A ribonuclease III involved in virulence of Mucorales fungi has evolved to cut exclusively single-stranded RNA.

Members of the ribonuclease III (RNase III) family regulate gene expression by processing double-stranded RNA (dsRNA). This family includes eukaryotic Dicer and Drosha enzymes that generate small dsRNAs in the RNA interference (RNAi) pathway. The fungus Mucor lusitanicus, which causes the deadly infection mucormycosis, has a complex RNAi system encompassing a non-canonical RNAi pathway (NCRIP) that regulates virulence by degrading specific mRNAs. In this pathway, Dicer function is replaced by R3B2, an atypical class I RNase III, and small single-stranded RNAs (ssRNAs) are produced instead of small dsRNA as Dicer-dependent RNAi pathways. Here, we show that R3B2 forms a homodimer that binds to ssRNA and dsRNA molecules, but exclusively cuts ssRNA, in contrast to all known RNase III. The dsRNA cleavage inability stems from its unusual RNase III domain (RIIID) because its replacement by a canonical RIIID allows dsRNA processing. A crystal structure of R3B2 RIIID resembles canonical RIIIDs, despite the low sequence conservation. However, the groove that accommodates dsRNA in canonical RNases III is narrower in the R3B2 homodimer, suggesting that this feature could be responsible for the cleavage specificity for ssRNA. Conservation of this activity in R3B2 proteins from other mucormycosis-causing Mucorales fungi indicates an early evolutionary acquisition.

NADP-dependent glutamate dehydrogenases in a dimorphic zygomycete Benjaminiella poitrasii: Purification, characterization and their evaluation as an antifungal drug target.

BACKGROUND: It has been reported that the genes coding for NADP-dependent glutamate dehydrogenases (NADP-GDHs) showed a cause-effect relationship with Yeast-Hypha (YH) reversible transition in a zygomycete Benjaminiella poitrasii. As YH transition is significant in human pathogenic fungi for their survival and proliferation in the host, the NADP-GDHs can be explored as antifungal drug targets. METHODS: The yeast-form specific BpNADPGDH I and hyphal-form specific BpNADPGDH II of B. poitrasii were purified by heterologous expression in E. coli BL-21 cells and characterized. The structural analogs of L-glutamate, dimethyl esters of isophthalic acid (DMIP) and its derivatives were designed, synthesized and screened for inhibition of NADP-GDH activity as well as YH transition in B. poitrasii, and also in human pathogenic Candida albicans strains. RESULTS: The BpNADPGDH I and BpNADPGDH II were found to be homo-hexameric proteins with native molecular mass of 282 kDa and 298 kDa, respectively and subunit molecular weights of 47 kDa and 49 kDa, respectively. Besides the distinct kinetic properties, BpNADPGDH I and BpNADPGDH II were found to be regulated by cAMP-dependent- and Calmodulin (CaM) dependent- protein kinases, respectively. The DMIP compounds showed a more pronounced effect on H-form specific BpNADPGDH II and inhibited YH transition as well as growth in B. poitrasii and C. albicans strains. CONCLUSION: The present study will be useful to design and develop antifungal drugs against dimorphic human pathogens using glutamate dehydrogenase as a target. SIGNIFICANCE: Glutamate dehydrogenases can be explored as a target against human pathogenic fungi.

Exploring Catalysis Specificity of Phytoene Dehydrogenase CrtI in Carotenoid Synthesis.

Carotenoids, a variety of natural products, have significant pharmaceutical and commercial potential. Phytoene dehydrogenase (CrtI) is the rate-limit enzyme for carotenoid synthesis, whose catalysis specificity results in various carotenoids. However, the structural characteristics of CrtI for controlling the catalysis specificity on dehydrogenation steps are still unclear, which limited the development of CrtI function. Here we confirmed two mutation sites H136 and H453 in the mutant library of CrtI from Blakeslea trispora, which markedly regulated catalytic specificity. Interestingly, the sequence alignment features at H136 and H453 were consistent with the phylogenetic analysis of CrtI families. Subsequently, the functions of saturated mutants at H136 and H453 were clustered by principal component analysis (PCA) and k-means. According to the clustering results, diversiform mutants with specific dehydrogenation function provided important application value for carotenoid product customization. Meanwhile, this study also enriched the theory of enzyme evolution and guided the functional development of enzymes.

A thermophilic fungal GH36 α-galactosidase from Lichtheimia ramosa and its synergistic hydrolysis of locust bean gum.

