Efficient secretion of heterologous proteins is essential for advancing yeast-based bioprocesses, yet signal peptide (SP) optimization in the thermotolerant methylotrophic yeast Ogataea polymorpha remains limited. This study integrates in-silico SP discovery, experimental validation, and bioprocess engineering to enhance secretion of the thermophilic xylanase X11P under sucrose-inducible expression. Genome-wide screening of 5184 O. polymorpha proteins using SignalP, Phobius, DeepLoc, WoLF PSORT, and ProP identified 11 high-confidence SP candidates. Comparative analysis with Komagataella phaffii endogenous proteins guided selection of seven SPs for experimental evaluation. Among these, the novel O. polymorpha α-mating factor-like peptide FUN_005010 exhibited strong secretion-promoting activity, with its prepro-sequence yielding the highest extracellular xylanase levels and outperforming the classical Saccharomyces cerevisiae α-MF. To evaluate industrial applicability, sucrose-based fermentation strategies were systematically optimized in a 5-L bioreactor. Controlled sucrose feeding and balanced C/N ratios were found to be critical for maximizing maltase (MAL) promoter-driven expression. A stepwise increasing sucrose feed combined with induction at 30°C enabled X11P titers up to 770 U/mL, representing a 15-fold improvement over shake-flask cultures. This work demonstrates that the combination of SP evaluation and optimized sucrose-inducible fed-batch operation significantly enhances X11P production in O. polymorpha. The identified FUN_005010-prepro SP and the refined process framework provide valuable tools for developing O. polymorpha as a high-performance industrial expression platform.

Two Gram-stain-negative, aerobic, non-motile, non-gliding, rod-shaped bacterial strains, designated as TBRC 19031T and TBRC 19032, were isolated from water samples collected from the Mekong River, Thailand. Strain TBRC 19031T was obtained from Chiang Saen in the upstream section near the borders with China and Myanmar, while TBRC 19032 originated from Khong Chiam, in the downstream section where the river exits Thailand. Colonies of both strains were circular, smooth and deep yellow on Reasoner's 2A agar and did not produce flexirubin-type pigments. Phylogenetic analysis with 16S rRNA gene sequences placed both strains within the genus Flavobacterium, showing the highest sequence similarity to Flavobacterium cheonhonense ARSA-15T (98.29% for TBRC 19031T and 98.22% for TBRC 19032). However, whole-genome comparisons between the strains and F. cheonhonense ARSA-15T revealed average nt identity (89.39% and 89.29%), average aa identity (92.84% and 92.95%) and digital DNA-DNA hybridization (35.00% and 34.70%). The predominant fatty acids were iso-C15:1, iso-C15:0 and iso-C15:0 3-OH, and menaquinone MK-6 was the major respiratory quinone. The major polar lipids of both strains included phosphatidylethanolamine, steryl ester and diacylglycerol. The genome sizes were 3.02 and 3.04 Mbp, with G+C contents of 38.3% and 38.2% for TBRC 19031T and TBRC 19032, respectively. Comparative genomic analyses revealed the absence of genes involved in sulphate reduction and denitrification pathways and the presence of a gene encoding phosphatidylinositol synthase, distinguishing them from other Flavobacterium within the clade. Ecological profiling using public metagenomic datasets showed that both strains were associated with lotic freshwater environments. This study not only introduces Flavobacterium mekongense sp. nov. as a new species but also provides broader insights into the ecology, metabolism and environmental distribution of freshwater Flavobacterium. The genomic features identified here offer promising leads for future studies in microbial ecology, comparative genomics and functional gene mining in aquatic ecosystems. The type strain is TBRC 19031T (TBRC 19031T=NBRC 117006T).

