Genome-Scale Model of Rhizopus microsporus: Metabolic integration of a fungal holobiont with its bacterial and viral endosymbionts.

Rhizopus microsporus often lives in association with bacterial and viral symbionts that alter its biology. This fungal model represents an example of the complex interactions established among diverse organisms in functional holobionts. We constructed a Genome-Scale Model (GSM) of the fungal-bacterial-viral holobiont (iHol). We employed a constraint-based method to calculate the metabolic fluxes to decipher the metabolic interactions of the symbionts with their host. Our computational analyses of iHol simulate the holobiont's growth and the production of the toxin rhizoxin. Analyses of the calculated fluxes between R. microsporus in symbiotic (iHol) versus asymbiotic conditions suggest that changes in the lipid and nucleotide metabolism of the host are necessary for the functionality of the holobiont. Glycerol plays a pivotal role in the fungal-bacterial metabolic interaction, as its production does not compromise fungal growth, and Mycetohabitans bacteria can efficiently consume it. Narnavirus RmNV-20S and RmNV-23S affected the nucleotide metabolism without impacting the fungal-bacterial symbiosis. Our analyses highlighted the metabolic stability of Mycetohabitans throughout its co-evolution with the fungal host. We also predicted changes in reactions of the bacterial metabolism required for the active production of rhizoxin. This iHol is the first GSM of a fungal holobiont.

Rhizopus arrhizus infection in mice causes brain histopathological alterations and exacerbates neuronal apoptosis.

Rhizopus arrhizus is a fungus that can cause central nervous system infections in animals, resulting in high morbidity and mortality, but the mechanism of injury is rarely reported. In this study, we investigated the mechanism of Rhizopus arrhizus damage to the central nervous system of mice by observing the clinical neurological symptoms and resolving the pathological changes in the ultrastructure of brain tissues, combined with the alteration of apoptosis-related genes and immunohistochemistry (IHC). The results showed that all the mice in the treated group died, the brain pyknosis of neurons, there were black mycelium aggregates around the blood vessels, and apoptotic vesicles were produced. The RT-qPCR results showed that, compared with the control group, the relative transcriptome levels of Caspase 8 and BcL-2 genes were significantly increased (P < 0.05), the relative transcriptome level of Caspase 9 gene was highly significant (P < 0.01), the relative transcriptome level of Caspase 3 and Bax gene was significantly decreased (P < 0.05), and the ratio of Bcl-2/Bax was significantly increased (P < 0.05) in the brains of the treated group. TUNEL staining showed that the rate of neuronal apoptosis in the treated group of mice was extremely significantly higher than that in the control group (P < 0.01). This study shows that Rhizopus arrhizus strain XMLO1 causes brain damage by triggering neuronal apoptosis. This study provided a theoretical basis for revealing the mechanism of Rhizopus arrhizus infection.

Clinical and Mycologic Characteristics of Emerging Mucormycosis Agent Rhizopus homothallicus.

We retrospectively reviewed consecutive cases of mucormycosis reported from a tertiary-care center in India to determine the clinical and mycologic characteristics of emerging Rhizopus homothallicus fungus. The objectives were ascertaining the proportion of R. homothallicus infection and the 30-day mortality rate in rhino-orbital mucormycosis attributable to R. homothallicus compared with R. arrhizus. R. homothallicus accounted for 43 (6.8%) of the 631 cases of mucormycosis. R. homothallicus infection was independently associated with better survival (odds ratio [OR] 0.08 [95% CI 0.02-0.36]; p = 0.001) than for R. arrhizus infection (4/41 [9.8%] vs. 104/266 [39.1%]) after adjusting for age, intracranial involvement, and surgery. We also performed antifungal-susceptibility testing, which indicated a low range of MICs for R. homothallicus against the commonly used antifungals (amphotericin B [0.03-16], itraconazole [0.03-16], posaconazole [0.03-8], and isavuconazole [0.03-16]). 18S gene sequencing and amplified length polymorphism analysis revealed distinct clustering of R. homothallicus.

