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.

Functional properties of Rhizopus oryzae strains isolated from agricultural soils as a potential probiotic for broiler feed fermentation.

The most crucial and expensive fragment in the broiler chicken production industry is the feed. Because of the rising demand, finding a cheap and effective feed is an urgent necessity. Fermentation of broiler feed by probiotic fungal starters can enhance the nutrient's availability and digestibility while preventing pathogenic growth. In this study different Rhizopus spp. have been isolated from agricultural soils around Izmir, Turkey, and tested for their probiotic potential and fermentative capacity. The isolated Rhizopus strains first underwent microscopical fluorescent investigation to exclude endofungal bacterial presence, then, those without endofungal bacteria (totally 82) were tested for antimicrobial activity counter bacterial and fungal pathogens. The ones with wide-spectrum antimicrobial activity (totally 10) were tested for gastrointestinal tolerance and antioxidant ability. Upon phenotypic and genotypic identification, the 10 isolates were found to belong to Rhizopus oryzae species. While all 10 strains showed variable gastrointestinal tolerance and antioxidant activities, three of them (92/1, 236/2, and 284) had relatively high antioxidant activity. Upon fermentative capacity assay, compared to unfermented commercial feed, there was a general decrease in crude fiber content by 56% after fermentation by 92/1 isolate for 4 days and 236/2 isolate for 2 days. The highest increase in crude protein content (by 14.5%) occurred after a 4-day fermentation period by 236/2 isolate. The highest increase in metabolizable energy was 8.64%, by the 284 isolate after 2 days of fermentation. In conclusion, the three strains showed good probiotic properties and fermentative capacities hence can be beneficial for the poultry industry.

Rhino-Orbital mucormycosis in an immunocompetent pediatric patient with hyperglycemia of the hospitalized patient.

INTRODUCTION: Acute invasive rhino-orbital mucormycosis usually affects diabetic or neutropenic patients, and only exceptionally develops in immunocompetent adults and children. METHODOLOGY: A 12-years-old immunocompetent female, presented with complicated rhinosinusitis with a subperiosteal orbital abscess, without improvement after initial medical and surgical management, the patient also developed hyperglycemia of the hospitalized patient that represented a challenging and potentially lethal clinical scenario. RESULTS: Diagnosed with an unsuspected rhino-orbital mucormycosis by direct microscopy and PCR, she survived after amphotericin B and surgical treatment. CONCLUSIONS: In cases with torpid clinical evolution, even in apparently immunocompetent patients, appropriate multidisciplinary workup must be performed to rule out opportunistic etiologies including mucormycosis to improve survival.

Development of a novel multi-strain wheat Qu with high enzyme activities for Huangjiu fermentation.

BACKGROUND: Wheat Qu has long been used as a fermentation starter to produce Huangjiu. Wheat Qu quality depends on its microbial community structure and the hydrolytic enzymes generated by the micro-organisms. RESULTS: Strain YF1 and YF2 were successfully screened as they exhibited high acidic protease (231.9 ± 1.4 U g-1 ) and cellulase (7.1 ± 0.6 U g-1 ) activities. Based on a morphological and sequence analysis of the internal transcribed spacer (ITS) gene, YF1 and YF2 were identified as Rhizopus oryzae and Aspergillus niger, respectively. Cooked wheat Qu was produced using mixed fungal starter fermentations with Aspergillus oryzae SU-16, YF1, and YF2. For Qu-making, the optimized conditions for fermentation time, water content, and inoculum size were 47.8 h, 69.4%, and 6.1%, respectively. Under these conditions, compared with single-strain cooked wheat Qu, enzyme activities of amylase, acidic protease, and cellulase increased by 27.4%, 657.1%, and 1276.2%, respectively. Short peptides and free amino acids contents increased by 19.6% and 131.8%, respectively. This wheat Qu was used for Huangjiu brewing, and the alcohol content increased by approximately 14.6% because of the increased starch hydrolysis efficiency mainly attributed to its high enzyme activity. CONCLUSION: Using mixed fungal strains as starter cultures may be an efficient strategy to improve wheat Qu quality, with great potential for application in industrial Huangjiu production. © 2021 Society of Chemical Industry.

Gastrointestinal Zygomycosis in a Preterm Neonate Associated With Contaminated Probiotics.

