CRISPR/Cas9-based genome engineering in the filamentous fungus Rhizopus oryzae and its application to L-lactic acid production.

The filamentous fungus Rhizopus oryzae is one of the main industrial strains for the production of a series of important chemicals such as ethanol, lactic acid, and fumaric acid. However, the lack of efficient gene editing tools suitable for R. oryzae makes it difficult to apply technical methods such as metabolic engineering regulation and synthetic biology modification. A CRISPR-Cas9 system suitable for efficient genome editing in R. oryzae was developed. Firstly, four endogenous U6 promoters of R. oryzae were identified and screened with the highest transcriptional activity for application to sgRNA transcription. It was then determined that the U6 promoter mediated CRISPR/Cas9 system has the ability to efficiently edit the genome of R. oryzae through NHEJ and HDR-mediated events. Furthermore, the newly constructed CRISPR-Cas9 dual sgRNAs system can simultaneously disrupt or insert different fragments of the R. oryzae genome. Finally, this CRISPR-Cas9 system was applied to the genome editing of R. oryzae by knocking out pyruvate carboxylase gene (PYC) and pyruvate decarboxylase gene (pdcA) and knocking in phosphofructokinase (pfkB) from Escherichia coli and L-lactate dehydrogenase (L-LDH) from Heyndrickxia coagulans, which resulted in a substantial increase in L-LA production. In summary, this study showed that the CRISPR/Cas9-based genome editing tool is efficient for manipulating genes in R. oryzae.

Ferulic acid triggering a co-production of 4-vinyl guaiacol and fumaric acid from lignocellulose-based carbon source by Rhizopus oryzae.

Plant secondary metabolites have attracted considerable attention due to the increasing demand for finite fossil resources and environmental concerns. However, the biosynthesis of aromatic aldehydes or alcohols from renewable resources remains challenging and costly. This study explores a novel approach performed by the aromatic catabolizing organism Rhizopus oryzae, which enables a ferulic acid-activated co-production of 4-vinyl guaiacol (4-VG) and fumaric acid. The strain produced 4.60 g/L 4-VG and 11.25 g/L fumaric acid from a mixed carbon source of glucose and xylose, suggesting that this new pathway allows the potential production of natural 4-VG from low-cost substrates. This green route, which utilizes Rhizopus oryzae's ability to efficiently convert various renewable resources into valuable chemicals, paves the way for improved catalytic efficiency in 4-VG production.

Enrichment of Fruit Peels' Nutritional Value by Solid-State Fermentation with Aspergillus ibericus and Rhizopus oryzae.

The fruit processing industry is responsible for disposing of huge amounts of byproducts, especially fruit peels (FPs), which are often discarded in landfills. Using FPs in biotechnological processes contributes to a circular economy, reducing the environmental burden of FPs and increasing the revenue of the fruit processing industry. This study was focused on upgrading the nutritional value of orange (OPs) and banana (BPs) peels by solid-state fermentation (SSF) with filamentous fungi. SSF factors (moisture, fermentation time, inoculum size, ammonium sulfate (AS), and corn steep liquor (CSL)) and fungi species (Aspergillus ibericus and Rhizopus oryzae) were studied by a variable screening Plackett-Burman design. Both fungi grew on untreated FPs, increasing their protein content and antioxidant activity. Moisture, AS, and CSL were further studied by a Box-Behnken design with A. ibericus. Fermented OPs at 70% moisture and 0.005 g/g AS increased their protein content by 200%, whereas BPs at 70% moisture and 0.005 g/g CSL increased by 123%. Fermented peels were enriched in protein, fiber, and minerals, with a low content of carbohydrates and soluble sugars. Fermented OPs and BPs showed higher antioxidant activity than unfermented peels. The SSF of these FPs is an innovative approach that contributes to obtaining rich nutrient-fermented peels for food.

Metabolomic analysis reveals the positive effects of Rhizopus oryzae fermentation on the nutritional and functional constituents of adlay millet seeds.

Adlay millet seeds are well known for excellent health benefits. However, using fungal fermentation to improve their nutritional and functional constituents and the underlying mechanisms has not been thoroughly investigated. Herein, we used Rhizopus oryzae as starter and applied metabolomics combining with quantitative verification to understand the changes of the nutritional and functional profiles of adlay millet seeds. Results showed that a total of 718 metabolites from 18 compound classes were identified. The fermentation with R. oryzae varied 203 differential metabolites, of which 184 became more abundant and 19 got less abundant, and many components such as amino acids, nucleotides, vitamins, flavonoids, terpenoids, and phenols significantly increased after the fermentation process. Interestingly, we found that R. oryzae synthesized high levels of two important beneficial compounds, S-adenosylmethionine (SAMe) and ß-Nicotinamide mononucleotide (ß-NMN), with their contents increased from 0.56 to 370.26 µg/g and 0.55 to 8.32 µg/g, respectively. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of enriched metabolites revealed the amino acid metabolic pathways were important for conversion of the primary and secondary metabolites. Specifically, aspartate can up-regulate the biosynthesis of SAMe and ß-NMN. These findings improved our understanding into the effects of R. oryzae fermentation on enhancing the nutritional and functional values of cereal foods.

