Enhanced poly-γ-glutamic acid synthesis in Corynebacterium glutamicum by reconstituting PgsBCA complex and fermentation optimization.

Previously, a novel Corynebacterium glutamicum strain for the de novo biosynthesis of tailored poly-γ-glutamic acid (γ-PGA) has been constructed by our group. The strain was based on the γ-PGA synthetase complex, PgsBCA, which is the only polyprotein complex responsible for γ-PGA synthesis in Bacillus spp. In the present study, PgsBCA was reconstituted and overexpressed in C. glutamicum to further enhance γ-PGA synthesis. First, we confirmed that all the components (PgsB, PgsC, and PgsA) of γ-PGA synthetase derived from B. licheniformis are necessary for γ-PGA synthesis, and γ-PGA was detected only when PgsB, PgsC, and PgsA were expressed in combination in C. glutamicum. Next, the expression level of each pgsB, pgsC, and pgsA was tuned in order to explore the effect of expression of each of the γ-PGA synthetase subunits on γ-PGA production. Results showed that increasing the transcription levels of pgsB or pgsC and maintaining a medium-level transcription level of pgsA led to 35.44% and 76.53% increase in γ-PGA yield (γ-PGA yield-to-biomass), respectively. Notably, the expression level of pgsC had the greatest influence (accounting for 68.24%) on γ-PGA synthesis, followed by pgsB. Next, genes encoding for PgsC from four different sources (Bacillus subtilis, Bacillus anthracis, Bacillus methylotrophicus, and Bacillus amyloliquefaciens) were tested in order to identify the influence of PgsC-encoding orthologues on γ-PGA production, but results showed that in all cases the synthesis of γ-PGA was significantly inhibited. Similarly, we also explored the influence of gene orthologues encoding for PgsB on γ-PGA production, and found that the titer increased to 17.14 ± 0.62 g/L from 8.24 ± 0.10 g/L when PgsB derived from B. methylotrophicus replaced PgsB alone in PgsBCA from B. licheniformis. The resulting strain was chosen for further optimization, and we achieved a γ-PGA titer of 38.26 g/L in a 5 L fermentor by optimizing dissolved oxygen level. Subsequently, by supplementing glucose, γ-PGA titer increased to 50.2 g/L at 48 h. To the best of our knowledge, this study achieved the highest titer for de novo production of γ-PGA from glucose, without addition of L-glutamic acid, resulting in a novel strategy for enhancing γ-PGA production.

Association of smart elderly care and quality of life among older adults: the mediating role of social support.

BACKGROUND: In the current context of ageing, the field of smart elderly care has gradually developed, contributing to the promotion of health among older adults. While the positive impact on health has been established, there is a scarcity of research examining its impact on the quality of life (QoL). This study aims to investigate the mediating role of social support in the relationship between smart elderly care and QoL among older adults. METHODS: A total of 1313 older adults from Zhejiang Province, China, participated in the study. Questionnaires were used to collect data on participants' basic demographic information, smart elderly care, social support, and QoL. The descriptive analyses of the demographic characteristics and correlation analyses of the three variables were calculated. Indirect effects were tested using bootstrapped confidence intervals (CI). RESULTS: The analysis revealed a positive association between smart elderly care and social support (ß = 0.42, p < 0.01), as well as a positive correlation between social support and QoL (ß = 0.65, p < 0.01). Notably, social support emerged as an important independent mediator (effect size = 0.28, 95% bootstrap CI 0.24 to 0.32) in the relationship between smart elderly care and QoL. CONCLUSIONS: The results of this study underscore the importance of promoting the utilization of smart elderly care and improving multi-faceted social support for older adults, as these factors positively contribute to the overall QoL.

An Ancient Respiratory System in the Widespread Sedimentary Archaea Thermoprofundales.

