Establishing the Weimberg pathway in Corynebacterium glutamicum as an alternative route for D-xylose utilization

Radek A. Establishing the Weimberg pathway in Corynebacterium glutamicum as an alternative route for D-xylose utilization. Bielefeld: Universität Bielefeld; 2018.In times of scarcity of fossil resources and climate change, the need of renewable resources for the biobased economy becomes increasingly important. Within this thesis a contribution was done to achieve the aim to produce chemicals biobased and not anymore only petrochemical based. To accomplish this, the industrial relevant microorganism Corynebacterium glutamicum was engineered to utilize the hemicellulose derived pentose sugar D-xylose via an alternative pathway. This utilization was made feasible via the Weimberg pathway without loss of carbon in terms of products derived from alpha-ketoglutarate. During the first investigation two major by-products, D-xylonate and xylitol, of the new synthetic pathway were found. The latter is known as inhibitor of different microorganisms, which could be also confirmed in this thesis for C. glutamicum. The inhibition studies showed different target genes, which are partly responsible for this inhibitory effect (e.g. xylB). Interestingly, the reference strain in this thesis, a strain expressing the isomerase pathway (including the xylB gene), was already inhibited by low amounts of extracellular xylitol and accumulates the highest amount of intracellular xylitol-5-phosphate. Within this thesis a high amount of engineered strains had to be screened and charaterized. Therefore, it was the initial aim to develop new robotic workflows and automated enzymatic assays on a robotic platform. The extended Mini Pilot Plant enables now the high-throughput microbial phenotyping with connected analytics in 384-well format. Further, it is now possible to run a complete process with separation of cell-free supernatants for analytics under sterile conditions without human interruption. Another new robotic workflow which was designed within this thesis is the miniaturized and automated repetitive batch cultivation. The developed workflow was used for fully automated adaptive laboratory evolution (ALE) of the engineered C. glutamicum strain expressing theWeimberg pathway for D-xylose utilization. The ALE process was successful and the final strain had an 260% increased maximal growth rate of max = 0.26 h-1on D-xylose as sole carbon source. The final D-xylose utilizing strain (WMB2evo) grows stable during lab-scale bioreactor operation, which demonstrates the high potential of this strain for future projects with biorefinery applications. Additionally, the occurred mutations during the ALE process were analyzed by genome sequencing and revealed about 15 potential key mutations for improved D-xylose assimilation. These found mutations can be used for rational strain engineering within further biorefinery projects. But for now the final D-xylose utilizing strain (WMB2evo) is the fastest growing C. glutamicum strain on D-xylose as sole carbon source in literature

Improved glycerol utilization by a triacylglycerol-producing Rhodococcus opacus strain for renewable fuels

Background: Glycerol generated during renewable fuel production processes is potentially an attractive substrate for the production of value-added materials by fermentation. An engineered strain MITXM-61 of the oleaginous bacterium Rhodococcus opacus produces large amounts of intracellular triacylglycerols (TAGs) for lipid-based biofuels on high concentrations of glucose and xylose. However, on glycerol medium, MITXM-61 does not produce TAGs and grows poorly. The aim of the present work was to construct a TAG-producing R. opacus strain capable of high-cell-density cultivation at high glycerol concentrations. Results: An adaptive evolution strategy was applied to improve the conversion of glycerol to TAGs in R. opacus MITXM-61. An evolved strain, MITGM-173, grown on a defined medium with 16 g L[superscript −1] glycerol, produced 2.3 g L[superscript −1] of TAGs, corresponding to 40.4% of the cell dry weight (CDW) and 0.144 g g[superscript −1] of TAG yield per glycerol consumed. MITGM-173 was able to grow on high concentrations (greater than 150 g L[superscript −1]) of glycerol. Cultivated in a medium containing an initial concentration of 20 g L[superscript −1] glycerol, 40 g L[superscript −1] glucose, and 40 g L[superscript −1] xylose, MITGM-173 was capable of simultaneously consuming the mixed substrates and yielding 13.6 g L[superscript −1] of TAGs, representing 51.2% of the CDM. In addition, when 20 g L[superscript −1] glycerol was pulse-loaded into the culture with 40 g L[superscript −1] glucose and 40 g L[superscript −1] xylose at the stationary growth phase, MITGM-173 produced 14.3 g L[superscript −1] of TAGs corresponding to 51.1% of the CDW although residual glycerol in the culture was observed. The addition of 20 g L[superscript −1] glycerol in the glucose/xylose mix resulted in a TAG yield per glycerol consumed of 0.170 g g[superscript −1] on the initial addition and 0.279 g g[superscript −1] on the pulse addition of glycerol. Conclusion: We have generated a TAG-producing R. opacus MITGM-173 strain that shows significantly improved glycerol utilization in comparison to the parental strain. The present study demonstrates that the evolved R. opacus strain shows significant promise for developing a cost-effective bioprocess to generate advanced renewable fuels from mixed sugar feedstocks supplemented with glycerol.Sweetwater Energy, Inc.MIT Energy Initiativ