A novel GH36 α-galactosidase gene (LrAgal36A) from Lichtheimia ramosa was synthesized and highly expressed in Pichia pastoris. The enzyme titer and protein yield for high-density fermentation in a 5 L fermentor were up to 953.6 U mL-1 and 4.36 g L-1. Purified recombinant LrAgal36A showed the maximum activity at pH 6.0 and 65 °C and was thermostable with a half-life of 70 min at 60 °C. LrAgal36A displayed the highest specific activity (353.17 ± 4.19 U mg-1) toward p-nitrophenyl-α-d-galactopyranoside (pNPGal) followed by galacto-oligosaccharides and could act slightly on galactomannans. The Km and catalytic efficiency (kcat/Km) of LrAgal36A for pNPGal were 0.33 mM and 1569.50 mM-1 s-1, respectively. LrAgal36A and GH5 ß-mannanase from L. ramosa showed a significant synergistic effect on the degradation of locust bean gum (LBG), resulting in release more reducing sugars (1.56 folds) and galactose (7.6 folds) by simultaneous or sequential reactions. Due to its hydrolysis properties, LrAgal36A might have potential applications in the area of pulp biobleaching, feed and food processing.

A Collagenolytic Aspartic Protease from Thermomucor indicae-seudaticae Expressed in Escherichia coli and Pichia pastoris.

Proteases are produced by the most diverse microorganisms and have a wide spectrum of applications. However, the use of wild microorganisms, mainly fungi, for enzyme production has some drawbacks. They are subject to physiological instability due to metabolic adaptations, causing complications and impairments in the production process. Thus, the objective of this work was to promote the heterologous expression of a collagenolytic aspartic protease (ProTiN31) from Thermomucor indicae seudaticae in Escherichia coli and Pichia pastoris. The pET_28a (+) and pPICZαA vectors were synthesized containing the gene of the enzyme and transformed into E. coli and P. pastoris, respectively. The recombinant enzymes produced by E. coli and P. pastoris showed maximum activity at pH 5.0 and 50 °C, and pH 5.0 and 60 °C, respectively. The enzyme produced by P. pastoris showed better thermostability when compared to that produced by E. coli. Both enzymes were stable at pH 6.0 and 6.5 for 24 h at 4 °C, and sensitive to pepstatin A, ß-mercaptoethanol, and Hg2+. Comparing the commercial collagen hydrolysate (Artrogen duo/Brazil) and gelatin degradation using protease from P. pastoris, they showed similar peptide profiles. There are its potential applications in a wide array of industrial sectors that use collagenolytic enzymes.

Molecular studies of NAD- and NADP-glutamate dehydrogenases decipher the conundrum of yeast-hypha dimorphism in zygomycete Benjaminiella poitrasii.

Benjaminiella poitrasii, a zygomycete, shows glucose- and temperature-dependent yeast (Y)-hypha (H) dimorphic transition. Earlier, we reported the biochemical correlation of relative proportion of NAD- and NADP-glutamate dehydrogenases (GDHs) with Y-H transition. Further, we observed the presence of one NAD-GDH and two form-specific NADP-GDH isoenzymes in B. poitrasii. However, molecular studies are necessary to elucidate the explicit role of GDHs in regulating Y-H reversible transition. Here, we report the isolation and characterization of one NAD (BpNADGDH, 2.643 kb) and two separate genes, BpNADPGDH I (Y-form specific, 1.365 kb) and BpNADPGDH II (H-form specific, 1.368 kb) coding for NADP-GDH isoenzymes in B. poitrasii. The transcriptional profiling during Y-H transition showed higher BpNADPGDH I expression in Y cells while expression of BpNADPGDH II was higher in H cells. Moreover, the yeast-form monomorphic mutant (Y-5) did not show BpNADPGDH II expression under normal dimorphism triggering conditions. Transformation with H-form specific BpNADPGDH II induced the germ tube formation in Y-5, which confirmed the cause-effect relationship between BpNADPGDH genes and morphological outcome in B. poitrasii. Interestingly, expression of H-form specific BpNADPGDH II also induced germ tube formation in human pathogenic, non-dimorphic yeast Candida glabrata, which further corroborated our findings.

Efficient expression of a novel thermophilic fungal ß-mannosidase from Lichtheimia ramosa with broad-range pH stability and its synergistic hydrolysis of locust bean gum.