Alteration of Chain-Length Selectivity and Thermostability of Rhizopus oryzae Lipase via Virtual Saturation Mutagenesis Coupled with Disulfide Bond Design.

Rhizopus oryzae lipase (ROL) is one of the most important enzymes used in the food, biofuel, and pharmaceutical industries. However, the highly demanding conditions of industrial processes can reduce its stability and activity. To seek a feasible method to improve both the catalytic activity and the thermostability of this lipase, first, the structure of ROL was divided into catalytic and noncatalytic regions by identifying critical amino acids in the crevice-like binding pocket. Second, a mutant screening library aimed at improvement of ROL catalytic performance by virtual saturation mutagenesis of residues in the catalytic region was constructed based on Rosetta's Cartesian_ddg protocol. A double mutant, E265V/S267W (with an E-to-V change at residue 265 and an S-to-W change at residue 267), with markedly improved catalytic activity toward diverse chain-length fatty acid esters was identified. Then, computational design of disulfide bonds was conducted for the noncatalytic amino acids of E265V/S267W, and two potential disulfide bonds, S61C-S115C and E190C-E238C, were identified as candidates. Experimental data validated that the variant E265V/S267W/S61C-S115C/E190C-E238C had superior stability, with an increase of 8.5°C in the melting temperature and a half-life of 31.7 min at 60°C, 4.2-fold longer than that of the wild-type enzyme. Moreover, the variant improved the lipase activity toward five 4-nitrophenyl esters by 1.5 to 3.8 times, exhibiting a potential to modify the catalytic efficiency. IMPORTANCE Rhizopus oryzae lipase (ROL) is very attractive in biotechnology and industry as a safe and environmentally friendly biocatalyst. Functional expression of ROL in Escherichia coli facilitates effective high-throughput screening for positive variants. This work highlights a method to improve both selectivity and thermostability based on a combination of virtual saturation mutagenesis in the substrate pocket and disulfide bond prediction in the noncatalytic region. Using the method, ROL thermostability and activity to diverse 4-nitrophenyl esters could be substantially improved. The strategy of rational introduction of multiple mutations in different functional domains of the enzyme is a great prospect in the modification of biocatalysts.

Primary cutaneous mucormycosis due to Rhizopus arrhizus in an immunosuppressed patient with paroxysmal nocturnal haemoglobinuria.

OBJECTIVE: We present a case of primary cutaneous mucormycosis in a patient with bone marrow failure secondary to paroxysmal nocturnal haemoglobinuria (PNH). CLINICAL CASE: A 60-year-old male patient with a history of PNH, complicated to a severe aplastic anaemia, presented to the emergency department complaining of papules on the lower limbs that rapidly turned into necrotic plaques within 2 months. Histopathological examination showed granulomatous and suppurative dermatitis with tissue necrosis and the presence of non-septate hyphae. Molecular identification was achieved by amplification and sequencing of the 18S-ITS1-5.8S-ITS2-28S rRNA region using the polymerase chain reaction. The sequence showed 100% identity with Rhizopus arrhizus. The patient received treatment with liposomal amphotericin B and surgical debridement. Nonetheless, the patient suffered from severe low red blood cells and platelets and also underwent septic shock; he died 6 days after admission to the hospital. CONCLUSION: Mucormycosis in the setting of immunosuppression is challenging. Upon suspicion of a diagnosis, immediate treatment is required. Adjunctive therapies may be considered; however, the case fatality rate remains high.

Comparative genomics predict specific genes in potential mucorales identification.

