Chemoenzymatic Synthesis of Complex Phenylpropanoid Derivatives by the Botrytis cinerea Secretome and Evaluation of Their Wnt Inhibition Activity

In this study, a series of complex phenylpropanoid derivatives were obtained by chemoenzymatic biotransformation of ferulic acid, caffeic acid, and a mixture of both acids using the enzymatic secretome of Botrytis cinerea. These substrates were incubated with fungal enzymes, and the reactions were monitored using state-of-the-art analytical methods. Under such conditions, a series of dimers, trimers, and tetramers were generated. The reactions were optimized and scaled up. The resulting mixtures were purified by high-resolution semi-preparative HPLC combined with dry load introduction. This approach generated a series of 23 phenylpropanoid derivatives, 11 of which are described here for the first time. These compounds are divided into 12 dimers, 9 trimers (including a completely new structural scaffold), and 2 tetramers. Elucidation of their structures was performed with classical spectroscopic methods such as NMR and HRESIMS analyses. The resulting compound series were analyzed for anti-Wnt activity in TNBC cells, with several derivatives demonstrating specific inhibition.

Prospective Application of Aspergillus Species: Focus on Enzyme Production Strategies, Advances and Challenges

Fungal enzymes that catalyze different types of biochemical reactions play a significant role in modern industry by improving existing processes. Also, the use of enzymes to replace some traditional toxic chemical or mechanical approaches helps decrease energy demand and environmental pollution. However, enzymes must be able to compete commercially with relatively low-priced traditional approaches. Meeting economical and commercial feasibility criteria depends on a number of enzymatic properties including the specificity to the substrate, stability in industrial enzymatic reaction conditions and catalytic efficiency. Fungi used as an enzyme manufacture host should be appropriate for industrial scale fermentation. Aspergillus species are being developed as one of the best enzyme manufacture factories due to their capability to secrete high quantities of enzymes suitable for industrial applications. The industrial importance of Aspergillus species also includes the progress and commercialization of new products derived from genetically engineered modified strains. Hence, the main aim of this chapter investigation is to analyze the secreted and cellular proteins from Aspergillus species and their application in industries.

Apoplastic CBM1-interacting proteins bind conserved carbohydrate binding module 1 motifs in fungal hydrolases to counter pathogen invasion

When infecting plants, fungal pathogens secrete cell wall degrading enzymes (CWDEs) that break down cellulose and hemicellulose, the primary components of plant cell walls. Some fungal CWDEs contain a unique domain, named the carbohydrate binding module (CBM), that facilitates their access to polysaccharides. However, little is known about how plants counteract pathogen degradation of their cell walls. Here, we show that the rice cysteine-rich repeat secretion protein OsCBMIP binds to and inhibits xylanase MoCel10A of the blast fungus pathogen Magnaporthe oryzae, interfering with its access to the rice cell wall and degradation of rice xylan. We found binding of OsCBMIP to various CBM1-containing enzymes, suggesting it has a general role in inhibiting the catalytic activities of fungal enzymes. OsCBMIP is localized to the apoplast, and its expression is strongly induced in leaves infected with M. oryzae. Remarkably, knockdown of OsCBMIP reduced rice defense against M. oryzae, demonstrating that inhibition of CBM1-containing fungal enzymes by OsCBMIP is crucial for rice defense. We also identified additional CBMIP-related proteins from Arabidopsis thaliana and Setaria italica, indicating that a wide range of plants counteract pathogens through this mechanism.

A Comprehensive Insight into Fungal Enzymes: Structure, Classification, and Their Role in Mankind’s Challenges

Enzymes have played a crucial role in mankind’s challenges to use different types of biological systems for a diversity of applications. They are proteins that break down and convert complicated compounds to produce simple products. Fungal enzymes are compatible, efficient, and proper products for many uses in medicinal requests, industrial processing, bioremediation purposes, and agricultural applications. Fungal enzymes have appropriate stability to give manufactured products suitable shelf life, affordable cost, and approved demands. Fungal enzymes have been used from ancient times to today in many industries, including baking, brewing, cheese making, antibiotics production, and commodities manufacturing, such as linen and leather. Furthermore, they also are used in other fields such as paper production, detergent, the textile industry, and in drinks and food technology in products manufacturing ranging from tea and coffee to fruit juice and wine. Recently, fungi have been used for the production of more than 50% of the needed enzymes. Fungi can produce different types of enzymes extracellularly, which gives a great chance for producing in large amounts with low cost and easy viability in purified forms using simple purification methods. In the present review, a comprehensive trial has been advanced to elaborate on the different types and structures of fungal enzymes as well as the current status of the uses of fungal enzymes in various applications.

