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.
Corynebacterium glutamicum is an important industrial strain for amino acids and a key model organism for human pathogens. The study of C. glutamicum oxidoreductases, such as mycoredoxin 1 (Mrx1), dithiol-disulfide isomerase DsbA, and DsbA-like Mrx1, is helpful for understanding the survival, pathogenic infection, and stress resistance of its homologous species. However, the action mode and enzymatic function of C. glutamicum NCgl0018 preserving the Cys-Pro-Phe-Cys motif, annotated as a putative DsbA, have remained enigmatic. Here, we report that the NCgl0018-deleted strain increased sensitivity to various oxidative stresses. The ncgl0018 expression was induced in the stress-responsive extracytoplasmic function-sigma (ECF-σ) factor SigH- and organic peroxide- and antibiotic-sensing regulator (OasR)-dependent manner by stress. NCgl0018 reduced S-mycothiolated mixed disulfides and intramolecular disulfides via a monothiol-disulfide mechanism preferentially linking the mycothiol/mycothione reductase/NADPH electron pathway. Site-directed mutagenesis confirmed Cys107 was the resolving Cys residue, while Cys104 was the nucleophilic cysteine that was oxidized to a sulfenic acid and then could form an intramolecular disulfide bond with Cys107 or a mixed disulfide with mycothiol under stress. Biochemical analyses indicated that NCgl0018 lacked oxidase properties like the classical DsbA. Further, enzymatic rates and substrate preferences of NCgl0018 were highly similar to those of DsbA-like Mrx1. Collectively, our study presented the first evidence that NCgl0018 protected against stresses by functioning as a novel DsbA-like Mrx1 but not DsbA and Mrx1.
In this study, two fibrous carbon anodes (namely, pleated non-woven graphite (PNWG) and carbon brush (CB) made from artificial carbon) and bamboo charcoal (BC) were evaluated for current recovery from sewage wastewater. When these anodes were polarized at 0.2 V vs. Ag/AgCl in sewage wastewater, CB produced a maximum current of 2.9 A/m2. This exceeded that produced by PNWG (1.5 A/m2) and BC (1.4 A/m2). The accumulative charge recovery achieved with CB was superior to those achieved with the other two (1.6- and 2.2-fold higher than that with PNWG and BC, respectively). During the cyclic voltammetry analysis, CB demonstrated the highest catalytic current with maximum potential in the range of –0.6 to 0.4 V vs. Ag/AgCl and the smallest anode resistance (0.20 Ωm2). Direct cell counting revealed that the fibrous anodes (CB and PNWG) attached most of the cells in the anodes (80%), whereas BC did not. In contrast, the proportion of Geobacter species, a representative electrogenic microorganism in the total bacteria, was observed to be similar among the three anodes (4.4–5.8%). The tubular microbial fuel cell (ø 5.0 cm) equipped with an air-chamber core wrapped with an anion exchange membrane (AEM) and the CB delivered a current of 1.8 A/m2. This is higher than those reported in the existing literature for the same microbial fuel cell (MFC) configuration. This indicates that the alteration of the anode from planar to brush can contribute toward improving the current recovery through the air-cathode-AEM-MFC. The BC needs improvement to have more specific surface area, whereas it showed superiority in cost efficiency considering material and processing.
Pressed sake cake, a by-product of sake brewing, is a rich dietary source of folates, which are important vitamins for humans. However, considerable losses of folates occur during storage and cooking. We have previously reported that Km67, the house sake yeast strain of Kiku-masamune sake brewery, can accumulate high folate levels. In this study, we found that the folate content of pressed sake cakes produced with Km67 remained at approximately their maximum level after the fermentation activity stopped. To elucidate the mechanisms of high folate accumulation in Km67, we analyzed the expression of 23 folate-metabolizing genes. The expression of ABZ1 and FOL3 was almost always higher in Km67 than in Kyokai no. 701 yeast (K701), which suggested that enhanced expression of the genes involved in folate biosynthesis was a mechanism of high folate accumulation in Km67. We found that the folates of Km67 pressed sake cakes were quantitatively stable at 4°C under refrigerated storage conditions. In addition, the homocysteine content of Km67 pressed sake cakes was almost always higher than that of K701 pressed sake cakes. This result suggests that a reason for high folate accumulation in Km67 yeast is the need to reduce the intracellular concentration of homocysteine. Our results provide biologically meaningful information on folate metabolism in yeast.
We describe a novel expression cassette that enables efficient and constitutive expression of the ZZ domain derived from Staphylococcus aureus protein A on the yeast cell surface to easily prepare yeast-based immunosorbents. Using this expression cassette containing the PGK1 promoter, a secretion signal derived from α-factor, and a Flo1-derived anchor protein, we successfully created a yeast-based immunosorbent for human serum albumin.