A neonate of 29 weeks' gestation who received probiotics developed clinical signs suggesting surgical necrotizing enterocolitis. A specimen of resected ileum revealed fungal forms within the bowel wall. Rhizopus oryzae was detected via DNA sequencing from probiotic powder and tissue specimens from the infant. To our knowledge, this is the first report linking gastrointestinal zygomycosis to the administration of contaminated probiotics.

Molecular phylogeny, identification and pathogenicity of Rhizopus oryzae associated with root rot of mulberry in India.

AIMS: Root rot caused by a group of fungi is a serious disease in mulberry. This study aims to identify and characterize Rhizopus oryzae and other fungal species associated with root rot of mulberry in India. METHODS AND RESULTS: Rotted root samples were collected from the mulberry gardens from four states of Southern India. The majority of the isolates identified were R. oryzae, and others were saprophytic fungi, less abundant to occasional. Two methods of inoculations were tested to confirm the pathogenicity of the selected isolates and R. oryzae was found to be pathogenic on susceptible mulberry genotypes RC2 and SRDC-1. Multi gene phylogenetic analyses using the internal transcribed spacer region (ITS), actin (ACT) and translation elongation factor 1-α (TEF), identified the isolates as R. oryzae. Additionally, Ovatospora brasiliensis, Amesia nigricolor, Gongronella butleri, Myrmecridium schulzeri, Scedosporium boydii, Graphium euwallacea, Clonostachys rosea andTalaromyces spp. were also identified. CONCLUSION: This study revealed the existence of eleven species of fungi including the first report of R. oryzae and the occurrence of weak pathogens or saprophytes that are associated with the root rot of mulberry in India. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of R. oryzae causing Rhizopus rot of mulberry in India. Moreover, the occurrence of saprophytes associated with root rot of mulberry was identified. Further studies should focus more on the ability of these species to generate secondary metabolites and extracellular lytic enzymes as they are beneficial for the management of root rot disease.

Domesticating a food spoilage yeast into an organic acid-tolerant metabolic engineering host: Lactic acid production by engineered Zygosaccharomyces bailii.

Lactic acid represents an important class of commodity chemicals, which can be produced by microbial cell factories. However, due to the toxicity of lactic acid at lower pH, microbial production requires the usage of neutralizing agents to maintain neutral pH. Zygosaccharomyces bailii, a food spoilage yeast, can grow under the presence of organic acids used as food preservatives. This unique trait of the yeast might be useful for producing lactic acid. With the goal of domesticating the organic acid-tolerant yeast as a metabolic engineering host, seven Z. bailii strains were screened in a minimal medium with 10 g/L of acetic, or 60 g/L of lactic acid at pH 3. The Z. bailii NRRL Y7239 strain was selected as the most robust strain to be engineered for lactic acid production. By applying a PAN-ARS-based CRISPR-Cas9 system consisting of a transfer RNA promoter and NAT selection, we demonstrated the targeted deletion of ADE2 and site-specific integration of Rhizopus oryzae ldhA coding for lactate dehydrogenase into the PDC1 locus. The resulting pdc1::ldhA strain produced 35 g/L of lactic acid without ethanol production. This study demonstrates the feasibility of the CRISPR-Cas9 system in Z. bailii, which can be applied for a fundamental study of the species.

Molecular epidemiology of zygomycosis and their related factors in tertiary referral centers in southern Iran.

INTRODUCTION: For the best management of the zygomycosis in immunocompromised patients, the present study aims to detect and identify the etiologic agents by DNA sequencing method and their related factors in clinical samples of patients. METHODOLOGY: Clinical samples from 1,058 patients admitted in 11 university hospitals in Shiraz, Southern Iran were collected between July 2015 and July 2018. All samples (bronchoalveolar lavage, sputum, blood, tissue) were examined by routine microscopic and culture tests for zygomycetes. The etiologic agents were identified by the molecular method and sequencing. RESULTS: Direct microscopic examinations or pathology smear, culture, and PCR were positive in 61 (5.8%), 15 (1.4%), and 103 (9.7%) patients, respectively. According to EORTC/MSG criteria, the rates of proven, probable, and possible zygomycosis were 59.2% (61/103), 14.6% (15/103), and 26.2% (27/103 patients), respectively. The most prevalent etiologic agents according to sequencing were Rhizopus oryzae (44 cases), Rhizopus microsporus (31 cases), Rhizopus stolonifer (15 cases). Twenty-two patients (21.4%) with positive PCR died. There were significant relations between zygomycosis and the underlying disease (p = 0.043) and prior antifungal therapy (p = 0.023). White blood cell count was in the normal range in 14.1% of patients, and the means of erythrocyte sedimentation rate (ESR) and C reactive protein (CRP) were 65 mm/hour and 57 mg/L, respectively. CONCLUSIONS: Molecular methods and sequencing may have considered as suitable tools to diagnose zygomycosis. Identification of the etiologic agents may be considered as the future antifungal therapy and management of the respective patients.

