Improvement of phytochemical production by plant cells and organ culture and by genetic engineering.

Plants display an amazing ability to synthesize a vast array of secondary metabolites that are an inexhaustible source of phytochemicals, bioactive molecules some of which impact the human health. Phytochemicals present in medicinal herbs and spices have long been used as natural remedies against illness. Plant tissue culture represents an alternative to whole plants as a source of phytochemicals. This approach spares agricultural land that can be used for producing food and other raw materials, thus favoring standardized phytochemical production regardless of climatic adversities and political events. Over the past 20 years, different strategies have been developed to increase the synthesis and the extraction of phytochemicals from tissue culture often obtaining remarkable results. Moreover, the availability of genomics and metabolomics tools, along with improved recombinant methods related to the ability to overexpress, silence or disrupt one or more genes of the pathway of interest promise to open new exciting possibilities of metabolic engineering. This review provides a general framework of the cellular and molecular tools developed so far to enhance the yield of phytochemicals. Additionally, some emerging topics such as the culture of cambial meristemoid cells, the selection of plant cell following the expression of genes encoding human target proteins, and the bioextraction of phytochemicals from plant material have been addressed. Altogether, the herein described techniques and results are expected to improve metabolic engineering tools aiming at improving the production of phytochemicals of pharmaceutical and nutraceutical interest.

Exploring the infiltrative and degradative ability of Fusarium oxysporum on polyethylene terephthalate (PET) using correlative microscopy and deep learning.

Managing the worldwide steady increase in the production of plastic while mitigating the Earth's global pollution is one of the greatest challenges nowadays. Fungi are often involved in biodegradation processes thanks to their ability to penetrate into substrates and release powerful catabolic exoenzymes. However, studying the interaction between fungi and plastic substrates is challenging due to the deep hyphal penetration, which hinders visualisation and evaluation of fungal activity. In this study, a multiscale and multimodal correlative microscopy workflow was employed to investigate the infiltrative and degradative ability of Fusarium oxysporum fungal strain on polyethylene terephthalate (PET) fragments. The use of non-destructive high-resolution 3D X-ray microscopy (XRM) coupled with a state-of-art Deep Learning (DL) reconstruction algorithm allowed optimal visualisation of the distribution of the fungus on the PET fragment. The fungus preferentially developed on the edges and corners of the fragment, where it was able to penetrate into the material through fractures. Additional analyses with scanning electron microscopy (SEM), Raman and energy dispersive X-ray spectroscopy (EDX) allowed the identification of the different phases detected by XRM. The correlative microscopy approach unlocked a more comprehensive understanding of the fungus-plastic interaction, including elemental information and polymeric composition.

Engineered yeasts and lignocellulosic biomaterials: shaping a new dimension for biorefinery and global bioeconomy.

The next milestone of synthetic biology research relies on the development of customized microbes for specific industrial purposes. Metabolic pathways of an organism, for example, depict its chemical repertoire and its genetic makeup. If genes controlling such pathways can be identified, scientists can decide to enhance or rewrite them for different purposes depending on the organism and the desired metabolites. The lignocellulosic biorefinery has achieved good progress over the past few years with potential impact on global bioeconomy. This principle aims to produce different bio-based products like biochemical(s) or biofuel(s) from plant biomass under microbial actions. Meanwhile, yeasts have proven very useful for different biotechnological applications. Hence, their potentials in genetic/metabolic engineering can be fully explored for lignocellulosic biorefineries. For instance, the secretion of enzymes above the natural limit (aided by genetic engineering) would speed-up the down-line processes in lignocellulosic biorefineries and the cost. Thus, the next milestone would greatly require the development of synthetic yeasts with much more efficient metabolic capacities to achieve basic requirements for particular biorefinery. This review gave comprehensive overview of lignocellulosic biomaterials and their importance in bioeconomy. Many researchers have demonstrated the engineering of several ligninolytic enzymes in heterologous yeast hosts. However, there are still many factors needing to be well understood like the secretion time, titter value, thermal stability, pH tolerance, and reactivity of the recombinant enzymes. Here, we give a detailed account of the potentials of engineered yeasts being discussed, as well as the constraints associated with their development and applications.

Metabolic pathways of an organism depict its chemical repertoire and its genetic makeup.Autonomous synthetic microbes can be developed for lignocellulose biorefinery (LCB).LCBs can be harnessed with synthetic microbes to boost global bioeconomy.Yeasts can be engineered to enhance downstream process of LCB.

Isolation and Screening from Soil Biodiversity for Fungi Involved in the Degradation of Recalcitrant Materials.