Thermoprofundales, formerly Marine Benthic Group D (MBG-D), is a ubiquitous archaeal lineage found in sedimentary environments worldwide. However, its taxonomic classification, metabolic pathways, and evolutionary history are largely unexplored because of its uncultivability and limited number of sequenced genomes. In this study, phylogenomic analysis and average amino acid identity values of a collection of 146 Thermoprofundales genomes revealed five Thermoprofundales subgroups (A-E) with distinct habitat preferences. Most of the microorganisms from Subgroups B and D were thermophiles inhabiting hydrothermal vents and hot spring sediments, whereas those from Subgroup E were adapted to surface environments where sunlight is available. H2 production may be featured in Thermoprofundales as evidenced by a gene cluster encoding the ancient membrane-bound hydrogenase (MBH) complex. Interestingly, a unique structure separating the MBH gene cluster into two modular units was observed exclusively in the genomes of Subgroup E, which included a peripheral arm encoding the [NiFe] hydrogenase domain and a membrane arm encoding the Na+/H+ antiporter domain. These two modular structures were confirmed to function independently by detecting the H2-evolving activity in vitro and salt tolerance to 0.2 M NaCl in vivo, respectively. The peripheral arm of Subgroup E resembles the proposed common ancestral respiratory complex of modern respiratory systems, which plays a key role in the early evolution of life. In addition, molecular dating analysis revealed that Thermoprofundales is an early emerging archaeal lineage among the extant MBH-containing microorganisms, indicating new insights into the evolution of this ubiquitous archaea lineage.

Amplicon-based sequencing and co-occurence network analysis reveals notable differences of microbial community structure in healthy and dandruff scalps.

BACKGROUND: Dandruff is a chronic, recurring, and common scalp problem that is caused by several etiopathogeneses with complex mechanisms. Management of this condition is typically achieved via antifungal therapies. However, the precise roles played by microbiota in the development of the condition have not been elucidated. Despite their omnipresence on human scalp little is known about the co-occurrence/co-exclusion network of cutaneous microbiota. RESULTS: We characterized the scalp and hair surface bacterial and fungal communities of 95 dandruff-afflicted and healthy individuals residing in China. The degree distributions of co-occurrence/co-exclusion network in fungi-bacteria and bacteria-bacteria were higher in the healthy group (P < 0.0001), whereas the betweenness values are higher in the dandruff group (P < 0.01). Meanwhile, the co-occurrence/co-exclusion network among fungi-fungi and fungi-bacteria showed that compared to the healthy group, the dandruff group had more positive links (P < 0.0001). In addition, we observed that Malassezia slooffiae, Malassezia japonica and Malassezia furfur, were more abundant in the dandruff group than in the healthy group. These microbiota were co-exclusion by either multiple bacterial genera or Malassezia sp. in healthy group. The lactic acid bacteria on the scalp and hair surface, especially the genera Lactobacillus and Lactococcus, exhibit a negative correlation with multiple bacterial genera on the scalp and hair surface. Lactobacillus plantarum and Pediococcus lactis isolated on the healthy human scalp can inhibit the growth of Staphylococcus epidermidis in vitro. CONCLUSIONS: We showed that microbial networks on scalp and hair surface with dandruff were less integrated than their healthy counterparts, with lower node degree and more positive and stronger links which were deemed to be unstable and may be more susceptible to environmental fluctuations. Lactobacillus bacteria have extensive interactions with other bacteria or fungi in the scalp and hair surface micro-ecological network and can be used as targets for improving scalp health.

Improving glutathione production by engineered Pichia pastoris: strain construction and optimal precursor feeding.

Glutathione (GSH) is a metabolite that plays an important role in the fields of pharmacy, food, and cosmetics. Thus, it is necessary to increase its production to meet the demands. In this study, ScGSH1, ScGSH2, and StGshF were heterologously expressed in Pichia pastoris GS115 to realize the dual-path synthesis of GSH in yeast. To explore the effects of ATP metabolism on the synthesis of GSH, enzymes (ScADK1, PpADK1, VsVHB) of the ATP-related metabolic pathway and the energy co-substrate sodium citrate were taken into account. We found that both ScADK1 and sodium citrate had a positive influence on the synthesis of GSH. Then, a fermentation experiment in Erlenmeyer flasks was performed using the G3-SF strain (containing ScGSH1, ScGSH2, StGshF, and ScADK1), with the highest GSH titer and yield of 999.33 ± 47.26 mg/L and 91.53 ± 4.70 mg/g, respectively. Finally, the fermentation was scaled up in a 5-L fermentor, and the highest titer and yield were improved to 5680 mg/L and 45.13 mg/g, respectively, by optimizing the addition conditions of amino acids (40 mM added after 40 h). Our work provides an alternative strategy by combining dual-path synthesis with energy metabolism regulation and precursor feeding to improve GSH production. Key Points • ScGSH1, ScGSH2, and StGshF were overexpressed to achieve dual-path synthesis of GSH in yeast. • ScADK1 was overexpressed, and sodium citrate was added to increase the energy supply for GSH synthesis. • The addition conditions of amino acids were optimized to realize the efficient synthesis of GSH.

