Anaerobic Fungal Mevalonate Pathway Genomic Biases Lead to Heterologous Toxicity Underpredicted by Codon Adaptation Indices

Anaerobic fungi are emerging biotechnology platforms with genomes rich in biosynthetic potential. Yet, the heterologous expression of their biosynthetic pathways has had limited success in model hosts like E. coli. We find one reason for this is that the genome composition of anaerobic fungi like P. indianae are extremely AT-biased with a particular preference for rare and semi-rare AT-rich tRNAs in E coli, which are not explicitly predicted by standard codon adaptation indices (CAI). Native P. indianae genes with these extreme biases create drastic growth defects in E. coli (up to 69% reduction in growth), which is not seen in genes from other organisms with similar CAIs. However, codon optimization rescues growth, allowing for gene evaluation. In this manner, we demonstrate that anaerobic fungal homologs such as PI.atoB are more active than S. cerevisiae homologs in a hybrid pathway, increasing the production of mevalonate up to 2.5 g/L (more than two-fold) and reducing waste carbon to acetate by ~90% under the conditions tested. This work demonstrates the bioproduction potential of anaerobic fungal enzyme homologs and how the analysis of codon utilization enables the study of otherwise difficult to express genes that have applications in biocatalysis and natural product discovery.

New Method for Identifying Fungal Kingdom Enzyme Hotspots from Genome Sequences

Fungal genome sequencing data represent an enormous pool of information for enzyme discovery. Here, we report a new approach to identify and quantitatively compare biomass-degrading capacity and diversity of fungal genomes via integrated function-family annotation of carbohydrate-active enzymes (CAZymes) encoded by the genomes. Based on analyses of 1932 fungal genomes the most potent hotspots of fungal biomass processing CAZymes are identified and ranked according to substrate degradation capacity. The analysis is achieved by a new bioinformatics approach, Conserved Unique Peptide Patterns (CUPP), providing for CAZyme-family annotation and robust prediction of molecular function followed by conversion of the CUPP output to lists of integrated “Function;Family” (e.g., EC 3.2.1.4;GH5) enzyme observations. An EC-function found in several protein families counts as different observations. Summing up such observations allows for ranking of all analyzed genome sequenced fungal species according to richness in CAZyme function diversity and degrading capacity. Identifying fungal CAZyme hotspots provides for identification of fungal species richest in cellulolytic, xylanolytic, pectinolytic, and lignin modifying enzymes. The fungal enzyme hotspots are found in fungi having very different lifestyle, ecology, physiology and substrate/host affinity. Surprisingly, most CAZyme hotspots are found in enzymatically understudied and unexploited species. In contrast, the most well-known fungal enzyme producers, from where many industrially exploited enzymes are derived, are ranking unexpectedly low. The results contribute to elucidating the evolution of fungal substrate-digestive CAZyme profiles, ecophysiology, and habitat adaptations, and expand the knowledge base for novel and improved biomass resource utilization.

Production of chemotherapeutic enzyme L-asparaginase from fungal source

Background: L-Asparaginase is an antineoplastic agent used in the treatment of acute myeloid and acute lymphoblastic leukemia. The present study deals with the production of this chemotherapeutic enzyme drug from Aspergillus flavus NCIM 526. The production of enzymes was carried out using oil-extracted cakes in shake flask culture. Process parameters like carbon and nitrogen sources were taken into account. Methods: A total of six isolates were used to screen out efficient microorganisms for enzyme production. Aspergillus flavus NCIM 526 exhibited 138 IU/ml of enzyme activity in oil extracted mix cake after 96 hours of the incubation period. Molasses and l-asparagine were proved the best carbon and nitrogen sources for enzyme production. The enzyme was purified by column chromatography and the finest enzyme exhibited specific activity of 28 IU/mg. Results and Discussion: The fungal enzyme exhibited low Km values as compared with standard E. coli L-asparaginase, proving more substrate affinity of fungal enzyme than bacterial enzymes. Conclusion: The study explored the Aspergillus flavus NCIM 526 as a potential fungal source for high yield production of antileukemic enzyme drugs.

Roles for Structural Biology in the Discovery of Drugs and Agrochemicals Targeting Sterol 14α-Demethylases

Antifungal drugs and antifungal agrochemicals have significant limitations. These include several unintended consequences of their use including the growing importance of intrinsic and acquired resistance. These problems underpin an increasingly urgent need to improve the existing classes of antifungals and to discover novel antifungals. Structural insights into drug targets and their complexes with both substrates and inhibitory ligands increase opportunity for the discovery of more effective antifungals. Implementation of this promise, which requires multiple skill sets, is beginning to yield candidates from discovery programs that could more quickly find their place in the clinic. This review will describe how structural biology is providing information for the improvement and discovery of inhibitors targeting the essential fungal enzyme sterol 14α-demethylase.