Cellulase production and efficient saccharification of biomass by a new mutant Trichoderma afroharzianum MEA-12

Background Cellulase plays a key role in converting cellulosic biomass into fermentable sugar to produce chemicals and fuels, which is generally produced by filamentous fungi. However, most of the filamentous fungi obtained by natural breeding have low secretory capacity in cellulase production, which are far from meeting the requirements of industrial production. Random mutagenesis combined with adaptive laboratory evolution (ALE) strategy is an effective method to increase the production of fungal enzymes. Results This study obtained a mutant of Trichoderma afroharzianum by exposures to N-methyl-N’-nitro-N-nitrosoguanidine (MNNG), Ethyl Methanesulfonate (EMS), Atmospheric and Room Temperature Plasma (ARTP) and ALE with high sugar stress. The T. afroharzianum mutant MEA-12 produced 0.60, 5.47, 0.31 and 2.17 IU/mL FPase, CMCase, pNPCase and pNPGase, respectively. These levels were 4.33, 6.37, 4.92 and 4.15 times higher than those of the parental strain, respectively. Also, it was found that T. afroharzianum had the same carbon catabolite repression (CCR) effect as other Trichoderma in liquid submerged fermentation. In contrast, the mutant MEA-12 can tolerate the inhibition of glucose (up to 20 mM) without affecting enzyme production under inducing conditions. Interestingly, crude enzyme from MEA-12 showed high enzymatic hydrolysis efficiency against three different biomasses (cornstalk, bamboo and reed), when combined with cellulase from T. reesei Rut-C30. In addition, the factors that improved cellulase production by MEA-12 were clarified. Conclusions Overall, compound mutagenesis combined with ALE effectively increased the production of fungal cellulase. A super-producing mutant MEA-12 was obtained, and its cellulase could hydrolyze common biomasses efficiently, in combination with enzymes derived from model strain T. reesei, which provides a new choice for processing of bioresources in the future.

Sesquiterpene Cyclases from the Basidiomycete Cerrena unicolor

Hundreds of terpenoids have been isolated from Basidiomycota, among them are volatile mono- and sesquiterpenes with amazing sensory qualities, representing a promising alternative to essential oils from endangered plant species. Sesquiterpene synthases (STS) appear to be an abundant class of enzymes in these fungi. The basidiomycete Cerrena unicolor, a known sesquiterpene producer, was in silico screened for sesquiterpene cyclases via homology Basic Local Alignment Search Tool searches. Cyclase genes identified were cloned and heterologously expressed in Escherichia coli Bl21 using pCOLD I as the expression vector. Ten cyclases were successfully produced and purified, and their identity was confirmed using amino acid sequencing of tryptic peptides by nano-liquid chromatography-high resolution-electrospray ionization-tandem mass spectrometry. Gas chromatography/mass spectrometry analysis was applied to characterize these cyclases according to the formation of sesquiterpene hydrocarbons and oxidized terpenoids. Bioinformatic characterization and phylogenetic determination allowed for the classification of these diverse fungal enzymes. A representative single and a multi-product STS, respectively, were further analyzed for their dependency from divalent metal cations as a cofactor for the catalytic activity.

Fungal Enzymes as Catalytic Tools for Polyethylene Terephthalate (PET) Degradation

The ubiquitous persistence of plastic waste in diverse forms and different environmental matrices is one of the main challenges that modern societies are facing at present. The exponential utilization and recalcitrance of synthetic plastics, including polyethylene terephthalate (PET), results in their extensive accumulation, which is a significant threat to the ecosystem. The growing amount of plastic waste ending up in landfills and oceans is alarming due to its possible adverse effects on biota. Thus, there is an urgent need to mitigate plastic waste to tackle the environmental crisis of plastic pollution. With regards to PET, there is a plethora of literature on the transportation route, ingestion, environmental fate, amount, and the adverse ecological and human health effects. Several studies have described the deployment of various microbial enzymes with much focus on bacterial-enzyme mediated removal and remediation of PET. However, there is a lack of consolidated studies on the exploitation of fungal enzymes for PET degradation. Herein, an effort has been made to cover this literature gap by spotlighting the fungi and their unique enzymes, e.g., esterases, lipases, and cutinases. These fungal enzymes have emerged as candidates for the development of biocatalytic PET degradation processes. The first half of this review is focused on fungal biocatalysts involved in the degradation of PET. The latter half explains three main aspects: (1) catalytic mechanism of PET hydrolysis in the presence of cutinases as a model fungal enzyme, (2) limitations hindering enzymatic PET biodegradation, and (3) strategies for enhancement of enzymatic PET biodegradation.