Material priority engineered metal-polyphenol networks: mechanism and platform for multifunctionalities.

Engineering the surface of materials with desired multifunctionalities is an effective way to fight against multiple adverse factors during tissue repair process. Recently, metal-polyphenol networks (MPNs) have gained increasing attention because of their rapid and simple deposition process onto various substrates (silicon, quartz, gold and polypropylene sheets, etc.). However, the coating mechanism has not been clarified, and multifunctionalized MPNs remain unexplored. Herein, the flavonoid polyphenol procyanidin (PC) was selected to form PC-MPN coatings with Fe3+, and the effects of different assembly parameters, including pH, molar ratio between PC and Fe3+, and material priority during coating formation, were thoroughly evaluated. We found that the material priority (addition sequence of PC and Fe3+) had a great influence on the thickness of the formed PC-MPNs. Various surface techniques (e.g., ultraviolet-visible spectrophotometry, quartz crystal microbalance, X-ray photoelectron spectroscopy, atomic force microscopy, and scanning electron microscopy) were used to investigate the formation mechanism of PC-MPNs. Then PC-MPNs were further engineered with multifunctionalities (fastening cellular attachment in the early stage, promoting long-term cellular proliferation, antioxidation and antibacterial activity). We believe that these findings could further reveal the coating formation mechanism of MPNs and guide the future design of MPN coatings with multifunctionalities, thereby greatly broadening their application prospects, such as in sensors, environments, drug delivery, and tissue engineering.

Knockout of cardiac troponin I-interacting kinase leads to cardiac dysfunction and remodelling.

Cardiac troponin I-interacting kinase (TNNI3K) is a cardiac-specific kinase that has been identified as a diagnostic marker and a therapeutic target in cardiovascular diseases. However, the biological function of TNNI3K in cardiac dysfunction and remodelling remains elusive. In the present study, a Tnni3k cardiomyocyte-specific knockout (Tnni3k-cKO) mouse model was established. Echocardiography was used to evaluate cardiac function in mice. Heart failure markers were detected using enzyme-linked immunosorbent assay. Haematoxylin and eosin staining, wheat germ agglutinin staining, Masson's trichrome staining, Sirius red staining and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining were used to assess histopathological changes, cardiac hypertrophy, collagen deposition and myocardial apoptosis, respectively. Expression levels of TNNI3K, apoptosis-related proteins, and p38 mitogen-activated protein kinase were measured using Western blot analysis. Compared to wild-type controls, cardiac dysfunction and cardiac remodelling of Tnni3k-cKO mice increased gradually with age. Tnni3k-cKO mice exhibited cardiac hypertrophy, cardiac fibrosis and cardiomyocyte apoptosis. Upregulation of cleaved caspase-3 in Tnni3k-cKO mice appeared to be related to phosphorylation and activation of the p38 mitogen-activated protein kinase signalling pathway. In conclusion, this study shows that TNNI3K is essential for cardiac development and function, providing new insights into the development of novel therapeutic strategies for cardiac diseases.

Extraordinary diversity of viruses in deep-sea sediments as revealed by metagenomics without prior virion separation.

