Characterization and Genomic Analysis of Affinirhizobium gouqiense sp. nov. Isolated from Seawater of Gouqi Island Located in the East China Sea and Reclassification of Rhizobium lemnae to the Genus Affinirhizobium as Affinirhizobium lemnae comb. nov.

A novel bacterium designated as SSA5.23T was isolated from seawater. Cells of SSA5.23T are Gram-stain-negative, short, rod-shaped, and exhibit motility via numerous peritrichous flagella. The strain could grow at temperatures ranging from 15 to 35 °C (optimum at 25 °C), in a salinity range of 0-5.0% (w/v) NaCl, and within a pH range of 6.0-9.0 (optimum at pH 7.0). The predominant cellular fatty acid of SSA5.23T was C18:1 ω7c/C18:1 ω6c, and the major respiratory quinones were Q-9 and Q-10. Diphosphatidylglycerol, phosphatidylethanolamine, and phosphatidylglycerol were identified as the primary polar lipids. The complete genome (5.47 Mb) of SSA5.23T comprises of a circular chromosome of 3.64 Mb and three plasmids, specifically sized at 59.73 kb, 227.82 kb, and 1.54 Mb, respectively. Certain genes located on the plasmids play roles in denitrification, oxidative stress resistance, and osmotic tolerance, which likely contribute to the adaptability of this strain in marine conditions. Core-proteome average amino acid identity analysis effectively identified the strain's affiliation with the genus Affinirhizobium, showing the highest value (89.9%) with Affinirhizobium pseudoryzae DSM 19479T. This classification was further supported by the phylogenetic analysis of concatenated alignment of 170 single-copy orthologous proteins. When compared to related reference strains, SSA5.23T displayed an average nucleotide identity ranging from 74.9 to 80.3% and digital DNA-DNA hybridization values ranging from 19.9 to 23.9%. Our findings confirmed that strain SSA5.23T represents a novel species of the genus Affinirhizobium, for which the name Affinirhizobium gouqiense sp. nov. (type strain SSA5.23T = LMG 32560T = MCCC 1K07165T) was suggested.

A Hadal Streptomyces-Derived Echinocandin Acylase Discovered through the Prioritization of Protein Families.

Naturally occurring echinocandin B and FR901379 are potent antifungal lipopeptides featuring a cyclic hexapeptide nucleus and a fatty acid side chain. They are the parent compounds of echinocandin drugs for the treatment of severe fungal infections caused by the Candida and Aspergilla species. To minimize hemolytic toxicity, the native fatty acid side chains in these drug molecules are replaced with designer acyl side chains. The deacylation of the N-acyl side chain is, therefore, a crucial step for the development and manufacturing of echinocandin-type antibiotics. Echinocandin E (ECE) is a novel echinocandin congener with enhanced stability generated via the engineering of the biosynthetic machinery of echinocandin B (ECB). In the present study, we report the discovery of the first echinocandin E acylase (ECEA) using the enzyme similarity tool (EST) for enzymatic function mining across protein families. ECEA is derived from Streptomyces sp. SY1965 isolated from a sediment collected from the Mariana Trench. It was cloned and heterologously expressed in S. lividans TK24. The resultant TKecea66 strain showed efficient cleavage activity of the acyl side chain of ECE, showing promising applications in the development of novel echinocandin-type therapeutics. Our results also provide a showcase for harnessing the essentially untapped biodiversity from the hadal ecosystems for the discovery of functional molecules.

Shinella sedimenti sp. nov., isolated from sediment of Zhairuo Island located in the East China Sea.