Mucoralean fungi could cause mucormycosis in humans, particularly in immunodeficient individuals and those with diabetes mellitus or trauma. With plenty of species and genera, their molecular identification and pathogenicity have a large deviation. Reported cases of mucormycosis showed frequent occurrence in Rhizopus species, Mucor species, and Lichtheimia species. We analyzed the whole genome sequences of 25 species of the top 10 Mucorales genera, along with another 22 important pathogenic non-Mucorales species, to dig the target genes for monitoring Mucorales species and identify potential genomic imprints of virulence in them. Mucorales-specific genes have been found in various orthogroups extracted by Python script, while genus-specific genes were annotated covering cellular structure, biochemistry metabolism, molecular processing, and signal transduction. Proteins related to the virulence of Mucorales species varied with distinct significance in copy numbers, in which Orthofinder was conducted. Based on our fresh retrospective analysis of mucormycosis, a comparative genomic analysis of pathogenic Mucorales was conducted in more frequent pathogens. Specific orthologs between Mucorales and non-Mucoralean pathogenic fungi were discussed in detail. Referring to the previously reported virulence proteins, we included more frequent pathogenic Mucorales and compared them in Mucorales species and non-Mucorales species. Besides, more samples are needed to further verify the potential target genes.

A TAL effector-like protein of an endofungal bacterium increases the stress tolerance and alters the transcriptome of the host.

Symbioses of bacteria with fungi have only recently been described and are poorly understood. In the symbiosis of Mycetohabitans (formerly Burkholderia) rhizoxinica with the fungus Rhizopus microsporus, bacterial type III (T3) secretion is known to be essential. Proteins resembling T3-secreted transcription activator-like (TAL) effectors of plant pathogenic bacteria are encoded in the three sequenced Mycetohabitans spp. genomes. TAL effectors nuclear-localize in plants, where they bind and activate genes important in disease. The Burkholderia TAL-like (Btl) proteins bind DNA but lack the N- and C-terminal regions, in which TAL effectors harbor their T3 and nuclear localization signals, and activation domain. We characterized a Btl protein, Btl19-13, and found that, despite the structural differences, it can be T3-secreted and can nuclear-localize. A btl19-13 gene knockout did not prevent the bacterium from infecting the fungus, but the fungus became less tolerant to cell membrane stress. Btl19-13 did not alter transcription in a plant-based reporter assay, but 15 R. microsporus genes were differentially expressed in comparisons both of the fungus infected with the wild-type bacterium vs. the mutant and with the mutant vs. a complemented strain. Southern blotting revealed btl genes in 14 diverse Mycetohabitans isolates. However, banding patterns and available sequences suggest variation, and the btl19-13 phenotype could not be rescued by a btl gene from a different strain. Our findings support the conclusion that Btl proteins are effectors that act on host DNA and play important but varied or possibly host genotype-specific roles in the M. rhizoxinica-R. microsporus symbiosis.

Endofungal Mycetohabitans rhizoxinica Bacteremia Associated with Rhizopus microsporus Respiratory Tract Infection.

We report Mycetohabitans rhizoxinica bacteremia in a 65-year-old woman in California, USA, who was undergoing chimeric antigen receptor T-cell therapy for multiple myeloma. Acute brain infarction and pneumonia developed; Rhizopus microsporus mold was isolated from tracheal suction. Whole-genome sequencing confirmed bacteria in blood as genetically identical to endofungal bacteria inside the mold.

Cloning and screening of the putative hexokinase genes from Rhizopus oryzae and their heterologous expression in Saccharomyces cerevisiae.

BACKGROUND: A filamentous fungus, Rhizopus oryzae (R. oryzae) is one of the ideal candidates for ethanol and lactic acid production due to its ability to grow on renewable carbon sources. METHODS AND RESULTS: In this study, the nucleotide sequence of hexokinases and glucokinase from S. cerevisiae was found on the NCBI site ( http://www.ncbi.nlm.nih.gov/blast/Blast.cgi ) were used. With these nucleotide sequences, a blast search was done on the R. oryzae genome database ( http://www.broad.mit.edu/annotation/genome/rhizopus_oryzae/Home.html ) and ten probable genes were obtained. cDNA was synthesized from the total RNA and PCR products of the seven of these putative genes were determined using the primers designed for them. CONCLUSION: The results of the sequences and the complementation studies revealed that three of these seven putative genes were expressed in R. oryzae and the growth was observed on selective media.

Rational Design of Lipase ROL to Increase Its Thermostability for Production of Structured Tags.