ß-Mannosidase (EC 3.2.1.25) is an exoglycosidase specific for the hydrolysis of terminal ß-1,4-glycosidic linkage in mannan which can be applied in the food manufacture, animal feed, bioethanol making and coffee extraction industries. A novel ß-mannosidase gene (Lrman5A) from Lichtheimia ramosa was synthesized and recombinantly expressed in Pichia pastoris X33. Lrman5A encodes 444 amino acids with a calculated molecular mass of 51.0 kDa which shares the highest identity (73%) with the ß-mannosidase from Rhizomucor miehei. Purified recombinant Lrman5A showed the maximal activity at pH 6.0 and 65°C, had broad-range pH stability (retaining >65% activity after incubation at pH 3.0-8.5 at 37°C for 24 h), and was highly thermostable (retaining >80% activity after incubation at 65°C for 10 min). The specific activity, and Km of Lrman5A was 17.5 U/mg and 1.377 mM, respectively. Lrman5A and GH5 ß-mannanase displayed significant synergistic effects on the degradation of locust bean gum (LBG) and released more mannose (up to 2.89 folds) by simultaneous or sequential addition. Due to its hydrolytic properties, Lrman5A may have potential applications in the area of bioenergy, feed and food processing.

Structure of Calcarisporiella thermophila Hsp104 Disaggregase that Antagonizes Diverse Proteotoxic Misfolding Events.

Hsp104 is an AAA+ protein disaggregase with powerful amyloid-remodeling activity. All nonmetazoan eukaryotes express Hsp104 while eubacteria express an Hsp104 ortholog, ClpB. However, most studies have focused on Hsp104 from Saccharomyces cerevisiae and ClpB orthologs from two eubacterial species. Thus, the natural spectrum of Hsp104/ClpB molecular architectures and protein-remodeling activities remains largely unexplored. Here, we report two structures of Hsp104 from the thermophilic fungus Calcarisporiella thermophila (CtHsp104), a 2.70Å crystal structure and 4.0Å cryo-electron microscopy structure. Both structures reveal left-handed, helical assemblies with all domains clearly resolved. We thus provide the highest resolution and most complete view of Hsp104 hexamers to date. We also establish that CtHsp104 antagonizes several toxic protein-misfolding events in vivo where S. cerevisiae Hsp104 is ineffective, including rescue of TDP-43, polyglutamine, and α-synuclein toxicity. We suggest that natural Hsp104 variation is an invaluable, untapped resource for illuminating therapeutic disaggregases for fatal neurodegenerative diseases.

Characterization of a novel exo-chitosanase, an exo-chitobiohydrolase, from Gongronella butleri.

An exo-chitosanase was purified from the culture filtrate of Gongronella butleri NBRC105989 to homogeneity by ammonium sulfate precipitation, followed by column chromatography using CM-Sephadex C-50 and Sephadex G-100. The enzyme comprised a monomeric protein with a molecular weight of approximately 47,000 according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme exhibited optimum activity at pH 4.0, and was stable between pH 5.0 and 11.0. It was most active at 45°C, but was stable at temperatures below 30°C. The enzyme hydrolyzed soluble chitosan and glucosamine (GlcN) oligomers larger than tetramers, but did not hydrolyze N-acetylglucosamine (GlcNAc) oligomers. To clarify the mode of action of the enzyme, we used thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) to investigate the products resulting from the enzyme-catalyzed hydrolysis of chitosan and N1-acetylchitohexaose [(GlcN)5-GlcNAc] with a GlcNAc residue at the reducing end. The results indicated that the enzyme is a novel exo-type chitosanase, exo-chitobiohydrolase, that releases (GlcN)2 from the non-reducing ends of chitosan molecules. Analyses of the hydrolysis products of partially N-acetylated chitooligosaccharides revealed that the enzyme cleaves both GlcN-GlcNAc and GlcNAc-GlcN bonds in addition to GlcN-GlcN bonds in the substrate.

Catalytic and thermodynamic properties of ß-glucosidases produced by Lichtheimia corymbifera and Byssochlamys spectabilis.