A Gram-negative, rod-shaped and aerobic bacterial strain B3.7T, was isolated from the sediment of Zhairuo Island, Zhoushan city, Zhejiang Province, PR China. Maximum growth of strain B3.7T was observed at 30 °C when cultured in a medium containing 0.5 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences demonstrated that strain B3.7T belonged to the genus Shinella; it showed the highest sequence similarity of 98.47 % to Shinella kummerowiae CCBAU 25048T. The average nucleotide identity and digital DNA-DNA hybridization values between strain B3.7T and its reference strains were 82.9-84.2 % and 26.1-27.3 %, respectively. Chemotaxonomic analysis indicated that the sole respiratory quinone was Q-10 and the predominant cellular fatty acids were C19 : 0 cyclo ω8c, C16 : 0, C18 : 1 ω7c 11-methyl and summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c). The polar lipid profile was composed of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, three unidentified phospholipids and two unidentified aminolipids. Collectively, strain B3.7T can be considered to represent a novel species, for which the name Shinella sedimenti sp. nov. is proposed. The type strain is B3.7T (=MCCC 1K07163T=LMG 32559T).

Unnatural tetradeoxy echinocandins produced by gene cluster design and heterologous expression.

The hydroxyl groups in the amino acid residues of echinocandin B were related to the biological activity, the instability, and the drug resistance. The modification of hydroxyl groups was expected to obtain the new lead compounds for next generation of echinocandin drug development. In this work one method for heterologous production of the tetradeoxy echinocandin was achieved. A reconstructed biosynthetic gene cluster for tetradeoxy echinocandins composed of ecdA/I/K and htyE was designed and successfully hetero-expressed in Aspergillus nidulans. The target product of echinocandin E (1) together with one unexpected derivative echinocandin F (2), were isolated from the fermentation culture of engineered strain. Both of compounds were unreported echinocandin derivatives and the structures were identified on the basis of mass and NMR spectral data analysis. Compared with echinocandin B, echinocandin E demonstrated superior stability and comparable antifungal activity.

Atopomonas sediminilitoris sp. nov., isolated from beach sediment of Zhairuo Island, China.

A novel bacterium designated A3.4T was isolated from the beach sediment of Zhairuo Island, which is located in the East China Sea. Strain A3.4T was found to be Gram-stain negative, cream coloured, rod-shaped, aerobic and motile via a single monopolar flagellum. The isolate grows at 20-37 °C (optimum 25-30 °C), at pH 6.0-8.0 (optimum pH 7.0-8.0), and in the presence of 0-5.0% (w/v) NaCl (optimum 0.5-1%). A3.4T has catalase and oxidase activity. The predominant fatty acids (≥ 10%) of the strain were identified as C16:0, summed feature 3 (C16:1 ω7c /C16:1 ω6c) and summed feature 8 (C18:1 ω7c /C18:1 ω6c). Q-9 was identified as the major isoprenoid quinone, with trace levels of Q-8 present. The major polar lipids were identified as diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. The draft genome size is 3.55 Mb, with a DNA G + C content of 57.7 mol%. Analysis of the 16S rRNA gene sequence of strain A3.4T indicates that it belongs to the genus Atopomonas and shares high sequence similarity with Atopomonas hussainii JCM 19513T (97.60%). This classification was also supported by phylogenetic analysis using rpoB and several core genes. The genome of strain A3.4T shows an average nucleotide identity of 82.3%, an amino acid identity of 83.0%, and a digital DNA-DNA hybridization value of 22.1% with A. hussainii. In addition, 20 conserved signature indels (CSIs) were identified to be specific for A3.4T and A. hussainii, demonstrating that the strain A3.4T is closely related to A. hussainii rather than other species of family Pseudomonadaceae. Hundreds of unique genes were identified in the genomes of A3.4T and A. hussainii, which may underly multiple phenotypic differences between these strains. Based on phenotypic, chemotaxonomic, phylogenetic, and genomic investigations, strain A3.4T is concluded to represent a novel species of the genus Atopomonas, for which the name Atopomonas sediminilitoris sp. nov. is proposed. The type strain is A3.4T (= LMG 32563T = MCCC 1K07166T).

Ancylobacter gelatini sp. nov., isolated from beach sediment of Zhairuo Island, China.

