Dialysis patients with chronic renal failure receiving deferoxamine for treating iron overload are uniquely predisposed for mucormycosis, which is most often caused by Rhizopus oryzae. Although the ...deferoxamine siderophore is not secreted by Mucorales, previous studies established that Rhizopus species utilize iron from ferrioxamine (iron-rich form of deferoxamine). Here we determined that the CBS domain proteins of Fob1 and Fob2 act as receptors on the cell surface of R. oryzae during iron uptake from ferrioxamine. Fob1 and Fob2 cell surface expression was induced in the presence of ferrioxamine and bound radiolabeled ferrioxamine. A R. oryzae strain with targeted reduced Fob1/Fob2 expression was impaired for iron uptake, germinating, and growing on medium with ferrioxamine as the sole source of iron. This strain also exhibited reduced virulence in a deferoxamine-treated, but not the diabetic ketoacidotic (DKA), mouse model of mucormycosis. The mechanism by which R. oryzae obtains iron from ferrioxamine involves the reductase/permease uptake system since the growth on ferrioxamine supplemented medium is associated with elevated reductase activity and the use of the ferrous chelator bathophenanthroline disulfonate abrogates iron uptake and growth on medium supplemented with ferrioxamine as a sole source of iron. Finally, R. oryzae mutants with reduced copies of the high affinity iron permease (FTR1) or with decreased FTR1 expression had an impaired iron uptake from ferrioxamine in vitro and reduced virulence in the deferoxamine-treated mouse model of mucormycosis. These two receptors appear to be conserved in Mucorales, and can be the subject of future novel therapy to maintain the use of deferoxamine for treating iron-overload.
Rhizopus delemar and associated species attack a wide range of fruit and vegetables after harvest. Host nutrients and acidic pH are required for optimal germination of R. delemar, and we studied how ...this process is triggered. Glucose induced spore swelling in an acidic environment, expressed by an up to 3-fold increase in spore diameter, whereas spore diameter was smaller in a neutral environment. When suspended in an acidic environment, the spores started to float, indicating a change in their density. Treatment of the spores with HgCl2, an aquaporin blocker, prevented floating and inhibited spore swelling and germ-tube emergence, indicating the importance of water uptake at the early stages of germination. Two putative candidate aquaporin-encoding genes-RdAQP1 and RdAQP2-were identified in the R. delemar genome. Both presented the conserved NPA motif and six-transmembrane domain topology. Expressing RdAQP1 and RdAQP2 in Arabidopsis protoplasts increased the cells' osmotic water permeability coefficient (Pf) compared to controls, indicating their role as water channels. A decrease in R. delemar aquaporin activity with increasing external pH suggested pH regulation of these proteins. Substitution of two histidine (His) residues, positioned on two loops facing the outer side of the cell, with alanine eliminated the pH sensing resulting in similar Pf values under acidic and basic conditions. Since hydration is critical for spore switching from the resting to activate state, we suggest that pH regulation of the aquaporins can regulate the initial phase of R. delemar spore germination, followed by germ-tube elongation and host-tissue infection.
Mucormycosis is a life-threatening infection caused by Mucorales fungi. Here we sequence 30 fungal genomes, and perform transcriptomics with three representative Rhizopus and Mucor strains and with ...human airway epithelial cells during fungal invasion, to reveal key host and fungal determinants contributing to pathogenesis. Analysis of the host transcriptional response to Mucorales reveals platelet-derived growth factor receptor B (PDGFRB) signaling as part of a core response to divergent pathogenic fungi; inhibition of PDGFRB reduces Mucorales-induced damage to host cells. The unique presence of CotH invasins in all invasive Mucorales, and the correlation between CotH gene copy number and clinical prevalence, are consistent with an important role for these proteins in mucormycosis pathogenesis. Our work provides insight into the evolution of this medically and economically important group of fungi, and identifies several molecular pathways that might be exploited as potential therapeutic targets.
