Improving furfural tolerance in a xylose-fermenting yeast Spathaspora passalidarum CMUWF1-2 via adaptive laboratory evolution.

BACKGROUND: Spathaspora passalidarum is a yeast with the highly effective capability of fermenting several monosaccharides in lignocellulosic hydrolysates, especially xylose. However, this yeast was shown to be sensitive to furfural released during pretreatment and hydrolysis processes of lignocellulose biomass. We aimed to improve furfural tolerance in a previously isolated S. passalidarum CMUWF1-2, which presented thermotolerance and no detectable glucose repression, via adaptive laboratory evolution (ALE). RESULTS: An adapted strain, AF2.5, was obtained from 17 sequential transfers of CMUWF1-2 in YPD broth with gradually increasing furfural concentration. Strain AF2.5 could tolerate higher concentrations of furfural, ethanol and 5-hydroxymethyl furfuraldehyde (HMF) compared with CMUWF1-2 while maintaining the ability to utilize glucose and other sugars simultaneously. Notably, the lag phase of AF2.5 was 2 times shorter than that of CMUWF1-2 in the presence of 2.0 g/l furfural, which allowed the highest ethanol titers to be reached in a shorter period. To investigate more in-depth effects of furfural, intracellular reactive oxygen species (ROS) accumulation was observed and, in the presence of 2.0 g/l furfural, AF2.5 exhibited 3.41 times less ROS accumulation than CMUWF1-2 consistent with the result from nuclear chromatins diffusion, which the cells number of AF2.5 with diffuse chromatins was also 1.41 and 1.24 times less than CMUWF1-2 at 24 and 36 h, respectively. CONCLUSIONS: An enhanced furfural tolerant strain of S. passalidarum was achieved via ALE techniques, which shows faster and higher ethanol productivity than that of the wild type. Not only furfural tolerance but also ethanol and HMF tolerances were improved.

Nonomuraea corallina sp. nov., isolated from coastal sediment in Samila Beach, Thailand: insights into secondary metabolite synthesis as anticancer potential.

A novel marine actinomycete, designated strain MCN248T, was isolated from the coastal sediment in Songkhla Province, Thailand. Based on the 16S rRNA gene sequences, the new isolate was closely related to Nonomuraea harbinensis DSM45887T (99.2%) and Nonomuraea ferruginea DSM43553T (98.6%). Phylogenetic analyzes based on the 16S rRNA gene sequences showed that strain MCN248T was clustered with Nonomuraea harbinensis DSM45887T and Nonomuraea ferruginea DSM43553T. However, the digital DNA-DNA hybridization analyzes presented a low relatedness of 40.2% between strain MCN248T and the above closely related strains. This strain contained meso-diaminopimelic acid. The acyl type of the peptidoglycan was acetyl, and mycolic acids were absent. The major menaquinones were MK-9(H2) and MK-9(H4). The whole cell sugars consisted of madurose, ribose, mannose, and glucose. Diphosphatidylglycerol, hydroxyl-phosphatidylethanolamine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylglycerol were detected as the major phospholipids. The predominant cellular fatty acids were iso-C16:0 (40.4%), 10-methyl-C17:0 (22.1%), and C17:1ω8c (10.9%). The DNA G + C content of the genomic DNA was 71.7%. With in silico analyzes, the antiSMASH platform uncovered a diverse 29 secondary metabolite biosynthesis arsenal, including non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) of strain MCN248T, with a high prevalence of gene cluster encoding pathways for the production of anticancer and cytotoxic compounds. Consistently, the crude extract could inhibit colorectal HCT-116 cancer cells at a final concentration of 50 µg/mL. Based on the polyphasic approach, strain MCN248 was designated as a novel species of the genus Nonomuraea, for which the name Nonomuraea corallina sp. nov. is proposed. The type strain of the type species is MCN248T (=NBRC115966T = TBRC17110T).

Bioactive Metabolites from Terrestrial and Marine Actinomycetes.

