Biochemical characterization of recombinant isocitrate dehydrogenase and its putative role in the physiology of an acidophilic micrarchaeon

Despite several discoveries in recent years, the physiology of acidophilic Micrarchaeota, such as “Candidatus Micrarchaeum harzensis A_DKE”, remains largely enigmatic, as they highly express numerous genes encoding hypothetical proteins. Due to a lacking genetic system, it is difficult to elucidate the biological function of the corresponding proteins and heterologous expression is required. In order to prove the viability of this approach, A_DKE’s isocitrate dehydrogenase (MhIDH) was recombinantly produced in Escherichia coli and purified to electrophoretic homogeneity for biochemical characterization. MhIDH showed optimal activity around pH 8 and appeared to be specific for NADP$^{+}$ yet promiscuous regarding divalent cations as cofactors. Kinetic studies showed K$_{M}$-values of 53.03 ± 5.63 µM and 1.94 ± 0.12 mM and k$_{cat}$-values of 38.48 ± 1.62 and 43.99 ± 1.46 s$^{-1}$ resulting in k$_{cat}$/K$_{M}$-values of 725 ± 107.62 and 22.69 ± 2.15 mM$^{-1}$ s$^{-1}$ for DL-isocitrate and NADP$^{+}$, respectively. MhIDH’s exceptionally low affinity for NADP$^{+}$, potentially limiting its reaction rate, can likely be attributed to the presence of a proline residue in the NADP$^{+}$ binding pocket, which might cause a decrease in hydrogen bonding of the cofactor and a distortion of local secondary structure

Electrode-assisted acetoin production in a metabolically engineered Escherichia coli strain

Background This paper describes the metabolic engineering of Escherichia coli for the anaerobic fermentation of glucose to acetoin. Acetoin has well-established applications in industrial food production and was suggested to be a platform chemical for a bio-based economy. However, the biotechnological production is often hampered by the simultaneous formation of several end products in the absence of an electron acceptor. Moreover, typical production strains are often potentially pathogenic. The goal of this study was to overcome these limitations by establishing an electrode-assisted fermentation process in E. coli. Here, the surplus of electrons released in the production process is transferred to an electrode as anoxic and non-depletable electron acceptor. Results In a first step, the central metabolism was steered towards the production of pyruvate from glucose by deletion of genes encoding for enzymes of central reactions of the anaerobic carbon metabolism (ΔfrdA-D ΔadhE ΔldhA Δpta–ack). Thereafter, the genes for the acetolactate synthase (alsS) and the acetolactate decarboxylase (alsD) were expressed in this strain from a plasmid. Addition of nitrate as electron acceptor led to an anaerobic acetoin production with a yield of up to 0.9 mol acetoin per mol of glucose consumed (90% of the theoretical maximum). In a second step, the electron acceptor nitrate was replaced by a carbon electrode. This interaction necessitated the further expression of c-type cytochromes from Shewanella oneidensis and the addition of the soluble redox shuttle methylene blue. The interaction with the non-depletable electron acceptor led to an acetoin formation with a yield of 79% of the theoretical maximum (0.79 mol acetoin per mol glucose). Conclusion Electrode-assisted fermentations are a new strategy to produce substances of biotechnological value that are more oxidized than the substrates. Here, we show for the first time a process in which the commonly used chassis strain E. coli was tailored for an electrode-assisted fermentation approach branching off from the central metabolite pyruvate. At this early stage, we see promising results regarding carbon and electron recovery and will use further strain development to increase the anaerobic metabolic turnover rate

Nanowired electrodes as outer membrane cytochrome-independent electronic conduit in Shewanella oneidensis

Extracellular electron transfer (EET) from microorganisms to inorganic electrodes is a unique ability of electrochemically active bacteria. Despite rigorous genetic and biochemical screening of the c-type cytochromes that make up the EET network, the individual electron transfer steps over the cell membrane remain mostly unresolved. As such, attempts to transplant entire EET chains from native into non-native exoelectrogens have resulted in inferior electron transfer rates. In this study we investigate how nanostructured electrodes can interface with Shewanella oneidensis to establish an alternative EET pathway. Improved biocompatibility was observed for densely packed nanostructured surfaces with a low cell-nanowire load distribution during applied external forces. External gravitational forces were needed to establish a bioelectrochemical cell-nanorod interface. Bioelectrochemical analysis showed evidence of nanorod penetration beyond the outer cell membrane of a deletion mutant lacking all outer membrane cytochrome encoding genes that was only electroactive on a nanostructured surface and under external force