A Gram-negative, aerobic, non-motile, oxidase-positive, catalase-positive, methyl red-positive, and lipase-negative bacterium, designated A5.8T, was isolated from beach sediment of Zhairuo Island located in the East China Sea. Growth occurred at 10-40 °C (optimum, 30 °C), pH 5.5-9.5 (optimum, 7.5), and 0-2% NaCl (optimum, 1.5%). Based on 16S rRNA gene sequence analysis, strain A5.8T belongs to the genus Ancylobacter, sharing the highest similarity with Ancylobacter aquaticus JCM 20518T (98.0%). Its polar lipids mainly consist of phosphatidylethanolamine (PE) and phosphatidylcholine (PC). The predominant fatty acids are summed feature 8 (C18:1ω7c and/or C18:1ω6c, 91.0%), and the major respiratory quinone is Q-10. The DNA G + C content is 67.2 mol%. Based on above analysis, as well as digital DNA-DNA hybridization (22.5-22.9%) and average nucleotide identity (83.0-83.6%) of strain A5.8T with reference type strains of the genus Ancylobacter, strain A5.8T was suggested to represent a novel species of the genus Ancylobacter, for which the name Ancylobacter gelatini sp. nov. is proposed. The type strain is A5.8T (= MCCC 1K07167T = LMG 32566T).

Acinetobacter sedimenti sp. nov., isolated from beach sediment.

A Gram-stain-negative, aerobic, non-motile, non-haemolytic, oxidase-negative, catalase-positive bacillus strain (A3.8T) was isolated from beach sediment from Zhairuo Island, PR China. The strain grew at pH 6.0-9.0 (optimum, 7.0), with 0-4.5 % NaCl (optimum, 2 %) and at 10-35 °C (optimum, 30 °C). Its whole-genome sequence was 2.5 Mb in size, with a DNA G+C content of 41.6 mol%. On the basis of the results of core genome phylogenetic analysis, A3.8T represents a separate branch within the clade formed by five species of the genus Acinetobacter with 'Acinetobacter marinus' as the most closely related species. The average nucleotide identity compared with the closely related species of the genus Acinetobacter was below 83.66 % and digital DNA-DNA hybridization values were less than 28.80 %. The predominant fatty acids included C18 : 1ω9c, C16 : 0 and summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c). Q-9 was the major respiratory quinone. The polar lipids are mainly composed of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, two phospholipids, an aminolipid and four unknown lipids. A3.8T cannot assimilate dl-lactate and weakly utilizes l-glutamate, l-leucine, l-phenylalanine and l-tartrate, which distinguishes it from other species of the genus Acinetobacter. On the basis of the genotype, phenotype and biochemical data, strain A3.8T represents a novel species of the genus Acinetobacter, for which the name Acinetobacter sedimenti sp. nov. is proposed. The type strain is A3.8T (=MCCC 1K07161T=LMG 32568T).

Tanzawaic Acids from a Deep-Sea Derived Penicillium Species.

Twelve new tanzawaic acid derivatives, penitanzacids A-F (1-6), and G-J (9-12), and hatsusamides C-D (13-14), together with two revised structures [tanzawaic acids I-J (7-8)] and three known compounds (15-17) were isolated from the deep-sea-derived fungus Penicillium sp. KWF32. Their structures including absolute configurations were elucidated by spectroscopic data analysis, HRESIMS data, modified Mosher's method, chemical degradation studies, ECD calculations, single crystal X-ray diffraction, and biogenic considerations in comparison with reported known analogues. Penitanzacids H-J (10-12) represent the first examples of this family with a C3 side chain and support the proposed biosynthetic pathway in which the side chain is connected to the decalin backbone. Hatsusamides C-D (13-14) have a hybrid skeleton formed by linking a tanzawaic acid and a diketopiperazine through an ester bond. Compounds 13 and 14 exhibit weak cytotoxicity against the A549 cell line.

Recent Advances in the Heterologous Expression of Biosynthetic Gene Clusters for Marine Natural Products.

