Applied Microbiology and Biotechnology

Published by Springer Nature
Online ISSN: 1432-0614
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  • Aslıhan Kurt-KızıldoğanAslıhan Kurt-Kızıldoğan
  • Gözde ÇelikGözde Çelik
  • Eser ÜnsaldıEser Ünsaldı
  • [...]
  • Gülay ÖzcengizGülay Özcengiz
Clavulanic acid (CA) is a clinically important secondary metabolite used to treat infectious diseases. We aimed to decipher complex regulatory mechanisms acting in CA biosynthesis by analyzing transcriptome- and proteome-wide alterations in an industrial CA overproducer Streptomyces clavuligerus strain, namely DEPA and its wild-type counterpart NRRL3585. A total of 924 differentially expressed genes (DEGs) and 271 differentially produced proteins (DPPs) were obtained by RNA-seq and nanoLC-MS/MS analyses, respectively. In particular, CA biosynthetic genes, namely, car (cad), cas2, oat2, pah, bls, ceas2, orf12, and claR, a cluster situated regulatory (CSR) gene, were significantly upregulated as shown by RNA-seq. Enzymes of clavam biosynthesis were downregulated considerably in the DEPA strain, while the genes involved in the arginine biosynthesis, one of the precursors of CA pathway, were overexpressed. However, the biosynthesis of the other CA precursor, glyceraldehyde-3-phosphate (G3P), was not affected. CA overproduction in the DEPA strain was correlated with BldD, BldG, BldM, and BldN (AdsA) overrepresentation. In addition, TetR, WhiB, and Xre family transcriptional regulators were shown to be significantly overrepresented. Several uncharacterized/unknown proteins differentially expressed in the DEPA strain await further studies for functional characterization. Correlation analysis indicated an acceptable degree of consistency between the transcriptome and proteome data. The study represents the first integrative-omics analysis in a CA overproducer S. clavuligerus strain, providing insights into the critical control points and potential rational engineering targets for a purposeful increase of CA yields in strain improvement. Key points ∙ Transcriptome and proteome-wide alterations in industrial CA overproducer strain DEPA ∙ An acceptable degree of consistency between the transcriptome and proteome data ∙ New targets to be exploited for rational engineering Graphical abstract
  • Helmy Ahmed TorkyHelmy Ahmed Torky
  • Samy Abd-Elsalam KhalielSamy Abd-Elsalam Khaliel
  • Eman Khalifa SedeekEman Khalifa Sedeek
  • [...]
  • Eman Moneer ElghazalyEman Moneer Elghazaly
A total no. of 65 Salmonella enterica isolates recovered from food samples, feces of diarrheic calves, poultry, and hospital patient in large five cities at Northern West Egypt were obtained from the Department of Microbiology, Faculty of Veterinary Medicine, Alexandria University, Alexandria, Egypt. The 65 Salmonella enterica isolates had the inv A gene were grouped into 11 Salmonella enterica serovars with dominance of S. Enteritidis and S. Kentucky serovars. Their resistance pattern were characterized by using 18 antibiotics from different classes. Approximately 80% of the isolates were multidrug resistant (MDR). Enterobacterial repetitive intergenic consequences polymerase chain reaction (ERIC-PCR) typing of 7 strains of S. Enteritidis showed 5 clusters with dissimilarity 25%. S. Enteritidis clusters in 2 main groups A and B. Group A have 2 human strain (HE2 and HE3) and one food origin (FE7) with a similarity 99%. Group B divided into B1 (FE2) and B2 (FE3) with a similarity ratio ≥ 93%, while ERIC-PCR analysis of 5 strains of S. Kentucky revealed 4 ERIC types, clustered in 2 main groups A and B with similarity 75%. We studied the effect of silver nanoparticles (Ag-NPs) on 10 antibiotic resistant strains of S . Enteritidis and S. Kentucky. The broth microdilution minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were detected. Evaluation of the affection using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) showed different ratios of Ag-NPs and microorganism as well as at different contact time ended finally with morphological alteration of the bacteria. We submitted new method in vivo to explore the activity of nanosilver in chicken. Key points • Importance of ERIC-PCR to determine the relatedness between Salmonella isolates. • Effect of silver nanoparticles to confront the antibacterial resistance . • Studying the effect of silver nanoparticles in vivo on infected chicken with Salmonella .
Characteristics of mAbs against PCV2. a Immunoreactivity analysis of mAbs by Western blotting. PCV2b/MDJ (oval dimension) and PCV2d/SDRS (triangle dimension) were separated by SDS-PAGE, transferred to nitrocellulose membranes, and then incubated with mAbs (as the primary antibody) and horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (H + L) (1:4000, as the secondary antibody). MAb 5F2 and SP2/0 was used as positive and negative controls. Lane M is a protein molecular weight marker. b Neutralizing activity assays of mAbs for PCV2b/MDJ (mAb 1G5, 2C8, 4B3, 4C9, 6H9, 7E2, 9H4, and 10G8) or PCV2d/SDRS (mAb 2G8, 7B9, 7C7, 7D1, 3A5, and 5F2) by the sensitive neutralization assay. MAb 3A5 and 5F2 was used as positive and negative controls. The neutralizing activities of mAbs were expressed as the percentage reduction in the number of infected cells in comparison with negative control. A mean neutralizing activity of > 50% was considered to represent neutralization. Error bars represent the SD of neutralizing activities
Identification of purified mAb 4B3, 9H4, 7C7, and PCV2 pAb by SDS-PAGE
Sensitivity test of PCV2 red latex microsphere immunochromatographic strip
Specificity test of PCV2 red latex microsphere immunochromatographic strip
  • Chong YuChong Yu
  • Yanwu WeiYanwu Wei
  • Hao ZhangHao Zhang
  • [...]
  • Liping HuangLiping Huang
To establish a rapid and specific antigen detection method for porcine circovirus type 2 (PCV2), monoclonal antibodies (mAbs) were produced against the PCV2 epidemic strains and a red latex microsphere immunochromatographic strip was established. A total of eight anti-PCV2b and four anti-PCV2d mAbs were produced, and seven mAbs were confirmed to react with PCV2a, PCV2b, and PCV2d strains using an immunoperoxidase monolayer assay. The results of micro-neutralization tests showed that the mAbs 2C8, 9H4, 10G7, 7B9, and 7C7 had good neutralizing activity, whereas the neutralizing activity of the mAbs 4B3, 4C9, 6H9, and 7E2 was lower than 50%. Three mAbs, 4B3, 7C7, and 9H4, and PCV2 pAb were selected for the establishment of a red latex microsphere immunochromatographic strip, and the combination of mAb 7C7 labeled with red latex microspheres and mAb 9H4 exhibited the greatest detection ability. The immunochromatographic strip had minimum detection limits of 102.5 TCID50/0.1 ml, 100.7 TCID50/0.1 ml, and 101.5 TCID50/0.1 ml for PCV2a/CL, PCV2b/MDJ, and PCV2d/LNHC, respectively. Furthermore, no cross-reactivity was found for African swine fever virus, classical swine fever virus, porcine respiratory and reproductive syndrome virus, porcine parvovirus, porcine pseudorabies virus, porcine circovirus type 1, transmissible gastroenteritis virus, porcine epidemic diarrhea virus, porcine rotavirus, or porcine deltacoronavirus using the immunochromatographic strip. Using PCR as a reference standard, the detection sensitivity, specificity, and overall coincidence rate of the immunochromatographic strip were 81.13%, 100%, and 90.00%. Additionally, the detection ability of the immunochromatographic strip was correlated with that of virus titration. The immunochromatographic strip was used to detect 183 clinical disease samples, and the average positive detection rate was 22.95%. In summary, this method has good sensitivity and specificity and is simple, convenient, and quick to operate. It has high application value for on-site diagnosis of PCV2 and virus quantification. Key points • A red latex microsphere immunochromatographic strip for PCV2 detection was developed. • The method was not only simple to operate, but also takes less time. • The method had good sensitivity and specificity.
Solubility of 103Q-htt is enhanced in Δfps1 cells. A Yeast cells were grown as described in “Methods” and after induction, cells were pelleted down and lysed. Native PAGE analysis of soluble fraction of wild-type and Δfps1 yeast cell lysates was carried out. Arrow indicates the position of the soluble protein. Equal protein load was confirmed by Coomassie staining of the gel. B Densitometric analysis of signals for monomeric 103Q-htt seen on native PAGE (panel A) was carried out using ImageJ software. Intensity of signal for soluble protein in wild-type cells grown under N-DR was assigned an arbitrary value of 100%. ***p < 0.001, **p < 0.01 against wild-type cells expressing 103Q-htt under DR; ##p < 0.01 against Δfps1 cells expressing 103Q-htt under DR; ns, non-significant. C Aggregation of 103Q-htt was monitored by filter retardation assay using cellulose acetate membrane as described in “Methods”. A representative blot is shown in the upper panel. Lower panel shows densitometric analysis of signal intensity of aggregates carried out using ImageJ software. Intensity of signal for aggregated protein in wild-type cells grown under N-DR was assigned an arbitrary value of 100%. ***p < 0.001, **p < 0.01 against wild-type cells expressing 103Q-htt under DR; ##p < 0.01, #p < 0.05 against Δfps1 cells expressing 103Q-htt under DR; ns, non-significant. D Solubility of aggregates was monitored with formic acid using nitrocellulose membrane as described in “Methods”. A representative blot is shown in the upper panel. Lower panel shows densitometric analysis of signal intensity of aggregates carried out using ImageJ software. Intensity of signal for aggregated protein in wild-type cells grown under N-DR was assigned an arbitrary value of 100%. ***p < 0.001, **p < 0.01 against wild-type cells expressing 103Q-htt under DR; ##p < 0.01, #p < 0.05 against Δfps1 cells expressing 103Q-htt under DR; ns, non-significant. In panels B, C and D, values shown are mean ± SEM of three independent experiments
Increased solubilisation of 103Q-htt under DR condition in Δfps1 cells is due to increased glycerol content. A Estimation of level of glycerol in yeast cells expressing 103Q-htt. **p < 0.01 against wild-type cells expressing 103Q-htt under DR; ##p < 0.01 against wild-type cells expressing 103Q-htt under N-DR; ns, non-significant. B Analysis of FPS1 expression in wild-type yeast cells under DR and N-DR conditions. Expression level of FPS1 in uninduced wild-type cells under N-DR was assigned an arbitrary value of 1 and gene expression levels under other conditions were calculated with respect to this value. ns, non-significant, *p < 0.05. C Western blot analysis of wild-type and Δfps1 cells decorated with Hog1 (first panel) and phosphorylated Hog1 (second panel) using the corresponding antibodies. D Toxicity assay for wild-type and Δfps1 cells grown under N-DR and DR when exposed to sodium arsenite. The protocol followed was as described in the “Methods” section. Decreasing order (threefold) of cell count is indicated with the black gradient. In panels A and B, values shown are mean ± SEM of three independent experiments
Thermo-tolerance increases the solubilisation of 103Q-htt in Δfps1 cells. A Wild-type and Δfps1 cells were subjected to heat shock at 39 °C for 2 h and expression of 103Q-htt was induced with galactose. The extent of solubilisation of 103Q-htt expressed in wild-type and Δfps1 cells was monitored by native PAGE. Arrow indicates the position of the soluble protein. Equal protein load was confirmed by Coomassie staining of the gel. B Densitometric analysis of signals for monomeric 103Q-htt seen on native PAGE (panel A) was carried out using ImageJ software. Intensity of signal for soluble protein in untreated wild-type cells was assigned an arbitrary value of 100%. ***p < 0.001, **p < 0.01 against untreated wild-type cells expressing 103Q-htt; ###p < 0.001 against untreated Δfps1 cells expressing 103Q-htt; ns, non-significant. C Aggregation of 103Q-htt was monitored by filter retardation assay. A representative blot is shown in the upper panel. Lower panel shows densitometric analysis of signals for monomeric 103Q-htt seen on the membrane carried out using ImageJ software. Intensity of signal for aggregation in untreated wild-type cells was assigned an arbitrary value of 100%. ***p < 0.001, **p < 0.01 against untreated wild-type cells expressing 103Q-htt; ##p < 0.01 against untreated Δfps1 cells expressing 103Q-htt; ns, non-significant. D Formic acid solubilisation assay. A representative blot is shown in the upper panel. Lower panel shows densitometric analysis of signals for monomeric 103Q-htt seen on the membrane carried out using ImageJ software. Intensity of signal for aggregation in untreated wild-type cells is assigned an arbitrary value of 100%. **p < 0.01 against untreated wild-type cells expressing 103Q-htt; ##p < 0.01 against untreated Δfps1 cells expressing 103Q-htt; ns, non-significant. E Estimation of level of glycerol in yeast cells expressing 103Q-htt. ***p < 0.001, **p < 0.01, *p < 0.05 against untreated wild-type cells expressing 103Q-htt; ns, non-significant. In panels B–E, values shown are mean ± SEM of three independent experiments
  • Kuljit SinghKuljit Singh
  • Ratnika SethiRatnika Sethi
  • Eshita DasEshita Das
  • Ipsita RoyIpsita Roy
In response to osmotic shock, the components of high-osmolarity glycerol (HOG) pathway regulate the level of intracellular glycerol in yeast and ensure cell survival. Glycerol is a compatible solute and a stabiliser of proteins. Its role in maintaining proteostasis is less explored. We show that mild stress in the form of dietary restriction leads to increased glycerol level which increases cell viability. However, dietary restriction coupled with protein aggregation decreases intracellular glycerol level and attenuates cell viability. The transcript level of FPS1, the glycerol transporter channel, remains unchanged. However, its activity is altered under enhanced proteotoxic stress. Our results provide evidence for a probable role of the Fps1p channel in the cellular proteostasis network. Key points • Dietary restriction led to increased accumulation of glycerol in Fps1-deleted yeast cells. • This led to lower protein aggregation in these cells. • Increased production of glycerol under dietary restriction was not linked to increased level of Fps1.
Regulatory mechanism of high-osmolarity glycerol (HOG) and the cell wall integrity (CWI) pathway under heat stress. Heat shock stimulates intracellular glycerol outflow through the HOG pathway and CWI pathway, thus decreasing expansion pressure
The observing morphology and microstructure change of cells after heat shock. Under external heat stress, some fungi transition between the yeast and mycelium states. Simultaneously, the spore volume of some fungi increases, the organelles inside the spore also markedly change, and unknown structures (electron-translucent structure) even appear
Outline of the process of fungal thermal adaptation mechanism. The fungal cell membrane may be the first sensor of a sudden increase in external temperature; it subsequently transmits heat signals into the cell via lipid rafts and other substances that act as signaling molecules. After receiving a heat signal, cells control the expression of heat-resistance genes and secrete a variety of substances (such as heat shock proteins, trehalose, and glycerin) through the regulation of a series of transcription factors, thus helping cells resist the damage caused by heat stress. (a) When the concentration of unfolded proteins exceeds the capacity of Hsp70 at elevated temperature, Hsp70 is released from Hsf1, and the released Hsf1 induces more Hsp70. After sufficient Hsp70 is produced to restore protein homeostasis, Hsp70 binds and inactivates Hsf1. Experiments have indicated that Hsf1 expression is also inhibited by HS90 in vitro. However, further evidence is needed to explore the specific mechanism
  • Wei XiaoWei Xiao
  • Jinping ZhangJinping Zhang
  • Jian HuangJian Huang
  • [...]
