Revisiting chloroplast genomic landscape and annotation towards comparative chloroplast genomes of Rhamnaceae.

BACKGROUND: Massive parallel sequencing technologies have enabled the elucidation of plant phylogenetic relationships from chloroplast genomes at a high pace. These include members of the family Rhamnaceae. The current Rhamnaceae phylogenetic tree is from 13 out of 24 Rhamnaceae chloroplast genomes, and only one chloroplast genome of the genus Ventilago is available. Hence, the phylogenetic relationships in Rhamnaceae remain incomplete, and more representative species are needed. RESULTS: The complete chloroplast genome of Ventilago harmandiana Pierre was outlined using a hybrid assembly of long- and short-read technologies. The accuracy and validity of the final genome were confirmed with PCR amplifications and investigation of coverage depth. Sanger sequencing was used to correct for differences in lengths and nucleotide bases between inverted repeats because of the homopolymers. The phylogenetic trees reconstructed using prevalent methods for phylogenetic inference were topologically similar. The clustering based on codon usage was congruent with the molecular phylogenetic tree. The groups of genera in each tribe were in accordance with tribal classification based on molecular markers. We resolved the phylogenetic relationships among six Hovenia species, three Rhamnus species, and two Ventilago species. Our reconstructed tree provides the most complete and reliable low-level taxonomy to date for the family Rhamnaceae. Similar to other higher plants, the RNA editing mostly resulted in converting serine to leucine. Besides, most genes were subjected to purifying selection. Annotation anomalies, including indel calling errors, unaligned open reading frames of the same gene, inconsistent prediction of intergenic regions, and misannotated genes, were identified in the published chloroplast genomes used in this study. These could be a result of the usual imperfections in computational tools, and/or existing errors in reference genomes. Importantly, these are points of concern with regards to utilizing published chloroplast genomes for comparative genomic analysis. CONCLUSIONS: In summary, we successfully demonstrated the use of comprehensive genomic data, including DNA and amino acid sequences, to build a reliable and high-resolution phylogenetic tree for the family Rhamnaceae. Additionally, our study indicates that the revision of genome annotation before comparative genomic analyses is necessary to prevent the propagation of errors and complications in downstream analysis and interpretation.

Development of Robust Yeast Strains for Lignocellulosic Biorefineries Based on Genome-Wide Studies.

Lignocellulosic biomass has been widely studied as the renewable feedstock for the production of biofuels and biochemicals. Budding yeast Saccharomyces cerevisiae is commonly used as a cell factory for bioconversion of lignocellulosic biomass. However, economic bioproduction using fermentable sugars released from lignocellulosic feedstocks is still challenging. Due to impaired cell viability and fermentation performance by various inhibitors that are present in the cellulosic hydrolysates, robust yeast strains resistant to various stress environments are highly desired. Here, we summarize recent progress on yeast strain development for the production of biofuels and biochemical using lignocellulosic biomass. Genome-wide studies which have contributed to the elucidation of mechanisms of yeast stress tolerance are reviewed. Key gene targets recently identified based on multiomics analysis such as transcriptomic, proteomic, and metabolomics studies are summarized. Physiological genomic studies based on zinc sulfate supplementation are highlighted, and novel zinc-responsive genes involved in yeast stress tolerance are focused. The dependence of host genetic background of yeast stress tolerance and roles of histones and their modifications are emphasized. The development of robust yeast strains based on multiomics analysis benefits economic bioconversion of lignocellulosic biomass.

Functional expression of a foreign gene in Aspergillus oryzae producing new pyrone compounds.

