Interplay of xenobiotic-degrading and antibiotic-resistant microorganisms among the microbiome found in the air, handrail, and floor of the subway station.

Investigating the quality of the subway environment, especially regarding antibiotic resistance genes (ARGs) and xenobiotics, conveys ecological and health impacts. In this study, compositions and relations of microorganisms harboring ARGs and xenobiotic degradation and metabolism genes (XDGs) in the Sukhumvit subway station (MRT-SKV) in Bangkok was assessed by analyzing the taxonomic and genetic diversity of the microbiome in the air and on the surfaces of floor and handrail. The major bacteria in the MRT-SKV (including Moraxella, which was abundant in the bioaerosol and handrail samples, and Staphylococcus, which was abundant in the bioaerosol samples) were found to contain both ARGs and XDGs. The co-abundance correlation network revealed notable relationships among bacteria harboring antibiotic resistance genes (ARGs) and xenobiotic degradation genes (XDGs). Significant associations were observed between ARGs linked to glycopeptide and fluoroquinolone resistance and genes associated with benzoate, styrene, and atrazine degradation pathways, as well as between ARGs related to cephamycin, cephalosporin, and MLS resistance and XDGs associated with the cytochrome P450-dependent drug metabolism pathway. These correlations suggested that selective pressure exerted by certain xenobiotics and antibiotics can simultaneously affect both ARGs and XDGs in the environment and should favor correlations and co-survival among ARG- and XDG-containing bacteria in the environments. The correlations may occur via shared mechanisms of resistance to both xenobiotics and antibiotics. Finally, different correlation pairs were seen in different niches (air, handrail, floor) of the subway environment or different geolocations. Thus, the relationship between ARG and XDG pairs most likely depends on the unique characteristics of the niches and on the prominent types of xenobiotics and antibiotics in the subway environment. The results indicated that interactions and connections between microbial communities can impact how they function. These microorganisms can have profound effects on accumulation of xenobiotics and ARGs in the MRT-SKV.

Elucidating potential bioindicators from insights in the diversity and assembly processes of prokaryotic and eukaryotic communities in the Mekong River.

Drivers for spatio-temporal distribution patterns of overall planktonic prokaryotes and eukaryotes in riverine ecosystems are generally not fully understood. This study employed amplicon metabarcoding to investigate the distributions and assembly mechanisms of bacterial and eukaryotic communities in the Mekong River. The prevailing bacteria taxa were found to be Betaproteobacteria, Actinobacteria, and Bacteroidetes, while the dominant eukaryotic organisms were cryptophytes, chlorophytes, and diatoms. The community assemblages were influenced by a combination of stochastic and deterministic processes. Drift (DR) and dispersal limitation (DL), signifying the stochastic mechanism, were the main processes shaping the overall prokaryotic and eukaryotic communities. However, homogeneous selection (HoS), indicating deterministic mechanism, played a major role in the assembly process of core prokaryotic communities, especially in the wet season. In contrast, the core eukaryotic communities including Opisthokonta, Sar, and Chlorophyta were dominated by stochastic processes. The significance of HoS within prokaryotic communities was also found to exhibit a decreasing trend from the upstream sampling sites (Chiang Saen and Chiang Khan, Nong Khai) towards the downstream sites (Mukdahan, and Khong Chiam) of the Mekong River. The environmental gradients resulting from the site-specific variations and the gradual decrease in elevation along the river may have a potential influence on the role of HoS in community assembly. Crucial environmental factors that shape the phylogenetic structure within distinct bins of the core prokaryotic communities including water depth, temperature, chloride, sodium, and sulphate were identified, as inferred by their correlation with the beta Net Relatedness Index (betaNRI) during the wet season. Overall, these findings enhance understanding of the complex mechanisms governing the spatio-temporal dynamics of prokaryotic and eukaryotic communities in the Mekong River. Finally, insights gained from this study could provide information on further use of specific core bacteria as microbial-based bioindicators that are effective for the assessment and conservation of the Mekong River ecosystem.

gcType: a high-quality type strain genome database for microbial phylogenetic and functional research.