Marine natural products (MNPs) are an important source of biologically active metabolites, particularly for therapeutic agent development after terrestrial plants and nonmarine microorganisms. Sequencing technologies have revealed that the number of biosynthetic gene clusters (BGCs) in marine microorganisms and the marine environment is much higher than expected. Unfortunately, the majority of them are silent or only weakly expressed under traditional laboratory culture conditions. Furthermore, the large proportion of marine microorganisms are either uncultivable or cannot be genetically manipulated. Efficient heterologous expression systems can activate cryptic BGCs and increase target compound yield, allowing researchers to explore more unknown MNPs. When developing heterologous expression of MNPs, it is critical to consider heterologous host selection as well as genetic manipulations for BGCs. In this review, we summarize current progress on the heterologous expression of MNPs as a reference for future research.

Presence, Mode of Action, and Application of Pathway Specific Transcription Factors in Aspergillus Biosynthetic Gene Clusters.

Fungal secondary metabolites are renowned toxins as well as valuable sources of antibiotics, cholesterol-lowering drugs, and immunosuppressants; hence, great efforts were levied to understand how these compounds are genetically regulated. The genes encoding for the enzymes required for synthesizing secondary metabolites are arranged in biosynthetic gene clusters (BGCs). Often, BGCs contain a pathway specific transcription factor (PSTF), a valuable tool in shutting down or turning up production of the BGC product. In this review, we present an in-depth view of PSTFs by examining over 40 characterized BGCs in the well-studied fungal species Aspergillus nidulans and Aspergillus fumigatus. Herein, we find BGC size is a predictor for presence of PSTFs, consider the number and the relative location of PSTF in regard to the cluster(s) regulated, discuss the function and the evolution of PSTFs, and present application strategies for pathway specific activation of cryptic BGCs.

Flavobacterium salilacus sp. nov., isolated from surface water of a hypersaline lake, and descriptions of Flavobacterium salilacus subsp. altitudinum subsp. nov. and Flavobacterium salilacus subsp. salilacus subsp. nov.

Two yellow-pigmented, Gram-stain-negative, aerobic, rod-shaped bacteria were isolated from the water of the hypersaline Chaka Salt Lake (strain SaA2.12T) and sediment of Qinghai Lake (strain LaA7.5T), PR China. According to the 16S rRNA phylogeny, the isolates belong to the genus Flavobacterium, showing the highest 16S rRNA sequence similarities to Flavobacterium arcticum SM1502T(97.6-97.7 %) and Flavobacterium suzhouense XIN-1T(96.5-96.6 %). Moreover, strains SaA2.12T and LaA7.5T showed 99.73 % 16S rRNA sequence similarity to each other. Major fatty acids, respiratory quinones and polar lipids detected in these isolates were iso-C15 : 0, menaquinone-6 and phosphatidylethanolamine, respectively. Strains SaA2.12T and LaA7.5T showed significant unique characteristics between them as well as between the closest phylogenetic members. The highest digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values between SaA2.12T and its closest neighbours were 25.3 and 82.8 %, respectively; whereas these values (highest) between LaA7.5T and its closest members were 25.2 and 82.8 %, respectively. The dDDH and ANI values between strains SaA2.12T and LaA7.5T were calculated as 75.9 and 97.2 %, respectively. Therefore, based on polyphasic data, we propose that strain SaA2.12T represents a novel species with the name Flavobacterium salilacus sp. nov., with the type strain SaA2.12T (=KCTC 72220T=MCCC 1K03618T) and strain LaA7.5T as a subspecies within novel Flavobacterium salilacus with the name Flavobacterium salilacus subsp. altitudinum subsp. nov., with the type strain LaA7.5T (=KCTC 72806T=MCCC 1K04372T). These propositions automatically create Flavobacterium salilacus subsp. salilacus subsp. nov. with SaA2.12T (=KCTC 72220T=MCCC 1K03618T) as the type strain.

Flavobacterium ajazii sp. nov., Isolated from Seaweed of Gouqi Island, China.