Biochemical characterization of a glycoside hydrolase family 43 β-D-galactofuranosidase from the fungus Aspergillus niger

β-D-Galactofuranose (Galf) and its polysaccharides are found in bacteria, fungi and protozoa but do not occur in mammalian tissues, and thus represent a specific target for anti-pathogenic drugs. Understanding the enzymatic degradation of these polysaccharides is therefore of great interest, but the identity of fungal enzymes with exclusively galactofuranosidase activity has so far remained elusive. Here we describe the identification and characterization of a galactofuranosidase from the industrially important fungus Aspergillus niger. Phylogenetic analysis of glycoside hydrolase family 43 subfamily 34 (GH43_34) members revealed the occurrence of three distinct clusters and, by comparison with specificities of characterized bacterial members, suggested a basis for prediction of enzyme specificity. Using this rationale, in tandem with molecular docking, we identified a putative β-D-galactofuranosidase from A. niger which was recombinantly expressed in Escherichia coli. The Galf-specific hydrolase, encoded by xynD demonstrates maximum activity at pH 5, 25 °C towards 4-Nitrophenyl-β-galactofuranoside (pNP-βGalf), with a Km of 17.9 ± 1.9 mM and Vmax of 70.6 ± 5.3 μmol min-1. The characterization of this first fungal GH43 galactofuranosidase offers further molecular insight into the degradation of Galf-containing structures and may inform clinical treatments against fungal pathogens.

The Fusarium graminearum FGSG_03624 Xylanase Enhances Plant Immunity and Increases Resistance against Bacterial and Fungal Pathogens

Fungal enzymes degrading the plant cell wall, such as xylanases, can activate plant immune responses. The Fusarium graminearum FGSG_03624 xylanase, previously shown to elicit necrosis and hydrogen peroxide accumulation in wheat, was investigated for its ability to induce disease resistance. To this aim, we transiently and constitutively expressed an enzymatically inactive form of FGSG_03624 in tobacco and Arabidopsis, respectively. The plants were challenged with Pseudomonas syringae pv. tabaci or pv. maculicola and Botrytis cinerea. Symptom reduction by the bacterium was evident, while no reduction was observed after B. cinerea inoculation. Compared to the control, the presence of the xylanase gene in transgenic Arabidopsis plants did not alter the basal expression of a set of defense-related genes, and, after the P. syringae inoculation, a prolonged PR1 expression was detected. F. graminearum inoculation experiments of durum wheat spikes exogenously treated with the FGSG_03624 xylanase highlighted a reduction of symptoms in the early phases of infection and a lower fungal biomass accumulation than in the control. Besides, callose deposition was detected in infected spikes previously treated with the xylanase and not in infected control plants. In conclusion, our results highlight the ability of FGSG_03624 to enhance plant immunity, thus decreasing disease severity.

Agro-industrial wastes for biotechnological production as potential substrates to obtain fungal enzymes

Agro-industrial wastes contain high moisture content and are rich in nutrients, and can be used as useful substrates by microbes with the supplementation of nitrogen sources, thus providing an alternative tool for the industrial production of many products of economic value, such as enzymes for example. These are proteins that function as biological catalysts, responsible for carrying out various biochemical reactions, being applied in detergent, food, paper and cellulose, cosmetics, textile industries, etc. However, they are expensive raw materials, and it is worth noting that an important part of the cost of manufacturing enzymes is mainly due to the expense of means and fermentation processes. Thus, to minimize the cost of this production and in order to reduce the degradation of the environment due to agricultural waste, a variety of microorganisms and agro-industrial “by-products” can be used to facilitate the economic production of enzymes.Therefore, in this work, a review was carried out on agro-industrial residues and the main enzymes in the industrial market, as well as the use of these materials as sources to obtain enzymes produced by fungi.