  • Zhangyong SongZhangyong Song
Both the increasing environmental temperature in nature and the defensive body temperature response to pathogenic fungi during mammalian infection cause heat stress during the fungal existence, reproduction, and pathogenic infection. To adapt and respond to the changing environment, fungi initiate a series of actions through a perfect thermal response system, conservative signaling pathways, corresponding transcriptional regulatory system, corresponding physiological and biochemical processes, and phenotypic changes. However, until now, accurate response and regulatory mechanisms have remained a challenge. Additionally, at present, the latest research progress on the heat resistance mechanism of pathogenic fungi has not been summarized. In this review, recent research investigating temperature sensing, transcriptional regulation, and physiological, biochemical, and morphological responses of fungi in response to heat stress is discussed. Moreover, the specificity thermal adaptation mechanism of pathogenic fungi in vivo is highlighted. These data will provide valuable knowledge to further understand the fungal heat adaptation and response mechanism, especially in pathogenic heat-resistant fungi. Key points • Mechanisms of fungal perception of heat pressure are reviewed. • The regulatory mechanism of fungal resistance to heat stress is discussed. • The thermal adaptation mechanism of pathogenic fungi in the human body is highlighted.
Worldwide distribution of G. superba (source:
Gloriosa superbaa habit (source: Wikimedia commons; Creative Commons Attribution-Share Alike 4.0 International license); b flower (source: Wikimedia commons; Creative Commons Attribution 3.0); c fruit (source: Wikimedia commons; Creative Commons Attribution-Share Alike 4.0); d leaf (source: Wikimedia commons; Creative Commons Attribution-Share Alike 4.0); e fruits with seeds (source: Wikimedia commons; Creative Commons Attribution-Share Alike 4.0); f seeds (source: Wikimedia commons; Creative Commons Attribution-Share Alike 4.0); g, h, i three major phytochemicals: colchicine, gloriosine, and chelidonic acid
  • Rupa SanyalRupa Sanyal
  • Saheli NandiSaheli Nandi
  • Sharmila PandeySharmila Pandey
  • [...]
  • Abhijit DeyAbhijit Dey
Gloriosa superba L., commonly known as “gloriosa lily,” “glory lily,” and “tiger claw,” is a perennial climber in the Liliaceae family. This plant is used in African and Southeast Asian cultures as an ayurvedic medicinal herb to treat various health conditions. Its main bioactive component is colchicine, which is responsible for medicinal efficacies as well as poisonous properties of the plant. A high market demand, imprudent harvesting of G. superba from natural habitat, and low seed setting have led scientists to explore micropropagation techniques and in vitro optimization of its phytochemicals. Plant growth regulators have been used to induce callus, root, and shoot organogenesis, and somatic embryogenesis in vitro. This review is aimed at presenting information regarding the occurrence, taxonomic description, phytochemistry, micropropagation, in vitro secondary metabolite, and synthetic seed production. The data collected from the existing literature, along with an analysis of individual study details, outcomes, and variations in the reports, will contribute to the development of biotechnological strategies for conservation and mass propagation of G. superba. Key points • Latest literature on micropropagation of Gloriosa superba. • Biotechnological production and optimization of colchicine. • Regeneration, somatic embryogenesis, and synthetic seed production.
Neighbor-joining phylogenetic tree showing the phylogenetic affiliations of high-abundance OTUs (1%) in mangrove sediments (NR, non-rhizosphere sediment samples; R, rhizosphere sediment samples)
Community structure variations of the nitrifiers in mangrove sediments. a Community similarity was analyzed based on NMDS; b community and environmental factors were analyzed based on PCA. c Correlation analysis between high-abundance OTUs and community based on PCA analysis. (AOA: rose; Comammox and Comammox Nitrospira: green; AOB: blue; Comammox (amoB): brown; NOB: purple)
Quantitative PCR analysis of the different nitrifiers in mangrove sediment of the rhizosphere (R, 0–20 cm) and non-rhizosphere samples (NR, 0–20 cm). The error bars indicate the standard deviation (n = 3)
  • Shanshan MengShanshan Meng
  • Tao PengTao Peng
  • Hui WangHui Wang
  • [...]
  • Zhong HuZhong Hu
Ammonia-oxidizing archaea and ammonia-oxidizing bacteria (AOA and AOB), complete ammonia oxidizers (Comammox), and nitrite-oxidizing bacteria (NOB) play a crucial role in the nitrification process during the nitrogen cycle. However, their occurrence and diversity in mangrove ecosystems are still not fully understood. Here, a total of 11 pairs of PCR primers were evaluated to study the distribution and abundances of these nitrifiers in rhizosphere and non-rhizosphere sediments of a mangrove ecosystem. The amplification efficiency of these 11 pairs of primers was first evaluated and their performances were found to vary considerably. The CamoA-19F/CamoA-616R primer pair was suitable for the amplification of AOA in mangrove sediments, especially on the surface of rhizosphere sediments. Primer pair amoA1F/amoA2R was better for the characterization of novel AOB in the bacterial community of non-rhizosphere sediments of mangroves. In contrast, primer nxrB169F/nxrB638R showed a low abundance of NOB in mangrove sediments (except for R1). Comammox bacteria were abundant and diverse in mangrove sediments, as indicated by both the amoB gene for Comammox clade A and the amoA gene for Comammox Nitrospira clade B. However, the amoA gene for Comammox Nitrospira clade A was not successful in detecting them in the mangrove sediments. Furthermore, 568 operational taxonomic units (OTUs) were obtained by generating a clone library and a high abundance of OTUs was correlated with ammonium, pH, NO2⁻, and NO3⁻. Comammox and Comammox Nitrospira were identified by phylogenetic tree analysis, indicating that mangrove sediments harbor newly discovered nitrifiers. Additionally, many AOA and NOB were mainly distributed in the surface layer of the rhizosphere, whereas AOB and Comammox Nitrospira were in the subsurface of non-rhizosphere, as determined by qPCR analysis. Collectively, our findings highlight the limitations of some primers for the identification of specific nitrifying bacteria. Therefore, primers must be carefully selected to gain accurate insights into the ecological distribution of nitrifiers in mangroves. Key points • Several sets of PCR primers perform well for the detection of nitrifiers in mangroves. • Mangroves are an important source of newly discovered nitrifiers. • Ammonium, pH, NO2⁻, and NO3⁻ are important shapers of nitrifier communities in mangroves.
  • NoorafshaNoorafsha
  • Anil Kumar KashyapAnil Kumar Kashyap
  • Anupama KashyapAnupama Kashyap
  • [...]
  • Damini VishwakarmaDamini Vishwakarma
Copper oxide nanoparticles (CuO NPs) synthesis using an environmentally benign approach, as well as their antibacterial properties. Copper sulphate pentahydrate (CuSO4.5H2O) of different concentrations (2 mM, 5 mM and 10 mM) and aqueous Nyctanthes arbor-tristis leaf extract were used to make the CuO NPs. The synthesised CuO NPs are characterised by UV–vis spectroscopy, X-ray diffractometer (XRD), Fourier-transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). UV–vis spectroscopy confirmed the presence of CuO NPs. The functional groups of the active components were identified using the FTIR spectra of the control (leaf extract) and CuO NPs. SEM pictures revealed that the particles were rectangular, truncated triangle and spherical in shape, with sizes ranging between 4.9 nm, 18.4 nm and 23.8 nm determined using X-ray diffraction. The antibacterial activity of the produced CuO NPs was further evaluated using the well diffusion method. By observing inhibition zones around each well, the nanoparticles were revealed to have broad antibacterial action against human pathogenic bacterial strains Escherichia coli and Staphylococcus aureus withs the 7 ± 0.70-mm and 7 ± 0.21-mm inhibitory zone size respectively followed by 08 μg/mL and 2.5 μg/mL MIC respectively. Thus, these outputs concluded that the CuO NPs exhibited miraculous effect and it might be boon towards nanomedical science, pharmaceuticals and health industries. Graphical abstract Key points • Biosynthesis of CuO nanoparticle • Multifaceted utilization • Broad spectrum antimicrobial activity
Resting and operational stabilities of different transaminases. A RS, resting stability; OS, operational stability. Residual activities were measured after 8 h of incubation at 40 °C in storage buffer (50 mM sodium phosphate, pH 8, 0.1 mM PLP) or under operating conditions (storage buffer supplemented with 200 mM β-alanine, 20 mM cyclohexanone) using the acetophenone assay at 30 °C. Relative residual activities were calculated in relation to the initial activity after 1 min incubation. B Time- and temperature-dependent residual activities of TA 10 under resting conditions (storage buffer) and C operating conditions
Effect of co-solvents on the stability of TA-10. The enzyme was incubated for 16 h at operating conditions in the presence of different co-solvents at 30 °C and 60 °C. Relative activities were calculated compared to a sample which was incubated without co-solvent. The enzyme displayed full stability at 60 °C and 0–10% (v/v) organic co-solvent. For the enzyme activity measurement, the sample was diluted 10 times into the reagents of the acetophenone assay. The remaining 5% (v/v) of the different solvent showed no influence on enzyme activity compared to the reference reaction without co-solvent
Amine transaminases (ATA) convert ketones into optically active amines and are used to prepare active pharmaceutical ingredients and building blocks. Novel ATA can be identified in protein databases due to the extensive knowledge of sequence-function relationships. However, predicting thermo- and operational stability from the amino acid sequence is a persisting challenge and a vital step towards identifying efficient ATA biocatalysts for industrial applications. In this study, we performed a database mining and characterized selected putative enzymes of the β-alanine:pyruvate transaminase cluster (3N5M) — a subfamily with so far only a few described members, whose tetrameric structure was suggested to positively affect operational stability. Four putative transaminases (TA-1: Bilophilia wadsworthia, TA-5: Halomonas elongata , TA-9: Burkholderia cepacia , and TA-10: Burkholderia multivorans ) were obtained in a soluble form as tetramers in E. coli . During comparison of these tetrameric with known dimeric transaminases we found that indeed novel ATA with high operational stabilities can be identified in this protein subfamily, but we also found exceptions to the hypothesized correlation that a tetrameric assembly leads to increased stability. The discovered ATA from Burkholderia multivorans features a broad substrate specificity, including isopropylamine acceptance, is highly active (6 U/mg) in the conversion of 1-phenylethylamine with pyruvate and shows a thermostability of up to 70 °C under both, storage and operating conditions. In addition, 50% (v/v) of isopropanol or DMSO can be employed as co-solvents without a destabilizing effect on the enzyme during an incubation time of 16 h at 30 °C. Key points • Database mining identified a thermostable amine transaminase in the β-alanine:pyruvate transaminase subfamily . • The tetrameric transaminase tolerates 50% DMSO and isopropanol under operating conditions at 30 °C . • A tetrameric structure is not necessarily associated with a higher operational stability Graphical abstract
Spermidine is an important polyamine that can be used for the synthesis of various bioactive compounds in the food and pharmaceutical fields. In this study, a novel efficient whole-cell biocatalytic method with an NADPH self-sufficient cycle for spermidine biosynthesis was designed and constructed by co-expressing homoserine dehydrogenase (HSD), carboxyspermidine dehydrogenase (CASDH), and carboxyspermidine decarboxylase (CASDC). First, the enzyme–substrate coupled cofactor regeneration system from co-expression of NADP⁺-dependent ScHSD and NADPH-dependent AfCASDH exactly provides an efficient method for cofactor cycling. Second, we identified and characterized a putative CASDC with high decarboxylase activity from Butyrivibrio crossotus DSM 2876; it showed an optimum temperature of 35 °C and an optimum pH of 7.0, which make it better suited for the designed synthetic route. Subsequently, the protein expression level of each enzyme was optimized through the variation of the gene copy number, and a whole-cell catalyst with high catalytic efficiency was constructed successfully. Finally, a yield of 28.6 mM of spermidine was produced in a 1-L scale of E. coli whole-cell catalytic system with a 95.3% molar conversion rate after optimization of temperature, the ratio of catalyst-to-substrate, and the amount of NADP⁺, and a productivity of 0.17 g·L⁻¹·h⁻¹ was achieved. In summary, this novel pathway of constructing a whole-cell catalytic system from L-homoserine and putrescine could provide a green alternative method for the efficient synthesis of spermidine. Key points • A novel pathway for spermidine biosynthesis was developed in Escherichia coli. • The enzyme–substrate coupled system provides an NADPH self-sufficient cycle. • Spermidine with 28.6 mM was obtained using an optimized whole-cell system.