Fungi from the genus Xylaria produce a wide range of polyketides with diverse structures, which provide important sources for pharmaceutical agents. At least seven polyketide synthase (PKS) genes, including pksmt, were found in Xylaria sp. BCC 1067. The multifunctional enzyme pksmt contains the following catalytic motifs: ß-ketosynthase (KS), acyltransferase (AT), dehydratase (DH), methyltransferase (MT), enoylreductase (ER), ketoreductase (KR), and acyl carrier region (ACP). The presence of multiple domains indicated that pksmt was an iterative type I highly-reduced-type PKS gene. To identify the gene function, pksmt was fused with a gene encoding green fluorescent protein (GFP) and introduced into a surrogate host, Aspergillus oryzae, and expressed under the control of a constitutive gpdA promoter. In the transformant, the pksmt gene was functionally expressed and translated as detected by a green fluorescence signal. This transformant produced two new 2-pyrone compounds, 4-(hydroxymethyl)-5,6-dihydro-pyran-2-one and 5-hydroxy-4-methyl-5,6-dihydro-pyran-2-one, as well as a previously identified 4-methyl-5,6-dihydro-pyran-2-one. Our results suggested that pksmt from Xylaria sp. BCC 1067 represents a family of fungal PKSs that can synthesize 2-pyrone-containing compounds.

Insights into the genome of Methylobacterium sp. NMS14P, a novel bacterium for growth promotion of maize, chili, and sugarcane.

A novel methylotrophic bacterium designated as NMS14P was isolated from the root of an organic coffee plant (Coffea arabica) in Thailand. The 16S rRNA sequence analysis revealed that this new isolate belongs to the genus Methylobacterium, and its novelty was clarified by genomic and comparative genomic analyses, in which NMS14P exhibited low levels of relatedness with other Methylobacterium-type strains. NMS14P genome consists of a 6,268,579 bp chromosome, accompanied by a 542,519 bp megaplasmid and a 66,590 bp plasmid, namely pNMS14P1 and pNMS14P2, respectively. Several genes conferring plant growth promotion are aggregated on both chromosome and plasmids, including phosphate solubilization, indole-3-acetic acid (IAA) biosynthesis, cytokinins (CKs) production, 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, sulfur-oxidizing activity, trehalose synthesis, and urea metabolism. Furthermore, pangenome analysis showed that NMS14P possessed the highest number of strain-specific genes accounting for 1408 genes, particularly those that are essential for colonization and survival in a wide array of host environments, such as ABC transporter, chemotaxis, quorum sensing, biofilm formation, and biosynthesis of secondary metabolites. In vivo tests have supported that NMS14P significantly promoted the growth and development of maize, chili, and sugarcane. Collectively, NMS14P is proposed as a novel plant growth-promoting Methylobacterium that could potentially be applied to a broad range of host plants as Methylobacterium-based biofertilizers to reduce and ultimately substitute the use of synthetic agrochemicals for sustainable agriculture.

Soil microbiome analysis reveals effects of periodic waterlogging stress on sugarcane growth.

Sugarcane is one of the major agricultural crops with high economic importance in Thailand. Periodic waterlogging has a long-term negative effect on sugarcane development, soil properties, and microbial diversity, impacting overall sugarcane production. Yet, the microbial structure in periodically waterlogged sugarcane fields across soil compartments and growth stages in Thailand has not been documented. This study investigated soil and rhizosphere microbial communities in a periodic waterlogged field in comparison with a normal field in a sugarcane plantation in Ratchaburi, Thailand, using 16S rRNA and ITS amplicon sequencing. Alpha diversity analysis revealed comparable values in periodic waterlogged and normal fields across all growth stages, while beta diversity analysis highlighted distinct microbial community profiles in both fields throughout the growth stages. In the periodic waterlogged field, the relative abundance of Chloroflexi, Actinobacteria, and Basidiomycota increased, while Acidobacteria and Ascomycota decreased. Beneficial microbes such as Arthrobacter, Azoarcus, Bacillus, Paenibacillus, Pseudomonas, and Streptomyces thrived in the normal field, potentially serving as biomarkers for favorable soil conditions. Conversely, phytopathogens and growth-inhibiting bacteria were prevalent in the periodic waterlogged field, indicating unfavorable conditions. The co-occurrence network in rhizosphere of the normal field had the highest complexity, implying increased sharing of resources among microorganisms and enhanced soil biological fertility. Altogether, this study demonstrated that the periodic waterlogged field had a long-term negative effect on the soil microbial community which is a key determining factor of sugarcane growth.