Our current knowledge of the virosphere in deep-sea sediments remains rudimentary. Here we investigated viral diversity at both gene and genomic levels in deep-sea sediments of Southwest Indian Ocean. Analysis of 19 676 106 non-redundant genes from the metagenomic DNA sequences revealed a large number of unclassified viral groups in these samples. A total of 1106 high-confidence viral contigs were obtained after two runs of assemblies, and 217 of these contigs with sizes up to ~120 kb were shown to represent complete viral genomes. These contigs are clustered with no known viral genomes, and over 2/3 of the ORFs on the viral contigs encode no known functions. Furthermore, most of the complete viral contigs show limited similarity to known viral genomes in genome organization. Most of the classified viral contigs are derived from dsDNA viruses belonging to the order Caudovirales, including primarily members of the families Myoviridae, Podoviridae and Siphoviridae. Most of these viruses infect Proteobacteria and, less frequently, Planctomycetes, Firmicutes, Chloroflexi, etc. Auxiliary metabolic genes (AMGs), present in abundance on the viral contigs, appear to function in modulating the host ability to sense environmental gradients and community changes, and to uptake and metabolize nutrients.

Novel Alcaligenes ammonioxydans sp. nov. from wastewater treatment sludge oxidizes ammonia to N2 with a previously unknown pathway.

Heterotrophic nitrifiers are able to oxidize and remove ammonia from nitrogen-rich wastewaters but the genetic elements of heterotrophic ammonia oxidation are poorly understood. Here, we isolated and identified a novel heterotrophic nitrifier, Alcaligenes ammonioxydans sp. nov. strain HO-1, oxidizing ammonia to hydroxylamine and ending in the production of N2 gas. Genome analysis revealed that strain HO-1 encoded a complete denitrification pathway but lacks any genes coding for homologous to known ammonia monooxygenases or hydroxylamine oxidoreductases. Our results demonstrated strain HO-1 denitrified nitrite (not nitrate) to N2 and N2 O at anaerobic and aerobic conditions respectively. Further experiments demonstrated that inhibition of aerobic denitrification did not stop ammonia oxidation and N2 production. A gene cluster (dnfT1RT2ABCD) was cloned from strain HO-1 and enabled E. coli accumulated hydroxylamine. Sub-cloning showed that genetic cluster dnfAB or dnfABC already enabled E. coli cells to produce hydroxylamine and further to 15 N2 from (15 NH4 )2 SO4 . Transcriptome analysis revealed these three genes dnfA, dnfB and dnfC were significantly upregulated in response to ammonia stimulation. Taken together, we concluded that strain HO-1 has a novel dnf genetic cluster for ammonia oxidation and this dnf genetic cluster encoded a previously unknown pathway of direct ammonia oxidation (Dirammox) to N2 .

Silanization of a Metal-Polyphenol Coating onto Diverse Substrates as a Strategy for Controllable Wettability with Enhanced Performance to Resist Acid Corrosion.

Wettability is a crucial characteristic of materials that plays a vital role in surface engineering. Surface modification is the key to changing the wettability of materials, and a simple and universal modification approach is being extensively pursued by researchers. Recently, metal-phenolic networks (MPNs) have been widely studied because they impart versatility and functionality in surface modification. However, an MPN is not stable for long periods, especially under acidic conditions, and is susceptible to pollution by invasive species. Spurred by the versatility of MPNs and various functionalities achieved by silanization, we introduce a general strategy to fabricate functionally stable coatings with controllable surface wettability by combining the two methods. The formation process of MPN and silane-MPN coatings was characterized by spectroscopic ellipsometry (SE), UV-visible-near-infrared (UV-vis-NIR) spectroscopy, X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), atomic force microscopy (AFM), water contact angle (WCA), etc. We found that the stability of the MPN was greatly enhanced after silanization, which is attributed to the cross-linking effect that occurs between silane and the MPN, namely, the cross-linking protection produced in this case. Additionally, the wettability of an MPN can be easily changed through our strategy. We trust that our strategy can further extend the applications of MPNs and points toward potential prospects in surface modification.

Prospective comparison of integrated on-site CT-fractional flow reserve and static CT perfusion with coronary CT angiography for detection of flow-limiting coronary stenosis.