Long rDNA amplicon sequencing of insect-infecting nephridiophagids reveals their affiliation to the Chytridiomycota and a potential to switch between hosts

Nephridiophagids are unicellular eukaryotes that parasitize the Malpighian tubules of numerous insects. Their life cycle comprises multinucleate vegetative plasmodia that divide into oligonucleate and uninucleate cells, and sporogonial plasmodia that form uninucleate spores. Nephridiophagids are poor in morphological characteristics, and although they have been tentatively identified as early-branching fungi based on the SSU rRNA gene sequences of three species, their exact position within the fungal tree of live remained unclear. In this study, we describe two new species of nephridiophagids (Nephridiophaga postici and Nephridiophaga javanicae) from cockroaches. Using long-read sequencing of the nearly complete rDNA operon of numerous further species obtained from cockroaches and earwigs to improve the resolution of the phylogenetic analysis, we found a robust affiliation of nephridiophagids with the Chytridiomycota—a group of zoosporic fungi that comprises parasites of diverse host taxa, such as microphytes, plants, and amphibians. The presence of the same nephridiophagid species in two only distantly related cockroaches indicates that their host specificity is not as strict as generally assumed

New insights into the coevolutionary history of termites and their gut flagellates: Description of Retractinympha glossotermitis gen. nov. sp. nov. (Retractinymphidae fam. nov.)

Lower termites harbor diverse consortia of symbiotic gut flagellates. Despite numerous evidence for co-cladogenesis, the evolutionary history of these associations remains unclear. Here, we present Retractinymphidae fam. nov., a monogeneric lineage of Trichonymphida from Serritermitidae. Although Retractinympha glossotermitis gen. nov. sp. nov. morphologically resembles members of the genus Pseudotrichonympha, phylogenetic analysis identified it as sister group of the Teranymphidae. We compared morphology and ultrastructure of R. glossotermitis to that of Pseudotrichonympha and other Teranymphidae, including the so-far undescribed Pseudotrichonympha solitaria sp. nov. from Termitogeton planus (Rhinotermitidae). Like all Teranymphidae, R. glossotermitis is a large, elongated flagellate with a bilaterally symmetric rostrum, an anterior, flagella-free operculum, and an internal rostral tube. However, it is readily distinguished by the length of its rostral flagella, which never exceeds that of the postrostral flagella, and its retractable anterior end. Inclusion of the hitherto unstudied Stylotermes halumicus (Stylotermitidae) in our survey of trichonymphid flagellates in Neoisoptera confirmed that the combined presence of Heliconympha and Retractinympha and absence of Pseudotrichonympha is unique to Serritermitidae. The close phylogenetic relatedness of Heliconympha in Serritermitidae to the spirotrichosomid flagellates in Stolotermitidae provides strong support for their acquisition by horizontal transmission

New insights into the coevolutionary history of termites and their gut flagellates: Description of Retractinympha glossotermitis gen. nov. sp. nov. (Retractinymphidae fam. nov.)

Lower termites harbor diverse consortia of symbiotic gut flagellates. Despite numerous evidence for co-cladogenesis, the evolutionary history of these associations remains unclear. Here, we present Retractinymphidae fam. nov., a monogeneric lineage of Trichonymphida from Serritermitidae. Although Retractinympha glossotermitis gen. nov. sp. nov. morphologically resembles members of the genus Pseudotrichonympha, phylogenetic analysis identified it as sister group of the Teranymphidae. We compared morphology and ultrastructure of R. glossotermitis to that of Pseudotrichonympha and other Teranymphidae, including the so-far undescribed Pseudotrichonympha solitaria sp. nov. from Termitogeton planus (Rhinotermitidae). Like all Teranymphidae, R. glossotermitis is a large, elongated flagellate with a bilaterally symmetric rostrum, an anterior, flagella-free operculum, and an internal rostral tube. However, it is readily distinguished by the length of its rostral flagella, which never exceeds that of the postrostral flagella, and its retractable anterior end. Inclusion of the hitherto unstudied Stylotermes halumicus (Stylotermitidae) in our survey of trichonymphid flagellates in Neoisoptera confirmed that the combined presence of Heliconympha and Retractinympha and absence of Pseudotrichonympha is unique to Serritermitidae. The close phylogenetic relatedness of Heliconympha in Serritermitidae to the spirotrichosomid flagellates in Stolotermitidae provides strong support for their acquisition by horizontal transmission