Enhancing the Thermostability and Catalytic Activity of the Lipase from Rhizopus oryzae via Introducing N-Glycosylation.

Lipase from Rhizopus oryzae (ROL) exhibits remarkable sn-1,3 stereoselectivity and catalytic activity, but its poor thermostability limits its applications in the production of 1,3-dioleoyl-2-palmitoyl glycerol (OPO, a high-quality substitute for human milk fat). In this work, a semirational method was proposed to engineer the thermostability and catalytic activity of 4M (ROL mutant in our previous study). First, a computer-aided design is performed using 4M as a template, and N-glycosylation mutants are then recombinantly expressed and screened in Pichia pastoris, the optimal mutant N227 exhibited a half-life of 298.8 h at 45 °C, which is 7.23-folds longer than that of 4M. Its catalytic activity also reached 1043.80 ± 61.98 U/mg, representing a 29.2% increase compared to 4M (808.02 ± 47.02 U/mg). Molecular dynamics simulations of N227 suggested that the introduction of glycan enhanced the protein rigidity, while the strong hydrogen bonds formed between the glycan and the protein stabilized the lipase structure, thereby improving its thermostability. The acidolysis reaction between oleic acid (OA) and glycerol tripalmitate (PPP) was successfully carried out using immobilized N227, achieving a molar conversion rate of 90.2% for PPP. This engineering strategy guides the modification of lipases, while the glycomutants obtained in this study have potential applications in the biosynthesis of OPO.

Polyphasic identification of Rhizopus oryzae and evaluation of physical fermentation parameters in potato starch processing liquid waste for ß-glucan production.

Β-glucans are polysaccharide macromolecules that can be found in the cell walls of molds, such as Rhizopus oryzae. They provide functional properties in food systems and have immunomodulatory activity, anticancer, and prebiotic effects; reduce triglycerides and cholesterol; and prevent obesity, among others benefits. Furthermore, potato starch production requires a large amount of water, which is usually discharged into the environment, creating problems in soils and bodies of water. The physical parameters to produce ß-glucans were determined, liquid waste from potato starch processing was used and native Rhizopus oryzae was isolated and identified from cereal grains. The isolates grew quickly on the three types of agars used at 25 °C and 37 °C, and they did not grow at 45 °C. Rhizopus oryzae M10A1 produced the greatest amount of ß-glucans after six days of culture at 30 °C, pH 6, a stirring rate of 150 rpm and a fermentation volume of 250 mL. By establishing the physical fermentation parameters and utilizing the liquid waste from potato starch, Rhizopus oryzae M10A1 yielded 397.50 mg/100 g of ß-glucan was obtained.

Utility of Itraconazole in Combination with Liposomal Amphotericin B in Rhizopus oryzae Associated Mucormycosis-An Exploratory Study.

BACKGROUND: During the COVID-19 pandemic-associated mucor epidemic, acute antifungal drug shortage necessitated the exploration of other antifungals based on culture sensitivity. Itraconazole is a cheap, safe, and effective antifungal in sensitive cases. METHODOLOGY: We enrolled itraconazole-sensitive COVID-19-associated mucormycosis during the mucormycosis pandemic. After the intensive phase course of liposomal amphotericin B, Itraconazole was offered in susceptible cases during the maintenance phase along with standard of care. These patients were clinically and radiologically followed for 6 months. RESULTS: We enrolled 14 patients (Male: Female-11:3) of Rhino-orbito-cerebral mucormycosis (ROCM) which included 12 diabetics. All patients had facial swelling, orbital swelling, visual impairment, and headache. MRI showed involvement of bilateral sinus (10/14), orbital extension (13/14), cavernous sinus (5/14), cerebral part of the internal carotid artery (3/14), and brain infarcts (4/14). All 14 patients showed sensitivity to Itraconazole with 12 having minimum inhibitory concentration (MIC) ≤ 1 µg/ml and 2 having MIC ≤ 2 µg/ml. Follow-up at 6 months showed clinical improvement in the majority (11/14) and radiological improvement in six out of seven scanned patients. CONCLUSION: Our study shows the potential therapeutic role of oral Itraconazole in ROCM.