Actinomycetes inhabit both terrestrial and marine ecosystems and are highly proficient in producing a wide range of natural products with diverse biological functions, including antitumor, immunosuppressive, antimicrobial, and antiviral activities. In this review, we delve into the life cycle, ecology, taxonomy, and classification of actinomycetes, as well as their varied bioactive metabolites recently discovered between 2015 and 2023. Additionally, we explore promising strategies to unveil and investigate new bioactive metabolites, encompassing genome mining, activation of silent genes through signal molecules, and co-cultivation approaches. By presenting this comprehensive and up-to-date review, we hope to offer a potential solution to uncover novel bioactive compounds with essential activities.

Potential antihypertensive activity of novel peptides from green basil leaves.

Hypertension is among the risk factors of death globally. Novel antihypertensive peptides are alternative choices of antihypertensive assistance. This study aimed to discover novel antihypertensive peptides from green basil leaves. Two bioactive peptides with high angiotensin-converting enzyme inhibition (Asp-Leu-Ser-Ser-Ala-Pro; peptide 1) and antioxidant (Asp-Ser-Val-Ser-Ala-Ser-Pro; peptide 2) activities were gavaged to male Wistar rats induced with NG-nitro-l-arginine methyl-ester (L-NAME). L-NAME-treated rats (HT) had decreased body weights and levels of nitrite and nitrate, which are metabolites of nitric oxide. The levels of their glucose and liver function indicators increased as compared to normal rats. HT rats receiving antihypertensive drugs (HTD) showed higher low-density lipoprotein and low-density lipoprotein/high-density lipoprotein levels than HT rats. Peptide 1 seems to benefit the rat lipid profiles, liver functions, antioxidant, nitrite, nitrate, and angiotensin II peptide levels but not peptide 2. In conclusion, our findings indicate the antihypertensive potential related to vasodilation of peptides from green basil leaves.

Amycolatopsis iheyensis sp. nov., isolated from soil on Iheya Island, Japan.

A novel actinomycete, designated strain OK19-0408T, was isolated from soil collected on Iheya island, Okinawa prefecture, Japan. Using the polyphasic taxonomic approach, comparing 16S rRNA gene sequences, the new isolate was found to be most closely related to Amycolatopsis vancoresmycina JCM12675T (98.71 %). Phylogenetic analyses using 16S rRNA sequences indicated that strain OK19-0408T was clustered with Amycolatopsis australiensis JCM15587T. However, digital DNA-DNA hybridization analyses indicated a low relatedness, in the range of 33.9-34.7 %, between strain OK19-0408T and these closely related strains. Strain OK19-0408T contained meso-diaminopimelic acid and whole-cell sugars consisting of arabinose and galactose. The acyl type of the peptidoglycan was acetyl and mycolic acids were absent in strain OK19-0408T. The major menaquinone was MK-9(H4) and hydroxy-phosphatidylethanolamine was detected as the predominant phospholipid. The predominant cellular fatty acid was iso-C16 : 0. The DNA G+C content of the genomic DNA was 71.5 mol%. Based on the polyphasic approach, strain OK19-0408T represents a novel species of the genus Amycolatopsis, for which the name Amycolatopsis iheyensis sp. nov. is proposed. The type strain of the type species is OK19-0408T (=NBRC115671T=TBRC16040T).

Plant growth-promoting properties of Streptomyces spp. isolates and their impact on mung bean plantlets' rhizosphere microbiome.

Phytophthora is an important, highly destructive pathogen of many plants, which causes considerable crop loss, especially durians in Thailand. In this study, we selectively isolated Streptomyces from the rhizosphere soil with a potent anti-oomycete activity against Phytophthora palmivora CbP03. Two strains (SNN087 and SNN289) demonstrated exceptional plant growth-promoting properties in pot experiment. Both strains promoted mung bean (Vigna radiate) growth effectively in both sterile and non-sterile soils. Metagenomic analysis revealed that Streptomyces sp. SNN289 may modify the rhizosphere microbial communities, especially promoting microbes beneficial for plant growth. The relative abundance of bacterial genera Bacillus, Sphingomonas, Arthrobacter, and Pseudarthrobacter, and fungal genera Coprinellus and Chaetomium were noticeably increased, whereas a genus Fusarium was slightly reduced. Interestingly, Streptomyces sp. SNN289 exhibited an exploratory growth, which allows it to survive in a highly competitive environment. Based on whole genome sequence analysis combined with an ANI and dDDH values, this strain should be classifiable as a new species. Functional annotation was also used to characterize plant-beneficial genes in SNN087 and SNN289 genomes for production of siderophores, 3-indole acetic acid (IAA), ammonia, and solubilized phosphate. AntiSMASH genome analysis and preliminary annotation revealed biosynthetic gene clusters with possible secondary metabolites. These findings emphasize the potential for application of strain SNN289 as a bioinoculant for sustainable agricultural practice.

