Rapid screening of <Emphasis Type="Italic">Aspergillus terreus</Emphasis> mutants for overproduction of lovastatin

Summary A novel rapid screening method is demonstrated for isolating lovastatin-overproducing strains of Aspergillus terreus. The screening methodology, based on the activity of lovastatin against the yeast Candida albicans, is nearly three times as fast as the selection methods used earlier. The new 6-h assay shows a linear correlation between the quantity of lovastatin generated by A. terreus isolates and the inhibition zones obtained on plates of C. albicans. The new technique is less expensive and requires less labour.     

Enhanced continuous production of lovastatin using pellets and siran supported growth of Aspergillus terreus in an airlift reactor

Lovastatin, a hypocholesterolemic agent, is a secondary metabolite produced by filamentous microorganism Aspergillus terreus in submerged batch cultivation. Lovastatin production by pellets and immobilized siran cells was investigated in an airlift reactor. The process was carried out by submerged cultivation in continuous mode with the objective of increasing productivity using pellet and siran supported growth of A terreus. The continuous mode of fermentation improves the rate of lovastatin production. The effect of dilution rate and aeration rate were studied in continuous culture. The optimum dilution rate for pellet was 0.02 h⁻¹ and for siran carrier was 0.025 h⁻¹. Lovastatin productivity using immobilized siran carrier (0.0255 g/L/h) was found to be greater than pellets (0.022 g/L/h). The productivity by both modes of fermentation was found higher than that of batch process which suggests that continuous cultivation is a promising strategy for lovastatin production.     

Lovastatin inhibits its own synthesis in<Emphasis Type="Italic"> Aspergillus terreus</Emphasis>

Lovastatin suppresses its own synthesis in the microfungus Aspergillus terreus. The inhibitory effect was documented by spiking identical batch cultures with pure lovastatin (0, 50, 100 and 250 mg/l) 24 h after initiation from spores.     

Aspects of the biosynthesis of non-aromatic fungal polyketides by iterative polyketide synthases

Lovastatin biosynthesis in Aspergillus terreus involves two unusual type I multifunctional polyketide syntheses (PKSs). Lovastatin nonaketide synthase (LNKS), the product of the lovB gene, is an iterative PKS that interacts with LovC, a putative enoyl reductase, to catalyze the 35 separate reactions in the biosynthesis of dihydromonacolin L, a lovastatin precursor. LNKS also displays Diels-Alderase activity in vitro. Lovastatin diketide synthase (LDKS) made by lovF, in contrast, acts non-iteratively like the bacterial modular PKSs to make (2R)-2–methylbutyric acid. Then, like LNKS, LDKS interacts closely with another protein, the LovD transesterase enzyme that catalyzes attachment of the 2–methylbutyric acid to monacolin J in the final step of the lovastatin pathway. Key features of the genes for these four enzymes and others, plus the regulatory and self-resistance factors involved in lovastatin production, are also described.     

In vitro determination of phagocytosis and intracellular killing of Aspergillus species by mononuclear phagocytes

We investigated phagocytosis and intracellular killing of clinical and environmental isolates of Aspergillus spp. by human monocyte-derived macrophages (MDMs). Serial pathogens such as Aspergillus fumigatus, Aspergillus flavus and Aspergillus terreus were examined with a microbiological assay. Phagocytosis for resting conidia of Aspergillus spp. was similar for all isolates tested. During 30 min of incubation phagocytosis ranged from 49.9% to 85.5% for clinical isolates and from 40.3% to 87.1% for environmental isolates. MDMs killed A. fumigatus, A. flavus and A. terreus conidia after ingestion for 120 min, as shown by a decrease in colony forming units (cfu) count of intracellular fungi. The killing index for all isolates of Aspergillus spp., ranged from 12.1 ± 1.1% to 90.3 ± 10.4%; isolate-dependent (P < 0.01) differences against the fungicidal action of MDMs were observed. In conclusion, significant differences were noted for killing indices between several strains of Aspergillus spp. whereas phagocytosis was similar for all isolates tested in vitro. No differences were observed within environmental and clinical isolates.     