As an important semi-essential amino acid, l-arginine (l-Arg) has important application prospects in medicine and health care. However, it remains a challenge to efficiently produce l-Arg by Escherichia coli (E. coli). In the present study, we obtained an E. coli A1 with l-Arg accumulation ability, and carried out a series of metabolic engineering on it, and finally obtained an E. coli strain A7 with high l-Arg production ability. First, genome analysis of strain A1 was performed to explore the related genes affecting l-Arg accumulation. We found that gene speC and gene speF played an important role in the accumulation of l-Arg. Second, we used two strategies to solve the feedback inhibition of the l-Arg pathway in E. coli. One was the combination of a mutation of the gene argA and the deletion of the gene argR, and the other was the combination of a heterologous insertion of the gene argJ and the deletion of the gene argR. The combination of exogenous argJ gene insertion and argR gene deletion achieved higher titer accumulation with less impact on strain growth. Finally, we inserted the gene cluster argCJBDF of Corynebacterium glutamicum (C. glutamicum) to enhance the metabolic flux of the l-Arg pathway in E. coli. The final strain obtained 70.1 g/L l-Arg in a 5-L bioreactor, with a yield of 0.326 g/g glucose and a productivity of 1.17 g/(L· h). This was the highest level of l-Arg production by E. coli ever reported. Collectively, our findings provided valuable insights into the possibility of the industrial production of l-Arg by E. coli. Key points • Genetic background of E. coli A1 genome analysis. • Heterologous argJ substitution of argA mutation promoted excessive accumulation of L-Arg in E. coli A1. • The overexpression of L-Arg synthesis gene cluster argCJBDF of Corynebacterium glutamicum (C. glutamate) promoted the accumulation of L-Arg, and 70.1 g/L L-Arg was finally obtained in fed-batch fermentation. Graphical abstract
Events in total phosphorus (TP) removal in SF
Relative abundance of polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms * (GAOs) among the samples. Being the samples: G03, 3 m; G08, 8 m; and G12, 12 m from the front of the SF (outlet of the effluent) in the different points: (A) geotextile placed on the sand layer and (B) on the sand surface just below the geotextile
The legislation for environment protection requires strict controls of the wastewater releasing in water bodies. The wastewater treatment plants (WWTP) have been used for organic matter degradation; however, the residual total phosphorus (TP) removal has not been efficient. TP and nitrogen present in wastewater are associated to eutrophication of water bodies and algae growth. Therefore, this study discusses the efficiency of phosphorus removal by a slow filter (SF), complementary to a WWTP and the microbial community involved. The results showed that the use of SF, with or without macrophytes, is not suitable to remove TP. Spatial variation in microbial communities distributed in three distinct zones was identified in the SF. Proteobacteria, Bacteroidetes, Chloroflexi and Firmicutes covered the hydrolytic and fermentative bacteria. The acetogenesis, nitrification, and denitrification, as well as the removal of phosphorus from the effluent, were performed by representatives affiliated to different groups. Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria among these, Dokdonella sp., Frateuria sp., Comamonas sp., Diaphorobacter sp., Nitrosospira sp., Ferruginibacter sp., Flavobacterium sp., and the uncultured OD1 were the most abundant bacteria in the SF. The low efficiency for TP removing from SF effluents can be explained by the low abundance of phosphorus accumulating organisms (PAOs), with the association of the low concentration of biodegradable organic matter in the inlet effluent. Therefore, the alternative to using SF as a complement to WWTPs, as recommended by some Brazilian environmental agencies, did not prove to be viable and new approaches must be evaluated. Key points • The phosphorus removal was performed by a slow filter system in a WWTP but obtained a low efficiency. • Microbial spatial variation was distributed into distinct zones from slow filter. • Low abundance of PAOs was observed due to the low availability of organic matter.
Colonies of Pseudomonas syringae pv. maculicola and P. cannabina pv. alisalensis on DTarTA, SPamt, and SPbc media after 5, 3, and 3 days, respectively. Each image shows a 1 × 1 cm² area
Ratio of the number of colonies of Pseudomonas syringae pv. maculicola and P. cannabina pv. Alisalensis grown on DTarTA medium to that on KB medium. The vertical bars indicate the standard error for three independent experiments
Ratio of the number of colonies of Pseudomonas syringae pv. maculicola and P. cannabina pv. alisalensis on KBC, SPbc, and SPamt to that on KB. Vertical bars indicate the standard error for three independent experiments. Asterisks indicate a significant difference following Tukey’s HSD test (* P < 0.05, ** P < 0.01)
Effect of Raphanus sativus seed-associated bacteria on the recovery of Pseudomonas syringae pv. maculicola and P. cannabina pv. alisalensis on SPamt and SPbc media. The sample contained seeds in group B (Hataro, Takii Seeds, Kyoto, Japan) mixed with MAFF 106156- or MAFF 106179-inoculated seeds. Arrows point to a typical MAFF 106156 or 106179 colony
Three semi-selective media, DTarTA, SPbc, and SPamt, were developed and tested to isolate Pseudomonas syringae pv. maculicola (Psm) and P. cannabina pv. alisalensis (Pca) from Raphanus sativus seeds. DTarTA contained D-tartaric acid as a carbon source and potassium tellurite, ampicillin sodium, and methyl violet as antibiotics. DTarTA suppressed growth in 19 of the 24 pathovars from the P. syringae complex, whereas Psm and Pca grew and formed gray to black colonies. SPamt contained sucrose and peptone as nutrient sources and was supplemented with bromothymol blue and the same antibiotics present in DTarTA and Psm and Pca formed yellowish to dark brown colonies on the SPamt medium. SPbc contained sucrose and peptone and was supplemented with cephalexin and boric acid as antibiotics and Psm and Pca formed semi-translucent to white colonies on the SPbc medium. SPamt and SPbc suppressed the growth of several plant-associated bacteria (except the P. syringae complex). The growth of saprophytic bacteria in seeds on the different media was compared with that on King’s B medium, using five types of commercially available Raphanus sativus seeds. The suppression rate of DTarTA was 85–99% and was lower for seeds with more saprophytic bacteria. The suppression rates of SPamt and SPbc were 90–99%. In detection tests using 10,000 seed samples mixed with Pca or Psm-contaminated seeds, it was possible to selectively isolate Psm and Pca using SPamt and SPbc, even when the colony numbers of the target bacterium constituted less than 10% of the total colonies. Key points • Bacterial leaf spot and blight pathogens were selectively isolated from seeds. • DTarTA medium distinguishes these pathogens from P. syringae complex pathovars. • SPamp and SPbc media have different selectivity for plant-associated bacteria.
Peridinin is a light–harvesting carotenoid–pigment present in phototrophic dinoflagellates and has great potential for new drug applications and cosmetics development. Herein, the effects of irradiance mediated by light–emitting diodes on growth performance, carotenoid and fatty acid profiles, and antioxidant activity of the endosymbiotic dinoflagellate Durusdinium glynnii were investigated. The results demonstrate that D. glynnii is particularly well adapted to low–light conditions, however, it can be high–light tolerant. In contrast to other light–harvesting carotenoids, the peridinin accumulation in D. glynnii occurred during high–light exposure. The perdinin to chlorophyll–a ratio varied as a function of irradiance, while the peridinin to total carotenoids ratio remained stable. Under optimal irradiance for growth there was a peak in docosahexaenoic acid (DHA) bioaccumulation. This study contributes to the understanding of the photoprotective role of peridinin in endosymbiont dinoflagellates and highlights the strong antioxidant activity of peridinin–rich extracts.
Genetic technology applicable in oleaginous yeasts
As important chemical raw materials and potential nutritional supplements, microbial lipids play an important role in ensuring economic development, food security, and energy security. Compared with non-natural hosts, oleaginous yeasts exhibit obvious advantages in lipid yield and productivity and have great potential to be genetically engineered into an oil cell factory. The main bottleneck in the current oleaginous yeasts engineering is the lack of genetic manipulation tools. Fortunately, the rapid development of synthetic biology has provided numerous new approaches, resources, and ideas for the field. Most importantly, gene editing technology mediated by CRISPR/Cas systems has been successfully applied to some oleaginous yeasts, almost completely rewriting the development pattern of genetic manipulation technology applicable. This paper reviews recent progress in genetic technology with regard to oleaginous yeasts, with a special focus on transformation methods and genome editing tools, discussing the effects of some important genetic parts. Key points •Contribution of microbiotechnology in food safety and biofuel by oleaginous yeasts. •Advancement of genetic manipulation and transformation for oleaginous yeasts.
Principal coordinate analysis plots of Bray–Curtis dissimilarity distances. Comparison between a Al Fakher (light blue) and Fumari (light green) brands; b Fumari flavors: red, ambrosia (AMB); green, mint chocolate chill (MCC); and brown, white gummy bear (WGB); c Al Fakher flavors: dark blue, mint (MF); orange, two apple (TA); and purple, watermelon (WF) at baseline; d day 0 (gray) and day 14 (blue) in Fumari brand; e day 0 (gray) and day 14 (blue) in Al Fakher brand. Solid ellipses represent the 95% confidence intervals for brands (a), flavors (b and c), and days of incubation (d and e)
Average relative abundance of the top 25 bacterial taxa in a two brands of hookah tobacco products (Al Fakher and Fumari) and b different flavors of the two brands of hookah tobacco products (Al Fakher, watermelon (WF), two apple (TA), and mint (MF); Fumari, white gummy bear (WGB), ambrosia (AMB), and mint chocolate chill (MCC))
Longitudinal shifts in the average relative abundance (+ / − standard deviation) of the top 25 bacterial taxa over time (from D0 to D14) of incubation for Al Fakher (light blue) and Fumari (light green) brands
Bacterial profiles of shared and unique taxa between Hookah brands (A), Fumari flavors (B), and Al Fakher flavors (C) visualized by network plots in Cytoscape
Young adults are increasingly using non-cigarette products, such as hookahs, since they are perceived as healthier alternatives to cigarette smoking. However, hookah users are exposed to not only carcinogenic compounds but also microorganisms that may play an active role in the development of both infectious and chronic diseases among users. Nevertheless, existing hookah research in this area has focused only on microorganisms that may be transferred to users through the smoking apparatus and not on bacterial communities associated with hookah tobacco. To address this knowledge gap, we conducted time-series experiments on commercially available hookah brands (Al Fakher (flavors: two apple, mint, and watermelon) and Fumari (flavors: white gummy bear, ambrosia, and mint chocolate chill)) stored under three different temperature and relative humidity conditions over 14 days. To characterize bacterial communities, the total DNA was extracted on days 0, 5, 9, and 14, PCR-amplified for the V3V4 region of the bacterial 16S rRNA gene, sequenced on the Illumina HiSeq platform, and analyzed using R. Diversity (alpha and beta) analyses revealed that the microbiotas of Fumari and Al Fakher products differed significantly and that flavor had a significant effect on the hookah microbiota. Overall, Pseudomonas , Bacillus , Sphingomonas , and Methylobacterium were the predominant bacterial taxa across all products . Additionally, we observed compositional differences between hookah brands across the 14-day incubation . These data suggest that the bacterial communities of hookah tobacco are diverse and differ across brands and flavors, which may have critical implications regarding exposures to specific bacteria among hookah users. Key points • Commercial hookah products harbor diverse bacterial communities . • Brands and flavors impact the diversity of these communities . • Research on their viability and transmission to users’ respiratory tracts is needed . Graphical abstract
Growth curves of coculture systems (a) and speculative growth of mutant cells in each coculture system determined by qPCR (b). “TY + TYΔndpA,” “TY + TYΔndpB,” “TY + TYΔndpC,” “TY + TYΔndpD,” “TY + TYΔndpT,” and “TY + TYΔsRNA-E” means the coculture of wild-type TY with corresponding mutant strains. The black arrows in graph B indicate the two sampling points, SP1 and SP2, for comparative transcriptome analysis. The result shown in a is the average OD600 at each sampling point from four biological replicates. Error bars indicate the standard error
Principal component analysis (PCA) and principal co-ordinates analysis (PCoA) of transcriptome data. Each point in the graph represents a biological sample. SP1.1, SP1.2, and SP1.3 are three biological replicates of the SP1 group. SP2.1, SP2.2, and SP2.3 are three biological replications of the SP2 group. PC1 indicates principal component 1, and PC2 indicates principal component 2
Relative expression of genes validated by qRT-PCR. These 16 genes show the same trend of change with transcriptome results. The error bars represent the standard errors from the analysis of three biological replicates. The asterisk between two groups indicates that the two groups show significant differences (p < 0.05). “ns” means non-significant differences
Growth curve of TYΔndpD and wild-type TY in the supernatant medium. The result shown here is the average OD600 at each sampling point from three biological replicates. Error bars indicate the standard error
Schematic of competition mechanisms of S. melonis TY employed during coculture with the isogenic mutant TYΔndpD under nutritional stress. This graph shows the brief changes in cellular processes and functions in wild-type TY cell (left) and TYΔndpD cell (right) according to DEGs when comparing SP2 with SP1. Nicotine metabolism pathway, the illustrated nicotine degradation pathway and molecular mechanism in S. melonis TY, wild-type TY cells hold the complete pathway, whereas the TYΔndpD cells are deficient in catalyzing 6-hydroxy-3-succinoyl-pyridine to 2,5-dihydroxy-pyridine. Catabolic genes involved in nicotine degradation are ndpA, nicotine hydroxylase; ndpB, 6-hydroxy-l-nicotine oxidase; ndpC, 6-hydroxypseudooxynicotine oxidase; ndpD, 6-hydroxy-3-succinoyl-pyridine monooxygenase; ndpE, 2,5-dihydroxypyridine dioxygenase; ndpF, N-formylmaleamic acid deformylase; ndpG, maleamate amidase; and ndpH, maleate isomerase. Genes involved in the carbon and nitrogen metabolism response in wild-type TY include AmtB, ammonium transporter; P-II, P-II family nitrogen regulator; Crp/Fnr, Crp/Fnr family transcriptional regulator; and sarcosine oxidase. Genes involved in the DNA repair response in wild-type TY include RecA, DNA recombination/repair protein, and YbaB/EbfC, YbaB/EbfC family nucleoid–associated protein. Upregulated antitoxin genes in wild-type TY include phd, vapB, mazE, and parD. CvpA indicates colicin V production protein. Genes involved in the stress response in wild-type TY include Ohr, organic hydroperoxide resistance protein; PspB, envelope stress response membrane protein; CckA, PAS domain S-box protein; SOD, superoxide dismutase; and OmpR, DNA-binding response regulator. Genes involved in the DNA replication in wild-type TY include NrdA, ribonucleoside-diphosphate reductase subunit alpha, and ParA, chromosome partitioning protein. TadE/G and PilZ represent pilus assembly protein. EF-Tu represents elongation factor Tu. ParE, Doc, VapC, and MazF are corresponding toxin proteins of ParD, Phd, VapB, and MazE. The bactericidal effects of these toxin proteins on TYΔndpD cell include blocking DNA replication, inhibiting protein synthesis, and cleavage of tRNA, mRNA, and 23S rRNA. The plus and minus signs in the extracellular and intracellular sides of TYΔndpD cell membrane indicate the normal membrane potential, colicin V will disrupt membrane potential, resulting in depolarization. The toxin proteins in the yellow dotted box in the middle of the diagram mean that the toxin proteins may be delivered to TYΔndpD from wild-type TY. The yellow dotted box inside the TYΔndpD cell means that the bactericidal effect may be caused by endogenous toxin protein, because labile antitoxins are easily degraded by proteases under stress condition
Bacteria have developed various mechanisms by which they can compete or cooperate with other bacteria. This study showed that in the cocultures of wild-type Sphingomonas melonis TY and its isogenic mutant TYΔndpD grow with nicotine, the former can outcompete the latter. TYΔndpD undergoes growth arrest after four days when cocultured with wild-type TY, whereas the coculture has just entered a stationary phase and the substrate was nearly depleted, and the interaction between the two related strains was revealed by transcriptomic analysis. Analysis of the differential expression genes indicated that wild-type TY inhibited the growth of TYΔndpD mainly through toxin-antitoxin (TA) systems. The four upregulated antitoxin coding genes belong to type II TA systems in which the bactericidal effect of the cognate toxin was mainly through inhibition of translation or DNA replication, whereas wild-type TY with upregulated antitoxin genes can regenerate cognate immunity protein continuously and thus prevent the lethal action of toxin to itself. In addition, colicin-mediated antibacterial activity against closely related species may also be involved in the competition between wild-type TY and TYΔndpD under nutritional stress. Moreover, upregulation of carbon and nitrogen catabolism related–, stress response related–, DNA repair related–, and DNA replication–related genes in wild-type TY showed that it triggered a series of response mechanisms when facing dual stress of competition from isogenic mutant cells and nutritional limitation. Thus, we proposed that S. melonis TY employed the TA systems and colicin to compete with TYΔndpD under nutritional stress, thereby maximally acquiring and exploiting finite resources. • Cross-feeding between isogenic mutants and the wild-type strain. • Nutrition stress caused a shift from cooperation to competition. • TYΔndpD undergo growth arrest by exogenous and endogenous toxins.