Taxonomic and functional research of microorganisms has increasingly relied upon genome-based data and methods. As the depository of the Global Catalogue of Microorganisms (GCM) 10K prokaryotic type strain sequencing project, Global Catalogue of Type Strain (gcType) has published 1049 type strain genomes sequenced by the GCM 10K project which are preserved in global culture collections with a valid published status. Additionally, the information provided through gcType includes >12 000 publicly available type strain genome sequences from GenBank incorporated using quality control criteria and standard data annotation pipelines to form a high-quality reference database. This database integrates type strain sequences with their phenotypic information to facilitate phenotypic and genotypic analyses. Multiple formats of cross-genome searches and interactive interfaces have allowed extensive exploration of the database's resources. In this study, we describe web-based data analysis pipelines for genomic analyses and genome-based taxonomy, which could serve as a one-stop platform for the identification of prokaryotic species. The number of type strain genomes that are published will continue to increase as the GCM 10K project increases its collaboration with culture collections worldwide. Data of this project is shared with the International Nucleotide Sequence Database Collaboration. Access to gcType is free at http://gctype.wdcm.org/.

Development of a CRISPR/Cpf1 system for targeted gene disruption in Aspergillus aculeatus TBRC 277.

BACKGROUND: CRISPR-Cas genome editing technologies have revolutionized biotechnological research particularly in functional genomics and synthetic biology. As an alternative to the most studied and well-developed CRISPR/Cas9, a new class 2 (type V) CRISPR-Cas system called Cpf1 has emerged as another versatile platform for precision genome modification in a wide range of organisms including filamentous fungi. RESULTS: In this study, we developed AMA1-based single CRISPR/Cpf1 expression vector that targets pyrG gene in Aspergillus aculeatus TBRC 277, a wild type filamentous fungus and potential enzyme-producing cell factory. The results showed that the Cpf1 codon optimized from Francisella tularensis subsp. novicida U112, FnCpf1, works efficiently to facilitate RNA-guided site-specific DNA cleavage. Specifically, we set up three different guide crRNAs targeting pyrG gene and demonstrated that FnCpf1 was able to induce site-specific double-strand breaks (DSBs) followed by an endogenous non-homologous end-joining (NHEJ) DNA repair pathway which caused insertions or deletions (indels) at these site-specific loci. CONCLUSIONS: The use of FnCpf1 as an alternative class II (type V) nuclease was reported for the first time in A. aculeatus TBRC 277 species. The CRISPR/Cpf1 system developed in this study highlights the feasibility of CRISPR/Cpf1 technology and could be envisioned to further increase the utility of the CRISPR/Cpf1 in facilitating strain improvements as well as functional genomics of filamentous fungi.

Heterologous expression of Aspergillus aculeatus endo-polygalacturonase in Pichia pastoris by high cell density fermentation and its application in textile scouring.

BACKGROUND: Removal of non-cellulosic impurities from cotton fabric, known as scouring, by conventional alkaline treatment causes environmental problems and reduces physical strength of fabrics. In this study, an endo-polygalacturonase (EndoPG) from Aspergillus aculeatus produced in Pichia pastoris was evaluated for its efficiency as a bioscouring agent while most current bioscouring process has been performed using crude pectinase preparation. RESULTS: The recombinant EndoPG exhibited a specific activity of 1892.08 U/mg on citrus pectin under the optimal condition at 50 °C, pH 5.0 with a V max and K m of 65,451.35 µmol/min/mL and 15.14 mg/mL, respectively. A maximal activity of 2408.70 ± 26.50 U/mL in the culture supernatant was obtained by high cell density batch fermentation, equivalent to a 4.8 times greater yield than that from shake-flask culture. The recombinant enzyme was shown to be suitable for application as a bioscouring agent, in which the wettability of cotton fabric was increased by treatment with enzyme at 300 U/mL scouring solution at 40 °C, pH 5.0 for 1 h. The bio-scoured fabric has comparable wettability to that obtained by conventional chemical scouring, but has higher tensile strength. CONCLUSION: The work has demonstrated for the first time functions of A. aculeatus EndoPG on bioscouring in eco-textile processing. EndoPG alone was shown to possess effective scouring activity. High expression level and homogeneity could be achieved in bench-scale bioreactor.

