S, Wendisch VF. Overexpression of the primary sigma factor gene sigA improved carotenoid production by Corynebacterium glutamicum: application to production of beta-carotene and the non-native linear C50 carotenoid bisanhydrobacterioruberin. Metabolic Engineering Communications. 2017;4:1-11.Corynebacterium glutamicum shows yellow pigmentation due to biosynthesis of the C50 carotenoid decaprenoxanthin and its glycosides. This bacterium has been engineered for production of various non-native cyclic C40 and C50 carotenoids such as β-carotene, astaxanthin or sarcinaxanthin. In this study, the effect of modulating gene expression more broadly by overexpression of sigma factor genes on carotenoid production by C. glutamicum was characterized. Overexpression of the primary sigma factor gene sigA improved lycopene production by recombinant C. glutamicum up to 8-fold. In C. glutamicum wild type, overexpression of sigA led to 2-fold increased accumulation of the native carotenoid decaprenoxanthin in the stationary growth phase. Under these conditions, genes related to thiamine synthesis and aromatic compound degradation showed increased RNA levels and addition of thiamine and the aromatic iron chelator protocatechuic acid to the culture medium enhanced carotenoid production when sigA was overexpressed. Deletion of the gene for the alternative sigma factor SigB, which is expected to replace SigA in RNA polymerase holoenzymes during transition to the stationary growth phase, also increased carotenoid production. The strategy of sigA overexpression could be successfully transferred to production of the non-native carotenoids β-carotene and bisanhydrobacterioruberin (BABR). Production of the latter is the first demonstration that C. glutamicum may accumulate a non-native linear C50 carotenoid instead of the native cyclic C50 carotenoid decaprenoxanthin

Sorafenib as a secondary treatment

Background and Aim: Currently, there is no molecular‐targeted agent that has demonstrated evidence of efficacy in patients with unresectable hepatocellular carcinoma (u‐HCC) who have developed resistance to treatment with lenvatinib (LEN). In this real‐world study, we aimed to investigate the therapeutic effect and safety of sorafenib (SOR) in patients with u‐HCC after progression on treatment with LEN. Methods (Patients) and Results: A total of 13 patients with u‐HCC (12 males and 1 female), who were treated with SOR after progression on LEN, were enrolled in this retrospective study. Therapeutic efficacy was evaluated via contrast‐enhanced computerized tomography at 8 weeks after the initiation of SOR therapy according to modified response evaluation criteria in solid tumors (mRECIST) and RECIST. According to mRECIST, the objective response rate (ORR) and disease control rate (DCR) were 15.3% (2/13) and 69.2% (9/13), respectively. According to RECIST, the ORR and DCR were 0% (0/13) and 69.2% (9/13), respectively. The median progression‐free survival was 4.1 months. The median albumin‐bilirubin scores did not deteriorate significantly at 4, 6, and 8 weeks after initiation of SOR, compared with the scores at the baseline. The most frequent grade 1 or 2 adverse events (AEs) were palmar–plantar erythrodysesthesia, fatigue, diarrhea, and hypertension. There was no incidence of grade 3 AEs. Conclusion: Treatment with SOR may be effective for u‐HCC after failure on LEN and may not worsen the liver reserve