A NMDS analysis based on the Bray–Curtis distance. Each point in the scatter plot represents one sample, and the distance between points represents relative dissimilarities in community structures. The closer in the distance, the higher their community similarity. The three samples used for whole-genome metagenomic analysis are labeled in red. B Comparison of the gut bacterial community at the phylum level. C The accumulated relative abundance of known pathogens listed in the NCBI Pathogen Detection System in the gut microbiota of the ten individuals
A Co-occurrence network generated 22 interacting modules based on the microbial relative abundance data. B Microbial interactions in module1 (M1) that was the most complex module, and species names were shown near the nodes, which were recognized as zoonotic pathogens
A Comparison of gut bacterial composition revealed at the phylum level. B The Venn diagram of functional distributions of the three samples based on the annotated KOs by the emapper pipeline. C Microbial composition of the VF-containing reads revealed at the family level
A Pathogenic and beneficial functional traits in sample 3 (harboring lowest abundance of pathogens), sample 2 and sample 5 (harboring relative high abundance of pathogens). B Accumulated abundance of VF encoding genes in the three samples. C Accumulated abundance of SCFA synthesis genes in the three samples. D Abundance of representative bacteriophages in the three samples. The abundance of VF, SCFA synthesis genes, and bacteriophages were displayed as mapped reads in each sample
Overview of the recovered MAGs and their relative abundance comparison among samples 2, 3, and 5. The neighbor-joining (NJ) phylogenetic trees were reconstructed using MEGA X (Kumar et al. 2018) based on full-length 16S rDNA sequences extracted from each MAG and their close relatives identified in EZBiocloud database (Yoon et al. 2017). The MAGs were recovered from the metagenomic data using MetaBAT2 (Kang et al. 2015), and the completeness and contamination rate were estimated using CheckM (Parks et al. 2015). A metabat41, with a genome size of 5.54 Mb spanning 69 contigs and completeness rate 99.27% and contamination rate 4.77%. B metabat35, 2.33 Mb spanning 30 contigs with completeness rate 98.77% and contamination rate 3.33%, exhibiting 99.25% ANI with reference genome GCF_000741295.1. C metabat52, 1.71 Mb spanning 27 contigs with completeness rate 85.82% and contamination rate 0%, showing 97.6% ANI with reference genome GCF_001436025.1
Certain animals harbor a high proportion of pathogens, particular the zoonotic pathogens, in their gut microbiome but are usually asymptomic; however, their carried pathogens may seriously threaten the public health. By understanding how the microbiome overcomes the negative effects of pathogens to maintain host health, we can develop novel solutions to control animal-mediated pathogen transmission including identification and application of beneficial microbes. Here, we analyzed the gut microbiota of 10 asymptomic captive sika deer individuals by full-length 16S rDNA sequencing. Twenty-nine known pathogens capable of infecting humans were identified, and the accumulated proportions of the identified pathogens were highly variable among individuals (2.33 to 39.94%). The relative abundances of several beneficial bacteria, including Lactobacillus and Bifidobacterium, were found to be positively correlated with the relative abundances of accumulated pathogens. Whole-genome metagenomic analysis revealed that the beneficial- and pathogenic-associated functions, such as genes involved in the synthesis of short chain fatty acids and virulence factors, were also positively correlated in the microbiome, indicating that the beneficial and pathogenic functions were maintained at a relatively balanced ratio. Furthermore, the bacteriophages that target the identified pathogens were found to be positively correlated with the pathogenic content in the microbiome. Several high-quality genomes of beneficial bacteria affiliated with Lactobacillus and Bifidobacterium and bacteriophages were recovered from the metagenomic data. Overall, this study provides novel insights into the interplay between beneficial and pathogenic content to ensure maintenance of a healthy gut microbiome, and also contributes to discovery of novel beneficial microbes and functions that control pathogens. Key points • Certain asymptomic captive sika deer individuals harbor relatively high amounts of zoonotic pathogens. • The beneficial microbes and the beneficial functions are balanced with the pathogenic contents in the gut microbiome. • Several high-quality genomes of beneficial bacteria and bacteriophages are recovered by metagenomics.
Amidophosphoribosyltransferase catalyzes the conversion of 5-phosphoribosyl-1-pyrophosphate into 5-phosphoribosyl-1-amine in the de novo purine biosynthetic pathway. Herein, we identified and characterized the functions of MoAde4, an orthologue of yeast Ade4 in Magnaporthe oryzae. MoAde4 is a 537-amino acid protein containing GATase_6 and pribosyltran domains. MoADE4 transcripts were highly expressed during the conidiation, early-infection, and late-infection stages of the fungus. Disruption of the MoADE4 gene resulted in ΔMoade4 exhibiting adenine, adenosine, and hypoxanthine auxotrophy on minimal medium. Conidia quantification assays showed that sporulation was significantly reduced in the ΔMoade4 mutant. The conidia of ΔMoade4 could still form appressoria but mostly failed to penetrate the rice cuticle. Pathogenicity tests showed that ΔMoade4 was completely nonpathogenic on rice and barley leaves, which was attributed to restricted infectious hyphal growth within the primary cells. The ΔMoade4 mutant was defective in the induction of strong host immunity. Exogenous adenine partially rescued conidiation, infectious hyphal growth, and the pathogenicity defects of the ΔMoade4 mutant on barley and rice leaves. Taken together, our results demonstrated that purine nucleotide biosynthesis orchestrated by MoAde4 is required for fungal development and pathogenicity in M. oryzae. These findings therefore act as a suitable target for antifungal development against recalcitrant plant fungal pathogens. Key points • MoAde4 is crucial for de novo purine nucleotide biosynthesis. • MoAde4 is pivotal for conidiogenesis and appressorium development of M. oryzae. • MoAde4 is involoved in the pathogenicity of M. oryzae.
Micropipette tips are currently among the most used disposable devices in bioresearch and development laboratories. Their main application is the fractionation of solutions. New functionalities have recently been added to this device, widening their applications. This paper analyzed disposable micropipette tips as reagent holders of PCR reagents. PCR has become a prevalent and often indispensable technique in biological laboratories for various applications, such as the detection of coronavirus and other infectious diseases. A functional micropipette tip was implemented to simplify PCR analysis and reduce the contamination chances of deoxynucleotides and specific primers. This disposable device is prepared by tip coating processes of reagents, using polyvinyl alcohol polymer and additives. The coated layer is optimized to load and release PCR reagents efficiently. As a proof of concept, we show that the detection of Bordetella pertussis, the etiological agent of whooping cough whose diagnostic relies on PCR, can be quickly done using practical-functional tips. This device is an excellent example of testing the functionality and contribution of molecular diagnostic PCR tips. KEY POINTS: • Functional micropipette tips are prepared by coating with dNTPs and primers. • Functional tips are used to replace dNTPs and primers in the PCR master mix. • PCR diagnostic of Bordetella pertussis is performed using functional tips.
Smokeless tobacco (SLT) alters the oral microbiome of smokeless tobacco users. Dysbiosis of oral bacteriome has been determined; however, the mycobiome of SLT users has not been characterized. The oral mycobiome was assayed by amplification and sequencing of the fungal internal transcribed spacer (ITS1) region from oral swab samples of non-SLT users, SLT users (with or without oral lesions), and SLT with alcohol users. We observed that the richness and diversity of oral mycobiome were significantly decreased in SLT with oral lesions users than in non-users. The β-diversity analysis showed significant dissimilarity of oral mycobiome between non-users and SLT with oral lesions users. Linear discriminant analysis effect size and random forest analysis of oral mycobiome affirm that the genus Pichia was typical for SLT with oral lesions users. Prevalence of the fungal genus Pichia correlates positively with Starmerella, Mortierella, Fusarium, Calonectria, and Madurella, but is negatively correlated with Pyrenochaeta, Botryosporium, and Alternaria. Further, the determination of oral mycobiome functionality showed a high abundance of pathotroph-saprotroph-symbiotroph and animal pathogen-endophyte-epiphyte-undefined saprotroph at trophic and guild levels, respectively, indicating possibly major changes in normal growth repression of types of fungi. The oral mycobiome in SLT users was identified and comprehensively analyzed for the first time. SLT intake is associated with oral mycobiome dysbiosis and such alterations of the oral mycobiome may contribute to oral carcinogenesis in SLT users. This study will provide a basis for further large-scale investigations on the potential role of the mycobiome in SLT-induced oral cancer. Key points • SLT induces dysbiosis of the oral microbiome that can contribute to oral cancer. • Oral mycobiome diversity is noticeably reduced in SLT users having oral lesions. • Occurrence of Pichia can be used as a biomarker for SLT users having oral lesions.
a Multiple sequence alignments of BcPLC and the selected proteins including BAD85570.1 (TkPLC). Only protein regions with conserved amino acids are shown. Conserved residues involved in binding the three zinc ions in the active site of BcPLC are highlighted. See Table S1, ESM_1 for accession numbers and further details of the selected sequences included in the alignment. b Schematic representation of the different domain arrangements in TkPLC and BcPLC. The CUB-like domain insertion in TkPLC is represented at the end of the PLC fold
TkPLC enzymatic activity with NPPC as substrate. aTkPLC activity was measured between 30 and 100 °C. bTkPLC thermal stability. Enzymatic activity was measured at 80 °C after pre-incubation of TkPLC between 30 and 100 °C for 30 min. cTkPLC enzymatic activity analysis in different reaction conditions (pH 4.0–8.0, 5 mM EDTA) at 80 °C. The results are expressed as means and standard deviations of at least three independent experiments
Effect of temperature and denaturing agents on TkPLC and BcPLC stability. Transition temperature analysis of TkPLC and BcPLC at different temperatures by CD spectroscopy
Enzymatic activity in crude soybean oil determined as a function of released phosphate. a Time course hydrolysis analysis at 80 °C (5, 10, 15 µg of TkPLC per gram crude oil). b Enzymatic activity of TkPLC measured after 120 min at different temperatures between 30 and 100 °C. c Enzymatic activity of TkPLC compared with BcPLC at 50 and 80 °C. The results are expressed as means and standard deviations of at least three independent experiments
TkPLC and BcPLC hydrolysis of phospholipids in oil. NMR spectra for crude soybean oil to which no enzyme was added as a control (a), treated with TkPLC for 120 min at 80 °C (b), treated with BcPLC for 120 min at 50 °C (c) and treated with BcPLC for 120 min at 80 °C (d). PC phosphatidylcholine, PE phosphatidylethanolamine, PI phosphatidylinositol, PA phosphatidic acid, pCho phosphocholine, pEt phosphoethanolamine
The implementation of cleaner technologies that minimize environmental pollution caused by conventional industrial processes is an increasing global trend. Hence, traditionally used chemicals have been replaced by novel enzymatic alternatives in a wide variety of industrial-scale processes. Enzymatic oil degumming, the first step of the oil refining process, exploits the conversion catalyzed by phospholipases to remove vegetable crude oils’ phospholipids. This enzymatic method reduces the gums’ volume and increases the overall oil yield. A thermostable phospholipase would be highly advantageous for industrial oil degumming as oil treatment at higher temperatures would save energy and increase the recovery of oil by facilitating the mixing and gums removal. A thermostable phosphatidylcholine (PC) (and phosphatidylethanolamine (PE))-specific phospholipase C from Thermococcus kodakarensis (TkPLC) was studied and completely removed PC and PE from crude soybean oil at 80 °C. Due to these characteristics, TkPLC is an interesting promising candidate for industrial-scale enzymatic oil degumming at high temperatures. Key points • A thermostable phospholipase C from T. kodakarensis (TkPLC) has been identified. • TkPLC was recombinantly produced in Pichia pastoris and successfully purified. • TkPLC completely hydrolyzed PC and PE in soybean oil degumming assays at 80 °C.