Exploratory Research for New Diagnostic Markers of Mucormycosis.

Mucormycosis is a fungal infectious disease caused by Rhizopus oryzae and other members of the order Mucorales, and it is known as one of the most lethal fungal infections. Early diagnosis of mucormycosis improves prognosis because of limited effective treatments and the rapid progression of the disease. On the other hand, the lack of characteristic clinical findings in mucormycosis and the challenge of early definitive diagnosis make early treatment difficult. Our goal was to establish a serodiagnostic method to detect Rhizopus specific antigen (RSA), and we have developed a diagnostic kit by Enzyme-linked immuno-sorbent assay (ELISA) using a monoclonal antibody against this antigen. RSA increased over time in the serum and alveolar lavage fluid of R. oryzae-infected mice. RSA was also detected in serum and alveolar fluid, even at an early stage (Day 1), when the tissue invasion of R. oryzae mycelium was not histopathologically detectable in the lungs of R. oryzae-infected mice. Further evaluation is needed to determine the feasibility of using this assay in clinical practice.

Morphological aspects and the effectiveness of photodynamic inactivation against Rhizopus oryzae in different life cycles.

Mucormycosis is an extremely aggressive fungal disease with a high mortality rate, especially in people with compromised immune systems. Most cases of mucormycosis are caused by the fungus Rhizopus oryzae. The treatments used are based on high doses of antifungals, associated with surgical resections, when it is possible. However, even with this aggressive treatment, the estimated attributable mortality rate is high. There is therefore a need to develop adjuvant treatments. Photodynamic Inactivation (PDI) may be an auxiliary therapeutic option for mucormycosis. Due to the lack of reports in the literature on the morphology and photodynamic inactivation of R. oryzae, characterization of the fungus using Confocal Microscopy and Transmission Electron Microscopy, and different protocols using Photodithazine® (PDZ), a chlorin e6 compound, as a photosensitizer, were performed. The fungus growth rate under different concentrations and incubation times of the photosensitizer and its association with the surfactant Sodium Dodecyl Sulphate (SDS) was evaluated. For the hyphae, both in the light and dark phases, in the protocols using only PDZ, no effective photodynamic response was observed. Meanwhile with the combination of SDS 0.05% and PDZ, inhibition growth rates of 98% and 72% were achieved for the white and black phase, respectively. In the conidia phase, only a 1.7 log10 reduction of the infective spores was observed. High concentration of melanin and the complex and resistant structures, especially at the black phase, results in a high limitation of the PDI inactivation response. The combined use of the SDS resulted in an improved response, when compared to the one obtained with the amphotericin B treatment.

A Patient With Type 1 Diabetes and Acute Rhinosinusitis.

A 41-year-old with type 1 diabetes had generalized weakness, muffled voice, and slurred speech. Neck computed tomography showed soft-tissue gas in the nasopharynx and prevertebral fascia; examination of sinus mucosal samples identified numerous broad, nonseptate right-angled hyphae and fruiting bodies. What is the diagnosis and what would you do next?

Airway necrosis and granulation tissue formation caused by Rhizopus oryzae leading to severe upper airway obstruction: a case report.

Pulmonary Mucormycosis is a fatal infectious disease with high mortality rate. The occurrence of Mucormycosis is commonly related to the fungal virulence and the host's immunological defenses against pathogens. Mucormycosis infection and granulation tissue formation occurred in the upper airway was rarely reported. This patient was a 60-year-old male with diabetes mellitus, who was admitted to hospital due to progressive cough, sputum and dyspnea. High-resolution computed tomography (HRCT) and bronchoscopy revealed extensive tracheal mucosal necrosis, granulation tissue proliferation, and severe airway stenosis. The mucosal necrotic tissue was induced by the infection of Rhizopus Oryzae, confirmed by metagenomic next-generation sequencing (mNGS) in tissue biopsy. This patient was treated with the placement of a covered stent and local instillation of amphotericin B via bronchoscope. The tracheal mucosal necrosis was markedly alleviated, the symptoms of cough, shortness of breath, as well as exercise tolerance were significantly improved. The placement of airway stent and transbronchial microtube drip of amphotericin B could conduce to rapidly relieve the severe airway obstruction due to Mucormycosis infection.

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.

Triple in silico targeting of IMPDH enzyme and RNA-dependent RNA polymerase of both SARS-CoV-2 and Rhizopus oryzae.