1,3-regiospecific lipases are important enzymes that are heavily utilized in the food industries to produce structured triacylglycerols (TAGs). The Rhizopus oryzae lipase (ROL) has recently gained interest because this enzyme possesses high selectivity and catalytic efficiency. However, its low thermostability limits its use towards reactions that work at lower temperature. Most importantly, the enzyme cannot be used for the production of 1,3-dioleoyl-2-palmitoylglycerol (OPO) and 1,3-stearoyl-2-oleoyl-glycerol (SOS) due to the high melting points of the substrates used for the reaction. Despite various engineering efforts used to improve the thermostability of ROL, the enzyme is unable to function at temperatures above 60 °C. Here, we describe the rational design of ROL to identify variants that can retain their activity at temperatures higher than 60 °C. After two rounds of mutagenesis and screening, we were able to identify a mutant ROL_10x that can retain most of its activity at 70 °C. We further demonstrated that this mutant is useful for the synthesis of SOS while minimal product formation was observed with ROL_WT. Our engineered enzyme provides a promising solution for the industrial synthesis of structured lipids at high temperature.

Investigation of presence of endofungal bacteria in Rhizopus spp. isolated from the different food samples.

Rhizopus species are opportunistic pathogens and cause infections which lead to deaths in individuals with the weakened immune system. Some strains of Rhizopus species have been detected to have a symbiotic relationship with bacteria. The toxicity of the Rhizopus species is important. Because strains harbouring endofungal bacteria are able to produce secondary metabolites and if endofungal bacteria are released from mycelium, serious problems can occur. We aimed to investigate the presence of endofungal bacteria in Rhizopus species isolated from food samples. Rhizopus species were isolated from different food samples. The presence of endofungal bacteria in the Rhizopus isolates was investigated. Rhizopus strains containing the endofungal bacteria were identified through phenotypic and genotypic methods. Universal primers amplifying bacterial 16S rRNA region were used to amplify 1.2-1.5-kb fragment from fungal metagenomic DNA. Sequence analysis of PCR products amplified from fungal metagenomic DNA was made. Fluorescence microscopy and scanning electron microscopy were used to visualize the presence of endofungal bacteria in fungal hyphae. According to our results, the Rhizopus strains is associated with Serratia marcescens, Pseudomonas fluorescens and Klebsiella pneumoniae. Until now there is no evidence that Pseudomonas fluorescens and Klebsiella pneumoniae were identified as endofungal. These species are opportunistic pathogen dangerous for humans. It is important for humans not only the presence of the fungi but also the presence of the endofungal bacteria in foods. Our work is important because it draws attention to the presence of endofungal bacteria in foods. Because there is danger releasing of a bacterium from the mycelium, it is likely to face sepsis or serious problems.

Diagnostic performance of real-time polymerase chain reaction assay on blood for invasive aspergillosis and mucormycosis.

OBJECTIVES: This study aimed to evaluate the diagnostic usefulness of real-time (RT) polymerase chain reaction (PCR) on blood samples for diagnosis of invasive aspergillosis and mucormycosis in patients with suspected invasive mould infection. METHODS: Adult patients with suspected invasive mould infection were prospectively enrolled at a tertiary referral hospital in Seoul, South Korea between 2017 and 2020. Standard tests for diagnosis of invasive mould infection and RT-PCR for Aspergillus, Mucor and Rhizopus using blood samples were performed. We evaluated the diagnostic performance of RT-PCR tests in patients diagnosed with proven and probable invasive aspergillosis or mucormycosis infection, according to the modified definitions of the EORTC/MSG 2019. RESULTS: A total of 102 patients with suspected invasive mould infection were enrolled. Of these patients, 46 (45%) were classified as having proven (n = 13) or probable (n = 33) invasive aspergillosis, 21 (21%) as proven (n = 17) or probable (n = 4) invasive mucormycosis and 18 (18%) as possible invasive mould infection. The remaining 13 (13%) were classified as not having invasive mould infection. Patients with possible invasive mould infection (n = 18) and coinfection of aspergillosis and mucormycosis (n = 4) were excluded from the final analysis. The sensitivity and specificity of the Aspergillus PCR were 54.3% ([25/46], 95% confidence interval [CI]: 40.2-67.9%) and 94.1% ([32/34], 95% CI: 80.9-98.4%), respectively. The sensitivity and specificity of the Mucor or Rhizopus PCR were 57.1% ([12/21], 95% CI: 36.6-75.5%) and 76.3% ([45/59], 95% CI: 64.0-85.3), respectively. CONCLUSIONS: Our study suggests that blood PCR can be a useful adjunct test for diagnosing patients with suspected invasive mould infection.