An overview of the biotransformation reactions occurring in yeast. Abbreviations: 3MH 3-mercaptohexanol, 3MHA 3-mercaptohexyl acetate, Cys cysteine, GSH glutathione, TPA terpene alcohol. Credit: Henrik Svedlund
(A) Cones of the hop cultivar Taurus. Cones are ~ 5 cm in length. (B) Longitudinal section of a hop cone showing lupulin glands at the base of bracteoles. (C) A light microscopy image of ripe lupulin glands. Bar = 500 µm. (D) Scanning electron micrograph of a ripe lupulin gland showing the peaked appearance of the filled subcuticular sac. Bar = 100 µm (Nagel et al. 2008). Reprinted with permission
A summary of hop- and grape-derived flavour compounds that can be released or formed through biotransformation reactions
There is a growing appreciation for the role that yeast play in biotransformation of flavour compounds during beverage fermentations. This is particularly the case for brewing due to the continued popularity of aromatic beers produced via the dry-hopping process. Here, we review the current literature pertaining to biotransformation reactions mediated by fermentative yeasts. These reactions are diverse and include the liberation of thiols from cysteine or glutathione-bound adducts, as well as the release of glycosidically bound terpene alcohols. These changes serve generally to increase the fruit and floral aromas in beverages. This is particularly the case for the thiol compounds released via yeast β-lyase activity due to their low flavour thresholds. The role of yeast β-glucosidases in increasing terpene alcohols is less clear, at least with respect to fermentation of brewer’s wort. Yeast acetyl transferase and acetate esterase also have an impact on the quality and perceptibility of flavour compounds. Isomerization and reduction reactions, e.g. the conversion of geraniol (rose) to β-citronellol (citrus), also have potential to alter significantly flavour profiles. A greater understanding of biotransformation reactions is expected to not only facilitate greater control of beverage flavour profiles, but also to allow for more efficient exploitation of raw materials and thereby greater process sustainability. Key points • Yeast can alter and boost grape- and hop-derived flavour compounds in wine and beer • β-lyase activity can release fruit-flavoured thiols with low flavour thresholds • Floral and citrus-flavoured terpene alcohols can be released or interconverted
Glaesserella parasuis is an important bacterial pathogen that affects the swine industry worldwide. Research on the pathogenic mechanism and genetically engineered vaccine remains undeveloped because an effective markerless and multiple-gene knockout system is unavailable for G. parasuis yet. To establish a markerless knockout, deleted allelic genes with kanamycin resistance (KanR) cassettes were introduced into the genome of G. parasuis by using natural transformation with suicide plasmids. Then, the KanR cassette was excised with a thermosensitive plasmid pGF conferring a constitutive Flp expression. To realize the markerless and multiple-gene knockout, plasmid pGAF was constructed by placing the Flp gene under the control of an arabinose-inducible promoter. Firstly, pGAF was introduced into G. parasuis by electroporation, and the marked mutants were produced following natural transformation. Finally, the KanR cassette was excised from the genome by the inducible expression of Flp upon arabinose action. Based on the natural transformation and the inducible expression of Flp, the markerless single-gene knockout mutants of ΔhsdR, ΔneuA2, ΔespP2, Δapd, and ΔnanH were constructed. In addition, a five-gene knockout mutant of ΔhsdRΔneuA2ΔespP2ΔapdΔnanH was generated by successive natural transformation with five suicide plasmids. Taken together, a markerless and multiple-gene deletion system was established for G. parasuis in the present study for the first time. This system is simple, efficient, and easy to manipulate for G. parasuis; thus, our technique will substantially aid the understanding of the etiology, pathogenesis, and genetic engineering of G. parasuis and other bacteria that can be naturally transformed in laboratory conditions. Key points • Flp recombinase excised the KanR gene flanked by FRT sites in Glaesserella parasuis. • The regulatory expression of Flp enabled a multiple-gene knockout forG. parasuis. • The technique will promote the understanding of Glässer’s disease pathogens. Graphical abstract
aHaslea ostrearia culture in airlift plan-photobioreactor system (Laboratoire GEPEA/Université de Nantes/CNRS) and b Sketche of lab-scale airlift plan-PBR for a controlled culture of H. ostrearia (batch mode)
Changes in photosynthetic pigment content (Chla: chlorophyll a; Chlc: chlorophyll c; carotenoids) and specific growth rate of Haslea ostrearia at different growth conditions a temperature, b light intensity, and c pH). When varying different growth conditions, 200 µmol photons m⁻² s⁻¹ and pH 7.0 were used for temperature treatment, 20 °C and pH 7.0 for light treatment, and 20 °C and 200 µmol photons m⁻² s.⁻¹) for pH treatment. Values are means ± SE (n = 3) and * indicates significant difference between treatments (one-way ANOVA, Scheffé test at p ≤ 0.05)
Variation of extracellular marennine (EMn) concentration in Haslea ostrearia grown under different growth conditions (a temperature, b light intensity, and c pH). During the variation of different growth conditions, 200 µmol photons m⁻² s⁻¹ and pH 7.0 were used for temperature treatment, 20 °C and pH 7.0 for light treatment, and 20 °C and 200 µmol photons m⁻² s.⁻¹ for pH treatment. Values are means ± SE (n = 3) and * indicates significant difference between means (one-way ANOVA, Scheffé test at p ≤ 0.05)
Effect of different growth conditions (a temperature, b light intensity, and c pH) in protein content of H. ostrearia. When varying temperature, light intensity, and pH, 200 µmol photons m⁻² s⁻¹ and pH 7.0 were used for temperature treatment, 20 °C and pH 7.0) for light treatment, and 20 °C and 200 µmol photons m⁻² s.⁻¹ for pH treatment. Values are means ± SE (n = 3) and * indicates significant difference between means (one-way ANOVA, Scheffé test at p ≤ 0.05)
Total fatty acid content of H. ostrearia in function of different growth conditions (a temperature, b light intensity, and c pH). On the variation of different growth conditions, 200 µmol photons m⁻² s⁻¹ and pH 7.0 were used for temperature treatment, 20 °C and pH 7.0 for light treatment, and 20 °C and 200 µmol photons m⁻² s.⁻¹ for pH treatment. Values are means ± SE (n = 3) and * indicates significant difference between means (one-way ANOVA, Scheffé test at p ≤ 0.05)
Haslea ostrearia is a pennate diatom that produces marennine, a water-soluble blue pigment responsible for the greening phenomenon and the increase of organoleptic quality of oysters. Apart from the oyster industry, there is a growing interest in the mass cultivation of this diatom due to the biological activities of marennine. To gain knowledge about the feasibility to upscale production of this diatom, in particular, in the context of global warming, the effects of different temperatures (20, 25, and 30 °C), irradiances (100, 200, and 300 μmol photons m⁻² s⁻¹), and pH (7.0, 8.0, and 9.0) on growth and biochemical composition were studied in H. ostrearia cultured in an airlift plan-photobioreactor. The maximum growth rate of H. ostrearia (0.9 ± 0.0 day⁻¹) was obtained at 20 °C, 200 μmol photons m⁻² s⁻¹, and pH 7.0, referred to as control conditions. The highest concentration in Chla (2.5 ± 0.1 µg 10⁻⁶ cells) and total fatty acids (71.6 ± 1.4 mg g⁻¹ of dry weight, DW) was observed at 20 °C, 300 μmol photons m⁻² s⁻¹, and pH 7.0. The highest concentration of carotenoids (1.4 ± 0.1 µg 10⁻⁶ cells), Chlc (1.3 ± 0.1 µg 10⁻⁶ cells), and extracellular marennine (33.1 ± 0.2 µg 10⁻⁶ cells) was observed at 30 °C, 200 μmol photons m⁻² s⁻¹, and pH 7.0, and a higher protein content (309.7 ± 24.5 mg g⁻¹ of DW) at 25 °C, 200 μmol photons m⁻² s⁻¹, and pH 7.0. The biomass of H. ostrearia was enriched with C14:0 fatty acid at 30 °C, 200 μmol photons m⁻² s⁻¹, and pH 7.0, and with C16:0 and C16:1n − 7 fatty acids at control conditions. However, DHA C22:6n − 3 (ω-3), C22:0, and C20:0 were only observed at 300 µmol photons m⁻² s⁻¹, 20 °C, and pH 7.0. A high abundance of essential polyunsaturated fatty acids C22:1n − 9 (ω-9) + C20:5n − 3 (EPA) was observed at 100 µmol photons m⁻² s⁻¹, 20 °C, and pH 7.0. It is thus possible to anticipate and tune the production of specific metabolites through the control of growth conditions of the benthic diatom H. ostrearia. Key points • Validation of H. ostrearia culture in a new photobioreactor in batch mode • Biochemical composition variation of H. ostrearia in function of growth conditions • Growth inhibition and unbalanced metabolites induced by the treatment conditions
In the present work, bioleaching of two valuable metals of cobalt (Co) and nickel (Ni) from spent lithium-ion batteries (LIBs) of laptop by Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans through a novel adaptation procedure was investigated. Different bioleaching methods including A. ferrooxidans and A. thiooxidans spent medium, A. ferrooxidans one-step and two-step bioleaching were carried out. The effect of silver ion on the bioleaching of Co and Ni in these methods was evaluated. Moreover, a novel strain adaptation approach to the toxic solid content of the battery powder was chosen, which resulted in a very short adaptation time and bioleaching (2 days). Even though silver ion did not have a significant effect on the spent medium method, it had an increasing effect of 26% and 7%, for Co and Ni recovery, respectively, on two-step bioleaching with silver ion–adapted A. ferrooxidans, in gradual addition of the battery powder. The highest extraction results in the spent medium method were 45.2% and 71.5% for Co and Ni, respectively, and a very high extraction yield of 99.95% for these metals was achieved in a short time of only 3 days by two-step bioleaching with gradual addition of the solid content and in the presence of Ag⁺. Key points • Mixed spent medium of acidophilic bacteria resulted in higher Ni and Co extraction. • Adaptation to Ag⁺has enhanced the strain capability for Co and Ni extraction. • With Ag⁺presence, Co and Ni extraction reached 99.95% in two-step bioleaching. Graphical abstract
Sexual cycle of the diploid and homothallic yeast Xanthophyllomyces dendrorhous (adapted from Webster and Weber 2007) starting from fusion of diploid vegetative cells to form a tetraploid zygote and meiotic transformation to diploid basidiospores, which germinate, into vegetative growing cells. The centre shows a scanning electron-microscopic picture of a budding cell (red circle)
Mitotic recombination affecting transformants of Xanthophyllomces dendrorhous.A Transformation of a yellow mutant and selection of red transformants with hygromycin; growth of a red cells in the absence of hygromycin resulting in a mixed culture with red and yellow colonies; repeated sub-cultivation and selection of the red phenotype yields homogenous stable cultures, in analogy to Breitenbach et al. (2019). B Illustration of mitotic recombination of the heterozygous diploid X. dendrorhous transformant and segregation into homozygous wild type with the transgenic phenotype
Formation of an intermediate phenotype due to heterozygosity after gene inactivation of Xanthophyllomces dendrorhous and generation of homozygous lines through a sexual cycle corresponding to Pollmann et al. (2017c). A Inactivation of the gene of the phytoene-metabolising enzyme, crtI, resulting in yellowish transformants (IT). B Demonstration of the presence of the wild type together with the inactivated gene (lane IT) in the transformant. C Different stages of the sexual cycle of the diploid heterozygous transformant and selection of segregated, white lines (ST) which are homozygous for the inactivated gene (part B, lane ST)
The nonconventional yeast Xanthophyllomyces dendrorhous is an established platform for genetic pathway modification. A genetic tool box is available and can be used extensively to select from for different engineering strategies. Due to the diploid nature of X. dendrorhous, genetic transformation typically results in heterozygous lines. They are genetically unstable and lose their phenotypes caused by mitotic recombination. In addition, targeted integration for inactivation of genes of the carotenoid pathway resulted in an intermediary phenotype of incomplete pathway disruption. This issue is the main scope of this review. It is illustrated by using genetic modification of the carotenoid pathway of X. dendrorhous as a model system with a focus on the demonstration of how to solve these problems by generation of homozygous lines. They can be selected from heterozygous transformants after spontaneous mitotic recombination and selection or after induced meiotic recombination. Corresponding methods of how to proceed including the initiation of a sexual cycle are described. The selected segregated lines are stable in fermenter cultures without the need of selection pressure. This is an essential requirement for any industrial application. Key points • Genetic interventions of diploid yeasts result in heterozygous transformants that are unstable without selection pressure. • This is due to mitotic recombination leading to the elimination of inserted DNA. • Stable homozygous lines can be obtained and selected after either meiotic or mitotic recombination.
Infliximab is a mouse/human chimeric IgG1 monoclonal antibody which recognizes the proinflammatory cytokine, tumor necrosis factor α (TNFα), and inhibits receptor interactions, thereby decreasing inflammation and autoimmune response in patients. This monoclonal antibody has been successfully used to treat rheumatoid arthritis, ankylosing spondylitis, and psoriatic arthritis. However, the high treatment cost limits patient access to this biotherapy. One alternative to this problem is the use of biosimilars. In this work, we describe the stable expression and physicochemical characterization of an anti-TNFα antibody. While infliximab is produced in recombinant murine SP2/0 cells, our anti-TNFα IgG antibody was expressed in recombinant murine NS0 myeloma cells. The best anti-TNFα antibody-expressing clone was selected from three clone candidates based on the stability of IgG expression levels, specific productivity as well as TNFα-binding activity compared to commercial infliximab. Our results indicate that the selected cell clone, culture medium, and fermentation mode allowed for the production of an anti-TNFα antibody with similar characteristics to the reference commercially available product. An optimization of the selected culture medium by metabolomics may increase the volumetric productivity of the process to satisfy the demand for this product. Further experiments should be performed to evaluate the biological properties of this anti-TNFα antibody. Key points • An anti-TNFα antibody was produced in NS0 cells using perfusion culture. • A proprietary chemically defined culture medium was used to replace commercially available protein-free medium. • The purified anti-TNFα antibody was comparable to the reference marketed product.
Beta-glucosidase (Bgl) is an enzyme with considerable food, beverage, and biofuel processing potential. However, as many Bgls are inhibited by their reaction end product glucose, their industrial applications are greatly limited. In this study, a novel Bgl gene (Bgl1973) was cloned from Leifsonia sp. ZF2019 and heterologously expressed in E. coli. Sequence analysis and structure modeling revealed that Bgl1973 was 748 aa, giving it a molecular weight of 78 kDa, and it showed high similarity with the glycoside hydrolase 3 (GH3) family Bgls with which its active site residues were conserved. By using pNPGlc (p-nitrophenyl-β-D-glucopyranoside) as substrate, the optimum temperature and pH of Bgl1973 were shown to be 50 °C and 7.0, respectively. Bgl1973 was insensitive to most metal ions (12.5 mM), 1% urea, and even 0.1% Tween-80. This enzyme maintained 60% of its original activity in the presence of 20% NaCl, demonstrating its excellent salt tolerance. Furthermore, it still had 83% residual activity in 1 M of glucose, displaying its outstanding glucose tolerance. The Km, Vmax, and kcat of Bgl1973 were 0.22 mM, 44.44 μmol/min mg, and 57.78 s⁻¹, respectively. Bgl1973 had a high specific activity for pNPGlc (19.10 ± 0.59 U/mg) and salicin (20.43 ± 0.92 U/mg). Furthermore, molecular docking indicated that the glucose binding location and the narrow and deep active channel geometry might contribute to the glucose tolerance of Bgl1973. Our results lay a foundation for the studying of this glucose-tolerant β-glucosidase and its applications in many industrial settings. Key points • A novel β-glucosidase from GH3 was obtained from Leifsonia sp. ZF2019. • Bgl1973 demonstrated excellent glucose tolerance. • The glucose tolerance of Bgl1973 was explained using molecular docking analysis.
The statistical result of GSMMs since 1999. A: the distribution of all GSMMs, and the increase in each year
The development of the first-generation GSMMs
The development of the second-generation GSMMs. GRN gene transcriptional regulatory network, PPI protein–protein interaction network, STN signal transduction network
Over the last two decades, thousands of genome-scale metabolic network models (GSMMs) have been constructed. These GSMMs have been widely applied in various fields, ranging from network interaction analysis, to cell phenotype prediction. However, due to the lack of constraints, the prediction accuracy of first-generation GSMMs was limited. To overcome these limitations, the next-generation GSMMs were developed by integrating omics data, adding constrain condition, integrating different biological models, and constructing whole-cell models. Here, we review recent advances of GSMMs from the first generation to the next generation. Then, we discuss the major application of GSMMs in industrial biotechnology, such as predicting phenotypes and guiding metabolic engineering. In addition, human health applications, including understanding biological mechanisms, discovering biomarkers and drug targets, are also summarized. Finally, we address the challenges and propose new trend of GSMMs. Key points •This mini-review updates the literature on almost all published GSMMs since 1999. •Detailed insights into the development of the first- and next-generation GSMMs. •The application of GSMMs is summarized, and the prospects of integrating machine learning are emphasized.