The terrestrial cyanobacterium Nostoc commune has a cosmopolitan distribution. It is edible, and dry thalli are sold as a food in China under the name of Di Pi Cai. The pigment composition and the genotypes were characterized to identify the cyanobacterium Di Pi Cai from China as N. commune. Myxol glycosides and ketocarotenoids were detected, as expected in Nostoc sp., but β-carotene and hydroxylated carotenoids were not detected. Nostoc-756, mycosporine-2-(4-deoxygadusoyl-ornitine), was found to be a main mycosporine-like amino acid, which indicates that Di Pi Cai belongs to the N. commune chemotype C. However, the 16S rRNA gene and the petH gene encoding ferredoxin-NADP+ oxidoreductase of Di Pi Cai did not exactly match those of genotype C found in Japan. These results suggest the unique molecular genetic features of Di Pi Cai and the global diversity of N. commune.
Strains of Lactococcus lactis subsp. cremoris are used to produce yogurt containing exopolysaccharides with a sticky texture. When strain G3-2 producing exopolysaccharides was grown at elevated temperatures, a spontaneous mutant EPSC, which had lost exopolysaccharides biosynthesis, was isolated. Genomes of the two strains were determined to be composed of a 2.4-Mb chromosome and up to eleven plasmids, and it was revealed that one of the plasmids encoding the gene cluster for exopolysaccharides biosynthesis was lost selectively in EPSC.
A unicellular cyanobacterium that produces a large amount of exopolysaccharide (EPS) was isolated. The isolate, named Chroococcus sp. FPU101, grew between 20 and 30°C and at light intensities between 10 and 80 μmol m–2 s–1. Purified EPS from Chroococcus sp. FPU101 had a molecular size of 5.9 × 103 kDa and contained galactose, rhamnose, fucose, xylose, mannose, glucose, galacturonic acid, and glucuronic acid at a molar ratio of 17.2:15.9:14.1:11.0:9.6:9.5:13.0:9.7. The EPS content significantly increased when the NaCl concentration in the medium was increased from 1.7 to 100 mM. However, high NaCl concentrations did not significantly affect the molecular size or chemical composition of the EPS. The genes wza, wzb, wzc, wzx, wzy, and wzz that are involved in EPS synthesis were conserved in the genome of Chroococcus sp. FPU101, which was sequenced in this study. These results suggest that the Wzy-dependent pathway is potentially involved in EPS production in this organism.
We clarified the roles of VPH1 in Cryptococcus neoformans serotype D by examining the detailed phenotypes of VPH1-deficient cells (Δvph1) in terms of their capability to grow in acidic and alkaline pH, at a high temperature, and under high osmotic conditions, in addition to the involvement of VPH1 in copper (Cu) homeostasis and the expression of some C. neoformans virulence factors. Δvph1 could grow well on minimal medium (YNB) but exhibited hypersensitivity to 20 μM Cu due to the failure to induce Cu-detoxifying metallothionein genes (CMT1 and CMT2). In contrast, Δvph1 exhibited defective growth on rich medium (YPD), and the induction of Cu transporter genes (CTR1 and CTR4) did not occur in this medium, implying that this strain was incapable of the uptake of Cu ions for growth. However, the addition of excess Cu promoted CTR gene expression and supported Δvph1 growth. These results suggested that the lack of the VPH1 gene disturbed Cu homeostasis in C. neoformans. Moreover, the loss of Vph1 function influenced the urease activity of C. neoformans.
L-Pipecolic acid is utilized as a vital component of specific chemical compounds, such as immunosuppressive drugs, anticancer reagents, and anesthetic reagents. We isolated and characterized a novel L-aminoacylase, N-acetyl-L-pipecolic acid-specific aminoacylase (LpipACY), from Pseudomonas sp. AK2. The subunit molecular mass of LpipACY was 45 kDa and was assumed to be a homooctamer in solution. The enzyme exhibited high substrate specificity toward N-acetyl-L-pipecolic acid and a high activity for N-acetyl-L-pipecolic acid and N-acetyl-L-proline. This enzyme was stable at a high temperature (60°C for 10 min) and under an alkaline pH (6.0–11.5). The N-terminal and internal amino acid sequences of the purified enzyme were STTANTLILRNG and IMASGGV, respectively. These sequences are highly consistent with those of uncharacterized proteins from Pseudomonas species, such as amidohydrolase and peptidase. We also cloned and overexpressed the gene coding LpipACY in Escherichia coli. Moreover, the recombinant LpipACY exhibited properties similar to native enzyme. Our results suggest that LpipACY is a potential enzyme for the enzymatic synthesis of L-pipecolic acid. This study provides the first description of the enzymatic characterization of L-pipecolic acid specific amino acid acylase.
