The Nma1 protein promotes long distance transport mediated by early endosomes in Ustilago maydis.

Early endosomes (EEs) are part of the endocytic transport pathway and resemble the earliest class of transport vesicles between the internalization of extracellular material, their cellular distribution or vacuolar degradation. In filamentous fungi, EEs fulfill important functions in long distance transport of cargoes as mRNAs, ribosomes, and peroxisomes. Formation and maturation of early endosomes is controlled by the specific membrane-bound Rab-GTPase Rab5 and tethering complexes as CORVET (class C core vacuole/endosome tethering). In the basidiomycete Ustilago maydis, Rab5a is the prominent GTPase to recruit CORVET to EEs; in rab5a deletion strains, this function is maintained by the second EE-associated GTPase Rab5b. The tethering- and core-subunits of CORVET are essential, buttressing a central role for EE transport in U. maydis. The function of EEs in long distance transport is supported by the Nma1 protein that interacts with the Vps3 subunit of CORVET. The interaction stabilizes the binding of Vps3 to the CORVET core complex that is recruited to Rab5a via Vps8. Deletion of nma1 leads to a significantly reduced number of EEs, and an increased conversion rate of EEs to late endosomes. Thus, Nma1 modulates the lifespan of EEs to ensure their availability for the various long distance transport processes.

A simple and efficient method for lyophilization of recombinant E. coli JM109 (DE3) whole-cells harboring active Rieske non-heme iron dioxygenases.

Rieske non-heme iron dioxygenases are a class of intriguing enzymes covering a broad reaction and substrate spectrum and have been studied extensively in the last decades. In nature, these biocatalysts are essential for the production of cis-dihydroxylated metabolites, as a first step during the degradation of aromatic compounds in microorganisms. The enzymes are able to produce relevant amounts of compounds in short reaction times, but the effort for constant cultivation of recombinant cells and production of cell mass for biotransformations is high. To overcome the steady production process, our task was to find a way to make the biocatalysts durable and storable. In this way, laboratories lacking equipment for microbiology, e.g. chemistry laboratories, can be supplied with the enzymes to open up new possibilities in the production of molecules. We present a quick and efficient method that uses lyophilization to freeze-dry recombinant whole-cells that harbor the enzyme of interest. By washing the cells with a cryoprotectant before lyophilization, we could conserve the enzyme activity to the level of freshly harvested cells. Moreover, this simple to apply method enables subsequent steps like storage of the cell powder for transportation and on demand use in biotransformations. The method was established with the cumene dioxygenase (CDO) of Pseudomonas fluorescens IP01 and its variant CDO M232A expressed in E. coli JM109 (DE3) cells, employing R-limonene and naphthalene, respectively, as substrates in biotransformations. The method could be successfully applied in the analytical and semi-preparative reaction scale.•Preservation of biocatalysts in recombinant whole-cells.•Ready-to-use enzymatic reaction.•Semi-preparative biotransformation with lyophilized whole-cells.

Assembly of a Rieske non-heme iron oxygenase multicomponent system from Phenylobacterium immobile E DSM 1986 enables pyrazon cis-dihydroxylation in E. coli.

Phenylobacterium immobile strain E is a soil bacterium with a striking metabolism relying on xenobiotics, such as the herbicide pyrazon, as sole carbon source instead of more bioavailable molecules. Pyrazon is a heterocyclic aromatic compound of environmental concern and its biodegradation pathway has only been reported in P. immobile. The multicomponent pyrazon oxygenase (PPO), a Rieske non-heme iron oxygenase, incorporates molecular oxygen at the 2,3 position of the pyrazon phenyl moiety as first step of degradation, generating a cis-dihydrodiendiol. The aim of this work was to identify the genes encoding for each one of the PPO components and enable their functional assembly in Escherichia coli. P. immobile strain E genome sequencing revealed genes encoding for RO components, such as ferredoxin-, reductase-, α- and ß-subunits of an oxygenase. Though, P. immobile E displays three prominent differences with respect to the ROs currently characterized: (1) an operon-like organization for PPO is absent, (2) all the elements are randomly scattered in its DNA, (3) not only one, but 19 different α-subunits are encoded in its genome. Herein, we report the identification of the PPO components involved in pyrazon cis-dihydroxylation in P. immobile, its appropriate assembly, and its functional reconstitution in E. coli. Our results contributes with the essential missing pieces to complete the overall elucidation of the PPO from P. immobile. KEY POINTS: • Phenylobacterium immobile E DSM 1986 harbors the only described pyrazon oxygenase (PPO). • We elucidated the genes encoding for all PPO components. • Heterologous expression of PPO enabled pyrazon dihydroxylation in E. coli JW5510.