WGP addition improved yeast cell growth and survival under osmotic stress. a Growth in 30% (w/v) sorbitol medium. b Survival in 30% (w/v) sorbitol medium. All data are presented as mean values from three independent experiments. Error bars indicate the standard deviations
WGP affects cell membrane fatty acids and ergosterol under 30% (w/v) sorbitol stress. a Cluster heatmap analysis of fatty acid composition. The transcriptional levels from bOLE1 and cERG1 genes. d Ergosterol contents in 30% (w/v) sorbitol medium. UFA, unsaturated fatty acids; SFA, saturated fatty acids; UI, unsaturation index. All data are presented as mean values from three independent experiments. Error bars indicate the standard deviations. *p < 0.05; **p < 0.01; ***p < 0.001
PI dying observation of yeast cells through fluorescence microscopy. a1, a2, a3 Control group, b1, b2, b3 WGP-LL group,c1, c2, c3 WGP-LML group, d1, d2, d3 WGP-LLL group. e WGP affects yeast cell membrane integrity, f membrane fluidity, g extracellular LDH contents, h membrane Na⁺/K⁺-ATPase activity, i and intracellular K.⁺ contents at logarithmic phase in 30% (w/v) sorbitol medium. *p < 0.05; **p < 0.01; ***p < 0.001
Scanning electron microphotographs of yeast supplemented with WGP under 30% (w/v) sorbitol stress. a Control group, b WGP-LL group, c WGP-LML group, d WGP-LLL group. Red arrows indicated the cells were wrinkled and sunken
Correlation between osmotic stress tolerance and membrane lipid component via analyzing yeast cells added with WGP under 30% (w/v) sorbitol stress. a Principal components analysis. b Schematic diagrams of yeast cell membrane affected by WGP addition under osmotic stress
The influences of three wheat gluten peptides (WGP-LL, WGP-LML, and WGP-LLL) on the osmotic stress tolerance and membrane lipid component in brewer’s yeast were investigated. The results demonstrated that the growth and survival of yeast under osmotic stress were enhanced by WGP supplementation. The addition of WGP upregulated the expressions of OLE1 (encoded the delta-9 fatty acid desaturase) and ERG1 (encoded squalene epoxidase) genes under osmotic stress. At the same time, WGP addition enhanced palmitoleic acid (C16:1) content, unsaturated fatty acids/saturated fatty acids ratio, and the amount of ergosterol in yeast cells under osmotic stress. Furthermore, yeast cells in WGP-LL and WGP-LLL groups were more resistant to osmotic stress. WGP-LL and WGP-LLL addition caused 25.08% and 27.02% increase in membrane fluidity, 22.36% and 29.54% reduction in membrane permeability, 18.38% and 14.26% rise in membrane integrity in yeast cells, respectively. In addition, scanning electron microscopy analysis revealed that the addition of WGP was capable of maintaining yeast cell morphology and reducing cell membrane damage under osmotic stress. Thus, alteration of membrane lipid component by WGP was an effective approach for increasing the growth and survival of yeast cells under osmotic stress. Key points •WGP addition enhanced cell growth and survival of yeast under osmotic stress. •WGP addition increased unsaturated fatty acids and ergosterol contents in yeast. •WGP supplementation improved membrane homeostasis in yeast at osmotic stress. Graphical abstract
Physiological comparison between the parent E3 and the mutant E3ΔsucC. a Schematic diagram of the TCA cycle in S. erythraea. PDH, pyruvate dehydrogenase; CS, citrate synthase; ACO, aconitase; ICDH, isocitrate dehydrogenase; SCS succinyl-CoA synthetase. b The pH profile of E3 and E3ΔsucC culture medium during the shake flask fermentation. c Targeted GS-MS analysis of extracellular organic acids of E3 and E3ΔsucC. d The relative concentrations of extracellular organic acids between E3ΔsucC and E3. Error bars depict the standard deviation of three independent experiments. e SDS-PAGE and Western blotting analysis of protein lysate of E3 and mutant E3ΔsucC at 36 and 72 h. One representative Western blot result was shown. f The succinylation levels of the protein lysates were assessed by Western blotting and quantified by ImageJ (on the right panel). The normalized value of E3 was set as 1.0
Pathway specificity analyses of the E3ΔsucC succinylome. The succinylation proteins were mainly enriched in the pathways of protein biosynthesis, carbon metabolism, and antibiotics biosynthesis by KEGG pathway analyses. a KEGG pathways that were significantly enriched among the succinylated proteins. b Schematic representation of lysine succinylation in the central carbon metabolism, glutamate metabolism, and erythromycin biosynthesis. The yellow cycles represent the number of succinylation sites on certain protein. SDH, succinate dehydrogenase; FH, fumarate hydratase; MDH, malate dehydrogenase; GDH, glutamate dehydrogenase; GS, glutamine synthetase; MCM, methylmalonyl-CoA mutase; PKS, polyketide synthases
Identify the relative activity of enzymes surrounding the pyruvate and citrate metabolism nodes. Error bars depict the standard deviation of three independent experiments. Differences were analyzed by Student’s t-test. ***p < 0.001, **p < 0.01, *p < 0.05; “n.s.” means not significant
Identify the affection of non-enzymatic succinylation on aconitase (SACE_3811) in vitro. a Time- and does-dependent non-enzymatic succinylation of SACE_3811 in vitro. The succinylation levels of the protein sample were assessed by Western blotting. Protein stained with Coomassie blue was set as the loading control. b The effect of succinylation on the activity of SACE_3811; error bars depict the standard deviation of three independent experiments. c Cartoon representation of protein structure shows the succinylation lysine sites on SACE_3811. The protein was colored in light green; succinylation lysine sites surrounding the active pocket were colored in yellow. The conserved K278 and K373 residues were colored in red, and the cluster of [4Fe-4S] is colored in brown. d The sequence logos of the two conserved lysine residues. The whole sequence alignment is provided on the supplemental Fig.S2. These sequence logos were constructed using Weblogo3
Physiological comparison between E3 and the mutants. a Design of the CRISPRi genome editing plasmid targeting sucC. Two strong promoters, PermE* and J23119, were used to drive the expression of dcas9 and sgRNA, respectively. Schematic representation of the sgRNA target sites for the targeted genomic loci with yellow lines for sucC. b Quantitative RT-PCR (qRT-PCR) analysis of the transcription levels of sucC during the early exponential phase. Relative transcript levels were obtained after normalization to the internal reference gene (hrdB). The relative expression values of sucC in E3 were set to 1.0 (arbitrary units). c The succinylation levels of whole cellular proteins were assessed by Western blotting using a pan-anti-succinyllysine antibody. d The succinylation level was further quantified by ImageJ and normalized against the protein loading. e The pH profile of E3 and the mutants culture medium during the shake flask fermentation. f The erythromycin titer in the E3 and the mutants culture medium. Error bars depict the standard deviation of three independent experiments. Differences were analyzed by Student’s t-test. ***p < 0.001, **p < 0.01, *p < 0.05; “n.s.” means not significant
As a novel protein post-translational modification (PTM), lysine succinylation is widely involved in metabolism regulation by altering the activity of catalytic enzymes. Inactivating succinyl-CoA synthetase in Saccharopolyspora erythraea HL3168 E3 was proved significantly inducing the global protein hypersuccinylation. To investigate the effects, succinylome of the mutant strain E3ΔsucC was identified by using a high-resolution mass spectrometry-based proteomics approach. PTMomics analyses suggested the important roles of succinylation on protein biosynthesis, carbon metabolism, and antibiotics biosynthesis in S. erythraea. Enzymatic experiments in vivo and in vitro were further conducted to determine the succinylation regulation in the TCA cycle. We found out that the activity of aconitase (SACE_3811) was significantly inhibited by succinylation in E3ΔsucC, which probably led to the extracellular accumulation of pyruvate and citrate during the fermentation. Enzyme structural analyses indicated that the succinylation of K278 and K373, conservative lysine residues locating around the protein binding pocket, possibly affects the activity of aconitase. To alleviate the metabolism changes caused by succinyl-CoA synthetase inactivation and protein hypersuccinylation, CRISPR interference (CRISPRi) was applied to mildly downregulate the transcription level of gene sucC in E3. The erythromycin titer of the CRISPRi mutant E3-sucC-sg1 was increased by 54.7% compared with E3, which was 1200.5 mg/L. Taken together, this work not only expands our knowledge of succinylation regulation in the TCA cycle, but also validates that CRISPRi is an efficient strategy on the metabolic engineering of S. erythraea. KEY POINTS: • We reported the first systematic profiling of the S. erythraea succinylome. • We found that the succinylation regulation on the activity of aconitase. • We enhanced the production of erythromycin by using CRISPRi to regulate the transcription of gene sucC.
Reports on chloroplast-targeted protein delivery using cell-penetrating peptides are scarce. In this study, a novel peptide-based macromolecule delivery strategy targeting chloroplasts was successfully developed in wheat mesophyll protoplasts. A peptide derived from the signal sequence of the chloroplast-targeted protein of ferredoxin-thioredoxin reductase catalytic chain of Spinacia oleracea with UniProtKB Id-P41348 exhibits properties of cellular internalization. DNase I was efficiently delivered into the chloroplast using 10 μM cTP with an efficiency of more than 90%. This cell-penetrating peptide-mediated approach offers various advantages over the existing chloroplast targeting methods, such as non-invasiveness, biocompatibility, low-toxicity, and target-specific delivery. The present study shows that peptide-based strategies hold tremendous potential in the field of chloroplast biotechnology. Key points • Screening of database of chloroplast targeting peptides in order to develop an efficient cell-penetrating peptide termed as cTP. • cTP efficiently crosses the cell barrier and demonstrated chloroplast-localization. • cTP can be incorporated as a promising strategy for delivering macromolecules for crop improvement.
Evolution of succinic acid production in vine-shoot hydrolysates with AActinobacillus succinogenes DSM 22257 and BBasfia succiniproducens DSM 22022. Average values and standard deviations are shown (n = 3). Note: Cellobiose, rhamnose, and arabinose are not represented in the plots for reasons of simplification
Metabolite production and sugar consumption for various initial sugar concentrations with red grape must after 96 h. AActinobacillus succinogenes DSM 22257. BBasfia succiniproducens DSM 22022. Average values and standard deviations are shown (n = 3)
Contour plot for the estimation of A succinic acid, and B total sugar consumption, as a function of nutrient concentrations (initial sugars and yeast extract), according to the mathematical RSM models for grape must fermented with A. succinogenes DSM 22257
Time evolution of succinic acid production with A. succinogenes DSM 22257 from 145.7 g/L sugars and 24.9 g/L yeast extract in A grape must and B control solution with NM-A salts. Average values and standard deviations are shown (n = 3)
Vine shoots and surplus grape must were assessed as feedstocks for succinic acid production with Actinobacillus succinogenes and Basfia succiniproducens. After acidic and enzymatic hydrolysis, vine shoots released 35–40 g/L total sugars. Both bacterial species produced 18–21 g/L succinic acid from this hydrolysate in 120 h. Regarding grape must fermentation, A. succinogenes clearly outperformed B. succiniproducens. Yeast extract (a source of organic nitrogen and vitamins) was the only additional nutrient needed by A. succinogenes to grow on grape must. Under mathematically optimized conditions (145.7 g/L initial sugars and 24.9 g/L yeast extract), A. succinogenes generated 88.9 ± 1.4 g/L succinic acid in 96 h, reaching a succinic acid yield of 0.66 ± 0.01 g/g and a sugar consumption of 96.64 ± 0.30%. Substrate inhibition was not observed in grape musts with 125–150 g/L initial sugars, provided that an adequate amount of yeast extract was available for bacteria. Alternative nitrogen sources to yeast extract (red wine lees, white wine lees, urea, NH4Cl, and choline chloride) were not suitable for A. succinogenes in grape must. Key Points • Vine shoots and surplus grape must were assessed for succinic acid bioproduction. • Succinic acid bioproduction was 21 g/L with vine shoots and 89 g/L with grape must. • Fermentation was efficient at high sugar loads if organic N supply was adequate. Graphical abstract
R. serpentina.a Habit (source: Wikimedia commons; Creative Commons Attribution-Share Alike 3.0). b Leaves (source: Wikimedia commons; Creative Commons Attribution-Share Alike 4.0). c Inflorescence (source: Wikimedia commons; Creative Commons Attribution-Share Alike 4.0). d Flower (source: Wikimedia commons; Creative Commons Attribution-Share Alike 3.0). e Fruits (source: Wikimedia commons; Creative Commons Attribution-Share Alike 3.0). f In vitro shoot multiplication. g In vitro liquid culture. h In vitro raised rooted plantlet. i Hardened plants (f–i, Abhijit Dey’s own photos)
Rauvolfia serpentina (L). Benth. ex Kurz. (Apocynaceae), commonly known as Sarpagandha or Indian snakeroot, has long been used in the traditional treatment of snakebites, hypertension, and mental illness. The plant is known to produce an array of indole alkaloids such as reserpine, ajmaline, amalicine, etc. which show immense pharmacological and biomedical significance. However, owing to its poor seed viability, lesser germination rate and overexploitation for several decades for its commercially important bioactive constituents, the plant has become endangered in its natural habitat. The present review comprehensively encompasses the various biotechnological tools employed in this endangered Ayurvedic plant for its in vitro propagation, role of plant growth regulators and additives in direct and indirect regeneration, somatic embryogenesis and synthetic seed production, secondary metabolite production in vitro, and assessment of clonal fidelity using molecular markers and genetic transformation. In addition, elicitation and other methods of optimization of its indole-alkaloids are also described herewith. Key points • Latest literature on in vitro propagation ofRauvolfia serpentina • Biotechnological production and optimization of indole alkaloids • Clonal fidelity and transgenic studies inR. serpentina
Microbially produced anticancer and antimicrobial drugs. Both anticancer and antimicrobial drugs can be derived from microbes and some drugs like actinomycin D and bleomycin have dual anticancer and antimicrobial properties
Mechanism of action of various microbe-derived anticancer drugs
Structures of microbe-derived anti-cancer drugs: a Doxorubicin; b Daunorubicin; c Calicheamicin; d Epothilone A (when R = H), Epothilone B (when R = CH3); e Bleomycin; f Actinomycin D; g Distamycin A; h Geldanamycin; i Chartreusin; j Staurosporine
Graph depicting the publications of antitumor antibiotics over the last three decades. Microbe-derived anticancer drugs: Anthracycline based, Carbohydrate based, Polyketide based, Polypeptide based, and Alkaloids based. Data collected from Google Scholar (31–05-22)
Recent years have seen an increased focus on the advancement of naturally derived products for the treatment of cancer. Since the beginning of recorded history, nature has provided a variety of medicinal agents, and an overwhelming number of drugs that we have today are derived from natural sources. Such natural agents are prominently used to treat several diseases such as diabetes, malaria, Alzheimer’s, pulmonary disorders, etc. with cancer being the highlight of this review. Due to the rapid development of resistance to chemotherapeutic drugs, the hunt for effective novel drugs is still a paramount concern in cancer treatment. Moreover, many chemotherapy drugs typically have high toxicity and adverse side effects, which necessitates the need to develop anti-tumor drugs that can be employed to treat deadly tumors with fewer negative effects on health and better efficacy. Isolation of several chemotherapeutic drugs has been conducted from a wide range of natural sources which include plants, microbes, fungi, and marine microorganisms. Considering the trends of previous decades, microbial diversity has grown to play a significant role in the formulation of pharmaceuticals and drugs, especially antibiotics and anti-cancer medications. Microbe-derived antitumor antibiotics such as anthracycline, epothilones, bleomycin, actinomycin, and staurosporine are amongst the widely used cancer chemotherapeutic agents. This review deals majorly with microbe-derived anticancer drugs taking into account their derivatives, mechanism of action, isolation procedures, limitations, and tumors targeted by them. This article also reports the phase of clinical study these drugs are undergoing. Moreover, it intends to portray the indispensable part that these microbes have been playing since time immemorial in the odyssey of chemotherapeutic agents. Key points • Microbial diversity contributes heavily towards the formulation of anticancer drugs. • Polypeptides, carbohydrates, and alkaloids are prevalent microbe-based drug classes. • Microbe-derived anticancer agents target various sarcomas, carcinomas, and lymphomas. Graphical abstract
Introducing proline to different proteins̕ structures as a stabilizing or destabilizing factor