OBJECTIVES: To compare the diagnostic power of separately integrating on-site computed tomography (CT)-derived fractional flow reserve (CT-FFR) and static CT stress myocardial perfusion (CTP) with coronary computed tomography angiography (CCTA) in detecting patients with flow-limiting CAD. The flow-limiting stenosis was defined as obstructive (≥ 50%) stenosis by invasive coronary angiography (ICA) with a corresponding perfusion deficit on stress single photon emission computed tomography (SPECT/MPI). METHODS: Forty-eight patients (74 vessels) were enrolled who underwent research-indicated combined CTA-CTP (320-row CT scanner, temporal resolution 137 ms) and SPECT/MPI prior to conventional coronary angiography. CT-FFR was computed on-site using resting CCTA data with dedicated workstation-based software. All five imaging modalities were analyzed in blinded independent core laboratories. Logistic regression and the integrated discrimination improvement (IDI) index were used to evaluate incremental differences in CT-FFR or CTP compared with CCTA alone. RESULTS: The prevalence of obstructive CAD defined by combined ICA-SPECT/MPI was 40%. Per-vessel sensitivity and specificity were 95 and 42% for CCTA, 76 and 89% for CCTA + CTP, and 81 and 96% for CCTA + CT-FFR, respectively. The diagnostic performance of CCTA (AUC = 0.82) was improved by combining it with CT-FFR (AUC = 0.92, p = 0.01; IDI = 0.27, p < 0.001) or CTP (AUC = 0.90, p = 0.02; IDI = 0.18, p = 0.003). CONCLUSION: On-site CT-FFR combined with CCTA provides an incremental diagnostic improvement over CCTA alone in identifying patients with flow-limiting CAD defined by ICA + SPECT/MPI, with a comparable diagnostic accuracy for integrated CTP and CCTA. KEY POINTS: • Both on-site CT-FFR and CTP perform well with high diagnostic accuracy in the detection of flow-limiting stenosis. • Comparable diagnostic accuracy between CCTA + CT-FFR and CCTA + CTP is demonstrated to detect flow-limiting stenosis. • Integrated CT-FFR and CCTA derived from a single widened CCTA data acquisition can accurately and conveniently evaluate both coronary anatomy and physiology in the future management of patients with suspected CAD, without the need for additional vasodilator administration and contrast and radiation exposure.

Feasibility of Cardiac Computed Tomography for the Evaluation of Ventricular Function in Postoperative Children With Congenital Heart Disease: Comparison With Cardiac Magnetic Resonance Imaging.

OBJECTIVE: We explored the feasibility of cardiac computed tomography (CCT) to evaluate postoperative ventricular function in children with congenital heart disease (CHD) and evaluated the accuracy and reproducibility of CCT using cardiac magnetic resonance (CMR) as a reference. METHODS: Thirty-two postoperative children with CHD (20 boys and 12 girls) who underwent CMR and CCT were enrolled. Left and right ventricular ejection fraction, end-diastolic volume, end-systolic volume, stroke volume, and cardiac index were measured using cardiac function analysis software. Cardiac function data were compared between CMR and CCT. The agreement between the 2 modalities was assessed using a Bland-Altman analysis. Intraclass correlation coefficients were used to assess intraobserver and interobserver reproducibility in CCT functional measurements. RESULTS: All functional parameters showed no significant difference (P > 0.05) and were well-correlated (r > 0.5, P < 0.05) between CMR and CCT. The mean values of all ventricular function parameters in CCT were higher compared with CMR. As indicated by 95% limits of agreement, left ventricular function parameters showed a better level of agreement compared with right ventricular function parameters between the 2 modalities. Intraobserver and interobserver reproducibility were excellent in CCT measurements for all functional parameters (intraclass correlation coefficient > 0.9). CONCLUSIONS: Compared with the criterion standard of CMR, CCT is feasible for assessing postoperative ventricular function with sufficient diagnostic accuracy and reproducibility in children with CHD. In addition to its important role regarding anatomical characterization, CCT is a suitable alternative and convenient follow-up tool that can be used to functional evaluation in children who are intolerant with CMR or have contraindications to CMR.

Psychometric properties of the critical thinking disposition assessment test amongst medical students in China: a cross-sectional study.