The supply of oven-fresh bakery products to consumers has been improved by frozen dough technology; however, freeze-thaw stress decreases the activity of yeast cells. To breed better baker's yeasts for frozen dough, it is important to understand the factors affecting freeze-thaw stress tolerance in baker's yeast. We analyzed the stress response in IB1411, a spore clone from Saccharomyces cerevisiae Shirakami kodama yeast, with an exceptionally high tolerance to freeze-thaw stress. Genes encoding trehalose-6-phosphate synthase (TPS1), catalase (CTT1), and disaggregase (HSP104) were highly expressed in IB1411 cells even under conditions of non-stress. The expression of Hsp104 protein was also higher in IB1411 cells even under non-stress conditions. Deletion of HSP104 (hsp104Δ) in IB1411 cells reduced the activity of the ubiquitin-proteasome system (UPS). By monitoring the accumulation of aggregated proteins using the ΔssCPY*-GFP fusion protein under freeze-thaw stress or treatment with proteasomal inhibitor, we found that IB1411 cells resolved aggregated proteins faster than the hsp104Δ strain. Thus, Hsp104 seems to contribute to freeze-thaw tolerance by maintaining UPS activity via the disaggregation of aggregated proteins. Lastly, we found that the IB1411 cells maintained high leavening ability in frozen dough as compared with the parental strain, Shirakami kodama yeast, and thus will be useful for making bread.
Probiotics have been shown to improve microbial compositions in animal intestine and feces, but the effects of probiotic administration on airborne microbial composition in animal houses remain unclear. In this study, we investigated the effects of dietary Enterococcus faecalis on the bacterial community structure in the air of piglet and layer hen houses. Indoor air and feces from piglet and layer hen houses were sampled after supplementing E. faecalis in feed for 60 days, and bacterial community structures were analyzed using Illumina high-throughput sequencing technology. Results showed that Chao1, ACE, Shannon, and Simpson indices of bacterial diversity did not significantly change in feces or indoor air of piglet or layer hen after supplementation with E. faecalis (P > 0.05). However, E. faecalis administration resulted in a decrease in the relative abundance of Proteobacteria (P < 0.05). In addition, E. faecalis significantly reduced the relative abundance of opportunistic pathogens such as Acinetobacter, Escherichia, and Shigella (P < 0.05), and beneficial bacterial genus such as Lactobacillus was significantly enriched in both feces and indoor air (P < 0.05). These changes should be of benefit to livestock, farm workers, and the surrounding environment.
Gamma-aminobutyric acid (GABA) plays a key role as an inhibitory neurotransmitter in the mammalian sympathetic nervous system and has other health benefits. Molecular characterization, genome analysis, and optimization were investigated to improve GABA production of a selected strain of lactic acid bacteria. Eleven isolates from plant materials were screened for GABA productivity and were identified based on phenotypic and genotypic characteristics. The most potent strain was chosen for genome analysis and GABA production optimization using the response surface methodology (RSM). Each of the two strains was closely related to Lactobacillus plantarum, Lactobacillus brevis, Weissella cibaria, Leuconostoc pseudomesenteroides while each strain was similar to Lactobacillus pentosus, Enterococcus, and Leuconostoc mesenteroides. They produced GABA ranging from 0.036 ± 0.000 to 17.315 ± 0.171 g/L at 72 h-cultivation. Among them, the most potent strain, SL9-6, showed the highest GABA production (17.315 g/L) when cultivated with 10% (v/v) inoculum for 48 h. The draft genome sequence of strain SL9-6 exhibited 96.90% average nucleotide identity value and 74.50% digital DNA-DNA hybridization to Lactobacillus brevis NCTC 13768T. This strain contained a glutamate decarboxylase gene system (gadA, gadB and gadC). Optimal culture conditions were determined as 40.00 g/L glucose, 49.90 g/L monosodium glutamate, pH 5.94, and 31.10°C by RSM, giving maximum GABA production of 32.48 g/L. Results from RSM also indicated that monosodium glutamate concentration, pH, and temperature were significant variables. GABA production significantly improved here could promise further application of strain SL9-6.