The thermotolerant yeast Ogataea polymorpha TBRC 4839 is a promising host for heterologous protein expression using sucrose and molasses as low-cost carbon sources, making it suitable for industrial applications. This study analyzed the genome and transcriptome of O. polymorpha under sucrose-induced conditions. The nuclear genome of strain TBRC 4839 measures 8.9 Mbp with a GC content of 47.87%, consistent with other Ogataea species. The genome encodes 5184 protein-coding genes, comparable to related strains. Additionally, the mitochondrial genome spans 49.4 Kbp and has a low GC content of approximately 20%. Transcriptomic analysis revealed that sucrose induction triggers a metabolic shift characterized by increased carbohydrate metabolism and decreased amino acid biosynthesis, stress signaling, and cell division, enabling efficient energy utilization in sucrose-rich environments. Among the identified genes with up-regulated expression, five were notable: FUN_000066 (hypothetical protein), FUN_001144 (maltose permease), FUN_001145 (maltase), FUN_002060 (mitochondrial NAD-dependent malic enzyme), and FUN_002263 (hypothetical protein). The promoter efficiency was evaluated by expressing the fungal xylanase gene under sucrose-inducing conditions using these promoters. The maltase (MAL) promoter exhibited the highest xylanase production efficiency, outperforming other promoters. Furthermore, the MAL promoter proved effective for xylanase production when molasses was used as the carbon source. These findings underscore the potential of O. polymorpha TBRC 4839 and the MAL promoter for industrial protein production.

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.

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.

Culture-independent and culture-dependent approaches were employed to investigate the taxonomic diversity and biosynthetic gene cluster potential of Actinomycetota in the Mekong River. Through 16S rRNA gene metabarcoding, 21,103 OTUs were revealed to represent 190 genera and at least 595 species of Actinomycetota, including putatively novel taxa. Conventional and in situ cultivation (IC) methods provided 75 Actinomycetota isolates representing 72 species from 21 genera. Of these, 45 species in 4 genera were new to the Thailand Bioresource Research Center (TBRC), a collection of 20,079 Actinomycetota strains from 660 species. Applying both culture-independent and culture-dependent approaches to the same sample revealed greater diversity among the Actinomycetota in the Mekong River than one approach alone.

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, β-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.

The thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC 656 is a potential host strain for industrial protein production. Heterologous proteins are often retained intracellularly in yeast resulting in endoplasmic reticulum (ER) stress and poor secretion, and despite efforts to engineer protein secretory pathways, heterologous protein production is often lower than expected. We hypothesized that activation of genes involved in the secretory pathway could mitigate ER stress. In this study, we created mutants defective in protein secretory-related functions using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) tools. Secretion of the model protein xylanase was significantly decreased in loss of function mutants for oxidative stress (sod1Δ) and vacuolar and protein sorting (vps1Δ and ypt7Δ) genes. However, xylanase secretion was unaffected in an autophagy related atg12Δ mutant. Then, we developed a system for sequence-specific activation of target gene expression (CRISPRa) in O. thermomethanolica and used it to activate SOD1, VPS1 and YPT7 genes. Production of both non-glycosylated xylanase and glycosylated phytase was enhanced in the gene activated mutants, demonstrating that CRISPR-Cas9 systems can be used as tools for understanding O. thermomethanolica genes involved in protein secretion, which could be applied for increasing heterologous protein secretion in this yeast.

Environmental microbiomes encompass massive biodiversity and genetic information with a wide-ranging potential for industrial and agricultural applications. Knowledge of the relationship between microbiomes and environmental factors is crucial for translating that information into practical uses. In this study, the integrated data of Southeast Asian soil bacteriomes were used as models to assess the variation in taxonomic and functional diversity of bacterial communities. Our results demonstrated that there were differences in soil bacteriomes across different geographic locality with different soil characteristics: soil class and pH level. Such differences were observed in taxonomic diversity, interspecific association patterns, and functional diversity of soil bacteriomes. The bacterial-mediated biogeochemical cycles of nitrogen, sulfur, carbon, and phosphorus illustrated the functional relationship of soil bacteriome and soil characteristics, as well as an influence from bacterial interspecific interaction. The insights from this study reveal the importance of microbiome data integration for future microbiome research.

The thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC656 is a potential host for heterologous protein production. However, overproduction of heterologous protein can induce cellular stress and limit the level of its secretion. To improve the secretion of heterologous protein, we identified the candidate proteins with altered production during production of heterologous protein in O. thermomethanolica by using a label-free comparative proteomic approach. Four hundred sixty-four proteins with various biological functions showed differential abundance between O. thermomethanolica expressing fungal xylanase (OT + Xyl) and a control strain. The induction of proteins in transport and proteasomal proteolysis was prominently observed. Eight candidate proteins involved in cell wall biosynthesis (Chs3, Gas4), chaperone (Sgt2, Pex19), glycan metabolism (Csf1), protein transport (Ypt35), and vacuole and protein sorting (Cof1, Npr2) were mutated by a CRISPR/Cas9 approach. An Sgt2 mutant showed higher phytase and xylanase activity compared with the control strain (13%-20%), whereas mutants of other genes including Cof1, Pex19, Gas4, and Ypt35 showed lower xylanase activity compared with the control strain (15%-25%). In addition, an Npr2 mutant showed defective growth, while overproduction of Npr2 enhanced xylanase activity. These results reveal genes that can be mutated to modulate heterologous protein production and growth of O. thermomethanolica TBRC656.

