Fungi: friends or foes-an outreach science initiative for the collection of airborne fungal spores by high school students.

Fungi mostly reproduce through spores that are adapted for airborne dispersal; hence, fungal spores (and fungi) are found virtually everywhere. Fungi can be "friends or foes." Our friends include fungi used in the food and biotech industries, fungi that contribute to the cycling of carbon and nutrients, and those involved in the decontamination of polluted soils and/or water, to mention just a few examples. Many species, however, are foes-they are detrimental to plants, animals, and/or humans. Annually, >1.5 million people die due to invasive fungal infections. With the aim of enhancing microbiology literacy and the understanding of microbial concepts, we set up a project for the collection of airborne spores (the principal agent through which human airways are exposed to fungi). Students from five high schools in the Oeiras municipality partnered with us as citizen scientists; they carried out sampling by collecting fungal spores on adhesive stickers. The fungal spores collected by the students were subsequently processed in the schools and our research laboratory. Results obtained by the students themselves revealed a large variety of fungal species capable of growing in a rich medium at 30°C. In the research laboratory, using selective isolation conditions, 40 thermotolerant fungi were isolated, 32 of which were taxonomically identified as aspergilla, mostly from within the Aspergillus fumigatus taxa, yet exhibiting high genetic heterogeneity. The protocols and results were presented to the students, who were made aware of the local dispersal of airborne fungal spores, including some from potentially pathogenic fungi. Through carrying out scientific activities, the students developed both the interest and the self-confidence needed to implement future environmental investigations.

NmrB (AN9181) expression is activated under oxidative stress conditions acting as a metabolic repressor of Aspergillus nidulans.

Aspergilli comprise a diversity of species that have been extensively studied due to their catabolic diversity, biotechnological and ecological value, and pathogenicity. An impressive level of structural and functional conservation has been shown for aspergilli, regardless of many (yet) cryptic genomic elements. We have hypothesized the existence of conserved genes responsive to stress in aspergilli. To test the hypothesis of such conserved stress regulators in aspergilli, a straightforward computational strategy integrating well-established bioinformatic tools was used as the starting point. Specifically, five transcriptome-based datasets on exposure to organic compounds were used, covering three distinct Aspergillus species. Among the identified up-regulated genes, only one gene showed the same response in all conditions, AN9181. This gene encodes a protein containing a phenylcoumaran benzylic ether reductase-like domain and a Nitrogen metabolite repressor regulator domain (NmrA). Deletion of this gene caused significant phenotypic alterations compared to that of the parental strain across diverse conditions. Specifically, the deletion of AN9181 raised the mutant's metabolic activity in different nitrogen sources. The acquired data supports that AN9181 acts by repressing (slowing down) A. nidulans growth when exposed to aromatic compounds in a concentration dependent manner. The same phenotype was observed for amphotericin B. Finally, AN9181 underwent differential upregulation under oxidative stress conditions. Collectively, the data suggest that AN9181, herein assigned as NmrB (Nitrogen Metabolite Repression Regulator B), builds up the genetic machinery of perception of oxidative stress by negatively regulating growth under such conditions.

c-di-AMP determines the hierarchical organization of bacterial RCK proteins.

In bacteria, intracellular K+ is involved in the regulation of membrane potential, cytosolic pH, and cell turgor as well as in spore germination, environmental adaptation, cell-to-cell communication in biofilms, antibiotic sensitivity, and infectivity. The second messenger cyclic-di-AMP (c-di-AMP) has a central role in modulating the intracellular K+ concentration in many bacterial species, controlling transcription and function of K+ channels and transporters. However, our understanding of how this regulatory network responds to c-di-AMP remains poor. We used the RCK (Regulator of Conductance of K+) proteins that control the activity of Ktr channels in Bacillus subtilis as a model system to analyze the regulatory function of c-di-AMP with a combination of in vivo and in vitro functional and structural characterization. We determined that the two RCK proteins (KtrA and KtrC) are neither physiologically redundant or functionally equivalent. KtrC is the physiologically dominant RCK protein in the regulation of Ktr channel activity. In explaining this hierarchical organization, we found that, unlike KtrA, KtrC is very sensitive to c-di-AMP inactivation and lack of c-di-AMP regulation results in RCK protein toxicity, most likely due to unregulated K+ flux. We also found that KtrC can assemble with KtrA, conferring c-di-AMP regulation to the functional KtrA/KtrC heteromers and potentially compensating KtrA toxicity. Altogether, we propose that the central role of c-di-AMP in the control of the K+ machinery, by modulating protein levels through gene transcription and by regulating protein activity, has determined the evolutionary selection of KtrC as the dominant RCK protein, shaping the hierarchical organization of regulatory components of the K+ machinery.