BACKGROUND: Critical thinking disposition helps medical students and professionals overcome the effects of personal values and beliefs when exercising clinical judgment. The lack of effective instruments to measure critical thinking disposition in medical students has become an obstacle for training and evaluating students in undergraduate programs in China. The aim of this study was to evaluate the psychometric properties of the CTDA test. METHODS: A total of 278 students participated in this study and responded to the CTDA test. Cronbach's α coefficient, internal consistency, test-retest reliability, floor effects and ceiling effects were measured to assess the reliability of the questionnaire. Construct validity of the pre-specified three-domain structure of the CTDA was evaluated by explanatory factor analysis (EFA) and confirmatory factor analysis (CFA). The convergent validity and discriminant validity were also analyzed. RESULTS: Cronbach's alpha coefficient for the entire questionnaire was calculated to be 0.92, all of the domains showed acceptable internal consistency (0.81-0.86), and the test-retest reliability indicated acceptable intra-class correlation coefficients (ICCs) (0.93, p < 0.01). The EFA and the CFA demonstrated that the three-domain model fitted the data adequately. The test showed satisfactory convergent and discriminant validity. CONCLUSIONS: The CTDA is a reliable and valid questionnaire to evaluate the disposition of medical students towards critical thinking in China and can reasonably be applied in critical thinking programs and medical education research.

The associations between coping strategies, psychological health, and career indecision among medical students: a cross-sectional study in China.

BACKGROUND: Medical students experience difficulties in the process of making decisions about their careers, which is referred to as career indecision. This study aimed to examine the difficulties in the career decision-making processes of medical students and to explore the association of coping strategies and psychological health with career indecision. The findings may provide a reference for designing interventions to advance satisfying career decisions for medical students. METHODS: A cross-sectional survey of 359 medical students was conducted in 5 Chinese medical schools. Students completed an anonymous self-administered questionnaire measuring their career indecision, coping strategies, and psychological health. Independent t-test, F-test, bivariate Pearson's correlation analysis, and linear regression analysis were applied to test the relation between career indecision and the associated factors. Data were analyzed using SPSS V.22 for Windows. A p-value < 0.05 was considered to be statistically significant. RESULTS: Difficulties regarding lack of readiness frequently occurred in medical students when making career decisions, with the highest score of 2.48 ± 0.58. Among all the associated factors in this study, career indecision was positively associated with psychological distress problem (ß = 0.20, p < 0.05). This study also proved that being at a higher level of career indecision is negatively associated with using problem-focused coping strategies (ß = - 0.14, p < 0.05). For the maladaptive coping strategies, applying dysfunctional coping strategies showed a significantly positive association with career indecision among medical students (ß = 0.25, p < 0.05). CONCLUSIONS: Medical students experienced difficulties regarding lack of readiness frequently when making career decisions. Both coping strategies and psychological health were associated with career indecision among medical students. To prevent career indecision, it is necessary to promote earlier career awareness to medical students. Specifically, psychological health should be addressed in career intervention programs for medical students. Additionally, when helping medical students to cope with career indecision, cognitive techniques that reduce the use of maladaptive coping strategies and enhance the use of adaptive coping strategies should be adopted.

Interfacial Nanoinjection-Based Nanoliter Single-Cell Analysis.

Single-cell analysis offers unprecedented resolution for the investigation of cellular heterogeneity and the capture of rare cells from large populations. Here, described is a simple method named interfacial nanoinjection (INJ), which can miniaturize various single-cell assays to be performed in nanoliter water-in-oil droplets on standard microwell plates. The INJ droplet handler can adjust droplet volumes for multistep reactions on demand with high precision and excellent monodispersity, and consequently enables a wide range of single-cell assays. Importantly, INJ can be coupled with fluorescence-activated cell sorting (FACS), which is currently the most effective and accurate single-cell sorting and isolation method. FACS-INJ pipelines for high-throughput plate well-based single-cell analyses, including single-cell proliferation, drug-resistance testing, polymerase chain reaction (PCR), reverse-transcription PCR, and whole-genome sequencing are introduced. This FACS-INJ pipeline is compatible with a wide range of samples and can be extended to various single-cell analysis applications in microbiology, cell biology, and biomedical diagnostics.

Directed modification of a ruminal cellulase gene (CMC-1) from a metagenomic library isolated from Yunnan gayal (Bos frontalis).

