Christopher D. Skory

Genomic Analysis of the Basal Lineage Fungus Rhizopus oryzae Reveals a Whole-Genome Duplication

Rhizopus oryzae is the primary cause of mucormycosis, an emerging, life-threatening infection characterized by rapid angioinvasive growth with an overall mortality rate that exceeds 50%. As a representative of the paraphyletic basal group of the fungal kingdom called “zygomycetes,” R. oryzae is also used as a model to study fungal evolution. Here we report the genome sequence of R. oryzae strain 99–880, isolated from a fatal case of mucormycosis. The highly repetitive 45.3 Mb genome assembly contains abundant transposable elements (TEs), comprising approximately 20% of the genome. We predicted 13,895 protein-coding genes not overlapping TEs, many of which are paralogous gene pairs. The order and genomic arrangement of the duplicated gene pairs and their common phylogenetic origin provide evidence for an ancestral whole-genome duplication (WGD) event. The WGD resulted in the duplication of nearly all subunits of the protein complexes associated with respiratory electron transport chains, the V-ATPase, and the ubiquitin–proteasome systems. The WGD, together with recent gene duplications, resulted in the expansion of multiple gene families related to cell growth and signal transduction, as well as secreted aspartic protease and subtilase protein families, which are known fungal virulence factors. The duplication of the ergosterol biosynthetic pathway, especially the major azole target, lanosterol 14α-demethylase (ERG11), could contribute to the variable responses of R. oryzae to different azole drugs, including voriconazole and posaconazole. Expanded families of cell-wall synthesis enzymes, essential for fungal cell integrity but absent in mammalian hosts, reveal potential targets for novel and R. oryzae-specific diagnostic and therapeutic treatments.

Tolerance to furfural-induced stress is associated with pentose phosphate pathway genes ZWF1 , GND1 , RPE1 , and TKL1 in Saccharomyces cerevisiae

Engineering yeast to be more tolerant to fermentation inhibitors, furfural and 5-hydroxymethylfurfural (HMF), will lead to more efficient lignocellulose to ethanol bioconversion. To identify target genes involved in furfural tolerance, a Saccharomyces cerevisiae gene disruption library was screened for mutants with growth deficiencies in the presence of furfural. It was hypothesized that overexpression of these genes would provide a growth benefit in the presence of furfural. Sixty two mutants were identified whose corresponding genes function in a wide spectrum of physiological pathways, suggesting that furfural tolerance is a complex process. We focused on four mutants, zwf1, gnd1, rpe1, and tkl1, which represent genes encoding pentose phosphate pathway (PPP) enzymes. At various concentrations of furfural and HMF, a clear association with higher sensitivity to these inhibitors was demonstrated in these mutants. PPP mutants were inefficient at reducing furfural to the less toxic furfuryl alcohol, which we propose is a result of an overall decreased abundance of reducing equivalents or to NADPHs role in stress tolerance. Overexpression of ZWF1 in S. cerevisiae allowed growth at furfural concentrations that are normally toxic. These results demonstrate a strong relationship between PPP genes and furfural tolerance and provide additional putative target genes involved in furfural tolerance.

The iron chelator deferasirox protects mice from mucormycosis through iron starvation

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.

Isolation and Expression of Lactate Dehydrogenase Genes from Rhizopus oryzae

ABSTRACT Rhizopus oryzae is used for industrial production of lactic acid, yet little is known about the genetics of this fungus. In this study I cloned two genes, ldhA and ldhB, which code for NAD+-dependent l-lactate dehydrogenases (LDH) (EC 1.1.1.27 ), from a lactic acid-producing strain of R. oryzae. These genes are similar to each other and exhibit more than 90% nucleotide sequence identity and they contain no introns. This is the first description of ldh genes in a fungus, and sequence comparisons revealed that these genes are distinct from previously isolated prokaryotic and eukaryotic ldh genes. Protein sequencing of the LDH isolated from R. oryzae during lactic acid production confirmed that ldhA codes for a 36-kDa protein that converts pyruvate to lactate. Production of LdhA was greatest when glucose was the carbon source, followed by xylose and trehalose; all of these sugars could be fermented to lactic acid. Transcripts fromldhB were not detected when R. oryzae was grown on any of these sugars but were present when R. oryzae was grown on glycerol, ethanol, and lactate. I hypothesize thatldhB encodes a second NAD+-dependent LDH that is capable of converting l-lactate to pyruvate and is produced by cultures grown on these nonfermentable substrates. BothldhA and ldhB restored fermentative growth toEscherichia coli (ldhA pfl) mutants so that they grew anaerobically and produced lactic acid.