Characterization of the Gut Microbiota in Urban Thai Individuals Reveals Enterotype-Specific Signature.

Gut microbiota play vital roles in human health, utilizing indigestible nutrients, producing essential substances, regulating the immune system, and inhibiting pathogen growth. Gut microbial profiles are dependent on populations, geographical locations, and long-term dietary patterns resulting in individual uniqueness. Gut microbiota can be classified into enterotypes based on their patterns. Understanding gut enterotype enables us to interpret the capability in macronutrient digestion, essential substance production, and microbial co-occurrence. However, there is still no detailed characterization of gut microbiota enterotype in urban Thai people. In this study, we characterized the gut microbiota of urban Thai individuals by amplicon sequencing and classified their profiles into enterotypes, including Prevotella (EnP) and Bacteroides (EnB) enterotypes. Enterotypes were associated with lifestyle, dietary habits, bacterial diversity, differential taxa, and microbial pathways. Microbe-microbe interactions have been studied via co-occurrence networks. EnP had lower α-diversities than those in EnB. A correlation analysis revealed that the Prevotella genus, the predominant taxa of EnP, has a negative correlation with α-diversities. Microbial function enrichment analysis revealed that the biosynthesis pathways of B vitamins and fatty acids were significantly enriched in EnP and EnB, respectively. Interestingly, Ruminococcaceae, resistant starch degraders, were the hubs of both enterotypes, and strongly correlated with microbial diversity, suggesting that traditional Thai food, consisting of rice and vegetables, might be the important drivers contributing to the gut microbiota uniqueness in urban Thai individuals. Overall findings revealed the biological uniqueness of gut enterotype in urban Thai people, which will be advantageous for developing gut microbiome-based diagnostic tools.

Media optimization of antimicrobial activity production and beta-glucan content of endophytic fungi Xylaria sp. BCC 1067.

Fungi is a notable asset for drug discovery and production of pharmaceuticals; however, slow growth and poor product yields have hindered industrial utilization. Here, the mycelial biomass of Xylaria sp. BCC 1067 was examined in parallel with the assessment of antimicrobial properties by using media-type selection. To enhance both mycelial content and antifungal activity, the media replacement approach was successfully applied to stimulate fungal growth and successively switched to poorer malt-peptone extract media for metabolite production. This simple optimization reduced fungal cultivation time by 7 days and yielded 4-fold increased mycelial mass (32.59 g/L), with approximately 3-fold increased antifungal activity against the model yeast Saccharomyces cerevisiae strain. A high level of ß-glucan (115.84 mg/g of cell dry weight) and additive antibacterial effect against Propionibacterium acnes were also reported. This simple strategy of culture media optimization allows for investigation of novel and rich source of health-promoting substances for effective microbial utilization.

Comparative Metagenomics Reveals Microbial Signatures of Sugarcane Phyllosphere in Organic Management.

Converting conventional farms to organic systems to improve ecosystem health is an emerging trend in recent decades, yet little is explored to what extent and how this process drives the taxonomic diversity and functional capacity of above-ground microbes. This study was, therefore, conducted to investigate the effects of agricultural management, i.e., organic, transition, and conventional, on the structure and function of sugarcane phyllosphere microbial community using the shotgun metagenomics approach. Comparative metagenome analysis exhibited that farming practices strongly influenced taxonomic and functional diversities, as well as co-occurrence interactions of phyllosphere microbes. A complex microbial network with the highest connectivity was observed in organic farming, indicating strong resilient capabilities of its microbial community to cope with the dynamic environmental stressors. Organic farming also harbored genus Streptomyces as the potential keystone species and plant growth-promoting bacteria as microbial signatures, including Mesorhizobium loti, Bradyrhizobium sp. SG09, Lactobacillus plantarum, and Bacillus cellulosilyticus. Interestingly, numerous toxic compound-degrading species were specifically enriched in transition farming, which might suggest their essential roles in the transformation of conventional to organic farming. Moreover, conventional practice diminished the abundance of genes related to cell motility and energy metabolism of phyllosphere microbes, which could negatively contribute to lower microbial diversity in this habitat. Altogether, our results demonstrated the response of sugarcane-associated phyllosphere microbiota to specific agricultural managements that played vital roles in sustainable sugarcane production.