Environmental pollution is an increasing problem, and identifying fungi involved in the bioremediation process is an essential task. Soil hosts an incredible diversity of microbial life and can be a good source of these bioremediative fungi. This work aims to search for soil fungi with bioremediation potential by using different screening tests. Mineral culture media supplemented with recalcitrant substances as the sole carbon source were used as growth tests. First, soil dilutions were plated on Petri dishes with mineral medium amended with humic acids or lignocellulose. The growing fungal colonies were isolated and tested on different substrates, such as complex mixtures of hydrocarbons (petrolatum and used motor oil) and powders of different plastic polymers (PET, PP, PS, PUR, PVC). Qualitative enzymatic tests were associated with the growth tests to investigate the production of esterases, laccases, peroxidases, and proteases. These enzymes are involved in the main degradation processes of recalcitrant material, and their constitutive secretion by the examined fungal strains could have the potential to be exploited for bioremediation. More than 100 strains were isolated and tested, and several isolates with good bioremediation potential were found. In conclusion, the described screening tests are an easy and low-cost method to identify fungal strains with bioremediation potential from the soil. In addition, it is possible to tailor the screening tests for different pollutants, according to requirements, by adding other recalcitrant substances to minimal culture media.

Fungal Enzymes Involved in Plastics Biodegradation.

Plastic pollution is a growing environmental problem, in part due to the extremely stable and durable nature of this polymer. As recycling does not provide a complete solution, research has been focusing on alternative ways of degrading plastic. Fungi provide a wide array of enzymes specialized in the degradation of recalcitrant substances and are very promising candidates in the field of plastic degradation. This review examines the present literature for different fungal enzymes involved in plastic degradation, describing their characteristics, efficacy and biotechnological applications. Fungal laccases and peroxidases, generally used by fungi to degrade lignin, show good results in degrading polyethylene (PE) and polyvinyl chloride (PVC), while esterases such as cutinases and lipases were successfully used to degrade polyethylene terephthalate (PET) and polyurethane (PUR). Good results were also obtained on PUR by fungal proteases and ureases. All these enzymes were isolated from many different fungi, from both Basidiomycetes and Ascomycetes, and have shown remarkable efficiency in plastic biodegradation under laboratory conditions. Therefore, future research should focus on the interactions between the genes, proteins, metabolites and environmental conditions involved in the processes. Further steps such as the improvement in catalytic efficiency and genetic engineering could lead these enzymes to become biotechnological applications in the field of plastic degradation.

The Analysis of the Mycobiota in Plastic Polluted Soil Reveals a Reduction in Metabolic Ability.

Plastic pollution is a growing environmental issue that results in its accumulation and persistence in soil for many decades, with possible effects on soil quality and ecosystem services. Microorganisms, and especially fungi, are a keystone of soil biodiversity and soil metabolic capacity. The aim of this research was to study soil fungal biodiversity and soil microbial metabolic profiles in three different sites in northern Italy, where macro- and microplastic concentration in soil was measured. The metabolic analyses of soil microorganisms were performed by Biolog EcoPlates, while the ITS1 fragment of the 18S ribosomal cDNA was used as a target for the metabarcoding of fungal communities. The results showed an intense and significant decrease in soil microbial metabolic ability in the site with the highest concentration of microplastics. Moreover, the soil fungal community composition was significantly different in the most pristine site when compared with the other two sites. The metabarcoding of soil samples revealed a general dominance of Mortierellomycota followed by Ascomycota in all sampled soils. Moreover, a dominance of fungi involved in the degradation of plant residues was observed in all three sites. In conclusion, this study lays the foundation for further research into the effect of plastics on soil microbial communities and their activities.

Efficacy of Bovine Nail Membranes as In Vitro Model for Onychomycosis Infected by Trichophyton Species.

Onychomycosis is a fungal infection caused by different etiologic agents, including dermatophytes that specifically colonize keratin-rich substrates. The aim of this work was to investigate mechanical modifications of bovine membranes (used as an in vitro nail model) placed in contact with Trichophyton species. Trichophyton strains were isolated from toenails specimens. The procedure was set up by spreading T. rubrum,T. interdigitale, and T. mentagrophytes strains on Petri dishes with minimal and rich media; after that, bovine membranes were placed in the center. After 27 days, T. interdigitale and T. mentagrophytes significantly reduced the thickness of the colonized membranes, whereas two T. rubrum strains showed the highest degradation limited to the small colonized area. These results were confirmed by SEM images of the colonization profile on membranes. Mechanical analyses performed on membranes were used as an innovative method to evaluate the thickness and structural integrity of membranes variation following fungal colonization. In conclusion, mechanical analyses of substrate may be used as a procedure for the development of a new onychomycosis diagnosis test in order to develop personalized and strain-specific treatment.