Introduced simultaneous modulating factors to enhance thermostability
Thermostability is an essential requirement of enzymes in the industrial processes to catalyze the reactions at high temperatures; thus, enzyme engineering through directed evolution, semi-rational design and rational design are commonly employed to construct desired thermostable mutants. Several strategies are implemented to fulfill enzymes’ thermostability demand including decreasing the entropy of the unfolded state through substitutions Gly → Xxx or Xxx → Pro, hydrogen bond, salt bridge, introducing two different simultaneous interactions through single mutant, hydrophobic interaction, filling the hydrophobic cavity core, decreasing surface hydrophobicity, truncating loop, aromatic-aromatic interaction and introducing positively charged residues to enzyme surface. In the current review, horizons about compatibility between secondary structures and substitutions at preferable structural positions to generate the most desirable thermostability in industrial enzymes are broadened. Key points • Protein engineering is a powerful tool for generating thermostable industrial enzymes. • Directed evolution and rational design are practical approaches in enzyme engineering. • Substitutions in preferable structural positions can increase thermostability. Graphical abstract
Graphical representation of various A. vinelandii strains constructed previously and as part of this work to test the contributions of the Rnf1 and Fix complexes. The rnfABCDGEH cluster (Avin_50920-Avin_50980) is designated as rnfA1B1C1D1G1E1H in the genome (Setubal et al. 2009), but the numerical designators were not included in the figure for simplicity. The rnf1 gene cluster is inverted in the figure, such that rnfA is adjacent to nifL. The small ferredoxin fixFd that is associated with the fix operon (Ledbetter et al. 2017) was included in efforts to relocate the fix cluster in the reorganized strain AZBB516. The intergenic region that was targeted for relocation of the nifA gene is located between Avin_11360 and Avin_11370. In strain AZBB664, the sequence between the start codons of rnfA and nifL and between the stop codon of nifA and the start codon of nifB (downstream of nifA) were left intact, as indicated by the approximate locations of the vertical lines (top and bottom). The AZBB163 and all derivatives constructed contain a C to T mutation within the kanamycin cassette as previously described (Barney et al. 2015)
Comparison of ammonium yields and growth curves for strains containing disruptions for the RNF cluster genes (AZBB455) and the Fix cluster genes (AZBB454) along with the control strain AZBB163. Growth rates are plotted on a log2-based scale (y-axis). Replicates are represented by open symbols, and averages are shown as closed symbols. Error bars represent the standard deviation (n = 4)
Comparison of ammonium yields and growth curves for strains containing a reorganized nifA deregulation (AZBB664) and an rnf1 deletion strain with the fixFdABCX genes relocated to a stronger promoter system (AZBB516), as well as the wild-type (DJ) strain. Growth rates are plotted on a log2-based scale (y-axis). Replicates are represented by open symbols, and averages are shown as closed symbols. Error bars represent the standard deviation (n ≥ 5)
The enzyme nitrogenase performs the process of biological nitrogen fixation (BNF), converting atmospheric dinitrogen gas into the biologically accessible ammonia, which is rapidly protonated at physiological pH to yield ammonium. The reduction of dinitrogen requires both ATP and electrons. Azotobacter vinelandii is an aerobic nitrogen-fixing microbe that is a model organism for the study of BNF. Previous reports have described strains of A. vinelandii that are partially deregulated for BNF, resulting in the release of large quantities of ammonium into the growth medium. Determining the source of the electrons required to drive BNF is complicated by the existence of several protein complexes in A. vinelandii that have been linked to BNF in other species. In this work, we used the high-ammonium-accumulating strains of A. vinelandii to probe the source of electrons to nitrogenase by disrupting the Rnf1 and Fix complexes. The results of this work demonstrate the potential of these strains to be used as a tool to investigate the contributions of other enzymes or complexes in the process of BNF. These results provide strong evidence that the Rnf1 complex of A. vinelandii is the primary source of electrons delivered to the nitrogenase enzyme in this partially deregulated strain. The Fix complex under native regulation was unable to provide sufficient electrons to accumulate extracellular ammonium in the absence of the Rnf1 complex. Increased ammonium accumulation could be attained in a strain lacking the Rnf1 complex if the genes of the Fix protein complex were relocated behind the strong promoter of the S-layer protein but still failed to achieve the levels found with just the Rnf1 complex by itself. Key points • The Rnf1 complex is integral to ammonium accumulation in A. vinelandii. • The Fix complex can be deleted and still achieve ammonium accumulation in A. vinelandii. • A. vinelandii can be engineered to increase the contribution of the Fix complex to ammonium accumulation.
Target proteins in biotechnological applications are highly diverse. Therefore, versatile flexible expression systems for their functional overproduction are required. In order to find the right heterologous gene expression strategy, suitable host-vector systems, which combine different genetic circuits, are useful. In this study, we designed a novel Bacillus subtilis expression toolbox, which allows the overproduction and secretion of potentially toxic enzymes. This toolbox comprises a set of 60 expression vectors, which combine two promoter variants, four strong secretion signals, a translation-enhancing downstream box, and three plasmid backbones. This B. subtilis toolbox is based on a tailor-made, clean deletion mutant strain, which is protease and sporulation deficient and exhibits reduced autolysis and secondary metabolism. The appropriateness of this alternative expression platform was tested for the overproduction of two difficult-to-produce eukaryotic model proteins. These included the sulfhydryl oxidase Sox from Saccharomyces cerevisiae , which forms reactive hydrogen peroxide and undesired cross-linking of functional proteins, and the human interleukin-1β, a pro-inflammatory cytokine. For the best performing Sox and interleukin, overproducing and secreting variants of these new B. subtilis toolbox fermentation strategies were developed and tested. This study demonstrates the suitability of the prokaryotic B. subtilis host-vector system for the extracellular production of two eukaryotic proteins with biotechnological relevance. Key points • Construction of a versatile Bacillus subtilis gene expression toolbox. • Verification of the toolbox by the secretory overproduction of two difficult-to-express proteins. • Fermentation strategy for an acetoin-controlled overproduction of heterologous proteins.
Animal carcasses introduce large amounts of nitrates and ammonium into the soil ecosystem. Some of this ammonium is transformed from nitrite through the nrfA-type microbial community. However, it is unclear how nrfA-type microorganisms respond to the decomposition of corpses. This study applied high-throughput sequencing to characterize the ecological succession of nrfA-type microbial communities in grassland soil. Our results showed that Cyclobacterium and Trueperella were the predominant genera for nrfA-type communities in soil with a decomposing corpse (experimental group), while Cyclobacterium and Archangium were dominant in soil without a corpse (control group). The alpha diversity indexes and the resistance and resilience indexes of the microbial communities initially increased and then decreased during decomposition. Compared with the control group, nrfA-encoding community structure in the experimental group gradually became divergent with succession and temporal turnover accelerated. Network analysis revealed that the microbial communities of the experimental group had more complex interactions than those of the control groups. Moreover, the bacterial community assembly in the experimental group was governed by stochastic processes, and the communities of the experimental group had a weaker dispersal capacity than those of the control group. Our results reveal the succession patterns of the nrfA-type microbial communities during degradation of wild animal corpses, which can offer references for demonstrating the ecological mechanism underlying the changes in the nrfA-type microbial community during carcass decay. Key points • Corpse decay accelerates the temporal turnover of the nrfA-type community in soil. • Corpse decay changes the ecological succession of the nrfA-type community in soil. • Corpse decay leads to a complex co-occurrence pattern of the nrfA-type community in soil.
Groups of microbes involved in methanation of syngas. Hydrogenotrophic methanogens converting H2/CO2 to methane (CH4) also compete with chemolithotrophic acetogenic bacteria that consume H2/CO2 to produce acetate. Acetate is consumed by either acetoclastic methanogens for CH4 production or syntrophic acetate oxidising bacteria to produce H2/CO2
Schematic diagram of the trickle bed reactor (TBR) and anaerobic filter (AF). 1: Outlet for product gas. 2: Carrier sampling port. 3: Position of temperature probe. 4: Syngas inflow. 5: Inflow of liquid nutrient medium. 6: Sampling port for microbial analysis
Process data from trickle bed reactor (TBR) operation during three periods (1–3) operating with different nutrient medium: 1) defined medium (M1) 2) dewatered digestate from a thermophilic biogas plant operating with food waste (M2) and 3) reject water from a biogas plant at a wastewater treatment plant (M3). Each period was further divided into two sub phases (A,B) based on major changes in operating parameters, such as flow rate of nutrient medium (see Table 1). a Specific syngas inflow (black) and biogas outflow rate (blue). b Specific outflow gas rate. The gap seen in period 3A was due to gas analyser malfunction. c) Total volatile fatty acids (VFA) concentration and alkalinity. d pH. e Total amount of sulphur (S) and ammonium nitrogen (NH4-N) added via nutrient medium and by additional supply via external source. For details, see Supplementary Material
Microbial community structure at genus level during trickle bed reactor (TBR) operation in three periods (1–3) operating with different nutrient medium: (1) defined mineral medium (M1) (2) dewatered digestate from a thermophilic biogas plant operating with food waste (M2) and (3) reject water from a biogas plant at a wasterwater treatment plant (M3). Each operating period was further divided into two sub-phases (A,B) based on major changes in operating parameters, such as flow rate of nutrient medium (see Table 1)
Microbial community structure on genus level in biofilm recovered from plastic carriers in the trickle bed reactor (TBR) in the start-up phase and in operating periods 1 and 2. Carrier samples taken in period 2B were sequenced in triplicate. Carrier samples taken in start-up, period 1 and period 2A were sequenced without replicates, due to lack of extracted material
Microbial community development within an anaerobic trickle bed reactor (TBR) during methanation of syngas (56% H2, 30% CO, 14% CO2) was investigated using three different nutrient media: defined nutrient medium (241 days), diluted digestate from a thermophilic co-digestion plant operating with food waste (200 days) and reject water from dewatered digested sewage sludge at a wastewater treatment plant (220 days). Different TBR operating periods showed slightly different performance that was not clearly linked to the nutrient medium, as all proved suitable for the methanation process. During operation, maximum syngas load was 5.33 L per L packed bed volume (pbv) & day and methane (CH4) production was 1.26 L CH4/Lpbv/d. Microbial community analysis with Illumina Miseq targeting 16S rDNA revealed high relative abundance (20–40%) of several potential syngas and acetate consumers within the genera Sporomusa, Spirochaetaceae, Rikenellaceae and Acetobacterium during the process. These were the dominant taxa except in a period with high flow rate of digestate from the food waste plant. The dominant methanogen in all periods was a member of the genus Methanobacterium, while Methanosarcina was also observed in the carrier community. As in reactor effluent, the dominant bacterial genus in the carrier was Sporomusa. These results show that syngas methanation in TBR can proceed well with different nutrient sources, including undefined medium of different origins. Moreover, the dominant syngas community remained the same over time even when non-sterilised digestates were used as nutrient medium. Key points •Independent of nutrient source, syngas methanation above 1 L/Lpbv/D was achieved. •Methanobacterium and Sporomusa were dominant genera throughout the process. •Acetate conversion proceeded via both methanogenesis and syntrophic acetate oxidation. Graphical abstract
Hydrolysis products of rCsXyn10A, rCsAbf62A, and rCsAbf62A-cd. A Thin-layer chromatography (TLC) of soluble reaction products of beechwood xylan (BX) and low-viscosity wheat arabinoxylan (WAX-lv) treated with the recombinant enzymes, as indicated ( +). The lane with no enzymes corresponds to the substrate control. B TLC of soluble reaction products of rCsXyn10A on xylo-oligosaccharides (X3 to X6) with varying incubation times. TLC standards were loaded at 0.2 mg/mL: ARA (arabinose), X1 (xylose), X2 (xylobiose), X3 (xylotriose), X4 (xylotetraose), X5 (xylopentaose), and X6 (xylohexaose). C MALDI-TOF sprectra of reaction products of rCsXyn10A on BX. [a.u.]: arbitrary units. XnMeGlcA [M + Na +]: sodium adduct of xylo-oligosaccharides with 4-O-methyl glucuronic acid with n degree of polymerization
1D ¹H NMR of rCsAbf62A and rCsAbf62A-cd wheat arabinoxylan reaction products. In the schematic, linkages within the polysaccharide (a–c) are labeled to correspond with peaks in the NMR spectra (a: α-1,3 linked Araf in single substitutions; b: α-1,3 linked Araf in double substitutions; c: α-1,2 linked Araf in double substitutions). Spectra are vertically aligned and the yellow dotted line indicates the position of peak “a.” The control spectrum is arabinoxylan that was incubated with protein buffer
Activity profile of rCsXyn10A, rCsAbf62A, and rCsAbf62A-cd. Optimal A pH condition, B temperature, and C substrate concentration, for the activity of rCsXyn10A (left) and rCsAbf62A and rCsAbf62A-cd (right). Optimal pH was determined at 50 °C for rCsXyn10A and 30 °C for rCsAbf62A and rCsAbf62A-cd. Optimal temperature was tested at pH 6.0 for all enzymes. The curves shown in C were obtained at 50 °C and pH 6.0 for rCsXyn10A and 30 °C and pH 5.5 for rCsAbf62A and rCsAbf62A-cd. Error bars indicate standard deviation of triplicates
Hydrolysis of WAX-lv and AXOS by rCsXyn10A and rCsAbf62A or rCsAbf62A-cd. Soluble reaction products generated by recombinant proteins after incubation with WAX-lv (A) or AXOS (B) were visualized by TLC. Reactions were carried out using enzymes individually or combinations of rCsXyn10A with rCsAbf62A or rCsAbf62A-cd, as indicated ( +). XA³XX: xylotetraose with 1,3-linked Araf; XAXX mix: xylotetraose with 1,2- and 1,3-linked Araf
Wheat bran (WB) digestion by A rCsAbf62A or rCsAbf62A-cd and B rCsXyn10A. HPAEC-PAD of soluble reaction products generated by rCsXyn10A (0.1 μM) and rCsAbf62A or rCsAbf62A-cd (0.6 μM) after incubation with WB (5% w/v) (pH 6, 30 °C, 48 h). Retention times were compared against commercial arabinose (Ara) and xylobiose (X2) standards
Valorization of the hemicellulose fraction of plant biomass is crucial for the sustainability of lignocellulosic biorefineries. The Cellulomonas genus comprises Gram-positive Actinobacteria that degrade cellulose and other polysaccharides by secreting a complex array of enzymes. In this work, we studied the specificity and synergy of two enzymes, CsXyn10A and CsAbf62A, which were identified as highly abundant in the extracellular proteome of Cellulomonas sp. B6 when grown on wheat bran. To explore their potential for bioprocessing, the recombinant enzymes were expressed and their activities were thoroughly characterized. rCsXyn10A is a GH10 endo-xylanase (EC, active across a broad pH range (5 to 9), at temperatures up to 55 °C. rCsAbf62A is an α-l-arabinofuranosidase (ABF) (EC that specifically removes α-1,2 and α-1,3-l-arabinosyl substituents from arabino-xylo-oligosaccharides (AXOS), xylan, and arabinan backbones, but it cannot act on double-substituted residues. It also has activity on pNPA. No differences were observed regarding activity when CsAbf62A was expressed with its appended CBM13 module or only the catalytic domain. The amount of xylobiose released from either wheat arabinoxylan or arabino-xylo-oligosaccharides increased significantly when rCsXyn10A was supplemented with rCsAbf62A, indicating that the removal of arabinosyl residues by rCsAbf62A improved rCsXyn10A accessibility to β-1,4-xylose linkages, but no synergism was observed in the deconstruction of wheat bran. These results contribute to designing tailor-made, substrate-specific, enzymatic cocktails for xylan valorization. Key points • rCsAbf62A removes α-1,2 and α-1,3-L-arabinosyl substituents from arabino-xylo-oligosaccharides, xylan, and arabinan backbones. • The appended CBM13 of rCsAbf62A did not affect the specific activity of the enzyme. • Supplementation of rCsXyn10A with rCsAbf62A improves the degradation of AXOS and xylan.