Draft Genome Sequence of the Wood-Decaying Fungus Xylaria sp. BCC 1067.

Xylaria sp. BCC 1067 is a wood-decaying fungus which is capable of producing lignocellulolytic enzymes. Based on the results of a single-molecule real-time sequencing technology analysis, we present the first draft genome of Xylaria sp. BCC 1067, comprising 54.1 Mb with 12,112 protein-coding genes.

Production of the polyketide 6-MSA in yeast engineered for increased malonyl-CoA supply.

The heterologous production of fungal polyketides was investigated using 6-methylsalicylic acid synthase (6-MSAS) as a model polyketide synthase and Saccharomyces cerevisiae as a host. In order to improve the production of 6-MSA by enhancing the supply of precursors, the promoter of the gene (ACC1) encoding acetyl-CoA carboxylase, which catalyzes the conversion of acetyl-CoA to malonyl-CoA, was replaced with a strong, constitutive promoter (TEF1p) in a strain harboring two plasmids carrying the genes encoding 6-MSAS from Penicillium patulum and PPTase from Aspergillus nidulans, respectively. The strain was characterized in batch cultivations with a glucose minimal media (20 g/L), and a 60% increase in 6-MSA titer was observed compared to a strain having the native promoter in front of ACC1. The production of 6-MSA was scaled up by the cultivation in minimal media containing 50 g/L of glucose, and hereby a final titer of 554+/-26 mg/L of 6-MSA was obtained.

The reducing clade IIb polyketide synthase PKS14 acts as a virulence determinant of the entomopathogenic fungus Beauveria bassiana.

The reducing clade IIb polyketide synthase gene, pks14, is preserved throughout the evolution of entomopathogenic fungi. We examined the functions of pks14 in Beauveria bassiana using targeted gene disruption, and pks14 disruption was verified by Southern blot and PCR analyses. The radial growth, cell dry weight and conidial germination of Δpks14 were comparable to that of the wild type. Our sequence and gene expression analyses of the pks14 biosynthetic cluster demonstrated: (i) cotranscription and constitutive expression of nearly all the genes of the aforementioned cluster including the C2H2 zinc finger transcription regulator gene, but not pks14 and the cytochrome P450 gene; (ii) expression of the pks14 gene in the insect-containing culture condition only; and (iii) a KAR9-like gene in direct proximity with pks14 is the only gene showing co-regulation. The Δpks14-infected Spodoptera exigua larvae survived significantly longer than those infected by the wild type, indicating a marked reduction in the virulence of Δpks14 against the insect. LT50 of Δpks14 was increased by 1.55 days. Hyphal body formation was decreased in the hemolymph of insects infected by Δpks14 as compared with those inoculated by the wild type. Our results suggest that PKS14-catalyzed polyketide enhances virulence and pathogenicity of B. bassiana on insects.

Culture degeneration in conidia of Beauveria bassiana and virulence determinants by proteomics.

The quality of Beauveria bassiana conidia directly affects the virulence against insects. In this study, continuous subculturing of B. bassiana on both rice grains and potato dextrose agar (PDA) resulted in 55 and 49 % conidial yield reduction after 12 passages and 68 and 60 % virulence reduction after 20 and 12 passages at four d post-inoculation, respectively. The passage through Tenebrio molitor and Spodoptera exigua restored the virulence of rice and PDA subcultures, respectively. To explore the molecular mechanisms underlying the conidial quality and the decline of virulence after multiple subculturing, we investigated the conidial proteomic changes. Successive subculturing markedly increased the protein levels in oxidative stress response, autophagy, amino acid homeostasis, and apoptosis, but decreased the protein levels in DNA repair, ribosome biogenesis, energy metabolism, and virulence. The nitro blue tetrazolium assay verified that the late subculture's colony and conidia had a higher oxidative stress level than the early subculture. A 2A-type protein phosphatase and a Pleckstrin homology domain protein Slm1, effector proteins of the target of rapamycin (TOR) complex 1 and 2, respectively, were dramatically increased in the late subculture. These results suggest that TOR signalling might be associated with ageing in B. bassiana late subculture, in turn affecting its physiological characteristics and virulence.

