Reconstitution of the Early Stage of Chetomin Biosynthesis in Aspergillus fumigatus Leads to the Production of Epipolythiodioxopiperazines.

Using CRISPR-Cas9 technology and a microhomology-mediated end-joining repair system, we substituted genes of the gliotoxin pathway in Aspergillus fumigatus with genes responsible for chetomin biosynthesis from Chaetomium cochliodes, leading to the production of three new epipolythiodioxopiperazines (ETPs). This work represents the first successful endeavor to produce ETPs in a non-native host. Additionally, the simultaneous disruption of five genes in a single transformation marks the most extensive gene knockout event in filamentous fungi to date.

Urease of Aspergillus fumigatus Is Required for Survival in Macrophages and Virulence.

The number of patients suffering from fungal diseases has constantly increased during the last decade. Among the fungal pathogens, the airborne filamentous fungus Aspergillus fumigatus can cause chronic and fatal invasive mold infections. So far, only three major classes of drugs (polyenes, azoles, and echinocandins) are available for the treatment of life-threatening fungal infections, and all present pharmacological drawbacks (e.g., low solubility or toxicity). Meanwhile, clinical antifungal-resistant isolates are continuously emerging. Therefore, there is a high demand for novel antifungal drugs, preferentially those that act on new targets. We studied urease and the accessory proteins in A. fumigatus to determine their biochemical roles and their influence on virulence. Urease is crucial for the growth on urea as the sole nitrogen source, and the transcript and protein levels are elevated on urea media. The urease deficient mutant displays attenuated virulence, and its spores are more susceptible to macrophage-mediated killing. We demonstrated that this observation is associated with an inability to prevent the acidification of the phagosome. Furthermore, we could show that a nickel-chelator inhibits growth on urea. The nickel chelator is also able to reverse the effects of urease on macrophage killing and phagosome acidification, thereby reducing virulence in systemic and trachea infection models. IMPORTANCE The development of antifungal drugs is an urgent task, but it has proven to be difficult due to many similarities between fungal and animal cells. Here, we characterized the urease system in A. fumigatus, which depends on nickel for activity. Notably, nickel is not a crucial element for humans. Therefore, we went further to explore the role of nickel-dependent urease in host-pathogen interactions. We were able to show that urease is important in preventing the acidification of the phagosome and therefore reduces the killing of conidia by macrophages. Furthermore, the deletion of urease shows reduced virulence in murine infection models. Taken together, we identified urease as an essential virulence factor of A. fumigatus. We were able to show that the application of the nickel-chelator dimethylglyoxime is effective in both in vitro and in vivo infection models. This suggests that nickel chelators or urease inhibitors are potential candidates for the development of novel antifungal drugs.

A miniaturized bionic ocean-battery mimicking the structure of marine microbial ecosystems.

Marine microbial ecosystems can be viewed as a huge ocean-battery charged by solar energy. It provides a model for fabricating bio-solar cell, a bioelectrochemical system that converts light into electricity. Here, we fabricate a bio-solar cell consisting of a four-species microbial community by mimicking the ecological structure of marine microbial ecosystems. We demonstrate such ecological structure consisting of primary producer, primary degrader, and ultimate consumers is essential for achieving high power density and stability. Furthermore, the four-species microbial community is assembled into a spatial-temporally compacted cell using conductive hydrogel as a sediment-like anaerobic matrix, forming a miniaturized bionic ocean-battery. This battery directly converts light into electricity with a maximum power of 380 µW and stably operates for over one month. Reproducing the photoelectric conversion function of marine microbial ecosystems in this bionic battery overcomes the sluggish and network-like electron transfer, showing the biotechnological potential of synthetic microbial ecology.

BtsT/ BtsS is involved in glyoxylate transport in E. coli and its mutations facilitated glyoxylate utilization.

Glyoxylate is an important chemical and is also an intermediate involved in metabolic pathways of living microorganisms. However, it cannot be rapidly utilized by many microbes. We observed a very long lag phase (up to 120 h) when E. coli is growing in a mineral medium supplemented with 50 mM glyoxylate. To better understand this strange growth pattern on glyoxylate and accelerate glyoxylate utilization, a random genomic library of E. coli was transformed into E. coli BW25113, and mutants that showed significantly shortened lag phase on glyoxylate were obtained. Interestingly, mutations in BtsT/BtsS, a two component system that is involved in pyruvate transport, were found to be a common feature in all mutants retrieved. We further demonstrated, through reverse engineering, that the mutations in BtsT/BtsS can indeed increase glyoxylate uptake. Growth experiments with different concentration of glyoxylate also showed the higher the concentration of glyoxylate, the shorter the lag phase. These new findings thus increased our understanding on microbial utilization of glyoxylate.