Heterologous expression and biochemical characterization of a cold-active lipase from Rhizopus microsporus suitable for oleate synthesis and bread making.

OBJECTIVES: Cold-active lipases which show high specific activity at low temperatures are attractive in industrial applications in terms of product stability and energy saving. We aimed to identify novel cold-active lipase suitable for oleates synthesis and bread making. RESULTS: A novel lipase gene (RmLipA) from Rhizopus microsporus was cloned and heterologously expressed in Pichia pastoris. The encoding sequence displayed 75% identity to the lipase from R. niveus. The highest extracellular lipase activity of 7931 U/mL was achieved in a 5-L fermentation. The recombinant enzyme (RmLipA) was optimally active at pH 8.0 and 20-25 °C, respectively, and stable over a wide pH range of 2.0-11.0. The enzyme was a cold-active lipase, exhibiting > 80% of its maximal activity at 0 °C. RmLipA was a sn-1,3 regioselective lipase, and preferred to hydrolyze pNP esters and triglycerides with relatively long chain fatty acids. RmLipA synthesized various oleates using oleic acid and different alcohols as substrates (> 95%). Moreover, it significantly improved the quality of bread by increasing its specific volume (21.7%) and decreasing its crumb firmness (28.6%). CONCLUSIONS: A novel cold-active lipase gene from R. microsporus was identified, and its application potentials were evaluated. RmLipA should be a potential candidate in oleates synthesis and bread making industries.

Rhizopus microsporus Infections Associated with Surgical Procedures, Argentina, 2006-2014.

Rhizopus spp. fungi are ubiquitous in the environment and a rare but substantial cause of infection in immunosuppressed persons and surgery patients. During 2005-2017, an abnormally high number of Rhizopus infections in surgery patients, with no apparent epidemiologic links, were reported in Argentina. To determine the likelihood of a common source of the cluster, we performed whole-genome sequencing on samples collected during 2006-2014. Most isolates were separated by >60 single-nucleotide polymorphisms, and we found no evidence for recombination or nonneutral mutation accumulation; these findings do not support common source or patient-to-patient transmission. Assembled genomes of most isolates were ≈25 Mbp, and multiple isolates had substantially larger assembled genomes (43-51 Mbp), indicative of infections with strain types that underwent genome expansion. Whole-genome sequencing has become an essential tool for studying epidemiology of fungal infections. Less discriminatory techniques may miss true relationships, possibly resulting in inappropriate attribution of point source.

Next-generation sequencing technology for detecting pulmonary fungal infection in bronchoalveolar lavage fluid of a patient with dermatomyositis: a case report and literature review.

BACKGROUND: Invasive fungal pneumonia is a severe infectious disease with high mortality in immunocompromised patients. However, the clinical diagnosis of the pathogen(s) remains difficult since microbiological evidence is difficult to acquire. CASE PRESENTATION: Here, we report a case of pulmonary fungal infection detected by next-generation sequencing (NGS) of bronchoalveolar lavage fluid (BALF) in a 61-year-old male with corticosteroid-treated dermatomyositis. Cytomegalovirus and influenza A virus infections were confirmed by nucleic acid detection and treated with antiviral medicine. The patient had been diagnosed with severe pneumonia and treated with empiric broad-spectrum antibacterial and antifungal drugs before bronchoscopy was performed. The patient responded poorly to those empiric treatments. Three fungi were found by NGS in the BALF, namely, Pneumocystis jirovecii, Aspergillus fumigatus and Rhizopus oryzae. After adjusting the patient's treatment plan according to the NGS results, he improved significantly. CONCLUSIONS: This case highlights the combined application of NGS and traditional tests in the clinical diagnosis of pulmonary invasive fungal disease. NGS is proposed as an important adjunctive diagnostic approach for identifying uncommon pathogens.