Characterization of cellulolytic enzyme system of Schizophyllum commune mutant and evaluation of its efficiency on biomass hydrolysis.

Schizophyllum commune is a basidiomycete equipped with an efficient cellulolytic enzyme system capable of growth on decaying woods. In this study, production of lignocellulose-degrading enzymes from S. commune mutant G-135 (SC-Cel) on various cellulosic substrates was examined. The highest cellulase activities including CMCase, FPase, and ß-glucosidase were obtained on Avicel-PH101 while a wider range of enzymes attacking non-cellulosic polysaccharides and lignin were found when grown on alkaline-pretreated biomass. Proteomic analysis of SC-Cel also revealed a complex enzyme system comprising seven glycosyl hydrolase families with an accessory carbohydrate esterase, polysaccharide lyase, and auxiliary redox enzymes. SC-Cel obtained on Avicel-PH101 effectively hydrolyzed all agricultural residues with the maximum glucan conversion of 98.0% using corn cobs with an enzyme dosage of 5 FPU/g-biomass. The work showed potential of SC-Cel on hydrolysis of various herbaceous biomass with enhanced efficiency by addition external ß-xylosidase.

Culture-independent study of bacterial communities in tropical river sediment.

Ubiquitous microbial communities in river sediments actively govern organic matter decomposition, nutrient recycling, and remediation of toxic compounds. In this study, prokaryotic diversity in two major rivers in central Thailand, the Chao Phraya (CP) and the Tha Chin (TC) distributary was investigated. Significant differences in sediment physicochemical properties, particularly silt content, were noted between the two rivers. Tagged 16S rRNA sequencing on a 454 platform showed that the sediment microbiomes were dominated by Gammaproteobacteria and sulfur/sulfate reducing Deltaproteobacteria, represented by orders Desulfobacteriales and Desulfluromonadales together with organic degraders Betaproteobacteria (orders Burkholderiales and Rhodocyclales) together with the co-existence of Bacteroidetes predominated by Sphingobacteriales. Enrichment of specific bacterial orders was found in the clayey CP and silt-rich TC sediments, including various genera with known metabolic capability on decomposition of organic matter and xenobiotic compounds. The data represent one of the pioneered works revealing heterogeneity of bacteria in river sediments in the tropics.

Comparative Study of Bacterial Communities in Nepenthes Pitchers and Their Correlation to Species and Fluid Acidity.

Pitchers are specialized digestive organs of carnivorous plants which evolved for trapping prey and represent a unique environment harboring hidden diversity of unexplored microbes forming transient hydrolytic microcosms. In this study, the diversity of bacterial communities in the pitcher fluids of seven local Nepenthes found in Thailand was assessed by tagged 16S ribosomal RNA (rRNA) gene amplicon sequencing on an Ion PGM™ platform. A total of 1,101,000 filtered sequences were obtained which were taxonomically classified into 20 phyla, 48 classes, 72 orders, 153 families, and 442 genera while the remainder (1.43 %) could not be assigned to any existing taxa. Proteobacteria represented the predominant members in closed pitchers and more diversified bacterial taxa particularly Bacteriodetes and Actinobacteria, showed increasing abundance in open pitchers containing insect bodies. Principal coordinate analysis revealed that distribution of bacterial taxa was not significantly related to the Nepenthes species but strongly correlated to the pH of the pitcher fluids (pH 1.7-6.7). Acidicella was a highly dominant bacterial genus in acidic pitcher fluids while Dyella and Mycobacterium were also common genera in most pitchers. A unique microbial community structure was found in Nepenthes ampullaria which could reflect their adaptation to digest leaf litter, in addition to insect prey. The work revealed the highly unexplored nature of bacterial microcosms in Nepenthes pitcher fluids and provides insights into their community structure in this unique ecological system.

