Yeast extract stimulates production of glycolipid biosurfactants, mannosylerythritol lipids, by Pseudozyma hubeiensis SY62

We improved the culture conditions for a biosurfactant producing yeast, Pseudozyma hubeiensis SY62. We found that yeast extract greatly stimulates MEL production. Furthermore, we demonstrated a highly efficient production of MELs in the improved medium by fed-batch cultivation. The final concentration of MELs reached 129 ± 8.2g/l for one week.     

Production of glycolipid biosurfactants, mannosylerythritol lipids, by Pseudozyma siamensis CBS 9960 and their interfacial properties

The search for a novel producer of glycolipid biosurfactants, mannosylerythritol lipids (MELs), was undertaken on the basis of the analysis of ribosomal DNA sequences of yeast strains of the genus Pseudozyma. In the course of the investigation, Pseudozyma siamensis CBS 9960, which is closely related to Pseudozyma shanxiensis, a known MEL-C producer but with a different morphology, was found to accumulate a large amount of glycolipids. On thin layer chromatography, the extracellular glycolipids showed nearly the same spots as those of the MELs produced by P. shanxiensis. However, the result of high-performance liquid chromatography analysis revealed that the present strain has a much higher glycolipid production yield than P. shanxiensis. From the structural characterization by (1)H and (13)C NMR, the major glycolipid (more than 84% of the total) was identified as a mixture of 4-O-[(2',4'-di-O-acetyl-3'-O-alka(e)noyl)-beta-D-mannopyranosyl]-D-erythritol and 4-O-[(4'-O-acetyl-3'-O-alka(e)noyl-2'-O-butanoyl)-beta-D-mannopyranosyl]-D-erythritol, both of which are types of MEL-C. The present MEL-C possessed a short-chain acid (C(2) or C(4)) at the C-2' position and a long-chain acid (C(16)) at the C-3' position of the mannose moiety, and thus, the hydrophobic part was considerably different from that of conventional MELs, which mainly possess two medium-chain acids (C(10)) at the C-2' and C-3' positions. Under optimal growth conditions with safflower oil in a shake culture, the total amount of MELs reached approximately 19 g/l after 9 d at 25 degrees C. We further investigated the interfacial properties of the present MEL-C, considering its unique hydrophobic structure. The observed critical micelle concentration (CMC) and the surface tension at the CMC of the MEL were 4.5 x 10(-6) M and 30.7 mN/m, respectively. In addition, on a water penetration scan, the MEL efficiently formed the liquid crystal phases such as hexagonal (H) and lamella (L(a)) at a wide range of concentrations. These results demonstrated that the newly identified MEL-C produced by P. siamensis exhibits not only high surface activity but also excellent self-assembling properties, and should facilitate the development of promising yeast biosurfactants.     

Microbial conversion of glycerol into glycolipid biosurfactants, mannosylerythritol lipids, by a basidiomycete yeast, Pseudozyma antarctica JCM 10317(T)

Microbial conversion of glycerol into functional bio-based materials was investigated, aiming to facilitate the utilization of waste glycerol. A basidiomycete yeast, Pseudozyma antarctica JCM 10317, efficiently produced mannosylerythritol lipids (MELs) as glycolipid biosurfactants from glycerol. The amount of MEL yield reached 16.3 g l(-1) by intermittent feeding of glycerol.     

Identification of the gene PaEMT1 for biosynthesis of mannosylerythritol lipids in the basidiomycetous yeast Pseudozyma antarctica

The yeast Pseudozyma antarctica produces a large amount of glycolipid biosurfactants known as mannosylerythritol lipids (MELs), which show not only excellent surface‐active properties but also versatile biochemical actions. To investigate the biosynthesis of MELs in the yeast, we recently reported expressed sequence tag (EST) analysis and estimated genes expressing under MEL production conditions. Among the genes, a contiguous sequence of 938 bp, PA_004, showed high sequence identity to the gene emt1, encoding an erythritol/mannose transferase of Ustilago maydis, which is essential for MEL biosynthesis. The predicted translation product of the extended PA_004 containing the two introns and a stop codon was aligned with Emt1 of U. maydis. The predicted amino acid sequence shared high identity (72%) with Emt1 of U. maydis, although the amino‐terminal was incomplete. To identify the gene as PaEMT1 encoding an erythritol/mannose transferase of P. antarctica, the gene‐disrupted strain was developed by the method for targeted gene disruption, using hygromycin B resistance as the selection marker. The obtained ΔPaEMT1 strain failed to produce MELs, while its growth was the same as that of the parental strain. The additional mannosylerythritol into culture allowed ΔPaEMT1 strain to form MELs regardless of the carbon source supplied, indicating a defect of the erythritol/mannose transferase activity. Furthermore, we found that MEL formation is associated with the morphology and low‐temperature tolerance of the yeast. Copyright © 2010 John Wiley & Sons, Ltd.     