Aim: Mucormycosis has been associated with SARS-CoV-2 infections during the last year. The aim of this study was to triple-hit viral and fungal RNA-dependent RNA polymerases (RdRps) and human inosine monophosphate dehydrogenase (IMPDH). Materials & methods: Molecular docking and molecular dynamics simulation were used to test nucleotide inhibitors (NIs) against the RdRps of SARS-CoV-2 and Rhizopus oryzae RdRp. These same inhibitors targeted IMPDH. Results: Four NIs revealed a comparable binding affinity to the two drugs, remdesivir and sofosbuvir. Binding energies were calculated using the most abundant conformations of the RdRps after 100-ns molecular dynamics simulation. Conclusion: We suggest the triple-inhibition potential of four NIs against pathogenic RdRps and IMPDH, which is worth experimental validation.

Development of a Strategy for L-Lactic Acid Production by Rhizopus oryzae Using Zizania latifolia Waste and Cane Molasses as Carbon Sources.

As a biodegradable and renewable material, polylactic acid is considered a major environmentally friendly alternative to petrochemical plastics. Microbial fermentation is the traditional method for lactic acid production, but it is still too expensive to compete with the petrochemical industry. Agro-industrial wastes are generated from the food and agricultural industries and agricultural practices. The utilization of agro-industrial wastes is an important way to reduce costs, save energy and achieve sustainable development. The present study aimed to develop a method for the valorization of Zizania latifolia waste and cane molasses as carbon sources for L-lactic acid fermentation using Rhizopus oryzae LA-UN-1. The results showed that xylose derived from the acid hydrolysis of Z. latifolia waste was beneficial for cell growth, while glucose from the acid hydrolysis of Z. latifolia waste and mixed sugars (glucose and fructose) from the acid hydrolysis of cane molasses were suitable for the accumulation of lactic acid. Thus, a three-stage carbon source utilization strategy was developed, which markedly improved lactic acid production and productivity, respectively reaching 129.47 g/L and 1.51 g/L·h after 86 h of fermentation. This work demonstrates that inexpensive Z. latifolia waste and cane molasses can be suitable carbon sources for lactic acid production, offering an efficient utilization strategy for agro-industrial wastes.

Production of 11α-Hydroxysteroid Derivatives by Corynebacterium glutamicum Expressing the Rhizopus oryzae Hydroxylating System.

Hydroxylation of steroids has acquired special relevance for the pharmaceutical industry. Particularly, the 11α-hydroxylation of steroids is a process of biotechnological importance currently carried out at industrial scale for the production of contraceptive drugs and glucocorticoids. This process is performed by several fungal species including Rhizopus nigricans, Aspergillus ochraceus, Aspergillus niger, and Rhizopus oryzae that are used to produce by biotransformation hydroxylated steroids for pharmaceutical purposes (Wang et al., J Steroid Biochem Mol Biol 171:254-261, 2017). However, the development of more efficient biotransformation processes is essential since the steroidal derivatives obtained by the in vivo hydroxylation are often a mixture of hydroxylated compounds in different positions of the steroid molecule. This phenomenon is due to the large number of different CYPs contained in the fungal strains.The genes responsible for the 11α-hydroxylase activity in R. oryzae consisting in the cytochrome CYP509C12 and its redox partner, the reductase RoCPR1, have been chemically synthetized forming a synthetic operon named FUN optimized to be expressed in bacteria. To express this operon, we have selected the strain Corynebacterium glutamicum R31 that is a robust and GRAS bacterial strain widely used for industrial purposes. The synthetic operon has been cloned in the pECXK-99E vector, yielding pXKFUN plasmid, and transformed C. glutamicum R31 to generate C. glutamicum R31 (pXKFUN) strain. This strain is not a steroid degrader and can efficiently transport C19 and C21 steroids across the cytoplasmic membrane (García-Fernandez et al. Catalysts 316:1-12, 2017). C. glutamicum can be used as a clean host for steroid biotransformation, because it does not introduce additional undesired side reactions on the steroids, thus reducing the contamination of the final products (Felpeto-Santero et al., Microbiol Biotechnol 12:856-868, 2019). Here we show a proof of concept that C. glutamicum can be used as a suitable chassis to perform steroid biotransformation expressing eukaryotic cytochromes. The protocol below provides detailed information on steroid 11α-hydroxylations by Corynebacterium recombinant strain.

Production of Rhizopus oryzae lipase using optimized Yarrowia lipolytica expression system.