The objective of the present study was to optimize parameters for the cultivation of Lichtheimia corymbifera (mesophilic) and Byssochlamys spectabilis (thermophilic) for the production of ß-glucosidases and to compare the catalytic and thermodynamic properties of the partially purified enzymes. The maximum amount of ß-glucosidase produced by L. corymbifera was 39 U/g dry substrate (or 3.9 U/mL), and that by B. spectabilis was 77 U/g (or 7.7 U/mL). The optimum pH and temperature were 4.5 and 55 °C and 4.0 and 50 °C for the enzyme from L. corymbifera and B. spectabilis, respectively. ß-Glucosidase produced by L. corymbifera was stable at pH 4.0-7.5, whereas the enzyme from B. spectabilis was stable at pH 4.0-6.0. Regarding the thermostability, ß-glucosidase produced by B. spectabilis remained stable for 1 h at 50 °C, and that from L. corymbifera was active for 1 h at 45 °C. Determination of thermodynamic parameters confirmed the greater thermostability of the enzyme produced by the thermophilic fungus B. spectabilis, which showed higher values of ΔH, activation energy for denaturation (Ea), and half-life t(1/2). The enzymes were stable in the presence of ethanol and were competitively inhibited by glucose. These characteristics contribute to their use in the simultaneous saccharification and fermentation of vegetable biomass.

Components of a new gene family of ferroxidases involved in virulence are functionally specialized in fungal dimorphism.

Mucormycosis is an emerging angio-invasive infection caused by Mucorales that presents unacceptable mortality rates. Iron uptake has been related to mucormycosis, since serum iron availability predisposes the host to suffer this infection. In addition, iron uptake has been described as a limiting factor that determines virulence in other fungal infections, becoming a promising field to study virulence in Mucorales. Here, we identified a gene family of three ferroxidases in Mucor circinelloides, fet3a, fet3b and fet3c, which are overexpressed during infection in a mouse model for mucormycosis, and their expression in vitro is regulated by the availability of iron in the culture media and the dimorphic state. Thus, only fet3a is specifically expressed during yeast growth under anaerobic conditions, whereas fet3b and fet3c are specifically expressed in mycelium during aerobic growth. A deep genetic analysis revealed partially redundant roles of the three genes, showing a predominant role of fet3c, which is required for virulence during in vivo infections, and shared functional roles with fet3b and fet3c during vegetative growth in media with low iron concentration. These results represent the first described functional specialization of an iron uptake system during fungal dimorphism.

Application of α- and ß-naphthoflavones as monooxygenase inhibitors of Absidia coerulea KCh 93, Syncephalastrum racemosum KCh 105 and Chaetomium sp. KCh 6651 in transformation of 17α-methyltestosterone.

In this work, 17α-methyltestosterone was effectively hydroxylated by Absidia coerulea KCh 93, Syncephalastrum racemosum KCh 105 and Chaetomium sp. KCh 6651. A. coerulea KCh 93 afforded 6ß-, 12ß-, 7α-, 11α-, 15α-hydroxy derivatives with 44%, 29%, 6%, 5% and 9% yields, respectively. S. racemosum KCh 105 afforded 7α-, 15α- and 11α-hydroxy derivatives with yields of 45%, 19% and 17%, respectively. Chaetomium sp. KCh 6651 afforded 15α-, 11α-, 7α-, 6ß-, 9α-, 14α-hydroxy and 6ß,14α-dihydroxy derivatives with yields of 31%, 20%, 16%, 7%, 5%, 7% and 4%, respectively. 14α-Hydroxy and 6ß,14α-dihydroxy derivatives were determined as new compounds. Effect of various sources of nitrogen and carbon in the media on biotransformations were tested, however did not affect the degree of substrate conversion or the composition of the products formed. The addition of α- or ß-naphthoflavones inhibited 17α-methyltestosterone hydroxylation but did not change the percentage composition of the resulting products.

Identification of rotundic acid metabolites after oral administration to rats and comparison with the biotransformation by Syncephalastrum racemosum AS 3.264.

The objective of this study was to identify the metabolites of rotundic acid after oral administration to rats and compare the similarities with its biotransformation by Syncephalastrum racemosum AS 3.264 using ultra-high performance liquid chromatography coupled with quadrupole time of flight mass spectrometry. A total of fourteen metabolites were determined based on the mass spectrometry and chromatographic behaviors, among which eleven (M1-M3, M7-M14) and six (M2, M4-M8) metabolites were identified in rats and S. racemosum, respectively. Three identical metabolites (M2, M7 and M8) were found in rats and S. racemosum, indicating that there were metabolic similarities. Moreover, to confirm the results of mass spectrometry, three (M2, M4 and M7) metabolites were obtained by the means of amplifying incubation and their structures were determined by various spectroscopic analyses, and M4 was proved to be a previously undescribed compound. This results showed that in vitro assisted preparation by microbial transformation is a feasible and effective method of obtaining metabolites which are in low amounts and difficult to be prepared in vivo.