Da Vinci's yeast: Blastobotrys davincii f.a., sp. nov

A new species of the yeast genus Blastobotrys was discovered during a worldwide survey of culturable xerophilic fungi in house dust. Several culture-dependent and independent studies from around the world detected the same species from a wide range of substrates including indoor air, cave wall paintings, bats, mummies, and the iconic self-portrait of Leonardo da Vinci from ca 1512. However, none of these studies identified their strains, clones, or OTUs as Blastobotrys. We introduce the new species as Blastobotrys davincii f.a., sp. nov. (holotype CBS H-24879) and delineate it from other species using morphological, phylogenetic, and physiological characters. The new species of asexually (anamorphic) budding yeast is classified in Trichomonascaceae and forms a clade along with its associated sexual state genus Trichomonascus. Despite the decade-old requirement to use a single generic name for fungi, both names are still used. Selection of the preferred name awaits a formal nomenclatural proposal. We present arguments for adopting Blastobotrys over Trichomonascus and introduce four new combinations as Blastobotrys allociferrii (≡ Candida allociferrii), B. fungorum (≡ Sporothrix fungorum), B. mucifer (≡ Candida mucifera), and Blastobotrys vanleenenianus (≡ Trichomonascus vanleenenianus). We provide a nomenclatural review and an accepted species list for the 37 accepted species in the Blastobotrys/Trichomonascus clade. Finally, we discuss the identity of the DNA clones detected on the da Vinci portrait, and the importance of using appropriate media to isolate xerophilic or halophilic fungi.     

The role of PaAAC1 encoding a mitochondrial ADP/ATP carrier in the biosynthesis of extracellular glycolipids,mannosylerythritol lipids,in the basidiomycetous yeast Pseudozyma antarctica

Pseudozyma antarctica produces large amounts of the glycolipid biosurfactants known as mannosylerythritol lipids (MEL), which show not only excellent surface‐active properties but also versatile biochemical actions. A gene homologous with a mitochondrial ADP/ATP carrier was dominantly expressed in P. antarctica under MEL‐producing conditions on the basis of previous gene expression analysis. The gene encoding the mitochondrial ADP/ATP carrier of P. antarctica (PaAAC1) contained a putative open reading frame of 954 bp and encodes a polypeptide of 317 amino acids. The deduced translation product shared high identity of 66%, 70%, 69%, 74%, 75% and 52% with the mitochondrial ADP/ATP carrier of Saccharomyces cerevisiae (AAC1), S. cerevisiae (AAC2), S. cerevisiae (AAC3), Kluyveromyces lactis (KlAAC), Neurospora crassa (NcAAC) and human (ANT1), respectively, and conserved the consensus sequences of all ADP/ATP carrier proteins. The gene expression by introducing a plasmid pUXV1‐PaAAC1 into the yeast cells increased the MEL production. In addition, the expression of PaAAC1 in which the conserved arginine and leucine required for ATP transport activity were replaced with isoleucine and serine, respectively, failed to increase MEL production. Accordingly, these results suggest that PaAAC1 encoding a mitochondrial ADP/ATP carrier should be involved in MEL biosynthesis in the yeast. Copyright © 2010 John Wiley & Sons, Ltd.     