Herwig 7.1 Release Note

A new release of the Monte Carlo event generator Herwig (version 7.1) is now available. This version introduces a number of improvements, notably: multi-jet merging with the dipole shower at LO and NLO QCD; a new model for soft interactions and diffraction; improvements to mass effects and top decays in the dipole shower, as well as a new tune of the hadronisation parameters.Comment: 7 pages, 7 figures. Herwig is available from https://herwig.hepforge.org

Nosema pieriae sp. n. (Microsporida, Nosematidae): A New Microsporidian Pathogen of the Cabbage Butterfly Pieris brassicae L. (Lepidoptera: Pieridae)

A new microsporidian pathogen of the cabbage butterfly, Pieris brassicae is described based on light microscopy, ultrastructural characteristics and comparative small subunit rDNA analysis. The pathogen infects the gut of P. brassicae. All development stages are in direct contact with the host cell cytoplasm. Meronts are spherical or ovoid. Spherical meronts measure 3.68 ± 0.73 × 3.32 ± 1.09 µm and ovoid meronts 4.04 ± 0.74 × 2.63 ± 0.49 µm. Sporonts are spherical to elongate (4.52 ± 0.48 × 2.16 ± 0.27 µm). Sporoblasts are elongated and measure 4.67 ± 0.60 × 2.30 ± 0.30 µm in length. Fresh spores with nuclei arranged in a diplokaryon are oval and measure 5.29 ± 0.55 µm in length and 2.31 ± 0.29 µm in width. Spores stained with Giemsa’s stain measure 4.21 ± 0.50 µm in length and 1.91 ± 0.24 µm in width. Spores have an isofilar polar filament with six coils. All morphological, ultrastructural and molecular features indicate that the described microsporidium belongs to the genus Nosema and confirm that it has different taxonomic characters than other microsporidia infecting Pieris spp

Deazaflavin reductive photocatalysis involves excited semiquinone radicals

Flavin-mediated photocatalytic oxidations are established in synthetic chemistry. In contrast, their use in reductive chemistry is rare. Deazaflavins with a much lower reduction potential are even better suited for reductive chemistry rendering also deazaflavin semiquinones as strong reductants. However, no direct evidence exists for the involvement of these radical species in reductive processes. Here, we synthesise deazaflavins with different substituents at C5 and demonstrate their photocatalytic activity in the dehalogenation of p-halogenanisoles with best performance under basic conditions. Mechanistic investigations reveal a consecutive photo-induced electron transfer via the semiquinone form of the deazaflavin as part of a triplet-correlated radical pair after electron transfer from a sacrificial electron donor to the triplet state. A second electron transfer from the excited semiquinone to p-halogenanisoles triggers the final product formation. This study provides first evidence that the reductive power of excited deazaflavin semiquinones can be used in photocatalytic reductive chemistry. Flavins and deazaflavins are well suited for photoredox processes but their application in photoreductions is challenging. Here, the authors provide direct evidence of the high reductive power of excited deazaflavin semiquinones and their application in catalytic photodehalogenations

Defensive behavior is linked to altered surface chemistry following infection in a termite society

The care-kill response determines whether a sick individual will be treated or eliminated from an insect society, but little is known about the physiological underpinnings of this process. We exploited the stepwise infection dynamics of an entomopathogenic fungus in a termite to explore how care-kill transitions occur, and identify the chemical cues behind these shifts. We found collective responses towards pathogen-injected individuals to vary according to severity and timing of pathogen challenge, with elimination, via cannibalism, occurring sooner in response to a severe active infection. However, injection with inactivated fungal blastospores also resulted in increased albeit delayed cannibalism, even though it did not universally cause host death. This indicates that the decision to eliminate an individual is triggered before pathogen viability or terminal disease status has been established. We then compared the surface chemistry of differently challenged individuals, finding increased amounts of long-chained methyl-branched alkanes with similar branching patterns in individuals injected with both dead and viable fungal blastospores, with the latter showing the largest increase. This coincided with the highest amounts of observed cannibalism as well as signs of severe moribundity. Our study provides new mechanistic insight into the emergent collective behaviors involved in the disease defense of a termite society

Chassis organism from Corynebacterium glutamicum – a top-down approach to identify and delete irrelevant gene clusters

Unthan S, Baumgart M, Radek A, et al. Chassis organism from Corynebacterium glutamicum – a top-down approach to identify and delete irrelevant gene clusters. Biotechnology Journal. 2015;10(2):290-301