Gastrointestinal mucormycosis due to Rhizopus microsporus following Streptococcus pyogenes toxic shock syndrome in an HIV patient: a case report.

BACKGROUND: Gastrointestinal (GI) mucormycosis is a rare and often deadly form of mucormycosis. Delayed diagnosis can lead to an increased risk of death. Here, we report a case of GI mucormycosis following streptococcal toxic shock syndrome in a virologically suppressed HIV-infected patient. CASE PRESENTATION: A 25-year-old Thai woman with a well-controlled HIV infection and Grave's disease was admitted to a private hospital with a high-grade fever, vomiting, abdominal pain, and multiple episodes of mucous diarrhea for 3 days. On day 3 of that admission, the patient developed multiorgan failure and multiple hemorrhagic blebs were observed on all extremities. A diagnosis of streptococcal toxic shock was made before referral to Siriraj Hospital - Thailand's largest national tertiary referral center. On day 10 of her admission at our center, she developed feeding intolerance and bloody diarrhea due to bowel ischemia and perforation. Bowel resection was performed, and histopathologic analysis of the resected bowel revealed acute suppurative transmural necrosis and vascular invasion with numerous broad irregular branching non-septate hyphae, both of which are consistent with GI mucormycosis. Peritoneal fluid fungal culture grew a grayish cottony colony of large non-septate hyphae and spherical sporangia containing ovoidal sporangiospores. A complete ITS1-5.8S-ITS2 region DNA sequence analysis revealed 100% homology with Rhizopus microsporus strains in GenBank (GenBank accession numbers KU729104 and AY803934). As a result, she was treated with liposomal amphotericin B. However and in spite of receiving appropriate treatment, our patient developed recurrent massive upper GI bleeding from Dieulafoy's lesion and succumbed to her disease on day 33 of her admission. CONCLUSION: Diagnosis of gastrointestinal mucormycosis can be delayed due to a lack of well-established predisposing factors and non-specific presenting symptoms. Further studies in risk factors for abdominal mucormycosis are needed.

Microsatellite analysis of the population structure in Rhizopus arrhizus.

OBJECTIVES: Rhizopus arrhizus is recognized as an emergent agent of superficial and invasive mucormycosis. Despite an increasing number of these infections, the molecular epidemiology of Rhizopus species has not been well studied. MATERIALS AND METHODS: In this study, 43 R. arrhizus strains (25 environmental and 18 clinical isolates) were genotyped using six novel panels of microsatellite markers. RESULTS: Upon the analysis of 43 isolates, 4-8 distinct alleles were detected for each marker. The discriminatory power for the individual markers ranged from 0·522 to 0·830. The combination of all six markers yielded 33 different haplotypes with a high degree of discrimination (0·989 D value). A four-marker combination were selected as the most parsimonious panel achieving D > 0·95. One clinical isolate and one environmental isolate shared the same genotype suggesting the possible nosocomial outbreak of mucormycosis in hospitalized patients. We have noted that the strains isolated from cutaneous mucormycosis were different from the strains isolated from rhino-orbito-cerebral mucormycosis. Then, the hypothesis of particular tropism of infectious strains for a given site is not excluded. The standardized indices of association IA and rBarD were significantly different from zero (P < 0·01), suggesting a prevailing clonal reproduction. The environmental population was significantly differentiated from clinical populations (Fst = 0·2249). CONCLUSIONS: Microsatellite typing method described in our study showed an excellent degree of discriminatory power. It is a promising tool for illuminating the molecular epidemiology of R. arrhizus species, including strain relatedness and transmission pathways.