A novel bacterium designated SSM4.2T was isolated from seaweed of Gouqi Island, which is the center of the Zhoushan fishing ground in the East China Sea. Strain SSM4.2T was Gram-stain-negative, bright yellow-pigmented, short rod-shaped, non-flagellated, non-spore forming, aerobic and motile by gliding. Growth was observed at 4-37 °C (optimum 25-30 °C), pH 6.0-8.0 (optimum pH 7.0) and 0-2.0% (w/v) NaCl (optimum 0%) concentration. The strain was catalase- and oxidase-positive. Menaquinone-6 (MK-6) was found as the sole respiratory quinone and zeaxanthin as the main carotenoid pigment. The predominant fatty acids (≥ 10%) were iso-C15:0, iso-C15:1 G, iso-C17:0 3-OH and summed feature 3 (C16:1 ω7c /C16:1 ω6c). The major polar lipid was phosphatidylethanolamine (PE). The genome size was 5.7 Mbp. The DNA G + C content was 34.1 mol%. 16S rRNA gene sequence revealed that strain SSM4.2T belongs to the genus Flavobacterium and shares high-sequence similarity with F. limi KACC 18851T (98.1%), F. hydrophilum KACC 19591T (97.6%), F. defluvii KCTC 12612T (97.1%), F. cheongpyeongense KACC 19592T (97.0%) and F. fluviatile KCTC 52446T (96.9%). Strain SSM4.2T had 73.2-84.6% average nucleotide identity and 19.1-29.4% digital DNA-DNA hybridization values with its closest type strains. Based on its phenotypic, chemotaxonomic, phylogenetic and genomic features, strain SSM4.2T represents a novel species of the genus Flavobacterium, for which the name Flavobacterium ajazii sp. nov. is proposed. The type strain is SSM4.2T (= KCTC 72807T = MCCC 1K04370T).

Heteroexpression of Aspergillus nidulans laeA in Marine-Derived Fungi Triggers Upregulation of Secondary Metabolite Biosynthetic Genes.

Fungi are a prospective resource of bioactive compounds, but conventional methods of drug discovery are not effective enough to fully explore their metabolic potential. This study aimed to develop an easily attainable method to elicit the metabolic potential of fungi using Aspergillus nidulans laeA as a transcription regulation tool. In this study, functional analysis of Aspergillus nidulans laeA (AnLaeA) and Aspergillus sp. Z5 laeA (Az5LaeA) was done in the fungus Aspergillus sp. Z5. Heterologous AnLaeA-and native Az5LaeA-overexpression exhibited similar phenotypic effects and caused an increase in production of a bioactive compound diorcinol in Aspergillus sp. Z5, which proved the conserved function of this global regulator. In particular, heteroexpression of AnLaeA showed a significant impact on the expression of velvet complex genes, diorcinol synthesis-related genes, and different transcription factors (TFs). Moreover, heteroexpression of AnLaeA influenced the whole genome gene expression of Aspergillus sp. Z5 and triggered the upregulation of many genes. Overall, these findings suggest that heteroexpression of AnLaeA in fungi serves as a simple and easy method to explore their metabolic potential. In relation to this, AnLaeA was overexpressed in the fungus Penicillium sp. LC1-4, which resulted in increased production of quinolactacin A.

Global Analysis of Furfural-Induced Genomic Instability Using a Yeast Model.

Furfural is an important renewable precursor for multiple commercial chemicals and fuels; a main inhibitor existing in cellulosic hydrolysate, which is used for bioethanol fermentation; and a potential carcinogen, as well. Using a genetic system in Saccharomyces cerevisiae that allows detection of crossover events, we observed that the frequency of mitotic recombination was elevated by 1.5- to 40-fold when cells were treated with 0.1 g/liter to 20 g/liter furfural. Analysis of the gene conversion tracts associated with crossover events suggested that most furfural-induced recombination resulted from repair of DNA double-strand breaks (DSBs) that occurred in the G1 phase. Furfural was incapable of breaking DNA directly in vitro but could trigger DSBs in vivo related to reactive oxygen species accumulation. By whole-genome single nucleotide polymorphism (SNP) microarray and sequencing, furfural-induced genomic alterations that range from single base substitutions, loss of heterozygosity, and chromosomal rearrangements to aneuploidy were explored. At the whole-genome level, furfural-induced events were evenly distributed across 16 chromosomes but were enriched in high-GC-content regions. Point mutations, particularly the C-to-T/G-to-A transitions, were significantly elevated in furfural-treated cells compared to wild-type cells. This study provided multiple novel insights into the global effects of furfural on genomic stability.IMPORTANCE Whether and how furfural affects genome integrity have not been clarified. Using a Saccharomyces cerevisiae model, we found that furfural exposure leads to in vivo DSBs and elevation in mitotic recombination by orders of magnitude. Gross chromosomal rearrangements and aneuploidy events also occurred at a higher frequency in furfural-treated cells. In a genome-wide analysis, we show that the patterns of mitotic recombination and point mutations differed dramatically in furfural-treated cells and wild-type cells.