Insights from the genome of Ophiocordyceps polyrhachis-furcata to pathogenicity and host specificity in insect fungi.

BACKGROUND: Ophiocordyceps unilateralis is an outstanding insect fungus for its biology to manipulate host ants' behavior and for its extreme host-specificity. Through the sequencing and annotation of Ophiocordyceps polyrhachis-furcata, a species in the O. unilateralis species complex specific to the ant Polyrhachis furcata, comparative analyses on genes involved in pathogenicity and virulence between this fungus and other fungi were undertaken in order to gain insights into its biology and the emergence of host specificity. RESULTS: O. polyrhachis-furcata possesses various genes implicated in pathogenicity and virulence common with other fungi. Overall, this fungus possesses protein-coding genes similar to those found on other insect fungi with available genomic resources (Beauveria bassiana, Metarhizium robertsii (formerly classified as M. anisopliae s.l.), Metarhizium acridum, Cordyceps militaris, Ophiocordyceps sinensis). Comparative analyses in regard of the host ranges of insect fungi showed a tendency toward contractions of various gene families for narrow host-range species, including cuticle-degrading genes (proteases, carbohydrate esterases) and some families of pathogen-host interaction (PHI) genes. For many families of genes, O. polyrhachis-furcata had the least number of genes found; some genes commonly found in other insect fungi are even absent (e.g. Class 1 hydrophobin). However, there are expansions of genes involved in 1) the production of bacterial-like toxins in O. polyrhachis-furcata, compared with other entomopathogenic fungi, and 2) retrotransposable elements. CONCLUSIONS: The gain and loss of gene families helps us understand how fungal pathogenicity in insect hosts evolved. The loss of various genes involved throughout the pathogenesis for O. unilateralis would result in a reduced capacity to exploit larger ranges of hosts and therefore in the different level of host specificity, while the expansions of other gene families suggest an adaptation to particular environments with unexpected strategies like oral toxicity, through the production of bacterial-like toxins, or sophisticated mechanisms underlying pathogenicity through retrotransposons.

Methanol-inducible promoter of thermotolerant methylotrophic yeast Ogataea thermomethanolica BCC16875 potential for production of heterologous protein at high temperatures.

Methanol-utilizing metabolism is generally found in methylotrophic yeasts. Several potential promoters regulating enzymes in this pathway have been extensively studied, especially alcohol oxidase. Here, we characterized the alcohol oxidase gene promoter from thermotolerant Ogataea thermomethanolica (OthAOX). This promoter can be induced by methanol, and was shown to regulate expression of phytase up to 45 °C. The pattern of heterologous phytase N-glycosylation depends on the induction temperature. Unlike the AOX promoter from Pichia pastoris, this OthAOX initially turns on the expression of the heterologous protein at the de-repression stage in the presence of glycerol. Full induction of protein is observed when methanol is present. With this methanol-inducible promoter, target protein can be initially produced prior to the induction phase, which would help shorten the time for protein production. Being able to drive protein expression at various temperatures prompts this newly identified AOX promoter to be potential tool for heterologous protein production in high temperature conditions.

Fungal communities as dual indicators of river biodiversity and water quality assessment.