Fumaric acid, a dicarboxylic acid used as a food acidulant and in manufacturing synthetic resins, can be produced from glucose in fermentation by Rhizopus oryzae. However, the fumaric acid yield is ...limited by the co-production of ethanol and other byproducts. To increase fumaric acid production, overexpressing endogenous pyruvate carboxylase (PYC) and exogenous phosphoenolpyruvate carboxylase (PEPC) to increase the carbon flux toward oxaloacetate were investigated. Compared to the wild type, the PYC activity in the pyc transformants increased 56%–83%, whereas pepc transformants exhibited significant PEPC activity (3–6mU/mg) that was absent in the wild type. Fumaric acid production by the pepc transformant increased 26% (0.78g/g glucose vs. 0.62g/g for the wild type). However, the pyc transformants grew poorly and had low fumaric acid yields (<0.05g/g glucose) due to the formation of large cell pellets that limited oxygen supply and resulted in the accumulation of ethanol with a high yield of 0.13–0.36g/g glucose. This study is the first attempt to use metabolic engineering to modify the fumaric acid biosynthesis pathway to increase fumaric acid production in R. oryzae.
► Fumaric acid synthesis pathway in R. oryzae was engineered to increase flux toward oxaloacetate. ► Pyruvate carboxylase (PYC) and phosphoenolpyruvate carboxylase (PEPC) were overexpressed. ► Overexpressing PYC resulted in poor growth and fumaric acid production due to reduced ATP. ► Overexpressing PEPC increased fumaric acid production from glucose by 26%. ► This is the first metabolic engineering study to increase fumaric acid production in R. oryzae.
Production of lactic acid from renewable sugars has received growing attention as lactic acid can be used for making renewable and bio-based plastics. However, most prior studies have focused on ...production of lactic acid from glucose despite that cellulosic hydrolysates contain xylose as well as glucose. Microbial strains capable of fermenting both glucose and xylose into lactic acid are needed for sustainable and economic lactic acid production. In this study, we introduced a lactic acid-producing pathway into an engineered Saccharomyces cerevisiae capable of fermenting xylose. Specifically, ldhA from the fungi Rhizopus oryzae was overexpressed under the control of the PGK1 promoter through integration of the expression cassette in the chromosome. The resulting strain exhibited a high lactate dehydrogenase activity and produced lactic acid from glucose or xylose. Interestingly, we observed that the engineered strain exhibited substrate-dependent product formation. When the engineered yeast was cultured on glucose, the major fermentation product was ethanol while lactic acid was a minor product. In contrast, the engineered yeast produced lactic acid almost exclusively when cultured on xylose under oxygen-limited conditions. The yields of ethanol and lactic acid from glucose were 0.31 g ethanol/g glucose and 0.22 g lactic acid/g glucose, respectively. On xylose, the yields of ethanol and lactic acid were <0.01 g ethanol/g xylose and 0.69 g lactic acid/g xylose, respectively. These results demonstrate that lactic acid can be produced from xylose with a high yield by S. cerevisiae without deleting pyruvate decarboxylase, and the formation patterns of fermentations can be altered by substrates.
We have cloned a glucansucrase from the type strain of Leuconostoc mesenteroides (NRRL B-1118; ATCC 8293) and successfully expressed the enzyme in Escherichia coli. The recombinant processed enzyme ...has a putative sequence identical to the predicted secreted native enzyme (1,473 amino acids; 161,468 Da). This enzyme catalyzed the synthesis of a water-insoluble α-D-glucan from sucrose (K M 12 mM) with a broad pH optimum between 5.0 and 5.7 in the presence of calcium. Removal of calcium with dialysis resulted in lower activity in the acidic pH range, effectively shifting the pH optimum to 6.0–6.2. The enzyme was quickly inactivated at temperatures above approximately 45°C. The presence of dextran offered some protection from thermal inactivation between room temperature and 40°C but had little effect above 45°C. NMR and methylation analysis of the water-insoluble α-D-glucan revealed that it had approximately equal amounts of α(1 to 3)-linked and α(1 to 6)-linked d-glucopyranosyl units and a low degree of branching.