Activation of cryptic milbemycin A4 production in Streptomyces sp. BB47 by the introduction of a functional bldA gene.

Streptomycetes are characterized by their ability to produce structurally diverse compounds as secondary metabolites and by their complex developmental life cycle, which includes aerial mycelium formation and sporulation. The production of secondary metabolites is growth-stage dependent, and generally coincides with morphological development on a solid culture. Streptomyces sp. BB47 produces several types of bioactive compounds and displays a bald phenotype that is devoid of an aerial mycelium and spores. Here, we demonstrated by genome analysis and gene complementation experiments that the bald phenotype arises from the bldA gene, which is predicted to encode the Leu-tRNAUUA molecule. Unlike the wild-type strain producing jomthonic acid A (1) and antarlide A (2), the strain complemented with a functional bldA gene newly produced milbemycin (3). The chemical structure of compound 3 was elucidated on the basis of various spectroscopic analyses, and was identified as milbemycin A4, which is an insecticidal/acaricidal antibiotic. These results indicate that genetic manipulation of genes involved in morphological development in streptomycetes is a valuable way to activate cryptic biosynthetic pathways.

An in vivo selection system with tightly regulated gene expression enables directed evolution of highly efficient enzymes.

In vivo selection systems are powerful tools for directed evolution of enzymes. The selection pressure of the systems can be tuned by regulating the expression levels of the catalysts. In this work, we engineered a selection system for laboratory evolution of highly active enzymes by incorporating a translationally suppressing cis repressor as well as an inducible promoter to impart stringent and tunable selection pressure. We demonstrated the utility of our selection system by performing directed evolution experiments using TEM ß-lactamase as the model enzyme. Five evolutionary rounds afforded a highly active variant exhibiting 440-fold improvement in catalytic efficiency. We also showed that, without the cis repressor, the selection system cannot provide sufficient selection pressure required for evolving highly efficient TEM ß-lactamase. The selection system should be applicable for the exploration of catalytic perfection of a wide range of enzymes.

Potential benefits of L. acidophilus in dyslipidemic rats.

Several studies have shown that probiotics and synbiotics ameliorate dyslipidemia. However, the molecular mechanisms mediating their effects remain to be determined. Therefore, we aimed to compare the effects of a probiotic, a prebiotic, and a synbiotic in dyslipidemic Sprague-Dawley rats, and explore the mechanisms involved using a proteomic approach. The rats were allocated to five groups: a control group that was fed normal chow, and four high-fat diet-fed groups, three of which were administered a probiotic (Lactobacillus acidophilus), a prebiotic (inulin), or a combination of the two (a synbiotic) for 30 days. We showed that the administration of inulin, and especially L. acidophilus, improved the lipid profile and reduced the serum concentrations of inflammatory markers in high-fat diet-fed rats. Proteomic analysis showed changes in lipid elongation, glycerolipid metabolism, activation of antioxidants, and a reduction in the activation of the mitogen-activated protein kinase signaling pathway in the livers of rats administered L. acidophilus, which likely mediate its beneficial effects on inflammation and dyslipidemia by reduced the levels of 18.56% CRP, 35.71% TNF-α 25.6% LDL-C and 28.57% LDL-C/HDL-C ratio when compared to HF group. L. acidophilus and inulin may represent effective natural means of maintaining inflammation and dyslipidemia.

Colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) as a visual diagnostic platform for the detection of the emerging coronavirus SARS-CoV-2.