Yarrowia lipolytica is an alternative yeast for heterologous protein production. Based on auto-cloning vectors, a set of 18 chromogenic cloning vectors was developed, each containing one of the excisable auxotrophic selective markers URA3ex, LYS5ex, and LEU2ex, and one of six different promoters: the constitutive pTEF, the phase dependent hybrid pHp4d, and the erythritol-inducible promoters from pEYK1 and pEYL1 derivatives. These vectors allowed to increase the speed of cloning of the gene of interest. In parallel, an improved new rProt recipient strain JMY8647 was developed by abolishing filamentation and introducing an auxotrophy for lysine (Lys-), providing an additional marker for genetic engineering. Using this cloning strategy, the optimal targeting sequence for Rhizopus oryzae ROL lipase secretion was determined. Among the eight targeting sequences, the SP6 signal sequence resulted in a 23% improvement in the lipase activity compared to that obtained with the wild-type ROL signal sequence. Higher specific lipase activities were obtained using hybrid erythritol-inducible promoters pHU8EYK and pEYL1-5AB, 1.9 and 2.2 times, respectively, when compared with the constitutive pTEF promoter. Two copy strains produce a 3.3 fold increase in lipase activity over the pTEF monocopy strain (266.7 versus 79.7 mU/mg).

Selective pertraction of dicarboxylic acids from simulated Rhizopus oryzae fermentation broths.

Fumaric, malic and succinic acids have been selectively separated by facilitated pertraction with Amberlite LA-2, using n-heptane as liquid membrane. The feed phase consisted on viscous aqueous solution with similar mixture of carboxylic acids and viscosity as those of Rhizopus oryzae fermentation broth. Due to the differences between the acidities and molecule size of these acids, it is possible to selectively recover fumaric acid from the initial solution. The pH-gradient between the feed and stripping phases, as well as carrier concentration in the liquid membrane represent the main process parameters influencing the pertraction selectivity. Among them, Amberlite LA-2 concentration exhibits the most important control on the selectivity factor S, the maximum value of S being reached for carrier concentration of 30 g/l. The increase of feed phase viscosity amplified the magnitude of these influences on pertraction selectivity, due to the hindrance of acids diffusion towards the region where their reaction with Amberlite LA-2 occurs, effect more important for malic acid. Therefore, by modifying the viscosity from 1 to 24 cP, the maximum value of selectivity factor was increased from 12 to 18.8.

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.

Infection due to Aspergillus flavus and Rhizopus oryzae complex in a patient with diabetes mellitus / Infección por Aspergillus flavus y Rhizopus oryzae complex en paciente con diabetes mellitus

Fungal sinusitis is a pathology that can occur in patients with diabetes mellitus and be associated with a hyperglycemic crisis. It is an aggressive entity with local complications that include involvement of the orbit or the central nervous system, and vascular involvement. Despite surgical and antifungal treatment, mortality raises up to 75%. We report the case of a female patient with a diagnosis of diabetic ketoacidosis and signs of unilateral ophthalmoplegia, which led to the study with magnetic resonance imaging of the central nervous system, finding signs of sinusitis, meningitis, and cerebritis. Initial microbiological studies were negative, and biomarkers such as serum galactomannan and Cryptococcus antigen were also negative. After surgical management and the identification of Aspergillus flavus and Rhizopus spp. in sinus tissue, the patient received treatment with posaconazole and after two months of follow-up she presented clinical improvement. Dual fungal infection and infection by A. flavus are uncommon and clinically relevant entities, with no cases previously reported in our country, therefore this corresponds to a case of clinical interest.

La sinusitis micótica es una condición patológica que puede presentarse en pacientes con diabetes mellitus y estar asociada a una crisis hiperglucémica. Es una entidad agresiva con complicaciones locales que incluyen afectación de la órbita y el sistema nervioso central, y compromiso vascular. A pesar del tratamiento quirúrgico y antimicótico, la mortalidad es de hasta el 75 %. Se describe el caso de una paciente con diagnóstico de cetoacidosis diabética y signos de oftalmoplejía unilateral que llevaron al estudio con resonancia magnética del sistema nervioso central; se encontraron signos de sinusitis, meningitis y cerebritis. Los estudios microbiológicos iniciales fueron negativos, y los biomarcadores galactomanano sérico y el antígeno de Cryptococcus también fueron negativos. Tras el manejo quirúrgico, se llegó a la identificación de Aspergillus flavus y Rhizopus spp. en el tejido de los senos paranasales. La paciente recibió tratamiento con posaconazol y, tras dos meses de seguimiento, había presentado mejoría clínica. La infección fúngica dual y la infección por A. flavus son entidades poco frecuentes y de relevancia clínica, sin casos presentados previamente en nuestro país por lo que este corresponde a un caso de interés clínico.