The yeast community of Conotelus sp. (Coleoptera: Nitidulidae) in Brazilian passionfruit flowers (Passiflora edulis) and description of Metschnikowia amazonensis sp. nov., a large-spored clade yeast

Species of the nitidulid beetle Conotelus found in flowers of Convolvulaceae and other plants across the New World and in Hawaii consistently harbour a yeast community dominated by one or more large-spored Metschnikowia species. We investigated the yeasts found in beetles and flowers of cultivated passionfruit in Rondônia state, in the Amazon biome of Brazil, where a Conotelus species damages the flowers and hinders fruit production. A sample of 46 beetles and 49 flowers yielded 86 and 83 yeast isolates, respectively. Whereas the flower community was dominated by Kodamaea ohmeri and Kurtzmaniella quercitrusa, the major yeasts recovered from beetles were Wickerhamiella occidentalis, which is commonly isolated from this community, and a novel species of large-spored Metschnikowia in the arizonensis subclade, which we describe here as Metschnikowia amazonensis sp. nov. Phylogenetic analyses based on barcode sequences (ITS-D1/D2) and a multigene alignment of 11,917 positions (genes ura2, msh6, and pmt2) agreed to place the new species as a sister to Metschnikowia arizonensis, a rare species known only from one locality in Arizona. The two form sterile asci when mated, which is typical of related members of the clade. The α pheromone of the new species is unique but typical of the subclade. The type of M. amazonensis sp. nov. is UFMG-CM-Y6309^T (ex-type CBS 16156^T, mating type a ), and the designated allotype (mating type α) is UFMG-CM-Y6307^A (CBS 16155^A). MycoBank MB 833560.     

Candida theae sp. nov., a new anamorphic beverage-associated member of the Lodderomyces clade

Four strains representing a novel yeast species belonging to the genus Candida were independently isolated in Taiwan and Ecuador. Two strains (G17(T) and G31) were isolated in Taiwan, by pellet precipitation from plastic-bottled tea drinks produced in Indonesia, while two additional strains (CLQCA 10-049 and CLQCA 10-062) were recovered from ancient chicha fermentation vessels found in tombs in Quito, Ecuador. These four strains were morphologically, and phylogenetically identical to each other. No sexual reproduction was observed on common sporulation media. Large-subunit (LSU) rRNA gene sequence analysis revealed the four strains to belong to the Lodderomyces clade, closely related to members of the Candida parapsilosis species complex. The four strains, which have identical LSU D1/D2 sequences, differ from their closest phylogenetic neighbors, Candida orthopsilosis and Candida parapsilosis, by 6-9 nt substitutions, respectively. Physiologically, the four strains are similar to Candida parapsilosis, although they can be distinguished from their closest relative by the assimilation of arbutin, nitrite, and creatine. The Indonesian and Ecuadorian strain sets can also be distinguished from one another based on ITS sequencing, differing by 4 substitutions in ITS1 and 1 single nucleotide indel in ITS2. Collectively, the results indicate that the four strains represent a previously unrecognized species of Candida. The name Candida theae sp. nov. is proposed to accommodate these strains, with G-17(T) (BCRC 23242(T)=CBS 12239(T)=ATCC MYA-4746(T)) designated as the type strain.     

Paenibacillus bovis sp.nov.BD3526作为益生菌的潜力

对Paenibacillus bovis sp.nov.BD3526作为益生菌的潜力进行了体外活性初步研究。采用模拟胃液、肠液、不同浓度的胆盐环境,测定BD3526菌株在酸性及高胆盐环境的存活率,并测试BD3526对金黄色葡萄球菌、藤黄、枯草芽孢杆菌的抑制作用。结果表明,BD3526可以耐受pH值为2.0、3.0、4.0的人工胃液、pH8.0的模拟肠液以及0.1%、0.2%、0.3%的胆盐环境;对金黄色葡萄球菌、藤黄、枯草芽孢杆菌有显著的抑制能力。说明BD3526在人工消化液环境中具有良好的存活能力,不具有溶血作用;可引起敏感菌细胞渗漏,达到杀灭部分细菌,包括金色葡萄球菌的作用,因此,该菌具备作为益生菌应用的潜力。     

Isolation of a novel mutant strain of Saccharomyces cerevisiae by an ethyl methane sulfonate-induced mutagenesis approach as a high producer of bioethanol