COVID-19, caused by the infection of SARS-CoV-2, has emerged as a rapidly spreading infection. The disease has now reached the level of a global pandemic and as a result a more rapid and simple detection method is imperative to curb the spread of the virus. We aimed to develop a visual diagnostic platform for SARS-CoV-2 based on colorimetric RT-LAMP with levels of sensitivity and specificity comparable to that of commercial qRT-PCR assays. In this work, the primers were designed to target a conserved region of the RNA-dependent RNA polymerase gene (RdRp). The assay was characterized for its sensitivity and specificity, and validated with clinical specimens collected in Thailand. The developed colorimetric RT-LAMP assay could amplify the target gene and enabled visual interpretation in 60 min at 65 °C. No cross-reactivity with six other common human respiratory viruses (influenza A virus subtypes H1 and H3, influenza B virus, respiratory syncytial virus types A and B, and human metapneumovirus) and five other human coronaviruses (MERS-CoV, HKU-1, OC43, 229E and NL63) was observed. The limit of detection was 25 copies per reaction when evaluated with contrived specimens. However, the detection rate at this concentration fell to 95.8% when the incubation time was reduced from 60 to 30 min. The diagnostic performance of the developed RT-LAMP assay was evaluated in 2120 clinical specimens and compared with the commercial qRT-PCR. The results revealed high sensitivity and specificity of 95.74% and 99.95%, respectively. The overall accuracy of the RT-LAMP assay was determined to be 99.86%. In summary, our results indicate that the developed colorimetric RT-LAMP provides a simple, sensitive and reliable approach for the detection of SARS-CoV-2 in clinical samples, implying its beneficial use as a diagnostic platform for COVID-19 screening.

Micromonospora pelagivivens sp. nov., a new species of the genus Micromonospora isolated from deep-sea sediment in Japan.

A novel marine actinomycete, designated strain KJ-029T, was isolated from a marine sediment sample (water depth of 226 m) in Kagoshima, Japan. 16S rRNA gene sequence analysis revealed that the new isolate was most closely related to Micromonospora craniellae LHW 63014T (99.3 % similarity). Phylogenetic analyses of the genus Micromonospora based on 16S rRNA gene sequences showed that strain KJ-029T was clustered with Micromonospora craniellae LHW 63014T and Micromonospora endophytica 202201T. However, digital DNA-DNA hybridization analyses presented low levels of relatedness in the range of 24.8-32.9 % between strain KJ-029T and the above closely related strains. The novel strain contained meso-diaminopimelic acid and 3-OH-diaminopimelic acid, d-glutamic acid, glycine and d-alanine in the cell-wall peptidoglycan. The acyl type of the peptidoglycan was glycolyl and mycolic acids were absent. The major menaquinone was MK-9(H4). The whole-cell sugars consisted of glucose, mannose, xylose and ribose. Phosphatidylethanolamine was detected as the major phospholipid and corresponded to phospholipid type II. The predominant cellular fatty acid was iso-C16 : 0. The DNA G+C content of the genomic DNA was 71.5 mol%. Based on the present polyphasic study, strain KJ-029T represents a novel species of the genus Micromonospora, for which the name Micromonospora pelagivivens sp. nov. is proposed. The type strain is KJ-029T (=NBRC 113519T=TBRC 9233T).

Amphiphilic dextran-vinyl laurate-based nanoparticles: formation, characterization, encapsulation, and cytotoxicity on human intestinal cell line.

Dextran has been the model material for the therapeutic applications owing to its biodegradable and biocompatible properties, and the ability to be functionalized in variety of ways. In this study, the amphiphilic dextran was successfully synthesized through lipase-catalyzed transesterification between dextran and vinyl laurate. In aqueous solution, the produced dextran ester could self-assemble into spherical nanoparticles ("Dex-L NPs") with approximately 200-nm diameter, and could incorporate porcine placenta hydrolysate with 60% encapsulation efficiency. Furthermore, Dex-L NPs exhibited low cytotoxic effects on human intestinal cell line and, thus, were potentially safe for oral administration. Taken together, the findings illustrate the potential of the newly developed nanoparticles to serve as an efficient and safe drug delivery system.

Complete NMR assignment and absolute configuration of k4610422, a norditerpenoid inhibitor of testosterone-5α-reductase originally from Streptosporangium: rediscovery from a thermophilic Actinomadura.