Increased Production of Pathogenic, Airborne Fungal Spores upon Exposure of a Soil Mycobiota to Chlorinated Aromatic Hydrocarbon Pollutants.

Organic pollutants are omnipresent and can penetrate all environmental niches. We evaluated the hypothesis that short-term (acute) exposure to aromatic hydrocarbon pollutants could increase the potential for fungal virulence. Specifically, we analyzed whether pentachlorophenol and triclosan pollution results in the production of airborne fungal spores with greater virulence than those derived from an unpolluted (Control) condition. Each pollutant altered the composition of the community of airborne spores compared to the control, favoring an increase in strains with in vivo infection capacity (the wax moth Galleria mellonella was used as an infection model). Fungi subsisting inside larvae at 72 h postinjection with airborne spore inocula collected in polluted and unpolluted conditions exhibited comparable diversity (mainly within Aspergillus fumigatus). Several virulent Aspergillus strains were isolated from larvae infected with the airborne spores produced in a polluted environment. Meanwhile, strains isolated from larvae injected with spores from the control, including one A. fumigatus strain, showed no virulence. Potential pathogenicity increased when two Aspergillus virulent strains were assembled, suggesting the existence of synergisms that impact pathogenicity. None of the observed taxonomic or functional traits could separate the virulent from the avirulent strains. Our study emphasizes pollution stress as a possible driver of phenotypic adaptations that increase Aspergillus pathogenicity, as well as the need to better understand the interplay between pollution and fungal virulence. IMPORTANCE Fungi colonizing soil and organic pollutants often meet. The consequences of this encounter constitute an outstanding question. We scrutinized the potential for virulence of airborne fungal spores produced under unpolluted and polluted scenarios. The airborne spores showed increased diversity of strains with higher infection capacity in Galleria mellonella whenever pollution is present. Inside the larvae injected with either airborne spore community, the surviving fungi demonstrated a similar diversity, mainly within Aspergillus fumigatus. However, the isolated Aspergillus strains greatly differ since virulence was only observed for those associated with a polluted environment. The interplay between pollution and fungal virulence still hides many unresolved questions, but the encounter is costly: pollution stress promotes phenotypic adaptations that may increase Aspergillus pathogenicity.

The Off-Targets of Clustered Regularly Interspaced Short Palindromic Repeats Gene Editing.

The repurposing of the CRISPR/Cas bacterial defense system against bacteriophages as simple and flexible molecular tools has revolutionized the field of gene editing. These tools are now widely used in basic research and clinical trials involving human somatic cells. However, a global moratorium on all clinical uses of human germline editing has been proposed because the technology still lacks the required efficacy and safety. Here we focus on the approaches developed since 2013 to decrease the frequency of unwanted mutations (the off-targets) during CRISPR-based gene editing.

c-di-AMP, a likely master regulator of bacterial K+ homeostasis machinery, activates a K+ exporter.

bis-(3',5')-cyclic diadenosine monophosphate (c-di-AMP) is a second messenger with roles in virulence, cell wall and biofilm formation, and surveillance of DNA integrity in many bacterial species, including pathogens. Strikingly, it has also been proposed to coordinate the activity of the components of K+ homeostasis machinery, inhibiting K+ import, and activating K+ export. However, there is a lack of quantitative evidence supporting the direct functional impact of c-di-AMP on K+ transporters. To gain a detailed understanding of the role of c-di-AMP on the activity of a component of the K+ homeostasis machinery in B. subtilis, we have characterized the impact of c-di-AMP on the functional, biochemical, and physiological properties of KhtTU, a K+/H+ antiporter composed of the membrane protein KhtU and the cytosolic protein KhtT. We have confirmed c-di-AMP binding to KhtT and determined the crystal structure of this complex. We have characterized in vitro the functional properties of KhtTU and KhtU alone and quantified the impact of c-di-AMP and of pH on their activity, demonstrating that c-di-AMP activates KhtTU and that pH increases its sensitivity to this nucleotide. Based on our functional and structural data, we were able to propose a mechanism for the activation of KhtTU by c-di-AMP. In addition, we have analyzed the impact of KhtTU in its native bacterium, providing a physiological context for the regulatory function of c-di-AMP and pH. Overall, we provide unique information that supports the proposal that c-di-AMP is a master regulator of K+ homeostasis machinery.

Characterization of the molecular properties of KtrC, a second RCK domain that regulates a Ktr channel in Bacillus subtilis.