Given their ecological importance, bioindicators are used for the assessment of the health of river ecosystems. This study explored the fungal compositions and the potential of fungal taxa as bioindicators for indicating the water quality of the Mekong River, as the use of fungal indicators of the Mekong River was not previously well characterized. The Mekong River exhibited dynamic variations in both physicochemical/hydrochemical properties and fungal communities according to seasons and locations. The results revealed the dominance of alkaline earth metal ions and weak acids in the water. The magnesium-bicarbonate water type was found in the dry season, but the water became the chloride-calcium type or mixed type of magnesium-bicarbonate and chloride-calcium in the rainy season at downstream sites. Fungal composition analysis revealed the dominance of Chytridiomycota in the dry season and intermediate periods, and Ascomycota and Basidiomycota in the rainy season. The fungal communities were influenced by stochastic and deterministic assembly processes, mainly homogeneous selection, heterogeneous selection, and dispersal limitation. The extent of environmental filtering implied that some fungal taxa were affected by environmental conditions, suggesting the possibility of identifying certain fungal taxa suitable for being bioindicators of water quality. Subsequently, machine learning with recursive feature elimination identified specific fungal bins mostly consisting of Agaricomycetes (mainly Polyporales, Agaricales, and Auriculariales), Dothideomycetes (mainly Pleosporales), Saccharomycetes (mainly Saccharomycetales), Chytridiomycota, and Rozellomycota as bioindicators that could predict ambient and irrigation water quality with high selectivity and sensitivity. These results thus promote the use of fungal indicators to assess the health of the river.

Phylogenetic analysis and metabolic potential of microbial communities in an industrial bagasse collection site.

Industrial bagasse collection sites at sugar mills are an important resource for biomass-based industries and represent a unique ecological niche in lignocellulose degradation. In this study, microbial community structures at regions with varying microenvironmental conditions contained within a bagasse collection site were explored using tagged 16S rRNA gene pyrosequencing. Overall, remarkable differences in microbial community structures were found in aerobic surface and oxygen-limited interior regions of the pile. A variety of Alphaproteobacteria and Gammaproteobacteria represented the majority of bacteria in the aerobic upper-pile regions with the predominance of acetic acid bacteria towards the outer surface. Diverse Proteobacteria, Bacteroidetes, and Acidobacteria represented the predominant phyla at the exterior soil-contact pile base with an increasing abundance of anaerobic Spirochaetes with the increasing depth, where it shared similar community structures to that in the open-field soil from decomposed bagasse. Using complementary shotgun pyrosequencing, a variety of genes encoding various glycosyl hydrolases targeting cellulose and hemicellulose degradation were identified in the oxygen-limited interior pile base. Most were relevant to orders Clostridiales, Bacteroidales, Sphingobacteriales, and Cytophagales, suggesting their role in lignocellulose degradation in this region, as evidenced by the decrease in cellulose and respective increase in lignin fractions of the biomass. Partial carbon flux in the anoxic region was metabolized through mixed methanogenesis pathways as suggested by the annotated functional genes in methane synthesis. This study gives insights into native microbial community structures and functions in this unique lignocellulose degrading environment and provides the basis for controlling microbial processes important for utilization of bagasse in bio-industries.

Comparative metagenomic analysis of microcosm structures and lignocellulolytic enzyme systems of symbiotic biomass-degrading consortia.

Decomposition of lignocelluloses by cooperative microbial actions is an essential process of carbon cycling in nature and provides a basis for biomass conversion to fuels and chemicals in biorefineries. In this study, structurally stable symbiotic aero-tolerant lignocellulose-degrading microbial consortia were obtained from biodiversified microflora present in industrial sugarcane bagasse pile (BGC-1), cow rumen fluid (CRC-1), and pulp mill activated sludge (ASC-1) by successive subcultivation on rice straw under facultative anoxic conditions. Tagged 16S rRNA gene pyrosequencing revealed that all isolated consortia originated from highly diverse environmental microflora shared similar composite phylum profiles comprising mainly Firmicutes, reflecting convergent adaptation of microcosm structures, however, with substantial differences at refined genus level. BGC-1 comprising cellulolytic Clostridium and Acetanaerobacterium in stable coexistence with ligninolytic Ureibacillus showed the highest capability on degradation of agricultural residues and industrial pulp waste with CMCase, xylanase, and ß-glucanase activities in the supernatant. Shotgun pyrosequencing of the BGC-1 metagenome indicated a markedly high relative abundance of genes encoding for glycosyl hydrolases, particularly for lignocellulytic enzymes in 26 families. The enzyme system comprised a unique composition of main-chain degrading and side-chain processing hydrolases, dominated by GH2, 3, 5, 9, 10, and 43, reflecting adaptation of enzyme profiles to the specific substrate. Gene mapping showed metabolic potential of BGC-1 for conversion of biomass sugars to various fermentation products of industrial importance. The symbiotic consortium is a promising simplified model for study of multispecies mechanisms on consolidated bioprocessing and a platform for discovering efficient synergistic enzyme systems for biotechnological application.