In yeasts, Hac1 transcription factor of the unfolded protein response (UPR) regulates many genes involved in secretory pathways. The thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC656 is a host for heterologous protein secretion. To understand the role of OtHac1 on the secretome of O. thermomethanolica, a comparative proteomic analysis using LC–MS/MS was employed to identify proteins with altered secretion levels when OtHac1 was mutated. 268 proteins were detected in the extracellular medium of O. thermomethanolica wild-type control and Othac1 mutant strains. A number of metabolic enzymes functioning in amino acid, carbohydrate, glycan, and lipid metabolism showed altered secretion in the mutant suggesting that OtHac1 may play a role in mediating extracellular metabolism. Most of the extracellular proteins identified do not contain canonical signal sequences suggesting that they are secreted via unconventional protein secretion pathways. Collectively, the data provide insights into protein secretion and OtHac1 function in O. thermomethanolica which will be useful for developing efficient host for protein production.

Ogataea thermomethanolica TBRC656 is a thermotolerant methylotrophic yeast suitable for heterologous protein expression at various temperatures. However, the lack of efficient methods for targeted gene mutagenesis limits strain engineering in this yeast. In this study, we applied a CRISPR-Cas9-based tool for targeted gene mutagenesis in O. thermomethanolica. The putative unfolded protein response regulator OtHAC1, and the OtMAL1 (maltase) and OtMAL2 (maltose permease) genes involved with sucrose and maltose utilization were targeted for CRISPR-Cas9 mutagenesis. Plasmids were constructed for integrative and episomal expression of CRISPR-Cas9 elements in O. thermomethanolica in which Cas9 and gRNA are transcribed from the alcohol oxidase (AOX) promoter. The expression of these genome-editing elements is controlled by derepression with glycerol and gRNA are flanked by self-cleaving ribozymes. For integrative system, OtHAC1, OtMAL1 and OtMAL2 were disrupted at 63%, 97% and 93%, respectively. In addition, OtMAL1 was also disrupted with episomal system at 92%. These findings indicate that the CRISPR-Cas9 system described herein is thus applicable for studying gene function and strain engineering in yeast O. thermomethanolica.

In yeast, the accumulation of unfolded proteins in the ER triggers the unfolded protein response (UPR) pathway, which is mediated by Hac1 transcription factor. Here, we characterized the function of a gene encoding Hac1 in the thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC656 (OtHAC1). OtHAC1 mRNA contains a non-canonical intron of 176 nt, which was demonstrated to be spliced by RT-PCR. To characterize the function of this gene, we compared the proteome of a Othac1 mutant with wild-type. A total of 463 proteins with differential abundance were detected. The functions of these proteins were annotated in oxidative stress, metabolic pathways, transcription, translation, and of particular interest in secretory pathway. While many intracellular proteins differentially expressed in the mutant were similar to proteins with altered expression in UPR-stressed Saccharomyces cerevisiae, two novel OtHAC1-dependent proteins (Iml1 and Npr2) were identified that are potentially involved in the regulation of autophagy. The data show that OtHAC1 is an important regulator of several different processes in O. thermomethanolica TBRC656.

The high production cost of bio-based plastic polyhydroxyalkanoates (PHAs) limits their use as commercial products. Thus, systems for PHAs production from waste substrates could reduce production costs. Crude glycerol is a by-product of biodiesel fuel production and thus represents an inexpensive, abundant and promising carbon source for production of valorized fermentation products. In this study, industrial crude glycerol by-product from palm oil biodiesel production was used as the carbon source for production of PHAs by recombinant Escherichia coli. Crude glycerol supplemented at 1–5 % (v/v) supported production of poly(3-hydroxybutyrate) (P(3HB)) in E. coli-ABC Ah , which harbors the PHA synthetic genes for β-ketothiolase (PhaA Re ), acetoacetyl-CoA reductase (PhaB Re ) of Ralstonia eutropha and Polyhydroxyalkanoate (PHA) synthase (PhaC Ah ) of Aeromonas hydrophila. The highest P(3HB) content and productivity of 14 wt% of cell dry weight and 0.6 g/L, respectively, were obtained at 1 % (v/v) glycerol concentration. Production of P(3HB-co-3-hydroxyhexanoate) (P(3HB-co-3HHx)) was achieved using E. coli-ABC Ah J Ah , harboring genes for PhaA Re , PhaB Re , PhaC Ah , and the (R)-specific enoyl-CoA hydratase (PhaJ Ah ) of A. hydrophila. This led to the copolymer content of 3 wt% of cell dry weight with 1 mol% of 3HHx. Molecular weight and degradation temperature of the polymers were in the range of 110–130 kDa and 295–299 °C, respectively. These results indicated that crude glycerol could be an attractive carbon source for economical production of PHAs with properties for industrial application.