In order to obtain mutant strains showing higher bioethanol production than wild-type strains, a commercial Saccharomyces cerevisiae type was subjected to mutagenesis using ethyl methane sulfonate (EMS). After adding EMS to a shaken yeast suspension, the viability of yeast cells was assessed by diluted sample inoculation to solid yeast-extract peptone glucose (YEPG) medium at 15-min intervals. At 45 min, the viability of yeast cells was estimated to be about 40%. Mutagenized cells were recovered from YEPG broth after incubation at 30 degrees C for 18 h. After this period, EMS-treated yeast cells were grown on solid aerobic low-peptone (ALP) medium containing 2-12% (v/v) ethanol. All plates were incubated at 30 degrees C for 2-6 d in order to form colonies. The mutant strains that tolerated high concentrations of ethanol were selected for bioethanol production in microfuge tubes containing fermentation medium. Formation of bioethanol in small tubes was detected by the distillation-colorimetric method. In addition, trehalose content and invertase activity were determined in each mutant strain. Among many isolated mutant strains, there were six isolated colonies that grew on ALP medium supplemented with 10% (v/v) ethanol and one of them produced bioethanol 17.3% more than the wild type.     

Purification and characterization of a novel aspartic protease from basidiomycetous yeast Cryptococcus sp. S-2

An aspartic protease (Cap1) was purified from basidiomycetous yeast Cryptococcus sp. S-2 (FERM ABP-10961) using HiTrap DEAE FF column and HiTrap Q HP column chromatography with azocasein as a substrate. Cap1 has a molecular mass of 34 kDa on SDS-PAGE. It was stable up to 50°C with maximum activity at 30°C. Maximum proteolytic activity was observed at pH 5.0. Cap1 was stable in the pH range 3.0-7.0. Its enzyme activity was strongly inhibited by pepstatin A, an inhibitor of aspartic proteases, indicating that Cap1 is an aspartic protease. Cap1 hydrolyzed protein substrates, including BSA, hemoglobin, α-casein, β-casein, and κ-casein. It showed activity on synthetic substrates, such as MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(Dnp)-D-Arg-NH? and MOCAc-Ala-Pro-Ala-Lys-Phe-Phe-Arg-Leu-Lys(Dnp)-NH?. Hydrolysis of the oxidized insulin B chain followed by amino acid sequencing analysis of the cleavage products revealed that 9 of its 30 peptide bonds were hydrolyzed by Cap1. This result was similar to that observed with pig pepsin A and human pepsin A. Cap1 also exhibited milk-clotting activity. We cloned the cDNA of CAP1 gene, which contained a 1254 bp open reading frame encoding a protein of 417 amino acid residues. Homology search in the NCBI database revealed that the amino acid sequence of Cap1 showed less than 39% identity to other known proteins. Therefore, we proposed that Cap1 is a novel aspartic protease.     

Fusarium sibiricum sp. nov, a novel type A trichothecene-producing Fusarium from northern Asia closely related to F. sporotrichioides and F. langsethiae

Production of type A trichothecenes has been reported in the closely related species Fusarium langsethiae and F. sporotrichioides. Here, we characterized a collection of Fusarium isolates from Siberia and the Russian Far East (hereafter Asian isolates) that produce high levels of the type A trichothecene T-2 toxin and are similar in morphology to the type A trichothecene-producing F. langsethiae, and to F. poae which often produces the type B trichothecene nivalenol. The Asian isolates possess unique macroscopic and microscopic characters and have a unique TG repeat in the nuclear ribosomal intergenic spacer (IGS rDNA) region. In Asian isolates, the TRI1-TRI16 locus, which determines type A versus type B trichothecene production in other species, is more similar in organization and sequence to the TRI1-TRI16 locus in F. sporotrichioides and F. langsethiae than to that in F. poae. Phylogenetic analysis of the TRI1 and TRI16 gene coding regions indicates that the genes in the Asian isolates are more closely related to those of F. sporotrichioides than F. langsethiae. Phylogenetic analysis of the beta-tubulin, translation elongation factor, RNA polymerase II and phosphate permease gene sequences resolved the Asian isolates into a well-supported sister lineage to F. sporotrichioides, with F. langsethiae forming a sister lineage to F. sporotrichioides and the Asian isolates. The Asian isolates are conspecific with Norwegian isolate IBT 9959 based on morphological and molecular analyses. In addition, the European F. langsethiae isolates from Finland and Russia were resolved into two distinct subgroups based on analyses of translation elongation factor and IGS rDNA sequences. Nucleotide polymorphisms within the IGS rDNA were used to design PCR primers that successfully differentiated the Asian isolates from F. sporotrichioides and F. langsethiae. Based on these data, we formally propose that the Asian isolates together with Norwegian isolate IBT 9959 comprise a novel phylogenetic species, F. sibiricum, while the two subgroups of F. langsethiae only represent intraspecific groups.     