Gayal (Bos frontalis) of the Yunnan region is well adapted to harsh environmental conditions. Its diet consists predominantly of bamboo, reeds, and woody plants, suggesting that the rumen of this species contains many fiber-degrading bacteria and cellulases. The aim of this study was to identify and modify specific cellulases found in the gayal rumen. In the present study, a directed evolution strategy of error-prone PCR was employed to improve the activity or optimal temperature of a cellulase gene (CMC-1) isolated from gayal rumen. The CMC-1 gene was heterologously expressed in Escherichia coli (E. coli) BL21, and the recombinant CMC-1 protein hydrolyzed carboxyl methyl cellulose (CMC) with an optimal activity at pH 5.0 and 50 °C. A library of mutated ruminal CMC-1 genes was constructed and a mutant EP-15 gene was identified. Sequencing analysis revealed that EP-15 and CMC-1 belonged to the glycosyl hydrolase family 5 (GHF5) and had the highest homology to a cellulase (Accession No. WP_083429257.1) from Prevotellaceae bacterium, HUN156. There were similar predicted GH5 domains in EP-15 and CMC-1. The EP-15 gene was heterologously expressed and exhibited cellulase activity in E. coli BL21 at pH 5.0, but the optimum temperature for its activity was reduced from that of CMC-1 (50 °C) to 45 °C, which was closer to the physiological temperature of the rumen (40 °C). The cellulase activity of EP-15 was about two times higher than CMC-1 at 45 °C or PH 5.0, and also was more stable in response to temperature and pH changes compared to CMC-1. This study successfully isolated and modified a ruminal cellulase gene from metagenomics library of Yunnan gayal. Our findings may obtain a useful cellulase in future applications and present the first evidence of modified cellulases in the gayal rumen.

Gimesia benthica sp. nov., a planctomycete isolated from a deep-sea water sample of the Northwest Indian Ocean.

A Gram-stain-negative, stalked, oval-shaped and budding bacterial strain, designated E7T, was isolated from a deep-sea water sample collected from the Northwest Indian Ocean. The novel strain was strictly aerobic, and catalase- and oxidase-positive. It grew at 6-40 °C (optimum 30 °C) and pH 5.5-8.0 (optimum pH 7.0-7.5). The strain required 0.5-9.0 % (w/v) NaCl (optimum 3.0-5.0 %) for growth. Aesculin, starch, pectin and Tween 20 were hydrolysed. Based on 16S rRNA gene sequence analysis, strain E7T showed the highest similarity with Gimesia maris DSM 8797T (97.5 %). The average nucleotide identity and in silico DNA-DNA hybridization values between strain E7T and G. maris DSM 8797T were 78.0 and 19.3 %, respectively. The predominant cellular fatty acids of strain E7T were C16 : 0 and summed feature 3 (comprising C16 : 1ω7c and/or C16 : 1ω6c). The major respiratory quinone was menaquinone-6 (MK-6) and the major polar lipids were phosphatidylethanolamine (PE), phosphatidylmonomethylethanolamine (PMME), phosphatidyldimethylethanolamine (PDME), phosphatidylcholine (PC) and diphosphatidylglycerol (DPG). The genomic DNA G+C content of strain E7T was 52.8 mol%. On the basis of phylogenetic inference and phenotypic characteristics, it is proposed that strain E7T represents a novel species of the genus Gimesia, for which the name Gimesia benthica sp. nov. is proposed. The type strain is E7T (=CGMCC 1.16119T=KCTC 72737T).

Halovulum marinum sp. nov., isolated from deep-sea water of the Indian Ocean, and emended description of the genus Halovulum.