RCK (regulating conductance of K+) domains are common regulatory domains that control the activity of eukaryotic and prokaryotic K+ channels and transporters. In bacteria these domains play roles in osmoregulation, regulation of turgor and membrane potential and in pH homeostasis. Whole-genome sequencing unveiled RCK gene redundancy, however the biological role of this redundancy is not well understood. In Bacillus subtilis, there are two closely related RCK domain proteins (KtrA and KtrC) that regulate the activity of the Ktr cation channels. KtrA has been well characterized but little is known about KtrC. We have characterized the structural and biochemical proprieties of KtrC and conclude that KtrC binds ATP and ADP, just like KtrA. However, in solution KtrC exist in a dynamic equilibrium between octamers and non-octameric species that is dependent on the bound ligand, with ATP destabilizing the octameric ring relative to ADP. Accordingly, KtrC-ADP crystal structures reveal closed octameric rings similar to those in KtrA, while KtrC-ATP adopts an open assembly with RCK domains forming a super-helix. In addition, both KtrC-ATP and -ADP octamers are stabilized by the signaling molecule cyclic-di-AMP, which binds to KtrC with high affinity. In contrast, c-di-AMP binds with 100-fold lower affinity to KtrA. Despite these differences we show with an E. coli complementation assay that KtrC and KtrA are interchangeable and able to form functional transporters with both KtrB and KtrD. The distinctive properties of KtrC, in particular ligand-dependent assembly/disassembly, suggest that this protein has a specific physiological role that is distinct from KtrA.

Efficient Production of the Dicarboxylic Acid Glutarate by Corynebacterium glutamicum via a Novel Synthetic Pathway.

The dicarboxylic acid glutarate is an important building-block gaining interest in the chemical and pharmaceutical industry. Here, a synthetic pathway for fermentative production of glutarate by the actinobacterium Corynebacterium glutamicum has been developed. The pathway does not require molecular oxygen and operates via lysine decarboyxylase followed by two transamination and two NAD-dependent oxidation reactions. Using a genome-streamlined L-lysine producing strain as basis, metabolic engineering was performed to enable conversion of L-lysine to glutarate in a five-step synthetic pathway comprising lysine decarboxylase, putrescine transaminase and γ-aminobutyraldehyde dehydrogenase from Escherichia coli and GABA/5AVA amino transferase and succinate/glutarate semialdehyde dehydrogenase either from C. glutamicum or from three Pseudomonas species. Loss of carbon via formation of the by-products cadaverine and N-acetylcadaverine was avoided by deletion of the respective acetylase and export genes. As the two transamination reactions in the synthetic glutarate biosynthesis pathway yield L-glutamate, biosynthesis of L-glutamate by glutamate dehydrogenase was expected to be obsolete and, indeed, deletion of its gene gdh increased glutarate titers by 10%. Glutarate production by the final strain was tested in bioreactors (n = 2) in order to investigate stability and reliability of the process. The most efficient glutarate production from glucose was achieved by fed-batch fermentation (n = 1) with a volumetric productivity of 0.32 g L-1 h-1, an overall yield of 0.17 g g-1 and a titer of 25 g L-1.

A new metabolic route for the fermentative production of 5-aminovalerate from glucose and alternative carbon sources.

Here, a new metabolic pathway for the production of 5-aminovalerate (5AVA) from l-lysine via cadaverine as intermediate was established and this three-step-pathway comprises l-lysine decarboxylase (LdcC), putrescine transaminase (PatA) and γ-aminobutyraldehyde dehydrogenase (PatD). Since Corynebacterium glutamicum is used for industrial l-lysine production, the pathway was established in this bacterium. Upon expression of ldcC, patA and patD from Escherichia coli in C. glutamicum wild type, production 5AVA was achieved. Enzyme assays revealed that PatA and PatD also converted cadaverine to 5AVA. Eliminating the by-products cadaverine, N-acetylcadaverine and glutarate in a genome-streamlined l-lysine producing strain expressing ldcC, patA and patD improved 5AVA production to a titer of 5.1gL-1, a yield of 0.13gg-1 and a volumetric productivity of 0.12gL-1h-1. Moreover, 5AVA production from the alternative feedstocks starch, glucosamine, xylose and arabinose was established.

Improved fermentative production of gamma-aminobutyric acid via the putrescine route: Systems metabolic engineering for production from glucose, amino sugars, and xylose.