A norditerpenoid k4610422 (1), an inhibitor of testosterone-5α-reductase originally discovered from a mesophilic rare actinomycete of the genus Streptosporangium, was isolated from the culture extract of a thermophilic actinomycete Actinomadura sp. The complete 1H and 13C NMR assignment and absolute configuration of 1 were addressed by spectroscopic measurements including NOESY and CD spectra coupled with ECD calculation, which allowed to establish the (5 R,9 S,10 R,13 S)-configuration. Compound 1 was moderately cytotoxic against P388 murine leukemia cells with IC50 30 µM.

Probiotic Activity of Enterococcus faecium and Lactococcus lactis Isolated from Thai Fermented Sausages and Their Protective Effect Against Clostridium difficile.

Lactic acid bacteria, Enterococcus faecium and Lactococcus lactis, previously isolated from Thai fermented sausages were elucidated their probiotic properties especially in the control of Clostridium difficile 630. Both isolates survived in simulated gastric solution at pH 3 followed in simulated intestinal solution at pH 8. The presence of skimmed milk also helped the bacteria to survive through acidic and alkaline in gastrointestinal conditions. The adhesion properties of both isolates were tested using a human colon adenocarcinoma cell line. The result showed that both isolates exhibited desirable probiotic properties which adhered to Caco-2 cells. The neutralized cell-free supernatant of both isolates demonstrated that no cytotoxicity toward Caco-2 cells vice versa cell-free supernatant of C. difficile 630 toward Caco-2 cell demonstrated high toxicity. The immunomodulation effect in response to bacterial neutralized cell-free supernatant and cell-free supernatant was also studied. The expression level of pro-inflammatory cytokine of Caco-2 cell which are tumor necrosis factor-α and interleukin-8 was evaluated using quantitative reverse transcriptase PCR. Both isolates were able to diminish the expression level of TNF-α and IL-8 induced by the cell-free supernatant of C. difficile 630. Hence, these isolates would be able to improve the gut health through counteracting the C. difficile-associated intestinal inflammation in human cell lines. These results may contribute to the development of the isolates using as probiotics.

In vitro antiviral activity of spirotetronate compounds against dengue virus serotype 2.

Spirotetronate compounds are polyketide secondary metabolites with diverse biological functions, such as antibacterial, antitumor and antiviral activities. Three pure spirotetronate compounds (2EPS-A, -B, -C) isolated from Actinomadura strain 2EPS showed inhibitory activity against dengue virus serotype 2 (DENV-2). 2EPS-A, -B and -C demonstrated the LC50 values of 11.6, 27.5 and 12.0 µg/ml, respectively, in a test of cytotoxicity to Vero cells. The least cytotoxic, 2EPS-B, was further analyzed for its impact on viral propagation in a cell-based replication assay. At a concentration of 6.25 µg/ml, it could reduce the DENV-2 infection in Vero cells by about 94% when cells infected with DENV-2 were exposed to 2EPS-B, whereas direct treatment of DENV-2 with 2EPS-B at the same concentration prior to subsequent infection to Vero cell yielded no inhibition. 2EPS-A, -B an -C showed strong DENV-2 NS2B-NS3 protease inhibition in an in vitro assay, with IC50 values of 1.94 ± 0.18, 1.47 ± 0.15 and 2.51 ± 0.21 µg/ml, respectively. Therefore, the spirotetronate compounds appear to prevent viral replication and viral assembly by inhibition of the viral protease.

Saccharopolyspora rhizosphaerae sp. nov., an actinomycete isolated from rhizosphere soil in Thailand.