Tornadic Shear Stress Induces a Transient, Calcineurin-Dependent Hypervirulent Phenotype in Mucorales Molds.

Trauma-related necrotizing myocutaneous mucormycosis (NMM) has a high morbidity and mortality in victims of combat-related injuries, geometeorological disasters, and severe burns. Inspired by the observation that several recent clusters of NMM have been associated with extreme mechanical forces (e.g., during tornados), we studied the impact of mechanical stress on Mucoralean biology and virulence in a Drosophila melanogaster infection model. In contrast to other experimental procedures to exert mechanical stress, tornadic shear challenge (TSC) by magnetic stirring induced a hypervirulent phenotype in several clinically relevant Mucorales species but not in Aspergillus or Fusarium Whereas fungal growth rates, morphogenesis, and susceptibility to noxious environments or phagocytes were not altered by TSC, soluble factors released in the supernatant of shear-challenged R. arrhizus spores rendered static spores hypervirulent. Consistent with a rapid decay of TSC-induced hypervirulence, minimal transcriptional changes were revealed by comparative RNA sequencing analysis of static and shear-challenged Rhizopus arrhizus However, inhibition of the calcineurin/heat shock protein 90 (hsp90) stress response circuitry by cyclosporine and tanespimycin abrogated the increased pathogenicity of R. arrhizus spores following TSC. Similarly, calcineurin loss-of-function mutants of Mucor circinelloides displayed no increased virulence capacity in flies after undergoing TSC. Collectively, these results establish that TSC induces hypervirulence specifically in Mucorales and point out the calcineurin/hsp90 pathway as a key orchestrator of this phenotype. Our findings invite future studies of topical calcineurin inhibitor treatment of wounds as an adjunct mitigation strategy for NMM following high-energy trauma.IMPORTANCE Given the limited efficacy of current medical treatments in trauma-related necrotizing mucormycosis, there is a dire need to better understand the Mucoralean pathophysiology in order to develop novel strategies to counteract fungal tissue invasion following severe trauma. Here, we describe that tornadic shear stress challenge transiently induces a hypervirulent phenotype in various pathogenic Mucorales species but not in other molds known to cause wound infections. Pharmacological and genetic inhibition of calcineurin signaling abrogated hypervirulence in shear stress-challenged Mucorales, encouraging further evaluation of (topical) calcineurin inhibitors to improve therapeutic outcomes of NMM after combat-related blast injuries or violent storms.

Improving the thermostability and acid resistance of Rhizopus oryzae α-amylase by using multiple sequence alignment based site-directed mutagenesis.

Higher thermostability or acid resistance for fungal α-amylase will help to improve the sugar-making process and cut down the production costs. Here, the thermostability or acid resistance of Rhizopus oryzae α-amylase (ROAmy) was significantly enhanced by site-directed evolution based on multiple sequence alignment (MSA) method. For instance, compared with the wild-type ROAmy, the optimum temperature of mutants G136D and A144Y was increased from 50 to 55 °C, whereas for mutants V174R and I276P, the optimum temperature was increased from 50 to 60 °C. The optimum pH of mutants G136D and A144Y shifted from 5.5 to 5.0, whereas for mutants V174R and T253E, the optimum pH changed from 5.5 to 4.5. The results showed that mutant V174R had a 2.52-fold increase in half-life at 55 °C, a 2.55-fold increase in half-life at pH 4.5, and a 1.61-fold increase in catalytic efficiency (kcat /Km ) on soluble starch. The three-dimensional model simulation revealed that changes of hydrophilicity, hydrogen bond, salt bridge, or rigidity observed in mutants might mainly account for the improvement of thermostability and acid resistance. The mutants with improved catalytic properties attained in this work may render an accessible and operable approach for directed evolution of fungal α-amylase aimed at interesting functions.