A correlative evaluation of morphology and rheology of<Emphasis Type="Italic">Aspergillus terreus</Emphasis> during lovastatin fermentation

Lovastatin, a secondary metabolite, was produced by fermentation process usingAspergillus terreus in an internal loop airlift reactor. It is a highly aerobic fermentation process. Biomass concentration and cell morphology were evaluated and observed to contribute significantly to the high viscosity and pseudoplastic non-Newtonian behavior of the broth. Typical morphological changes over 10 days in the fermentation broth were studied. The viscosity increased from the start of the fermentation with an increasing cell mass content, reached to a maximum of 60 N/m²·s at 160 h and then declined after the branching of the hyphae with the formation of arthrospores. Rheological parameters like consistency index and fluidity index were evaluated. The consistency index was observed to increase from 9.8 to 66.85 N/m², while fluidity index decreased from 0.69 to 0.48 s⁻¹ during 10 days of lovastatin production. A correlation between growth and consistency index of the broth has been evaluated.     

Aspergillus alabamensis,a New Clinically Relevant Species in the Section Terrei

Phylogenetic analyses of sequences generated from portions of three genes coding for the proteins enolase (enoA), β-tubulin (benA), and calmodulin (calM) of a large number of isolates within the section Terrei, genus Aspergillus, revealed the presence of a new cryptic species within this section, Aspergillus alabamensis. Most members of this new cryptic species were recovered as colonizing isolates from immunocompetent patient populations, had decreased in vitro susceptibilities to the antifungal drug amphotericin B, and were morphologically similar to but genetically distinct from Aspergillus terreus isolates.Invasive infections caused by Aspergillus terreus are often disseminated with increased lethality compared with infections caused by other Aspergillus species and tend to be resistant to treatment with the antifungal drug amphotericin B (6, 14, 17). Despite the clinical significance of this organism, little is known about the epidemiology, genetic diversity, and population structure of A. terreus.Historically, A. terreus has been identified in the laboratory by conventional methods such as colony morphology and microscopic characteristics. Such morphological studies have placed A. terreus as a single homogenous species within the section Terrei along with two other varieties, A. terreus var. africanus and A. terreus var. aureus (11). Recent studies have shown that morphological characteristics may not be reliable for distinguishing Aspergillus species, as inferred from the demonstration of multiple cryptic species within the section Fumigati by molecular phylogenetic methods (3-5, 13, 18).In the past, molecular methods largely based on randomly amplified polymorphic DNA-PCR-based assays have shown that A. terreus isolates can have great strain diversity (1, 8, 16). One recent genotyping study of several A. terreus clinical isolates recovered from two different medical centers using this method concluded that nosocomial acquisition of A. terreus infections was highly unlikely given the great genetic diversity observed (7). Another study demonstrated that comparative sequence analyses of the D1 and D2 regions had limited utility to study relationships within the section Terrei, while the internal transcribed spacer regions were useful since there was more nucleotide diversity in this region (16). However, the authors of this study could not resolve species within the section Terrei using these molecular approaches.In the present study, we have developed a multilocus sequence approach employing three protein-coding regions to study species diversity of the section Terrei using a large panel of isolates from both clinical and environmental origins recovered from various parts of the world. The studies outlined below demonstrate the presence of a new, clinically relevant species, Aspergillus alabamensis, and clarify the taxonomic position of the A. terreus variant A. terreus var. aureus.     

Pulmonary aspergillosis due toAspergillus terreus combined with staphylococcal pneumonia and hepatic candidiasis

A female patient with systemic lupus erythematosus (SLE) developed pulmonary aspergillosis with staphylococcal pneumonia and hepatic candidiasis.Aspergillus terreus, which is a rare causative organism of pulmonary aspergilosis, was identified from a pulmonary lesion by culture. The aleurioconidium production, a characeristic of the genusAspergillus sect.terrei, was demonstrated on short and irregular hyphal features in tissue sections. This report is the first of a combined case of pulmonary aspergillosis due toA. terreus with infections caused by other microorganisms.     

洛伐他汀工业菌株土曲霉HZ01的次级代谢产物合成分析

【目的】分析洛伐他汀工业生产菌株土曲霉HZ01的次级代谢产物合成能力,为后期的遗传改造、次级代谢产物及其基因簇挖掘提供指导。【方法】对洛伐他汀发酵条件下的样品进行了转录组分析,同时运用色谱分离技术及波谱学方法对主要次级代谢产物进行了分离和结构鉴定。【结果】洛伐他汀合成相关基因转录水平非常高,还有4个聚酮合酶(PKS)、6个非核糖体多肽合成酶(NRPS)和1个PKS-NRPS杂合酶基因进行了转录,其他PKS和NRPS基因都处于沉默状态。此外,从该菌的发酵产物中分离鉴定了10个主要副产物并确定了其结构。【结论】土曲霉HZ01是一株优良的洛伐他汀生产菌株,在构建次级代谢产物异源合成细胞工厂和鉴定次级代谢产物生物合成途径方面具有很好的应用潜力。     