Comparative analysis of microbial profiles in cow rumen fed with different dietary fiber by tagged 16S rRNA gene pyrosequencing.

The ruminal microbiome of cattle plays an important role not only in animal health and productivity but also in food safety and environment. Microbial profiles of rumen fluid obtained from dairy cows fed on three different fiber/starch diet compositions were characterized. Tagged 16S rRNA gene pyrosequencing and statistical analysis revealed that the dominant ruminal bacteria shared by all three sample groups belonged to phyla Bacteroidetes, Firmicutes, and Proteobacteria. However, the relative abundance of these bacterial groups was markedly affected by diet composition. In animals fed with a high fiber diet, the fibrolytic and cellulolytic bacteria Lachnospiraceae, Ruminococcaceae, and Fibrobacteraceae were found in highest abundance compared with animals fed other diets with lower fiber content. The polysaccharide-degrading Prevotellaceae and Flavobacteriaceae bacteria were most abundant in the rumen of cows fed on diet with the highest starch content. These data highlight the ruminal microbiome's ability to adapt to feed composition and also provide a basis for the development of feed formulation systems designed to improve livestock productivity.

ß-Mannanase production by Aspergillus niger BCC4525 and its efficacy on broiler performance.

BACKGROUND: Mannan is a hemicellulose constituent commonly found in plant-derived feed ingredients. The gum-like property of mannan can obstruct digestive enzymes and bile acids, resulting in impaired nutrient utilisation. In this study, ß-mannanase production by Aspergillus niger strain BCC4525 was investigated using several agricultural residues under solid state condition. The biochemical properties of the target enzyme and the effects of enzyme supplementation on broiler performance and energy utilisation were assessed. RESULTS: Among five carbon sources tested, copra meal was found to be the best carbon source for ß-mannanase production with the maximum yield of 1837.5 U g(-1) . The crude ß-mannanase exhibited maximum activity at 80 °C within a broad range of pH from 2 to 6. In vitro digestibility assay, which simulates the gastrointestinal tract system of broilers, showed that ß-mannanase could liberate reducing sugars from corn/soybean diet. Surprisingly, ß-mannanase supplementation had no significant effect on broiler feed intake, feed conversion rate or energy utilisation. CONCLUSION: A high level of ß-mannanase was produced by A. niger BCC4525 under solid state condition using copra meal as carbon source. Although the enzyme has the desired properties of an enzyme additive for improving broiler performance, it does not appear to be beneficial.

AirDNA sampler: An efficient and simple device enabling high-yield, high-quality airborne environment DNA for metagenomic applications.

Analyzing temporal and spatial distributions of airborne particles of biological origins is vital for the assessment and monitoring of air quality, especially with regard to public health, environmental ecology, and atmospheric chemistry. However, the analysis is frequently impeded by the low levels of biomass in the air, especially with metagenomic DNA analysis to explore diversity and composition of living organisms and their components in the air. To obtain sufficient amounts of metagenomic DNA from bioaerosols, researchers usually need a long sampling time with an expensive high-volume air sampler. This work shows the utilization of an air sampling device containing an economical, high-volume portable ventilation fan in combination with customized multi-sheet filter holders to effectively obtain high yields of genomic DNA in a relatively short time. The device, named 'AirDNA' sampler, performed better than other commercial air samplers, including MD8 Airport and Coriolis compact air samplers. Using the AirDNA sampler, an average DNA yield of 40.49 ng (12.47-23.24 ng at 95% CI) was obtained in only 1 hour of air sampling with a 0.85 probability of obtaining ≥10 ng of genomic DNA. The genomic DNA obtained by the AirDNA system is of suitable quantity and quality to be further used for amplicon metabarcoding sequencing of 16S, 18S, and cytochrome c oxidase I (COI) regions, indicating that it can be used to detect various prokaryotes and eukaryotes. Our results showed the effectiveness of our AirDNA sampling apparatus with a simple setup and affordable devices to obtain metagenomic DNA for short-term or long-term spatiotemporal analysis. The technique is well suited for monitoring air in built environments, especially monitoring bioaerosols for health purposes and for fine-scale spatiotemporal environmental studies.