Mapping chromosomal instability induced by small-molecular therapeutics in a yeast model.

The yeast Saccharomyces cerevisiae has been widely used as a model system for studying the physiological and pharmacological action of small-molecular drugs. Here, a heterozygous diploid S. cerevisiae strain QSS4 was generated to determine whether drugs could induce chromosomal instability by determining the frequency of mitotic recombination. Using the combination of a custom SNP microarray and yeast screening system, the patterns of chromosomal instability induced by drugs were explored at the whole genome level in QSS4. We found that Zeocin (a member of the bleomycin family) treatment increased the rate of genomic alterations, including aneuploidy, loss of heterozygosity (LOH), and chromosomal rearrangement over a hundred-fold. Most recombination events are likely to be initiated by DNA double-stand breaks directly generated by Zeocin. Another remarkable finding is that G4-motifs and low GC regions were significantly underrepresented within the gene conversion tracts of Zeocin-induced LOH events, indicating that certain DNA regions are less preferred Zeocin-binding sites in vivo. This study provides a novel paradigm for evaluating genetic toxicity of small-molecular drugs using yeast models.

Flavobacterium sharifuzzamanii sp. nov., Isolated from the Sediments of the East China Sea.

A novel bacterial strain A7.6T was isolated from the sediments collected near the Zhairuo Island located in the East China Sea and characterized using a polyphasic approach. Cells were Gram-stain-negative, rod-shaped, non-spore forming, non-flagellated but motile by gliding. The strain was aerobic, positive for oxidase and catalase activities. The strain can grow at 4-35 °C, pH 5.5-9.0, and 0-3% (w/v) NaCl concentration. The major polar lipid was phosphatidylethanolamine, the predominant fatty acids (> 10%) were iso-C15:0 and summed feature 3 (C16:1 ω7c and/or C16:1 ω6c). The genomic G+C content was 33.6 mol% and the major respiratory quinone was menaquinone 6. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain A7.6T belonged to the genus Flavobacterium and was closely related to Flavobacterium tistrianum GB 56.1T (98.4% similarity), F. nitrogenifigens NXU-44T (98.4%), F. ginsenosidimutans THG 01T (98.0%) and F. anhuiense D3T (97.7%). Average nucleotide identities and digital DNA-DNA hybridizations values for genomes ranged from 75.9 to 91.4% and 21.4 to 43.9% between strain A7.6T and its closest phylogenetic neighbors. The polyphasic characterization indicated that strain A7.6T represented a novel species of the genus Flavobacterium, for which the name Flavobacterium sharifuzzamanii is proposed. The type strain is A7.6T (= KCTC 62405T = MCCC 1K03485T). The NCBI GenBank accession number for the 16S rRNA gene of A7.6T is MH396692, and for the genome sequence is QJGZ00000000. The digital protologue database (DPD) Taxon Number is TA00643.

New Polyketides from the Marine-Derived Fungus Letendraea sp. 5XNZ4-2.