Fungal peptides from pneumonitis hypersensitivity etiologic agents are able to induce specific cellular immune response.

PURPOSE: Hypersensitivity pneumonitis (HP) is an immunoallergic disease due to chronic exposure to high quantities of different microorganisms such as Mycobacterium immunogenum (Mi), a mycobacterium, and Lichtheimia corymbifera (Lc), a filamentous fungus. It has recently been demonstrated that the protein DLDH (dihydrolipoyl dehydrogenase), is common to these microorganisms. This study aimed to investigate the immune potential of overlapping peptide pools covering the MiDLDH and LcDLDH. EXPERIMENTAL DESIGN: A selection of 34 peptides, from the MiDLDH and LcDLDH, able to interact with Major Histocompatibility Complex (MHC) 1 and MHC 2, was obtained using three different epitope prediction websites. By means of ELISPOT assays, we compared the frequency of Interferon gamma (IFNγ) secreting peripheral blood mononuclear cells (PBMC) after stimulation with overlapping peptide pools. Tests were performed using cells from 35 healthy blood donors. RESULTS: One peptide pool containing five peptides from MiDLDH and able to interact with MHC 2 induced a marked IFNγ specific immune response (Pool F, p<0.001, Wilcoxon signed-rank test). CONCLUSION: This study demonstrated that peptides from microorganisms involved in HP were able to induce a high IFNγ specific immune response after stimulation of PBMCs from healthy blood donors which could be useful to develop an effective prevention strategy.

Production and Catalytic Properties of Amylases from Lichtheimia ramosa and Thermoascus aurantiacus by Solid-State Fermentation.

The present study compared the production and the catalytic properties of amylolytic enzymes obtained from the fungi Lichtheimia ramosa (mesophilic) and Thermoascus aurantiacus (thermophilic). The highest amylase production in both fungi was observed in wheat bran supplemented with nutrient solution (pH 4.0) after 96 hours of cultivation, reaching 417.2 U/g of dry substrate (or 41.72 U/mL) and 144.5 U/g of dry substrate (or 14.45 U/mL) for L. ramosa and T. aurantiacus, respectively. The enzymes showed higher catalytic activity at pH 6.0 at 60°C. The amylases produced by L. ramosa and T. aurantiacus were stable between pH 3.5-10.5 and pH 4.5-9.5, respectively. The amylase of L. ramosa was stable at 55°C after 1 hour of incubation, whereas that of T. aurantiacus maintained 60% of its original activity under the same conditions. Both enzymes were active in the presence of ethanol. The enzymes hydrolyzed starch from different sources, with the best results obtained with corn starch. The enzymatic complex produced by L. ramosa showed dextrinizing and saccharifying potential. The enzymatic extract produced by the fungus T. aurantiacus presented only saccharifying potential, releasing glucose monomers as the main hydrolysis product.

An integrated genomic and transcriptomic survey of mucormycosis-causing fungi.

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.

PUFA-induced cell death is mediated by Yca1p-dependent and -independent pathways, and is reduced by vitamin C in yeast.

Polyunsaturated fatty acids (PUFA) such as linoleic acid (LA, n-6, C18:2) and γ-linolenic acid (GLA, n-6, C18:3) are essential and must be obtained from the diet. There has been a growing interest in establishing a bio-sustainable production of PUFA in several microorganisms, e.g. in yeast Saccharomyces cerevisiae. However, PUFAs can also be toxic to cells because of their susceptibility to peroxidation. Here we investigated the negative effects of LA and GLA production on S. cerevisiae by characterizing a strain expressing active Δ6 and Δ12 desaturases from the fungus Mucor rouxii. Previously, we showed that the PUFA-producing strain has low viability, down-regulated genes for oxidative stress response, and decreased proteasome activity. Here we show that the PUFA strain accumulates high levels of reactive oxygen species (ROS) and lipid peroxides, and accumulates damaged proteins. The PUFA strain also showed great increase in metacaspase Yca1p activity, suggesting cells could die by caspase-mediated cell death. When treated with antioxidant vitamin C, ROS, lipid peroxidation and protein carbonylation were greatly reduced, and the activity of the metacaspase was significantly decreased too, ultimately doubling the lifespan of the PUFA strain. When deleting YCA1, the caspase-like activity and the oxidative stress decreased and although the lifespan was slightly prolonged, the phenotype could not be fully reversed, pointing that Yca1p was not the main executor of cell death.