Identification and characterization of a cellulase-encoding gene from the buffalo rumen metagenomic library.

Microorganisms residing in the rumens of cattle represent a rich source of lignocellulose-degrading enzymes, since their diet consists of plant-based materials that are high in cellulose and hemicellulose. In this study, a metagenomic library was constructed from buffalo rumen contents using pCC1FOS fosmid vector. Ninety-three clones from the pooled library of approximately 10,000 clones showed degrading activity against AZCL-HE-Cellulose, whereas four other clones showed activity against AZCL-Xylan. Contig analysis of pyrosequencing data derived from the selected strongly positive clones revealed 15 ORFs that were closely related to lignocellulose-degrading enzymes belonging to several glycosyl hydrolase families. Glycosyl hydrolase family 5 (GHF5) was the most abundant glycosyl hydrolase found, and a majority of the GHF5s in our metagenomes were closely related to several ruminal bacteria, especially ones from other buffalo rumen metagenomes. Characterization of BT-01, a selected clone with highest cellulase activity from the primary plate screening assay, revealed a cellulase encoding gene with optimal working conditions at pH 5.5 at 50 °C. Along with its stability over acidic pH, the capability efficiently to hydrolyze cellulose in feed for broiler chickens, as exhibited in an in vitro digestibility test, suggests that BT-01 has potential application as a feed supplement.

Efficient multiplex CRISPR/Cpf1 (Cas12a) genome editing system in Aspergillus aculeatus TBRC 277.

CRISPR/Cas technology is a versatile tool for genome engineering in many organisms, including filamentous fungi. Cpf1 is a multi-domain protein of class 2 (type V) RNA-guided CRISPR/Cas endonuclease, and is an alternative platform with distinct features when compared to Cas9. However, application of this technology in filamentous fungi is limited. Here, we present a single CRISPR/Cpf1 plasmid system in Aspergillus aculeatus strain TBRC 277, an industrially relevant cell factory. We first evaluated the functionality of three Cpf1 orthologs from Acidaminococcus sp. BV3L6 (AsCpf1), Francisella tularensis subsp. novicida U112 (FnCpf1), and Lachnospiraceae bacterium (LbCpf1), in RNA-guided site-specific DNA cleavage at the pksP locus. FnCpf1 showed the highest editing efficiency (93 %) among the three Cpf1s. It was further investigated for its ability to delete a 1.7 kb and a 0.5 kb from pksP and pyrG genes, respectively, using two protospacers targeting these gene loci in a single crRNA array. Lastly, simultaneous editing of three sites within TBRC 277 genome was performed using three guide sequences targeting these two genes as well as an additional gene, kusA, which resulted in combined editing efficiency of 40 %. The editing of the NHEJ pathway by targeting kusA to generate a NHEJ-deficient strain of A. aculeatus TBRC 277 improved gene targeting efficiency and yielded more precise gene-editing than that of using wild-type strain. This promising genome-editing system can be used for strain improvement in industrial applications such as production of valuable bioproducts.

Beneficial bacterial-Auricularia cornea interactions fostering growth enhancement identified from microbiota present in spent mushroom substrate.