Efficacy and Pharmacokinetics of Fosmanogepix (APX001) in the Treatment of Candida Endophthalmitis and Hematogenous Meningoencephalitis in Non-neutropenic Rabbits

Candida endophthalmitis is a serious sight-threatening complication of candidemia that may occur before or during antifungal therapy. Hematogenous Candida meningoencephalitis (HCME) is also a serious manifestation of disseminated candidiasis in premature infants, immunosuppressed children, and immunocompromised adults. We evaluated the antifungal efficacy and pharmacokinetics of the prodrug fosmanogepix (APX001) in a rabbit model of endophthalmitis/HCME. Manogepix (APX001A), the active moiety of prodrug fosmanogepix, inhibits the fungal enzyme Gwt1, and is highly active in vitro and in vivo against Candida spp., Aspergillus spp., and other fungal pathogens. Plasma pharmacokinetics of manogepix after oral administration of fosmanogepix on Day-6 at 25, 50, and 100 mg/kg resulted in plasma Cmax of 3.96±0.41, 4.14±1.1, and 11.5±1.1 μg/ml, respectively, and AUC0-12 of 15.8±3.1, 30.8±5.0, 95.9±14 μg·h/ml, respectively. Manogepix penetrated into the aqueous humor, vitreous, and choroid with liquid to plasma ratios ranging from 0.19 to 0.52, 0.09 to 0.12, and 0.02 to 0.04, respectively. These concentrations correlated with a significant decrease in Candida albicans burden in vitreous (>101-103) and choroid (>101-103) (P≤0.05 and P≤0.001, respectively). Aqueous humor had no detectable C. albicans in treatment and control groups. The tissue/plasma concentration ratios of manogepix in meninges, cerebrum, cerebellum, and spinal cord were approximately 1:1, which correlated with a >102-104 decline of C. albicans in tissue vs control (P≤0.05). Serum and CSF (1→3)-β-D-glucan levels demonstrated significant declines in response to fosmanogepix treatment. These findings provide an experimental foundation for fosmanogepix in treatment of Candida endophthalmitis and HCME and de-risk the clinical trials of candidemia and invasive candidiasis.

Application of Milk-Clotting Protease from Aspergillus oryzae DRDFS13 MN726447 and Bacillus subtilis SMDFS 2B MN715837 for Danbo Cheese Production

This study aimed to investigate the efficiency, biochemical composition, and sensory quality of Danbo cheese produced using proteases derived from the fungus and bacterium compared to the commercial product. A fungal enzyme from Aspergillus oryzae DRDFS13MN726447 and a bacterial enzyme from Bacillus subtilis SMDFS 2B MN715837 were produced by solid-state and submerged fermentation, respectively. The crude enzyme from A. oryzae DRDFS13 and B. subtilis SMDFS 2B was partially purified by dialysis and used for Danbo cheese production using commercial rennet (CHY-MAX® Powder Extract NB, Christian Hansen, 2235 IMCU/g) as a control. The Danbo cheese produced using dialyzed fungal enzyme (E1) (267 U/mL), dialyzed bacterial enzyme (E2) (522 U/mL), and commercial rennet (C) were analyzed for body property, organoleptic characteristics, and proximate and mineral composition when fresh and after 2 months of ripening. There was no significant difference in the cheese yield (C = 9 kg, E1 = 8.6 kg, and E2 = 8.9 kg) among the three treatments. The body properties of Danbo cheese produced with the fungal enzyme (E1) were firm and acceptable as the control (C), whereas the Danbo cheese produced by bacterial enzymes has shown a watery body. The overall organoleptic characteristics of Danbo cheese produced by the fungal enzyme (5.3) were similar to control cheese produced by commercial rennet (5.5). Both cheese types were significantly different in organoleptic properties from Danbo cheese produced by the bacterial enzyme (4.9). There was no significant difference (p>0.05) in the proximate composition between the ripened Danbo cheese produced by fungal enzyme and the control cheese except for crude protein content. However, the ripened cheese products showed a significant difference in their mineral composition except for sodium. In conclusion, this study demonstrated that the fungal enzyme from Aspergillus oryzae DRDFS 13 is more appropriate for Danbo cheese production than the bacterial enzyme from Bacillus subtilis SMDFS 2B. However, it requires further application of the enzymes for the production of other cheese varieties.