Metagenomic analysis reveals the impact of JIUYAO microbial diversity on fermentation and the volatile profile of Shaoxing-jiu.

This study focused on the microbial communities found in JIUYAO, the fermentation starter traditionally used in Shaoxing-jiu, and elucidated their relationship with the fermentation activities and volatile compounds involved in winemaking. The microbial communities found in all JIUYAO samples tested were dominated by Pediococcus and Weissella bacteria and Saccharomycopsis and Rhizopus fungi. Saccharifying power showed significant positive correlations with the presence of Pedioccoccus, Saccharomycopsis, and Rhizopus, whereas acid production capacity was strongly associated with Pedioccoccus, Weissella, and Rhizopus. Alcohol production capacity positively correlated with the presence of Pedioccoccus and Rhizopus. Fifteen important volatile compounds (odor-activity values ≥ 1) including esters, alcohols, acids, and aldehydes were identified in Huangjiu samples fermented with JIUYAO. Positive correlations were found between Saccharomycopsis and phenylethanol/ethyl butyrate, Rhizopus and ethyl propionate/ethyl laurate/ethyl butyrate, Pedioccoccus and ethyl laurate/acetic acid, and Weissella and decanoic acid/isopentanol. These results imply that these microorganisms significantly contribute to the fermentation activities and flavor of Shaoxing-jiu. Finally, the results showed that a combination of five core microbes with Saccharomyces cerevisiae could be used as a starter in winemaking. To conclude, this study provides a comprehensive overview of the core microbes found in JIUYAO and strategies for the selection of beneficial microorganisms to improve the quality and flavor of Shaoxing-jiu.

Semi-pilot scale production of biodiesel from waste frying oil by genetically improved fungal lipases.

This paper addresses the issue of combining the usage of waste frying oil (WFO), as a feedstock, and a lipase produced in solid-state fermentation (SSF), as a biocatalyst, for semi-pilot scale production of biodiesel as fatty acid methyl esters (FAME). Two fungal mutants namely; Rhizopus stolonifer 1aNRC11 mutant F (1F) and Aspergillus tamarii NDA03a mutant G (3G) were used as a cocatalyst. The two mutants were cultivated separately by SSF in a tray bioreactor. The dried fermented solid of 1F and 3G mutants were used in a ratio of 3:1, respectively, for WFO transesterification. Optimization of several semi-pilot process stages including SSF and WFO transesterification reaction conditions resulted in 92.3% conversion of WFO to FAME. This FAME yield was obtained after 48 h using 10% cocatalyst (w/w of WFO), 10% water (w/w of WFO) and 3:1 methanol/ WFO molar ratio at 30 °C and 250 rpm. A preliminary economic evaluation of produced biodiesel price (190 $/Ton) is less than half the price of petroleum diesel in Egypt (401$/Ton) and is about 40.3% the price of biodiesel produced using a pure enzyme, which is a promising result. This strategy makes the biodiesel synthesis process greener, economical and sustainable.

Mining Symbionts of a Spider-Transmitted Fungus Illuminates Uncharted Biosynthetic Pathways to Cytotoxic Benzolactones.

A spider-transmitted fungus (Rhizopus microsporus) that was isolated from necrotic human tissue was found to harbor endofungal bacteria (Burkholderia sp.). Metabolic profiling of the symbionts revealed a complex of cytotoxic agents (necroximes). Their structures were characterized as oxime-substituted benzolactone enamides with a peptidic side chain. The potently cytotoxic necroximes are also formed in symbiosis with the fungal host and could have contributed to the necrosis. Genome sequencing and computational analyses revealed a novel modular PKS/NRPS assembly line equipped with several non-canonical domains. Based on gene-deletion mutants, we propose a biosynthetic model for bacterial benzolactones. We identified specific traits that serve as genetic handles to find related salicylate macrolide pathways (lobatamide, oximidine, apicularen) in various other bacterial genera. Knowledge of the biosynthetic pathway enables biosynthetic engineering and genome-mining approaches.