Endophytic fungi from rhizomes of <Emphasis Type="Italic">Paris polyphylla</Emphasis> var. <Emphasis Type="Italic">yunnanensis</Emphasis>

About 63 fungal endophytic isolates were separated from rhizomes of Paris polyphylla var. yunnanensis, which is a traditional medicinal plant mainly distributed in China. The isolates were characterized and grouped based on the culture characteristics and the morphology of colony growth and conidia. Eleven representative ones were selected for further taxonomical identification. Five genera namely Fusarium, Gliocladiopsis, Gliomastix, Aspergillus and Cylindrocarpon were identified on the basis of their morphological characterizations. Of them, the most frequent genus was Fusarium (i.e. Ppf1, Ppf3 and Ppf14). Their ITS-rDNA sequences were compared with those available in the GeneBank databases to obtain the closest related species by BLAST analysis as well as to analyze their phylogenetic affiliation. The isolates were identified as Gliocladiopsis irregularis (Ppf2), Plectosphaerella cucumerina (Ppf4), Padospora sp. (Ppf6), Gliomastix murorum var. murorum (Ppf7), Aspergillus fumigatus (Ppf9), Pichia guilliermondii (Ppf10), Neonectria radicicola (anamorph: Cylindrocarpon) (Ppf12) and one uncultured mycorrhizal ascomycete (Ppf13) separately based on their morphological and molecular features. The molecular characters of the endophytic fungi were basically coincident with their morphology. The broad diversity and taxonomic spectrum were exhibited by the endophytic fungi from P. polyphylla var. yunnanensis.     

Biotechnological production and applications of statins

Statins are a group of extremely successful drugs that lower cholesterol levels in blood; decreasing the risk of heath attack or stroke. In recent years, statins have also been reported to have other biological activities and numerous potential therapeutic uses. Natural statins are lovastatin and compactin, while pravastatin is derived from the latter by biotransformation. Simvastatin, the second leading statin in the market, is a lovastatin semisynthetic derivative. Lovastatin is mainly produced by Aspergillus terreus strains, and compactin by Penicillium citrinum. Lovastatin and compactin are produced industrially by liquid submerged fermentation, but can also be produced by the emerging technology of solid-state fermentation, that displays some advantages. Advances in the biochemistry and genetics of lovastatin have allowed the development of new methods for the production of simvastatin. This lovastatin derivative can be efficiently synthesized from monacolin J (lovastatin without the side chain) by a process that uses the Aspergillus terreus enzyme acyltransferase LovD. In a different approach, A. terreus was engineered, using combinational biosynthesis on gene lovF, so that the resulting hybrid polyketide synthase is able to in vivo synthesize 2,2-dimethylbutyrate (the side chain of simvastatin). The resulting transformant strains can produce simvastatin (instead of lovastatin) by direct fermentation.     

Two novel <Emphasis Type="Italic">Aspergillus</Emphasis> species from hypersaline soils of The National Park of Lake Urmia,Iran

Two novel Aspergillus species, one belonging to the section Terrei and the other to section Flavipedes, were isolated from hypersaline soils of The National Park of Lake Urmia (Iran) and are here described as Aspergillus iranicus and Aspergillus urmiensis. A polyphasic taxonomic approach comprising extrolite profiles, phenotypic characters and molecular data (beta-tubulin, calmodulin and ribosomal polymerase II second largest subunit gene sequences) was applied to determine their novel taxonomic status. Aspergillus iranicus (CBS 139561^T) is phylogenetically related to A. carneus, A. niveus, A. allahabadii and A. neoindicus, and it can be differentiated from those species by a unique extrolite pattern (citrinin, gregatins, and a terrequinone) and its conidial colour. Aspergillus urmiensis (CBS 139558^T) shares a most recent common ancestor with A. templicola. The former species produces globose vesicles, and those of A. templicola are predominantly elongate. The Aspergillus urmiensis isolates produce several uncharacterized extrolites. Two other strains obtained during this study reside in a clade, together with the type strain of A. movilensis (CCF 4410^T), and are identified accordingly. Based on the phylogenetic data presented in this study, A. frequens is reduced to synonymy with A. micronesiensis and A. mangaliensis is considered to be a synonym of A. templicola.     