Short-chain-length medium-chain-length polyhydroxyalkanoate (SCL-MCL PHA) copolymers are promising as bio-plastics with properties ranging from thermoplastics to elastomers. In this study, the hybrid pathway for the biosynthesis of SCL-MCL PHA copolymers was established in recombinant Escherichia coli by co-expression of β-ketothiolase (PhaARe) and NADPH-dependent acetoacetyl-CoA reductase (PhaBRe) from Ralstonia eutropha together with PHA synthases from R. eutropha (PhaCRe), Aeromonas hydrophila (PhaCAh), and Pseudomonas putida (PhaC2Pp) and with (R)-specific enoyl-CoA hydratases from P. putida (PhaJ1Pp and PhaJ4Pp), and A. hydrophila (PhaJAh). When glycerol supplemented with dodecanoate was used as primary carbon source, E. coli harboring various combinations of PhaABCJ produced SCL-MCL PHA copolymers of various monomer compositions varying from C4 to C10. In addition, polymer property analysis suggested that the copolymers produced from this recombinant source have thermal properties (lower glass transition and melting temperatures) superior to polyhydroxybutyrate homopolymer.

A polyhydroxyalkanote depolymerase gene from Thermobifida sp. isolate BCC23166 was cloned and expressed as a C-terminal His6-tagged fusion in Pichia pastoris. Primary structure analysis revealed that the enzyme PhaZ-Th is a member of a proposed new subgroup of SCL-PHA depolymerase containing a proline–serine repeat linker. PhaZ-Th was expressed as two glycosylated forms with apparent molecular weights of 61 and 70 kDa, respectively. The enzyme showed esterase activity toward p-nitrophenyl alkanotes with V max and K m of 3.63 ± 0.16 μmol min−1 mg−1 and 0.79 ± 0.12 mM, respectively, on p-nitrophenyl butyrate with optimal activity at 50–55°C and pH 7–8. Surface plasmon resonance (SPR) analysis demonstrated that PhaZ-Th catalyzed the degradation of poly-[(R)-3-hydroxybutyrate] (PHB) films, which was accelerated in (R)-3-hydroxyvalerate copolymers with a maximum degradation rate of 882 ng cm−2 h−1 for poly[(R)-3-hydroxybutyrate-co-3-hydroxyvalerate] (12 mol% V). Surface deterioration, especially on the amorphous regions of PHB films was observed after exposure to PhaZ-Th by atomic force microscopy. The use of P. pastoris as an alternative recombinant system for bioplastic degrading enzymes in secreted form and a sensitive SPR analytical technique will be of utility for further study of bioplastic degradation.

A thermophilic Streptomyces sp. capable of degrading various aliphatic polyesters was isolated from a landfill site. The isolate, Streptomyces sp. BCC23167, demonstrated rapid aerobic degradation of several polyesters, including polyhydroxyalkanoate copolymers, poly(ɛ-caprolactone) and polybutylene succinate at 50°C and neutral pH. The degrading activity was repressed by glucose and cellobiose, but tolerant to repression by other carbon substrates. Degradation of a commercial poly[(R)-3-hydroxybutyrate-co-3-hydroxyhexanoate] (PHBHx) by Streptomyces sp. BCC23167 progressed from surface to bulk as suggested by the slight decrease in polymer molecular weight. Differential scanning calorimetry analysis of PHBHx film degradation by Streptomyces sp. BCC23167 showed that relative crystallinity of the film increased slightly in the early stage of degradation, followed by a marked decrease later on. The surface morphology of degraded films was analyzed by scanning electron microscopy, which showed altered surface structure consistent with the changes in crystallinity. The isolate is thus of potential for application in composting technology for bio-plastic degradation.