Paenibacillus tyraminigenes sp. nov. isolated from Myeolchi-jeotgal, a traditional Korean salted and fermented anchovy

A bacterial strain H3029, a gram-positive, rod-shaped, oxidase-negative, endospore-forming bacterium that characteristically produces tyramine from tyrosine, was isolated from a Myeolchi-jeotgal, a traditional Korean salted and fermented anchovy (Engraulis japonicus). The H3029 strain showed a high ability to produce 4140 microg/ml of tyramine from the culture broth containing 5000 microg/ml tyrosine. On the other hand, the strain produced a relatively low level of other putrefactive amines, at 973 microg/ml of putrescine and 147 microg/ml of cadaverine from the media, with each 5000 microg/ml of ornithine hydrochloride and lysine hydrochloride. The H3029 strain produced no detectable level of histamine (detection limit of 4 microg/ml) from the media containing 5000 microg/ml of histidine hydrochloride. Meanwhile, tyramine, the main product of the strain, showed the antimicrobial activity at the level of over 1 mg/disk against Staphylococcus aureus by agar diffusion test, and the mutagenicity in Ames test at 0.1 mg/plate using Salmonella typhimurium TA98 and TA1535. On the basis of the polyphasic taxonomic study, the H3029(T) strain was assigned a novel species of the genus Paenibacillus as Paenibacillus tyraminigenes sp. nov. The type strain of which is strain H3029(T) (=KCTC 10694BP(T)).     

Actinomycete Nonomuraea sp. isolated from Indonesian soil is a new producer of inulin fructotransferase

An actinomycete that excretes inulin fructotransferase to the culture supernatant was able to produce di-d-fructofuranose 1,2':2,3' dianhydride (DFA III) from inulin, with the greatest rate of enzyme activity at 65°C and at a pH of 5.5. Through chemotaxonomic and 16S rRNA gene analysis, this strain was identified as genus Nonomuraea in the Streptosporangiaceae family. This is the first report of an inulin fructotransferase producer in this family.     

一株产低温纤维素酶放线菌的分离与产酶研究

用羧甲基纤维素钠-刚果红培养基从内蒙大青沟采集的森林土壤中分离了一株产纤维素酶的耐低温菌,命名为ND2-1,其形态和16S r DNA序列分析结果表明ND2-1为链霉菌属（Streptomyces sp.）的放线菌。ND2-1能在10³5℃范围内生长,最适生长温度为25℃,属耐低温菌。滤纸崩解实验表明,ND2-1能高效降解纤维素。ND2-1在25℃条件下,在含有2 g/L羧甲基纤维素钠（CMC-Na）的培养基中培养3 d后酶活性最高。对其酶学性质的初步研究结果表明:该菌株所产纤维素酶在p H3.0⁹.0范围内稳定,且酶活性都较高,最适反应p H为7.0;最适反应温度为30℃,K+和Cu2+对酶活有抑制作用,Mn2+能提高相对酶活性至166.7%。      

1株产类胡萝卜素盐盒菌的分离鉴定及培养条件优化

对分离自运城盐湖黑泥中的一株产类胡萝卜素嗜盐菌G14进行分离鉴定,经菌落形态观察、生理生化特性及16S rRNA基因 序列分析,确定该菌株隶属于盐盒菌属（Haloarcula sp.）。 通过单因素与正交试验对菌株G14的培养条件进行优化。 结果表明,菌株G14 最佳培养条件为NaCl质量浓度0.23 g/mL,初始pH值为7.0,43 ℃光照培养。 在此优化条件下,培养的菌液OD600 nm值达2.24,类胡萝卜素 产量为0.65 mg/L。