Complex dynamic bacterial-fungal interactions play key roles during mushroom growth, ranging from mutualism to antagonism. These interactions convey a large influence on mushroom's mycelial and fruiting body formation during mushroom cultivation. In this study, high-throughput amplicon sequencing was conducted to investigate the structure of bacterial communities in spent mushroom substrates obtained from cultivation of two different groups of Auricularia cornea with (A) high yield and (B) low yield of fruiting body production. It was found that species richness and diversity of microbiota in group (A) samples were significantly higher than in group (B) samples. Among the identified 765 bacterial OTUs, 5 bacterial species found to exhibit high differential abundance between group (A) and group (B) were Pseudonocardia mangrovi, Luteimonas composti, Paracoccus pantotrophus, Sphingobium jiangsuense, and Microvirga massiliensis. The co-cultivation with selected bacterial strains showed that A. cornea TBRC 12900 co-cultivated with P. mangrovi TBRC-BCC 42794 promoted a high level of mycelial growth. Proteomics analysis was performed to elucidate the biological activities involved in the mutualistic association between A. cornea TBRC 12900 and P. mangrovi TBRC-BCC 42794. After co-cultivation of A. cornea TBRC 12900 and P. mangrovi TBRC-BCC 42794, 1,616 proteins were detected including 578 proteins of A. cornea origin and 1,038 proteins of P. mangrovi origin. Functional analysis and PPI network construction revealed that the high level of mycelial growth in the co-culture condition most likely resulted from concerted actions of (a) carbohydrate-active enzymes including hydrolases, glycosyltransferases, and carbohydrate esterases important for carbohydrate metabolism and cell wall generation/remodeling, (b) peptidases including cysteine-, metallo-, and serine-peptidases, (c) transporters including the ABC-type transporter superfamily, the FAT transporter family, and the VGP family, and (d) proteins with proposed roles in formation of metabolites that can act as growth-promoting molecules or those normally contain antimicrobial activity (e.g., indoles, terpenes, ß-lactones, lanthipeptides, iturins, and ectoines). The findings will provide novel insights into bacterial-fungal interactions during mycelial growth and fruiting body formation. Our results can be utilized for the selection of growth-promoting bacteria to improve the cultivation process of A. cornea with a high production yield, thus conveying potentially high socio-economic impact to mushroom agriculture.

Cloning and expression of recombinant shrimp PmRab7 (a virus-binding protein) in Pichia pastoris.

White spot syndrome virus (WSSV) is one of the most serious pathogens in shrimp aquaculture. A shrimp WSSV-binding protein called PmRab7 has been isolated and characterized. Since injection of bacterial expressed-rPmRab7 could reduce shrimp mortality caused by WSSV from approximately 95% to 15% mortality, there was potential for its use in protection against WSSV in shrimp aquaculture. To test the feasibility of this, the Pichia pastoris yeast expression system was used for production of rPmRab7 since its expression system has eukaryote post-translational modification capability and since P. pastoris is widely accepted for use in human food or animal feed. Moreover, ß-1,3-glucan, a major cell wall component of yeast, has been reported to act as an immunostimulant in shrimp. The recombinant protein was produced intracellularly and the resulting whole yeast cells were lyophilized and stored for supplementation in shrimp feed. The yield of rPmRab7 was 20-30 mg/l of culture medium or 2.67 mg/g yeast dry weight, which was 2-3 times higher than the yield obtained from an Escherichia coli expression system. A two-copy gene expression system was developed to enhance rPmRab7 expression using expression vector pAO815 containing a two-copy PmRab7 expression cassette constructed by site-directed mutagenesis of the PmRab7 gene and two-step overlap, extension PCR. This improved the yield of rPmRab7 2-3 times (40-60 mg/l of culture medium). ELISA was developed to show that the expressed rPmRab7 had WSSV-binding activity. Although some loss of rPmRab7 was found after lyophilization of the yeast cells, projected cost calculations indicated that this production level would make it feasible to use rPmRab7 in shrimp feed for protection against WSSV.