This study aimed to evaluate the composition of the gastric microbiota in the gastric mucosa and gastric fluid of patients with chronic antral gastritis. Specifically, we sought to determine whether Helicobacter pylori (Hp) infection changes the bacterial community in the gastric mucosa or alters the microbiota in the gastric fluid. The bacterial community at another site in the stomach was also investigated. DNA was extracted from 160 samples collected from 40 patients with chronic antral gastritis (20 Hp-positive and 20 Hp-negative cases). Three tissue samples of the gastric mucosa (gastric angle, body, and antral mucosa) and one tube of gastric fluid were collected from every patient. A 16S rRNA amplification library was created, and high-throughput sequencing was performed. A profile of the community composition was obtained using bioinformatics methods, including cluster, taxonomy, and diversity analyses. Analysis of the gastric bacterial community revealed that the community compositions of the gastric mucosa and gastric fluid of patients without Hp are similar to but show differences from those of Hp-positive patients. The microbiota in Hp-positive patients exhibited reduced microbial diversity, and the gastric fluid of these patients contained a small proportion of Hp. The richness of Leptotrichia in mucosal samples was greater than that in gastric fluid samples from Hp-negative patients with chronic antral gastritis. Hp changes the growth of other microbiota in the mucosa and affects the microbiota in the gastric fluid of patients with chronic antral gastritis. In addition to Hp, the presence of other bacteria might be related to the development of chronic antral gastritis.
Glycerol is an attractive raw material for the production of useful chemicals using microbial cells. We previously identified metabolic engineering targets for the improvement of glycerol assimilation ability in Saccharomyces cerevisiae based on adaptive laboratory evolution (ALE) and transcriptome analysis of the evolved cells. We also successfully improved glycerol assimilation ability by the disruption of the RIM15 gene encoding a Greatwall protein kinase together with overexpression of the STL1 gene encoding the glycerol/H+ symporter. To understand glycerol assimilation metabolism in the evolved glycerol-assimilating strains and STL1-overexpressing RIM15 disruptant, we performed metabolic flux analysis using 13C-labeled glycerol. Significant differences in metabolic flux distributions between the strains obtained from the culture after 35 and 85 generations in ALE were not found, indicating that metabolic flux changes might occur in the early phase of ALE (i.e., before 35 generations at least). Similarly, metabolic flux distribution was not significantly changed by RIM15 gene disruption. However, fluxes for the lower part of glycolysis and the TCA cycle were larger and, as a result, flux for the pentose phosphate pathway was smaller in the STL1-overexpressing RIM15 disruptant than in the strain obtained from the culture after 85 generations in ALE. It could be effective to increase flux for the pentose phosphate pathway to improve the glycerol assimilation ability in S. cerevisiae.
With the aid of a chain transfer (CT) reaction, hydroxyalkanoate (HA) oligomers can be secreted by recombinant Escherichia coli carrying the gene encoding a lactate-polymerizing enzyme (PhaC1PsSTQK) using Luria-Bertani (LB) medium supplemented with a carbon source and CT agent. In this study, HA oligomers were produced through microbial secretion using a mineral-based medium instead of LB medium, and the impact of medium composition on HA oligomer secretion was investigated. The focused targets were medium composition and NaCl concentration related to osmotic conditions. It was observed that 4.21 g/L HA oligomer was secreted by recombinant E. coli in LB medium, but the amount secreted in the mineral-based modified R (MR) medium was negligible. However, when the MR medium was supplemented with 5 g/L yeast extract, 3.75 g/L HA oligomer was secreted. This can be accounted for by the enhanced expression and activity of PhaC1PsSTQK upon supplementation with growth-activated nutrients as supplementation with yeast extract also promoted cell growth and intracellular growth-associated polymer accumulation. Furthermore, upon adding 10 g/L NaCl to the yeast extract-supplemented MR medium, HA oligomer secretion increased to 6.86 g/L, implying that NaCl-induced osmotic pressure promotes HA oligomer secretion. These findings may facilitate the secretory production of HA oligomers using an inexpensive medium.
Glutaredoxins (Grxs) and thioredoxins (Trxs) play a critical role in resistance to oxidative conditions. However, physiological and biochemical roles of Mycoredoxin 3 (Mrx3) that shared a high amino acid sequence similarity to Grxs remain unknown in Corynebacterium glutamicum. Here we showed that mrx3 deletion strains of C. glutamicum was involved in the protection against oxidative stress. Recombinant Mrx3 not only catalytically reduced the disulfide bonds in ribonucleotide reductase (RNR), insulin and 5,5'-dithiobis-(2-nitro-benzoicacid) (DTNB), but also reduced the mixed disulphides between mycothiol (MSH) and substrate, which was exclusively linked to the thioredoxin reductase (TrxR) electron transfer pathway by a dithiol mechanism. Site-directed mutagenesis confirmed that the conserved Cys17 and Cys20 in Mrx3 were necessary to maintain its activity. The mrx3 deletion mutant showed decreased resistance to various stress, and these sensitive phenotypes were almost fully restored in the complementary strain. The physiological roles of Mrx3 in resistance to various stress were further supported by the induced expression of mrx3 under various stress conditions, directly under the control of the stress-responsive extracytoplasmic function-sigma (ECF-σ) factor SigH. Thus, we presented the first evidence that Mrx3 protected against various oxidative stresses by acting as a disulfide oxidoreductase behaving like Trx.