Aspergillus taichungensis,a new species from Taiwan

Aspergillus taichungensis isolated from a soil sample collected in Taiwan is described as a new species. The new species is characterized by its restricted growth on Czapek's and malt extract agars and its white to light yellow colonies, radiate conidial heads, smooth and often diminutive conidiophores, hemispherical to elongate vesicles with biseriate aspergilla (conidiogenous cells), globose, micro-verrucose conidia and dark brown sclerotia. The species somewhat resemblesA. versicolor, A. terreus andA. flavipes, but differs in cultural and morphological details, and is considered to represent an interface species in the subgenusNidulantes.     

Use of the 6-methylsalicylic-acid-synthase gene as a discriminating marker betweenaspergillus terreus andAspergillus flavipes

In nineteen pathogenic and saprophytic isolates denoted asAspergillus terreus the presence and restriction pattern of the genepksM (6-methylsalicylic acid synthase) was determined. Five patterns (A−E) were found and in three isolates the gene was missing. The RAPD (random amplified polymorphic DNA) patterns with three primers were analyzed. The strains withpksM pattern possibly derived from the most common pattern A by single mutation (patterns B−D) were also related by RAPD. Among them, three clades were found. The first one contained saprophytes from Asia, the other two clades contained both European and American pathogens and each of them one saprophyte. The sequences of rDNA region containing 5.8S rDNA and spacers ITS1 and ITS2 were established for representatives of each group and the strains missing thepksM gene. The isolates possessingpksM (although with different restriction patterns) grouped asA. terreus group, whereas the isolates lacking this gene were close toFennellia flavipes.     

Penicillium andAspergillus species in the habitats of allergy patients in the Tulsa, Oklahoma area

Penicillium andAspergillus have been recognized as important aeroallergens for more than 30 years, and are especially significant in indoor environments. There are over 400 species ofPenicillium andAspergillus combined, but there is little information on which species occur most frequently in the environment, or if each exhibits unique allergenic properties. A preliminary study showed no overlap between those species isolated from an outdoor site in Tulsa, Oklahoma and the species used in immunotherapy at allergy clinics in the Tulsa area. Pursuing this line of research, air samples were collected as three seasonal samples (over a 6 month period) in the homes or offices of ten allergy patients known to be allergic toPenicillium and/orAspergillus. Twenty three species ofPenicillium and 12 species ofAspergillus were identified from these samples through isolation, macroscopic, and microscopic examination.Penicillium corylophilum, P. glabrum, Aspergillus niger, andA. flavipes were the most abundant species isolated, supporting the data obtained in a preliminary study. At least in the Tulsa area, it appears that atopic patients are being tested and treated with extracts ofPenicillium andAspergillus species that are either not present or not abundant in the local indoor or outdoor environments. Additional research is necessary to determine if the environmental isolates share allergens with those species used in immunotherapy.     

Lovastatin Biosynthesis by Aspergillus terreus in a Chemically Defined Medium

Lovastatin is a secondary metabolite produced by Aspergillus terreus. A chemically defined medium was developed in order to investigate the influence of carbon and nitrogen sources on lovastatin biosynthesis. Among several organic and inorganic defined nitrogen sources metabolized by A. terreus, glutamate and histidine gave the highest lovastatin biosynthesis level. For cultures on glucose and glutamate, lovastatin synthesis initiated when glucose consumption levelled off. When A. terreus was grown on lactose, lovastatin production initiated in the presence of residual lactose. Experimental results showed that carbon source starvation is required in addition to relief of glucose repression, while glutamate did not repress biosynthesis. A threefold-higher specific productivity was found with the defined medium on glucose and glutamate, compared to growth on complex medium with glucose, peptonized milk, and yeast extract.     

Population variation and phylogeny in the endangered <Emphasis Type="BoldItalic">Chamaesyce skottsbergii</Emphasis> (Euphorbiaceae) based on RAPD and ITS analyses

Chamaesyce skottsbergii var. skottsbergii is federally listed as an endangered taxon, and is found in small and isolated populations restricted to calcareous soils in dry shrubland habitats on the Hawaiian islands of Oahu and Molokai. Concern over the genetic relationship among these disjunct populations arose as a result of threats to the habitat of the Oahu population. The populations were examined using random amplified polymorphic DNA (RAPD) markers and sequence analysis of the internal transcribed spacer (ITS) region of the rDNA cistron. Chamaesyce skottsbergii var. vaccinioides, a closely related variety found in several small populations on Molokai, was used for baseline comparison of the genetic divergence among populations. RAPD analysis demonstrated that variation within and among populations is the highest for any Hawaiian species examined. Polymorphism was greater than 95% within populations and was 99.4% at the species level. Similarly, measures of genetic similarity indicate that differentiation among these populations is higher than is known for some species. Both RAPD and ITS sequence analysis indicate that populations of C. skottsbergii var. skottsbergii on Oahu and Molokai are genetically distinct, and the extent of this genetic differentiation supports the recognition of these populations as distinct varieties. The Molokai population is in fact much more closely related to var. vaccinioides than to var. skottsbergii on Oahu, and thus should be recognized by the previously used variety name, C. skottsbergii var. audens. Further conservation measures for each of the varieties are addressed.     