A novel actinomycete, designated as strain H219T, was isolated from rhizosphere soil collected under an Elephant ear plant (Colocasiaesculenta) in Bangkok, Thailand. Strain H219T was characterised using a polyphasic approach. Phylogenetic analysis of the 16S rRNA gene sequences revealed that this isolate was most closely related to Saccharopolyspora tripterygii JCM 32123T (97.6 %), Saccharopolyspora dendranthemae NBRC 108675T (97.5 %) and Saccharopolyspora flava NBRC 16345T (97.5 %). However, DNA-DNA hybridization analyses showed a low relatedness in the range of 39-48 % between the novel isolate and the above closely related strains. The cell-wall peptidoglycan of strain H219T contained meso-diaminopimelic acid. The diagnostic whole-cell sugars consisted of arabinose and galactose. The cellular fatty acid profile mainly comprised iso-C16 : 0, anteiso-C17 : 0, iso-C15 : 0, and 10-methyl C17 : 0. The major menaquinone was MK-9(H4). The detected phospholipids were diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylethanolamine-containing hydroxylated fatty acids and an unknown phospholipid. The DNA G+C content was 70.6 mol%. Strain H219T represented chemotaxonomic and morphological characteristics that were consistent with members of the genus Saccharopolyspora. However, strain H219T could be distinguished from closely related strains by several phenotypic properties. Based on the data from the polyphasic studies, we propose that strain H219T is a novel species within the genus Saccharopolyspora, Saccharopolysporarhizosphaerae sp. nov. The type strain is H219T (=TBRC 8564T=NBRC 113388T).

Display of Escherichia coli Phytase on the Surface of Bacillus subtilis Spore Using CotG as an Anchor Protein.

Escherichia coli phytase (AppA) has been widely used as an exogenous feed enzyme for monogastric animals; however, the production of this enzyme has been examined primarily in E. coli and yeast expression systems. As an alternative to production of soluble phytase, an enzyme immobilization method using the Bacillus subtilis spore outer-coat protein CotG as an anchoring motif for the display of the AppA was attempted. Using this motif, AppA was successfully produced on the spore surface of B. subtilis as verified by Western blot analysis and phytase activity measurements. Analysis of the pH stability indicated that more than 50% activity was retained after incubation at four different pH values (2.0, 4.0, 7.0, and 8.0) for up to 12 h, with maximum activity observed at pH 4.5. The highest enzyme activity seen at 55 °C and thermal stability measurements demonstrated that more than 30% activity remained after 30 min incubation at 60 °C. The spore surface-displayed AppA was resistant to pepsin, and more stable than phytase produced previously using a yeast expression system. Furthermore, we present data indicating that the use of peptide linkers may help improve the bioactivity of displayed enzymes on the spore surface of B. subtilis.

Cystargamide B, a cyclic lipodepsipeptide with protease inhibitory activity from Streptomyces sp.

We identified a new cyclic lipodepsipeptide, cystargamide B (1), from the mycelial extract of a Kaempferia galanga rhizome-derived actinomycete strain, Streptomyces sp. PB013. The planar structure was elucidated based on high resolution fast-atom bombardment mass spectrometry (HRFABMS) spectroscopy and one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) spectroscopic data. The absolute configurations of the constituent amino acids were determined using advanced Marfey's method. Cystargamide B (1) includes rare structural units: a 5-hydroxytryptophan residue and a 2,3-epoxy fatty acid side chain. Notably, cystargamide B (1) inhibited the protease activity of the NS2B/NS3 complex from dengue virus.

Improvement of extracellular bacterial protein production in Pichia pastoris by co-expression of endoplasmic reticulum residing GroEL-GroES.

Pichia pastoris is an established host system for heterologous protein expression. However, the potential productivity of this system can be limited. In this study, the Escherichia coli chaperones (GroES-GroEL) were expressed from the PGAP promoter and targeted to the secretory pathway through the endoplasmic reticulum (ER). The ability of the ER targeted chaperones to improve production of bacterial protein in P. pastoris was evaluated. The chaperones tagged with α-factor secretion- and ER retention-signal sequences were co-expressed with either extracellularly secreted phytase or intracellular d-phenylglycine aminotransferase (D-PhgAT) enzymes. The ER residing GroEL-GroES successfully increased the levels of active phytase extracellularly, 1.5-2.3-fold higher than the phytase expression alone, but did not enhance the formation of active, intracellular D-PhgAT. These results indicate that the chaperones have the potential to enhance production of active enzymes when present in the same trafficking pathway. This is the first report on the improvement of extracellular bacterial protein production through co-expression with ER residing bacterial chaperones in the Pichia system. The modified P. pastoris expression system may be beneficial for extracellular expression of other prokaryotic proteins.