Marine-derived fungi have been reported to have great potential to produce structurally unique metabolites. Our investigation on secondary metabolites from marine-derived fungi resulted in the isolation of seven new polyketides (phomopsiketones D-G (1-4) and letendronols A-C (5-7)) as well as one known xylarinol (8) in the cultural broth of Letendraea sp. Their structures and absolute configurations were elucidated using a set of spectroscopic and chemical methods, including HRESIMS, NMR, single-crystal X-ray diffraction, ECD calculation, and a modified version of Mosher's method. Compound 2 showed weak inhibition against nitric oxide production in lipopolysaccaride-activated macrophages with an IC50 value of 86 µM.

Genetic characterization and modification of a bioethanol-producing yeast strain.

Yeast Saccharomyces cerevisiae strains isolated from different sources generally show extensive genetic and phenotypic diversity. Understanding how genomic variations influence phenotypes is important for developing strategies with improved economic traits. The diploid S. cerevisiae strain NY1308 is used for cellulosic bioethanol production. Whole genome sequencing identified an extensive amount of single nucleotide variations and small insertions/deletions in the genome of NY1308 compared with the S288c genome. Gene annotation of the assembled NY1308 genome showed that 43 unique genes are absent in the S288c genome. Phylogenetic analysis suggested most of the unique genes were obtained through horizontal gene transfer from other species. RNA-Seq revealed that some unique genes were not functional in NY1308 due to unidentified intron sequences. During bioethanol fermentation, NY1308 tends to flocculate when certain inhibitors (derived from the pretreatment of cellulosic feedstock) are present in the fermentation medium. qRT-PCR and genetic manipulation confirmed that the novel gene, NYn43, contributed to the flocculation ability of NY1308. Deletion of NYn43 resulted in a faster fermentation rate for NY1308. This work disclosed the genetic characterization of a bioethanol-producing S. cerevisiae strain and provided a useful paradigm showing how the genetic diversity of the yeast population would facilitate the personalized development of desirable traits.

Two Novel Aspochalasins from the Gut Fungus Aspergillus sp. Z4.

Two novel aspochalasins, tricochalasin A (1) and aspochalasin A2 (2), along with three known compounds (3⁻5) have been isolated from the different culture broth of Aspergillus sp., which was found in the gut of a marine isopod Ligia oceanica. Compound 1 contains a rare 5/6/6 tricyclic ring fused with the aspochalasin skeleton. The structures were determined on the basis of electrospray ionisation mass spectroscopy (ESIMS), nuclear magnetic resonance (NMR) spectral data, and the absolute configurations were further confirmed by modified Mosher's method. Cytotoxicity against the prostate cancer PC3 cell line were assayed by the MTT method. Compound 3 showed strong activity while the remaining compounds showed weak activity.

TrpE feedback mutants reveal roadblocks and conduits toward increasing secondary metabolism in Aspergillus fumigatus.

Small peptides formed from non-ribosomal peptide synthetases (NRPS) are bioactive molecules produced by many fungi including the genus Aspergillus. A subset of NRPS utilizes tryptophan and its precursor, the non-proteinogenic amino acid anthranilate, in synthesis of various metabolites such as Aspergillus fumigatus fumiquinazolines (Fqs) produced by the fmq gene cluster. The A. fumigatus genome contains two putative anthranilate synthases - a key enzyme in conversion of anthranilic acid to tryptophan - one beside the fmq cluster and one in a region of co-linearity with other Aspergillus spp. Only the gene found in the co-linear region, trpE, was involved in tryptophan biosynthesis. We found that site-specific mutations of the TrpE feedback domain resulted in significantly increased production of anthranilate, tryptophan, p-aminobenzoate and fumiquinazolines FqF and FqC. Supplementation with tryptophan restored metabolism to near wild type levels in the feedback mutants and suggested that synthesis of the tryptophan degradation product kynurenine could negatively impact Fq synthesis. The second putative anthranilate synthase gene next to the fmq cluster was termed icsA for its considerable identity to isochorismate synthases in bacteria. Although icsA had no impact on A. fumigatus Fq production, deletion and over-expression of icsA increased and decreased respectively aromatic amino acid levels suggesting that IcsA can draw from the cellular chorismate pool.