Enhanced production of propionic acid through acidic hydrolysis by choice of inoculum

BACKGROUND In this study, the enhancement of propionic acid production from a model feedstock mimicking kitchen waste was investigated. For that purpose, two operational runs of a semicontinuous anaerobic hydrolysis reactor were carried out at pH 6.0 ± 0.1 and mesophilic (30 °C) temperature. Two different types of inocula, a mixed microbial culture selected over 24 months for growth on cellulose and a culture contained in goat cheese were compared. RESULTS The results show that the goat cheese inoculum was significantly more efficient for propionic acid (PA) production. The highest propionic acid concentration achieved amounted to 139 mmol L−1 at a yield of 23.3 mg g−1 volatile solids (VS), which was 55% greater than what was achieved with the mixed culture. Furthermore, it was observed that propionic acid production was enhanced by a combination of high hydraulic retention time (HRT) with low organic loading rate (OLR), ensuring sufficient time for complete processing of the complex organic substrates. The fermentation could be kept in a stable process of propionic acid production at HRT of 20 days and a rather low OLR of 11.1 g L−1 day−1 VS. CONCLUSION Our results give a better understanding of PA production in semicontinuous mode, applying optimized process parameters and selecting the adequate microbial community for inoculation. This study provides important information for the improvement of PA production from complex substrates for future industrial application. © 2020 The Authors. Journal of Chemical Technology and Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry (SCI)

N-methylformamide: antitumour activity and metabolism in mice.

The antitumour activities of N-methylformamide, N-ethylformamide and formamide against a number of murine tumours in vivo (Sarcoma 180, M5076 ovarian sarcoma and TLX5 lymphoma) have been estimated. In all cases N-methyl-formamide had significant activity, formamide had marginal or no activity and N-ethylformamide had no significant activity. N-methylformamide and N-ethylformamide were equitoxic to the TLX5 lymphoma in vitro. Formamide was found as a metabolite in the plasma and urine of animals given N-methylformamide and N-ethylformamide, but excretion profiles do not support the hypothesis that formamide is an active antitumour species formed from N-alkylformamides. No appreciable metabolism of N-methylformamide occurred under a variety of conditions with liver preparations in vitro. N-methylformamide, but not N-ethylformamide or formamide, reduced liver soluble non-protein thiols by 59.8% 1 h after administration of an effective antitumour dose

PPARδ status and mismatch repair mediated neoplasia in the mouse intestine

BACKGROUND: Therapeutic regulation of PPARδ activity using selective agonists has been proposed for various disorders. However, the consequences of altered peroxisome proliferator-activated receptor delta (PPARδ) activity in the context of intestinal tumourigenesis remain somewhat unclear. Contradictory evidence suggesting PPARδ either attenuates or potentiates intestinal neoplasia. To further investigate the PPARδ dependency of intestinal tumourigenesis, we have analysed the consequences of PPARδ deficiency upon intestinal neoplasia occurring in mice with impaired mismatch DNA repair. METHODS: Mice deficient for both PPARδ and the mismatch repair gene Mlh1 were produced and the incidence and severity of intestinal neoplasia recorded. RESULTS: No significant differences between the control genotypes and the double mutant genotypes were recorded indicating that deficiency of PPARδ does not modify impaired mismatch repair induced neoplasia. CONCLUSION: In contrast with the previously observed acceleration of intestinal neoplasia in the context of the Apc(Min/+ )mouse, PPARδ deficiency does not alter the phenotype of mismatch repair deficiency. This data supports the notion that PPARδ is not required for adenoma formation and indicate that any pro-tumourigenic effect of PPARδ inactivation may be highly context dependent