Mucormycosis causes mortality in at least 50% of cases despite current first-line therapies. Clinical and animal data indicate that the presence of elevated available serum iron predisposes the host ...to mucormycosis. Here we demonstrate that deferasirox, an iron chelator recently approved for use in humans by the US FDA, is a highly effective treatment for mucormycosis. Deferasirox effectively chelated iron from Rhizopus oryzae and demonstrated cidal activity in vitro against 28 of 29 clinical isolates of Mucorales at concentrations well below clinically achievable serum levels. When administered to diabetic ketoacidotic or neutropenic mice with mucormycosis, deferasirox significantly improved survival and decreased tissue fungal burden, with an efficacy similar to that of liposomal amphotericin B. Deferasirox treatment also enhanced the host inflammatory response to mucormycosis. Most importantly, deferasirox synergistically improved survival and reduced tissue fungal burden when combined with liposomal amphotericin B. These data support clinical investigation of adjunctive deferasirox therapy to improve the poor outcomes of mucormycosis with current therapy. As iron availability is integral to the pathogenesis of other infections (e.g., tuberculosis, malaria), broader investigation of deferasirox as an antiinfective treatment is warranted.
•Novel use of amylose inclusion complex to control pathogenic microbes.•Control of fungi, gram(+) bacteria and algae – some control of gram(-) bacteria.•Amylose complexes formed from inexpensive corn ...starch and fatty ammonium salts.•Process used to produce complex is inexpensive and globally utilized.•Complexes contain no new chemical bonds and do not lyse blood cells.
Amylose-fatty (C12-C16) ammonium salt inclusion complexes are effective antimicrobial polymers causing growth inhibition of microbes at concentrations as low as 40 μg/mL of the complex (2 μg/mL active cationic ligand). The complex was more effective at controlling microbes than the uncomplexed ligand. The complexes were found to be particularly effective at inhibiting the growth of fungi, yeast, gram (+) bacteria, and algae; its performance was affected by pH. The complexes were not hemolytic at concentrations up to 2000 μg/mL. These agents were determined to be surface active polymers and their antimicrobial mode of action may involve cell membrane thinning or disruption, causing moderate leakage. Increased ligand concentration provided increased antimicrobial activity. Solutions of amylose complexes were found to be stable, retaining their antimicrobial efficacy after autoclaving, or after storage at room temperature for 6 months. Antimicrobial amylose complexes were produced using readily available inexpensive materials via an easily scalable process.
Control of bacterial contamination in bioethanol fermentation facilities has traditionally relied on chemical-based products such as hop acids and use of antibiotics. Recent emphasis on antibiotic ...stewardship has prompted new research into the development of alternative approaches to microbial remediation strategies. We recently described a recombinant peptidoglycan hydrolase, endolysin LysKB317, which inhibited
strains in corn mash fermentation. Here,
EBY100 was used to anchor recombinant LysKB317 using cell surface display with the a-agglutinin proteins Aga1p-Aga2p. Immunostaining and confocal fluorescence were used for localization of the extracellular interface of the cells. Yeast surface-expressed endolysin demonstrated an 83.8% decrease in bacterial cell counts compared to a 9.5% decrease in control yeast. Recombinant
expressing LysKB317 used for small-scale corn mash fermentation, when infected with
, could proactively control bacterial infection for 72 h with at least 1-log fold reduction. Analysis of fermentation products showed improved ethanol concentrations from 3.4% to at least 5.9% compared to the infection-only control and reduced levels of lactic and acetic acid from 34.7 mM to 13.8 mM and 25.5 mM to 18.1 mM, respectively. In an optimized yeast surface display system, proactive treatment of bacterial contaminants by endolysin LysKB317 can improve fermentation efficiency in the presence of
contamination.
•A method for overcoming bacterial contamination in ethanol fermentation is described.•The method utilizes beneficial microbes originally isolated as process contaminants.•This approach could ...eliminate the use of antibiotics in fuel ethanol fermentations.
Fuel ethanol fermentations are not performed under aseptic conditions and microbial contamination reduces yields and can lead to costly “stuck fermentations”. Antibiotics are commonly used to combat contaminants, but these may persist in the distillers grains co-product. Among contaminants, it is known that certain strains of lactic acid bacteria are capable of causing stuck fermentations, while other strains appear to be harmless. However, it was not previously known whether or how these strains interact one with another. In this study, more than 500 harmless strains of lactic acid bacteria were tested in a model system in combination with strains that cause stuck fermentations. Among these harmless strains, a group of beneficial strains was identified that restored ethanol production to near normal levels. Such beneficial strains may serve as an alternative approach to the use of antibiotics in fuel ethanol production.
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