A novel Gram-stain-negative, aerobic, motile by peritrichous flagella, oval to rod-shaped bacterium, designated strain 2CG4T, was isolated from a deep-sea water sample collected from the Northwest Indian Ocean. The results of phylogenetic analysis of both 16S rRNA gene and RpoC protein sequences indicated that this strain was affiliated with the genus Halovulum in the Amaricoccus clade of the family Rhodobacteraceae of the class Alphaproteobacteria, sharing 95.3 % similarity at the 16S rRNA gene sequence level with the type strain of Halovulum dunhuangense YYQ-30T, the only species in the genus Halovulum. The predominant fatty acids (>10 %) of 2CG4T were summed feature 8 (C18 : 1ω7c and/ or C18 : 1ω6c; 61.1 %) and cyclo-C19 : 0ω8c (15.6 %). The polar lipids of 2CG4T were phosphatidylethanolamine, phosphatidylmethylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine and sulfoquinovosyldiacylglycerol. The only isoprenoid quinone of 2CG4T was ubiquinone-10. The DNA G+C content of 2CG4T was determined to be 69.4 %. The central gene pufLM for the photosynthetic reaction was not detected. No growth occurred for 2CG4T in the absence of NaCl. On the basis of these data, it is concluded that the 2CG4T represents a novel species of the genus Halovulum, for which the name Halovulum marinum sp. nov. is proposed. The type strain is 2CG4T (=CGMCC 1.16468T=JCM 32611T).

The cysteine protease ATG4B of Trichinella spiralis promotes larval invasion into the intestine of the host.

The cysteine proteases of parasites are vital contributors that induce parasite migration to and invasion of host tissue. In this study, we analysed the cysteine protease ATG4B of Trichinella spiralis (TsATG4B) isolated from the soluble proteins of Trichinella spiralis (T. spiralis) adult worms to ascertain its biochemical properties and functions during invasion into the intestine of the host. The 43 kDa recombinant cysteine protease ATG4B protein (rTsATG4B) consists of a conserved peptidase_C54 domain and was expressed in Escherichia coli. Gelatine zymography showed that rTsATG4B could hydrolyse gelatine and that the hydrolytic activity was prevented by the cysteine protease inhibitor E-64 (pH 5.2). Immunofluorescence assays showed that TsATG4B is expressed at different stages and is localized at the cuticles and stichosomes of worms. Far-Western blotting and confocal microscopy revealed that rTsATG4B interacts with intestinal epithelial cells (IECs) and that it was subcellularly localized to the membrane and cytoplasm in IECs. Real­time quantitative PCR (qPCR) results indicated that the transcription level of the TsATG4B gene was the higher in 6-day-old adult worms (6 days AW) than in any other stage. An in vitro larval invasion assay verified that rTsATG4B promoted larval invasion and that invasion was inhibited when rTsATG4B was pre-incubated with E-64, whereas anti-rTsATG4B serum inhibited larval invasion in a dose-dependent manner. Collectively, these results suggested that the enzymatic activity of TsATG4B significantly influences the hydrolysis process, which is necessary for larval invasion of the host intestinal epithelium.

Engineering Corynebacterium glutamicum for the de novo biosynthesis of tailored poly-γ-glutamic acid.

γ-Polyglutamic acid (γ-PGA) is a biodegradable polymer naturally produced by Bacillus spp. that has wide applications. Fermentation of γ-PGA using Bacillus species often requires the supplementation of L-glutamic acid, which greatly increases the overall cost. Here, we report a metabolically engineered Corynebacterium glutamicum capable of producing γ-PGA from glucose. The genes encoding γ-PGA synthase complex from B. subtilis (pgsB, C, and A) or B. licheniformis (capB, C, and A) were expressed under inducible promoter Ptac in a L-glutamic acid producer C. glutamicum ATCC 13032, which led to low levels of γ-PGA production. Subsequently, C. glutamicum F343 with a strong L-glutamic acid production capability was tested. C. glutamicum F343 carrying capBCA produced γ-PGA up to 11.4 g/L, showing a higher titer compared with C. glutamicum F343 expressing pgsBCA. By introducing B. subtilis glutamate racemase gene racE under Ptac promoter mutants with different expression strength, the percentage of L-glutamic acid units in γ-PGA could be adjusted from 97.1% to 36.9%, and stayed constant during the fermentation process, while the γ-PGA titer reached 21.3 g/L under optimal initial glucose concentrations. The molecular weight (Mw) of γ-PGA in the engineered strains ranged from 2000 to 4000 kDa. This work provides a foundation for the development of sustainable and cost-effective de novo production of γ-PGA from glucose with customized ratios of L-glutamic acid in C. glutamicum.