Coalho Cheese Made with Protease from Thermomucor indicae-seudaticae N31: Technological Potential of the New Coagulant for the Production of High-Cooked Cheese.

The aim of this study was to explore the use of a new coagulant from Thermomucor indicae-seudaticae N31 for the manufacture of a high-cooked starter-free cheese variety, by evaluating its physicochemical and functional characteristics in comparison to cheeses made with a traditional commercial coagulant. Coalho cheese was successfully produced with the new protease as it exhibited comparable characteristics to the one produced using the commercial enzyme: pH behavior during manufacture; cheese composition; protein and fat recovery; and cheese yield. In addition, during storage, melting was low and not affected by storage time; the increase of TCA 12% soluble nitrogen (% of total nitrogen) was lower than half of that of pH 4.6 soluble nitrogen (% of total nitrogen); concentration of ß-CN significantly decreased, whereas αs1 -CN concentration was not affected by storage time.

Syncephalastrum racemosum amine oxidase with high catalytic efficiency toward ethanolamine and its application in ethanolamine determination.

Our screening study yielded a copper amine oxidase (SrAOX) from Syncephalastrum racemosum, which showed much higher affinity and catalytic efficiency toward ethanolamine (EA) than any other amine oxidase (AOX). Following purification of the enzyme to electrophoretic homogeneity from a cell-free extract, the maximum activity toward EA was detected at pH 7.2-7.5 and 45 °C. The SrAOX complementary DNA (cDNA) was composed of a 2052-bp open reading frame encoding a 683-amino acid protein with a molecular mass of 77,162 Da. The enzyme functions as a homodimer. The deduced amino acid sequence of SrAOX showed 55.3 % identity to Rhizopus delemar AOX and contains two consensus sequences of Cu-AOX, NYDY, and HHQH, suggesting SrAOX is a type 1 Cu-AOX (i.e., a topaquinone enzyme). Structural homology modeling showed that residues (112)ML(113), (141)FADTWG(146) M158, and N318 are unique, and T144 possibly characterizes the substrate specificity of SrAOX. The recombinant enzyme (rSrAOX) was produced using Escherichia coli. Steady-state kinetic analysis of rSrAOX activity toward EA (pH 7.5 and 45 °C) gave K m and k cat values of 0.848 ± 0.009 mM and 9.11 ± 0.13 s(-1), respectively. The standard curves were linear between 0.1 and 2 mM EA, and 10 µg mL(-1)-2.5 mg mL(-1) (15 µM-3.6 mM) phosphatidylethanolamine using Streptomyces chromofuscus phospholipase D, respectively, was sufficiently sensitive for clinical use.

The role of oxidative stress on carotene production by Blakeslea trispora in submerged fermentation.

In aerobic metabolism, reactive oxygen species (ROS) are formed during the fermentation that can cause oxidative stress in microorganisms. Microbial cells possess both enzymatic and non-enzymatic defensive systems that may protect cells from oxidative damage. The antioxidant enzymes superoxide dismutase and catalase are the two key defensive enzymes to oxidative stress. The factors that induce oxidative stress in microorganisms include butylated hydroxytoluene (BHT), hydrogen peroxide, metal ions, dissolved oxygen tension, elevated temperature, menadione, junglone, paraquat, liquid paraffin, introduction to bioreactors of shake flask inocula and synthetic medium sterilized at initial pH 11.0. Carotenes are highly unsaturated isoprene derivatives. They are used as antioxidants and as coloring agents for food products. In fungi, carotenes are derived via the mevalonate biosynthesis pathway. The key genes in carotene biosynthesis are hmgR, ipi, isoA, carG, carRA and carB. Among microorganisms, Βlakeslea trispora is the main microorganism used for the production of carotenes on the industrial scale. Currently, the synthetic medium is considered the superior substrate for the production of carotenes in a pilot plant scale. The fermentation systems used for the production of carotenes include shake flasks, stirred tank fermentor, bubble column reactor and flat panel photobioreactor. This review summarizes the oxidative stresses in microorganisms and it is focused on the current status of carotene production by B. trispora including oxidative stress induced by BHT, enhanced dissolved oxygen levels, iron ions, liquid paraffin and synthetic medium sterilized at an initial pH 11.0. The oxidative stress induced by the above factors increases significantly the production of carotenes. However, to further reduce the cost of carotene production, new biotechnological methods with higher productivity still need to be explored.