Cloning of a gene associated with aflatoxin B1 biosynthesis in Aspergillus parasiticus

SummaryA cosmid library was constructed by inserting genomic DNA isolated from a wild-type aflatoxin-producing strain of Aspergillus parasiticus (SU-1) into a cosmid vector containing an homologous nitrate reductase (niaD) gene as a selectable marker. One cosmid was isolated which complemented an aflatoxin-deficient, nitrate-nonutilizing mutant strain, A. parasiticus B62 (nor-1, niaD), to aflatoxin production. Deletion and complementation analyses showed that, a 1.7 kb BglII-SphI DNA fragment isolated form this cosmid was responsible for renewed aflatoxin production. Northern hybridization analyses revealed that the major RNA transcribed from this DNA fragment, was 1.4 kilonucleotides in size. Genetic complementation, proved to be a useful strategy for cloning a gene associated with aflatoxin biosynthesis in A. parasiticus.

Lactic acid production by Saccharomyces cerevisiae expressing a Rhizopus oryzae lactate dehydrogenase gene

Abstract. This work demonstrates the first example of a fungal lactate dehydrogenase (LDH) expressed in yeast. A L(+)-LDH gene, ldhA, from the filamentous fungus Rhizopus oryzae was modified to be expressed under control of the Saccharomyces cerevisiae adh1 promoter and terminator and then placed in a 2µ-containing yeast-replicating plasmid. The resulting construct, pLdhA68X, was transformed and tested by fermentation analyses in haploid and diploid yeast containing similar genetic backgrounds. Both recombinant strains utilized 92xa0g glucose/l in approximately 30xa0h. The diploid isolate accumulated approximately 40% more lactic acid with a final concentration of 38xa0g lactic acid/l and a yield of 0.44xa0g lactic acid/g glucose. The optimal pH for lactic acid production by the diploid strain was pHxa05. LDH activity in this strain remained relatively constant at 1.5xa0units/mg protein throughout the fermentation. The majority of carbon was still diverted to the ethanol fermentation pathway, as indicated by ethanol yields between 0.25–0.33xa0g/g glucose. S. cerevisiae mutants impaired in ethanol production were transformed with pLdhA68X in an attempt to increase the lactic acid yield by minimizing the conversion of pyruvate to ethanol. Mutants with diminished pyruvate decarboxylase activity and mutants with disrupted alcohol dehydrogenase activity did result in transformants with diminished ethanol production. However, the efficiency of lactic acid production also decreased.

Fermentation of corn fibre sugars by an engineered xylose utilizing Saccharomyces yeast strain

The ability of a recombinant Saccharomyces yeast strain to ferment the sugars glucose, xylose, arabinose and galactose which are the predominant monosaccharides found in corn fibre hydrolysates has been examined. Saccharomyces strain 1400 (pLNH32) was genetically engineered to ferment xylose by expressing genes encoding a xylose reductase, a xylitol dehydrogenase and a xylulose kinase. The recombinant efficiently fermented xylose alone or in the presence of glucose. Xylose-grown cultures had very little difference in xylitol accumulation, with only 4 to 5g/l accumulating, in aerobic, micro-aerated and anaerobic conditions. Highest production of ethanol with all sugars was achieved under anaerobic conditions. From a mixture of glucose (80g/l) and xylose (40g/l), this strain produced 52g/l ethanol, equivalent to 85% of theoretical yield, in less than 24h. Using a mixture of glucose (31g/l), xylose (15.2g/l), arabinose (10.5g/l) and galactose (2g/l), all of the sugars except arabinose were consumed in 24h with an accumulation of 22g ethanol/l, a 90% yield (excluding the arabinose in the calculation since it is not fermented). Approximately 98% theoretical yield, or 21g ethanol/l, was achieved using an enzymatic hydrolysate of ammonia fibre exploded corn fibre containing an estimated 47.0g mixed sugars/l. In all mixed sugar fermentations, less than 25% arabinose was consumed and converted into arabitol.