The present study was conducted to investigate the influence of microgravity on human gut microbiota using 16S rRNA gene sequencing in vitro. The diamagnetic material magnetic levitation method was used to simulate weightless environment. The human gut microbiota was cultured under two different conditions: normal gravity (1 g), and simulated microgravity (0 g), which showed that both the richness (P = 0.04) and diversity (P = 0.0002) of human gut microbiota were significantly altered. As compared to the normal gravity, the simulated microgravity significantly reduced abundance of bacteria related to anti-inflammatory effects, such as Subdoligranulum, Faecalibacterium, Fusicatenibacter, Butyricicoccus, and Lachnospiraceae-NK4A136-0 group (P < 0.05), while significantly increased that of Alistipes and Eubacterium-Ventriosum-group (P < 0.05). Moreover, the Spearman’s correlation analysis showed that there were more significantly correlated species (|r|≥ 0.5, P < 0.05) in normal gravity than that in the simulated microgravity. KEGG pathway analysis revealed that the microgravity significantly (P < 0.05) affected the metabolism of gut microbiota, such as the metabolism of pyrimidine, fatty acids, glyoxylate and dicarboxylate, peptidoglycan biosynthesis, and carbon fixation in photosynthetic organisms. These results suggested that the exposure to a microgravity environment might induce disturbances in human gut microbiota. Key points • Using 16sRNA gene sequencing technology, it was found that magnetic levitation-simulated microgravity had varying degrees of influence on the abundance, diversity, species correlation, and KEGG pathways of human intestinal microbes. • Digital PCR can improve the detection rate of microorganisms with low abundance.
Water quality deterioration of drinking water distribution systems (DWDSs) caused by water source switching has been reported previously. However, systematic investigation of the biostability of DWDS under water source switching is limited. Aged pipes, including three commonly used pipe materials dug out from a practical DWDS, were used to systematically investigate the biofilm stability mechanism of DWDS under water source switching to quality-improved water. An increase in adenosine triphosphate (ATP) concentration in the bulk water during the initial stage of the switching period was observed, indicating the risk of biofilm release through aged pipe surfaces after water source switching. Sloughing of biofilms might contribute to temporary instability. From day 35, the ATP concentration in the polyethylene (PE) and plastic stainless steel composite (PS) pipes were maintained at approximately 2.40 and 2.56 ng/L, respectively. In contrast, the ATP concentration in the ductile iron (DI) pipes was higher, at approximately 3.43 ng/L from day 42. The water quality variation could cause areas of the biofilm to slough and reduce the biomass of biofilm, causing partial alteration of the microbial community. 16S rRNA gene amplicon sequencing-based functional prediction revealed that the biofilm could increase the abundance of chlorine-resistant bacteria attributed to the increase in Pseudomonas and Methylobacterium after switching to quality-improved water. Moreover, the profiles of specific pathways linked to human diseases, antibiotic resistance, and antibiotic biosynthesis revealed that the safety of the biofilm could improve after switching to quality-improved water. KEY POINTS: • The PE and PS biofilm showed improved resistance to water quality perturbation. • Greater number of Methylobacterium was found in the biofilm after water source switching. • 3.16S gene-based metagenomics prediction revealed that the safety of the biofilm under water source switching.
Mature lysostaphin (mLst) is a glycineglycine endopeptidase, capable of specifically cleaving penta-glycine crosslinker in the peptidoglycan of Staphylococcus aureus cell wall. It is a very effective therapeutic enzyme to kill the multidrug-resistant S. aureus often encountered in hospital acquired infections. Fusing cellulose binding domain (CBD) to mLst significantly reduced the insoluble expression of mLst in E. coli. Employing mLst-cleavable peptides as fusion linkers leaded to an effective self-cleavage expression that CBD and mLst could be completely cleaved off from the fusions during the expression process. The presence of residue linker fragment at N-terminus of the cleaved-off mLst strongly inhibited the cell lytic activity of the recovered recombinant mLst, and only ~ 50% of the wild-type mLst activity could be retained. Intact CBD-Lst fusions were obtained when uncleavable peptide linkers were employed. With CBD at N-terminus of mLst, the intact fusion completely lost its cell lytic activity but the dipeptidase activity still remained. In contrast, approximately 10% cell lytic activity of mLst still could be maintained for the fusion with CBD at C-terminus of mLst. Key points • CBD fusion enhanced soluble expression of recombinant lysostaphin. • In vivo self-cleavage of fusion linkers by the expressed lysostaphin fusions. • Self-cleaved lysostaphin fusions retain most of dipeptidase but lose 50% cell lytic activity.
Chinese hamster ovary (CHO) cells are the most commonly used host cells for the production of recombinant monoclonal antibodies (mAbs) due to their several advantages. Although the yields of recombinant mAbs can be greatly increased by some strategies, such as medium formulation, culture conditions, and cell engineering, most studies focused on either upstream design or downstream processes. In the present study, we first expressed recombinant adalimumab through combination of the vector design and production process optimization in CHO cells. Bicistronic vector, monocistronic vector, and dual promoter vector were constructed, and the production process was optimized using low-temperature and fed-batch culture. The results showed that the dual promoter vector exhibited the highest yield under the transient and stable transfected cells among three different vector systems in CHO cells. In addition, low-temperature and fed-batch culture could further improve the yields of adalimumab. The purified antibody displayed tumor necrosis factor-α (TNF-α) binding activity. In conclusion, combination of expression vector design and production process optimization can achieve higher expression of recombinant mAbs in CHO cells. Key points • The dual promoter vector is more effective for expressing recombinant antibodies. • The yields of antibodies are related to the LC chain expression level. • Low-temperature and feed addition can promote antibody production.
Although the importance of intestinal microbes to aquaculture animals has been recognized, the intestinal bacteria of Sinonovacula constricta and its culture environment are rarely studied. In this study, high-throughput sequencing was used to explore the intestinal bacterial communities of pond water, sediment, and S. constricta intestine. Significance analysis and principal coordinates analysis (PCoA) showed that there were significant differences in bacterial communities among animals’ intestine, pond water, and sediment (p < 0.05). Venn analysis showed that intestinal bacteria shared a considerable number of OTUs (operational taxonomic units) with the sediment and water. SourceTracker analysis suggested that the contribution of sediment to the intestinal bacteria of S. constricta was much larger than that of rearing water. The Kruskal–Wallis test showed that the dominant bacterial taxa differed significantly between animals’ intestines and the pond environment, and each of them has a unique bacterial composition. A network diagram indicated the complex positive and negative interactions between intestinal bacteria at the OTU level. Furthermore, BugBase analysis indicated that the bacterial contribution to potential pathogens in the animals’ intestines is similar to that in sediments, suggesting that sediment was the main source of potential pathogens in S. constricta intestine. This study provided a theoretical basis for environmental regulation and disease prevention of S. constricta in aquaculture. Key points • Culture environment had a significant effect on the intestinal bacterial community in S. constricta. • Sediment was a major source of intestinal bacteria and potentially pathogenic bacteria. • Complex positive and negative interactions existed between intestinal bacteria.
Illustration of different factors responsible for free radical generation resulting in aging
D-glactose metabolism pathway (Umbayev et al. 2020)
Schematic representation of effect of D-galactose on different body organs of animal model resulting in aging
Diagrammatic illustration of the cell wall of Lactobacillus sp
Aging is a progressive, unalterable physiological degradation process of living organisms, which leads to deterioration of biological function and eventually to senescence. The most prevalent factor responsible for aging is the accumulation of damages resulting from oxidative stress and dysbiosis. D-galactose-induced aging has become a hot topic, and extensive research is being conducted in this area. Published literature has reported that the continuous administration of D-galactose leads to the deterioration of motor and cognitive skills, resembling symptoms of aging. Hence, this procedure is employed as a model for accelerated aging. This review aims to emphasize the effect of D-galactose on various bodily organs and underline the role of the Lactobacillus sp. in the aging process, along with its anti-oxidative potential. A critical consideration to the literature describing animal models that have used the Lactobacillus sp. in amending D-galactose-induced aging is also given. Key points • D-Galactose induces the aging process via decreasing the respiratory chain enzyme activity as well as ATP synthesis, mitochondrial dysfunction, and increased ROS production. • D-Galactose induced aging primarily affects the brain, heart, lung, liver, kidney, and skin. • The anti-oxidative potential of Lactobacillus sp. in improving D-galactose-induced aging in animal models via direct feeding and feeding of Lactobacillus-fermented food.
Scheme of an analytical procedure for the extraction of methylmercury from fungal materials
The aim of the study was to develop an efficient method for the determination of monomethyl-mercury (MeHg) and total mercury (THg) content in materials such as fungal sporocarps and sclerotia. Certified Reference Materials (CRMs) with the assigned values of MeHg and THg as well as the control materials (dried mushrooms) with known content of THg were evaluated for method validation. Recovery of MeHg from reference materials was at the following levels: from tuna fish at 87.0 ± 2.3% (THg at 101.9 ± 1.2%), from fish protein at 99.4 ± 1.3% (THg at 92.70 ± 0.41%), and from dogfish liver at 96.45 ± 0.73%. Recovery of THg from the fungal control material CS-M-5 was at 104.01 ± 0.60% (contribution of MeHg in THg content was at 6.2%), from CS-M-4 at 101.1 ± 2.0% (contribution at 3.2%), from CS-M-3 at 100.55 ± 0.67% (contribution at 0.6%), and from CS-M-2 at 101.5 ± 2.7% (contribution at 3.7%). The content of MeHg in randomly selected wild fungi and their morphological parts was in the range from 0.006 to 0.173 mg kg⁻¹ dry weight (dw). In the case of THg, the concentration values were in the range from 0.0108 to 10.27 mg kg⁻¹ dw. The MeHg content in the control materials with the assigned THg values was determined. Since the control materials play an important role in all elements of the quality assurance system of measurement results, they can be used to analyse MeHg as the first control material for fungi. Key points • An extraction procedure for MeHg analysis in fungi was developed and optimized. • Recovery of MeHg from the certified reference non-fungal materials was > 87%. • Fungal control materials with assigned THg concentration can serve also for MeHg analysis. Graphical abstract
The exploitation of active ingredients from plant volatile organic compounds as natural gaseous fungicides shows remarkable potential for controlling fungal decay in postharvest agroproducts. Although 1-octanol is a common component of cereal volatiles, its antifungal potency against spoilage fungi in postharvest grains remains unclear. In this study, we studied the effectiveness of 1-octanol against Aspergillus flavus growth in postharvest grains and its mechanisms of action. 1-Octanol vapor and liquid contact dose-dependently inhibited A. flavus spore germination and mycelial growth at a low concentration. The simulated storage experiment demonstrated that 300 μL/L of 1-octanol vapor completely controlled A. flavus growth in wheat, corn, and paddy grains with 20% moisture content. 1-Octanol treatment irreversibly damaged the conidial and mycelial morphology of A. flavus and caused electrolyte leakage due to reduced plasma membrane integrity. It induced apoptosis along with morphological abnormalities, phosphatidylserine externalization, mitochondrial membrane potential depolarization, intracellular reactive oxygen species accumulation, and DNA fragmentation in A. flavus cells. Metabolomic analysis revealed that 1-octanol treatment disrupted the biosynthesis of unsaturated fatty acids, ATP-binding cassette transporters, amino acid metabolism, and glycerophospholipid metabolism. This study demonstrated the promising application potential of 1-octanol as a biofumigant for preventing fungal spoilage of postharvest cereal grains. Key points • (1) 1-Octanol inhibits Aspergillus flavus growth in the vapor phase and liquid contact; • (2) 1-Octanol damages membrane integrity and induces apoptosis of A. flavus; • (3) Metabolomic changes in A. flavus mycelia were analyzed after 1-octanol treatment.
Top-cited authors
Tong Zhang
  • University of Hong Kong
Peter Weiland
  • Thünen Institute
Willy Verstraete
  • Ghent University
Ji-Dong Gu
  • Guangdong Technion - Israel Institute of Technology
Bruce E Logan
  • Pennsylvania State University