Gamma-aminobutyric acid (GABA) is a non-protein amino acid widespread in Nature. Among the various uses of GABA, its lactam form 2-pyrrolidone can be chemically converted to the biodegradable plastic polyamide-4. In metabolism, GABA can be synthesized either by decarboxylation of l-glutamate or by a pathway that starts with the transamination of putrescine. Fermentative production of GABA from glucose by recombinant Corynebacterium glutamicum has been described via both routes. Putrescine-based GABA production was characterized by accumulation of by-products such as N-acetyl-putrescine. Their formation was abolished by deletion of the spermi(di)ne N-acetyl-transferase gene snaA. To improve provision of l-glutamate as precursor 2-oxoglutarate dehydrogenase activity was reduced by changing the translational start codon of the chromosomal gene for 2-oxoglutarate dehydrogenase subunit E1o to the less preferred TTG and by maintaining the inhibitory protein OdhI in its inhibitory form by changing amino acid residue 15 from threonine to alanine. Putrescine-based GABA production by the strains described here led to GABA titers up to 63.2 g L-1 in fed-batch cultivation at maximum volumetric productivities up to 1.34 g L-1 h-1 , the highest volumetric productivity for fermentative GABA production reported to date. Moreover, GABA production from the carbon sources xylose, glucosamine, and N-acetyl-glucosamine that do not have competing uses in the food or feed industries was established. Biotechnol. Bioeng. 2017;114: 862-873. © 2016 Wiley Periodicals, Inc.

Roles of export genes cgmA and lysE for the production of L-arginine and L-citrulline by Corynebacterium glutamicum.

L-arginine is a semi-essential amino acid with application in cosmetic, pharmaceutical, and food industries. Metabolic engineering strategies have been applied for overproduction of L-arginine by Corynebacterium glutamicum. LysE was the only known L-arginine exporter of this bacterium. However, an L-arginine-producing strain carrying a deletion of lysE still accumulated about 10 mM L-arginine in the growth medium. Overexpression of the putative putrescine and cadaverine export permease gene cgmA was shown to compensate for the lack of lysE with regard to L-arginine export. Moreover, plasmid-borne overexpression of cgmA rescued the toxic effect caused by feeding of the dipeptide Arg-Ala to lysE-deficient C. glutamicum and argO-deficient Escherichia coli strains. Deletion of the repressor gene cgmR improved L-arginine titers by 5 %. Production of L-lysine and L-citrulline was not affected by cgmA overexpression. Taken together, CgmA may function as an export system not only for the diamine putrescine and cadaverine but also for L-arginine. The major export system for L-lysine and L-arginine LysE may also play a role in L-citrulline export since production of L-citrulline was reduced when lysE was deleted and improved by 45 % when lysE was overproduced.

A new metabolic route for the production of gamma-aminobutyric acid by Corynebacterium glutamicum from glucose.

Gamma-aminobutyric acid (GABA), a non-protein amino acid widespread in nature, is a component of pharmaceuticals, foods, and the biodegradable plastic polyamide 4. Corynebacterium glutamicum shows great potential for the production of GABA from glucose. GABA added to the growth medium hardly affected growth of C. glutamicum, since a half-inhibitory concentration of 1.1 M GABA was determined. As alternative to GABA production by glutamate decarboxylation, a new route for the production of GABA via putrescine was established in C. glutamicum. A putrescine-producing recombinant C. glutamicum strain was converted into a GABA producing strain by heterologous expression of putrescine transaminase (PatA) and gamma-aminobutyraldehyde dehydrogenase (PatD) genes from Escherichia coli. The resultant strain produced 5.3 ± 0.1 g L-1 of GABA. GABA production was improved further by adjusting the concentration of nitrogen in the culture medium, by avoiding the formation of the by-product N-acetylputrescine and by deletion of the genes for GABA catabolism and GABA re-uptake. GABA accumulation by this strain was increased by 51 % to 8.0 ± 0.3 g L-1, and the volumetric productivity was increased to 0.31 g L-1 h-1; the highest volumetric productivity reported so far for fermentative production of GABA from glucose in shake flasks was achieved.

Updates on industrial production of amino acids using Corynebacterium glutamicum.

L-Amino acids find various applications in biotechnology. L-Glutamic acid and its salts are used as flavor enhancers. Other L-amino acids are used as food or feed additives, in parenteral nutrition or as building blocks for the chemical and pharmaceutical industries. L-amino acids are synthesized from precursors of central carbon metabolism. Based on the knowledge of the biochemical pathways microbial fermentation processes of food, feed and pharma amino acids have been developed. Production strains of Corynebacterium glutamicum, which has been used safely for more than 50 years in food biotechnology, and Escherichia coli are constantly improved using metabolic engineering approaches. Research towards new processes is ongoing. Fermentative production of L-amino acids in the million-ton-scale has shaped modern biotechnology and its markets continue to grow steadily. This review focusses on recent achievements in strain development for amino acid production including the use of CRISPRi/dCas9, genome-reduced strains, biosensors and synthetic pathways to enable utilization of alternative carbon sources.