Fungicide Xylaria sp. BCC 1067 extract induces reactive oxygen species and activates multidrug resistance system in Saccharomyces cerevisiae.

AIM: To investigate antifungal potential of Xylaria sp. BIOTEC culture collection (BCC) 1067 extract against the model yeast Saccharomyces cerevisiae. MATERIALS & METHODS: Antifungal property of extract, reactive oxygen species levels and cell survival were determined, using selected deletion strains. RESULTS: Extract showed promising antifungal effect with minimal inhibitory concentration100 and minimal fungicidal concentration of 500 and 1000 mg/l, respectively. Strong synergy was observed with fractional inhibitory concentration index value of 0.185 for the combination of 60.0 and 0.5 mg/l of extract and ketoconazole, respectively. Extract-induced intracellular reactive oxygen species levels in some oxidant-prone strains and mediated plasma membrane rupture. Antioxidant regulator Yap1, efflux transporter Pdr5 and ascorbate were pivotal to protect S. cerevisiae from extract cytotoxicity. CONCLUSION: Xylaria sp. BCC 1067 extract is a potentially valuable source of novel antifungals.

In silico analysis of plant and animal transposable elements.

Transposable Elements (TEs) play important roles in the evolution of eukaryotic organisms. TEs widely distribute depending on their properties present in the genome. This study elucidated the molecular characteristics of TEs in land plants and animals using bioinformatics and in silico mutational approach. We discovered that the GC-rich class I TEs is the predominant class of TEs in animal, but the AT-rich class II TEs is prevalent in plants. The GC-rich class I TEs appears to be evolved within the animals. On contrary, the preserved in AT-rich in class II TEs is believed to be contributed in host defence systems.

Assessing medium constituents for optimal heterologous production of anhydromevalonolactone in recombinant Aspergillus oryzae.

Anhydromevalonolactone (AMVL) is a bioactive natural product that arises from a molecular biology technique using Aspergillus oryzae as a heterologous host. AMVL has been used as a precursor for the synthesis of insect pest control reagents and has numerous applications in the biotechnological and medical industries. In this study, the Plackett-Burman Design and the Central Composite Design, which offer efficient and feasible approaches, were complemented to screen significant parameters and identify the optimal values for maximum AMVL production. The results suggested that sucrose, NaNO3, yeast extract and K2HPO4 were the key factors affecting AMVL production in a complex medium, whereas the major components required for a defined medium were NaNO3, K2HPO4, KH2PO4 and trace elements. These factors were subsequently optimized using the response surface methodology. Under optimal conditions, a maximum AMVL production of 250 mg/L in the complex medium and 200 mg/L in the defined medium was achieved, which represents an increase of approximately 3-4-fold compared to the commonly used malt extract medium.

Optimization of heterologous production of the polyketide 6-MSA in Saccharomyces cerevisiae.

Polyketides are a group of natural products that have gained much interest due to their use as antibiotics, cholesterol lowering agents, immunosuppressors, and as other drugs. Many organisms that naturally produce polyketides are difficult to cultivate and only produce these metabolites in small amounts. It is therefore of general interest to transfer polyketide synthase (PKS) genes from their natural sources into heterologous hosts that can over-produce the corresponding polyketides. In this study we demonstrate the heterologous expression of 6-methylsalicylic acid synthase (6-MSAS), naturally produced by Penicillium patulum, in the yeast Saccharomyces cerevisiae. In order to activate the PKS a 4'-phosphopantetheinyl transferase (PPTase) is required. We therefore co-expressed PPTases encoded by either sfp from Bacillus subtilis or by npgA from Aspergillus nidulans. The different strains were grown in batch cultures. Growth and product concentration were measured and kinetic parameters were calculated. It was shown that both PPTases could be efficiently used for activation of PKS's in yeast as good yields of 6-MSA were obtained with both enzymes.