The high affinity iron permease is a key virulence factor required for Rhizopus oryzae pathogenesis.

Rhizopus oryzae is the most common cause of mucormycosis, an angioinvasive fungal infection that causes more then 50% mortality rate despite first‐line therapy. Clinical and animal model data clearly demonstrate that the presence of elevated available serum iron predisposes the host to mucormycosis. The high affinity iron permease gene (FTR1) is required for R. oryzae iron transport in iron‐depleted environments. Here we demonstrate that FTR1 is required for full virulence of R. oryzae in mice. We show that FTR1 is expressed during infection in diabetic ketoacidosis (DKA) mice. In addition, we disrupted FTR1 by double cross‐over homologous recombination, but multinucleated R. oryzae could not be forced to segregate to a homokaryotic null allele. Nevertheless, a reduction of the relative copy number of FTR1 and inhibition of FTR1 expression by RNAi compromised the ability of R. oryzae to acquire iron in vitro and reduced its virulence in DKA mice. Importantly, passive immunization with anti‐Ftr1p immune sera protected DKA mice from infection with R. oryzae. Thus, FTR1 is a virulence factor for R. oryzae, and anti‐Ftr1p passive immunotherapy deserves further evaluation as a strategy to improve outcomes of deadly mucormycosis.

Production of L-lactic acid by Rhizopus oryzae under oxygen limiting conditions

Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.During aerobic growth, Rhizopus oryzae produces L-lactic acid from lactate dehydrogenase mediated reduction of pyruvate, while O2 limiting conditions yield primarily ethanol. A mutant was isolated that expressed only 5% of the wild type alcohol dehydrogenase activity under O2 limiting conditions and produced nearly 40 g lactic acid/l in 70 h. This is almost a ten-fold increase in lactic acid production when compared to the parent strain.

Novel Family of Carbohydrate Esterases, Based on Identification of the Hypocrea jecorina Acetyl Esterase Gene

ABSTRACT Plant cell walls have been shown to contain acetyl groups in hemicelluloses and pectin. The gene aes1, encoding the acetyl esterase (Aes1) of Hypocrea jecorina, was identified by amino-terminal sequencing, peptide mass spectrometry, and genomic sequence analyses. The coded polypeptide had 348 amino acid residues with the first 19 serving as a secretion signal peptide. The calculated molecular mass and isoelectric point of the secreted enzyme were 37,088 Da and pH 5.89, respectively. No significant homology was found between the predicated Aes1 and carbohydrate esterases of known families, but putative aes1 orthologs were found in genomes of many fungi and bacteria that produce cell wall-degrading enzymes. The aes1 transcript levels were high when the fungal cells were induced with sophorose, cellulose, oat spelt xylan, lactose, and arabinose. The recombinant Aes1 produced by H. jecorina transformed with aes1 under the cellobiohydrolase I promoter displayed properties similar to those reported for the native enzyme. The enzyme hydrolyzed acetate ester bond specifically. Using 4-nitrophenyl acetate as substrate, the activity of the recombinant enzyme was enhanced by d-xylose, d-glucose, cellobiose, d-galactose, and xylooligosaccharides but not by arabinose, mannose, or lactose. With the use of 4-nitrophenyl-β-d-xylopyranoside monoacetate as substrate in a β-xylosidase-coupled assay, Aes1 hydrolyzed positions 3 and 4 with the same efficiency while the H. jecorina acetylxylan esterase 1 exclusively deacetylated the position 2 acetyl group. Aes1 was capable of transacetylating methylxyloside in aqueous solution. The data presented demonstrate that Aes1 and other homologous microbial proteins may represent a new family of esterases for lignocellulose biodegradation.