An insight into the distribution,genetic diversity,and mycotoxin production of <Emphasis Type="Italic">Aspergillus</Emphasis> section <Emphasis Type="Italic">Flavi</Emphasis> in soils of pistachio orchards

In the present study, 193 Aspergillus strains were isolated from a total of 100 soil samples of pistachio orchards, which all of them were identified as Aspergillus flavus as the most abundant species of Aspergillus section Flavi existing in the environment. Approximately 59%, 81%, and 61% of the isolates were capable of producing aflatoxins (AFs), cyclopiazonic acid (CPA), and sclerotia, respectively. The isolates were classified into four chemotypes (I to IV) based on the ability to produce AFs and CPA. The resulting dendrogram of random amplified polymorphic DNA (RAPD) analysis of 24 selected A. flavus isolates demonstrated the formation of two separate clusters. Cluster 1 contained both aflatoxigenic and non-aflatoxigenic isolates (17 isolates), whereas cluster 2 comprised only aflatoxigenic isolates (7 isolates). All the isolates of cluster 2 produced significantly higher levels of AFs than those of cluster 1 and the isolates that produced both AFB₁ and AFB₂ were found only in cluster 2. RAPD genotyping allowed the differentiation of A. flavus from Aspergillus parasiticus as a closely related species within section Flavi. The present study has provided for the first time the relevant information on distribution and genetic diversity of different A. flavus populations from nontoxigenic to highly toxigenic enable to produce hazardous amounts of AFB₁ and CPA in soils of pistachio orchards. These fungi, either toxigenic or not-toxigenic, should be considered as potential threats for agriculture and public health.     

<Emphasis Type="SmallCaps">l</Emphasis>-Methioninase Production by Filamentous Fungi: I-Screening and Optimization Under Submerged Conditions

Findings show 21 fungal isolates belonging to eight genera recovered from Egyptian soils that have the potential to attack l-methionine under submerged conditions. Aspergillus flavipes had the most methioninolytic activity, giving the highest yield of l-methioninase (10.78 U/mg protein), rate of methionine uptake (93.0%), and growth rate (5.0 g/l), followed by Scopulariopsis brevicaulis and A. carneus. The maximum l-methioninase productivity (11.60 U/mg protein) by A. flavipes was observed using l-methionine (0.8%) as an enzyme-inductive agent and glucose (1%) as a co-dissimilated carbon source. A significant reduction in l-methioninase biosynthesis by A. flavipes was detected using carbon-free medium, suggesting the lack of ability to use l-methionine as a carbon and nitrogen source. Potassium dihydrogen phosphate (0.25%), the best source of phosphorus, favors enzyme biosynthesis and enhances the level of methionine uptake by A. flavipes. The maximum l-methioninase productivity (12.58 U/mg protein) and substrate uptake (95.6%) were measured at an initial pH of 7.0.     

Aerobiological,biochemical and immunological studies on some of the dominant <Emphasis Type="Italic">Aspergillus</Emphasis> species of South Assam (India)

Two years atmospheric survey of air-borne Aspergillus was carried out in the environmental conditions of South Assam. The survey revealed a total of 16 different species of Aspergillus with marked seasonal and annual variations. Aspergillus fumigatus was found to be the dominant atmospheric fungal species followed by Aspergillus flavus, Aspergillus niger, etc. Among the sample extracts tested, highest quantity of soluble protein was recorded in Aspergillus fumigatus (95.0 mg/g) whereas highest quantity of soluble carbohydrate (40.8 mg/g) and free amino acid (135.0 mg/g) was recorded in the sample extract of Aspergillus niger per gram of dry weight, respectively. The highest numbers of protein polypeptide bands were detected in the sample extract of Aspergillus fumigatus followed by Aspergillus flavus and lowest in Aspergillus niger. The maximum numbers of immunoglobulin E binding protein fractions were found in Aspergillus fumigatus, followed by Aspergillus flavus, Aspergillus clavatus, etc.