A New-Generation Base Editor with an Expanded Editing Window for Microbial Cell Evolution In Vivo Based on CRISPR‒Cas12b Engineering.

Base editors (BEs) are widely used as revolutionary genome manipulation tools for cell evolution. To screen the targeted individuals, it is often necessary to expand the editing window to ensure highly diverse variant library. However, current BEs suffer from a limited editing window of 5-6 bases, corresponding to only 2-3 amino acids. Here, by engineering the CRISPR‒Cas12b, the study develops dCas12b-based CRISPRi system, which can efficiently repress gene expression by blocking the initiation and elongation of gene transcription. Further, based on dCas12b, a new-generation of BEs with an expanded editing window is established, covering the entire protospacer or more. The expanded editing window results from the smaller steric hindrance compared with other Cas proteins. The universality of the new BE is successfully validated in Bacillus subtilis and Escherichia coli. As a proof of concept, a spectinomycin-resistant E. coli strain (BL21) and a 6.49-fold increased protein secretion efficiency in E. coli JM109 are successfully obtained by using the new BE. The study, by tremendously expanding the editing window of BEs, increased the capacity of the variant library exponentially, greatly increasing the screening efficiency for microbial cell evolution.

The association between indoor air pollution from solid fuels and cognitive impairment: a systematic review and meta-analysis.

This study aimed to comprehensively and methodically evaluate the correlation between cognitive impairment and indoor air pollution from solid fuel used for cooking/heating. PubMed, Web of Science, EMBASE, and Cochrane Library databases were searched up to December January 2023. 13 studies from three countries with a total of 277,001 participants were enrolled. A negative correlation was discovered between solid fuel usage for cooking and total cognitive score (ß=-0.73, 95 % CI: -0.90 to -0.55) and episodic memory score (ß=-0.23, 95 % CI: -0.30 to -0.17). Household solid fuel usage for cooking was considerably associated with a raised risk of cognitive impairment (HR=1.31, 95 % CI: 1.09-1.57) and cognitive decline (HR=1.24, 95 % CI: 1.18-1.30). Compared to continuous solid fuel use for cooking, sustained use of clean fuel and switching from solid fuel to clean fuel were associated with a lower risk of cognitive decline (OR=0.55, 95 % CI: 0.42-0.73; OR=0.81, 95 % CI: 0.71-0.93). A negative association was found between solid fuel usage for heating and total cognitive score (ß=-0.43, 95 % CI: -0.59 to -0.26) and episodic memory score (ß=-0.22, 95 % CI: -0.34 to -0.10). Our research provided evidence that exposure to indoor air pollution from solid fuel is a potential cause of cognitive impairment and cognitive decline. Making the switch from solid fuels to cleaner fuels could be an important step in preventing cognitive impairment in the elderly.

Long-term abuse of caffeine sodium benzoate induces endothelial cells injury and leads to coagulation dysfunction.

Our hospital admitted a patient who had difficulty in coagulation even after blood replacement, and the patient had abused caffeine sodium benzoate (CSB) for more than 20 years. Hence, we aimed to explore whether CSB may cause dysfunction in vascular endothelial cells and its possible mechanism. Low, medium, and high concentrations of serum of long-term CSB intake patients were used to treat HUVECs, with LPS as the positive control. MTT and CCK8 were performed to verify CSB's damaging effect on HUVECs. The expression of ET-1, ICAM-1, VCAM-1, and E-selectin were measured by ELISA. TUNEL assay and Matrigel tube formation assay were carried out to detect apoptosis and angiogenesis of HUVECs. Flow cytometry was applied to analyze cell cycles and expression of CD11b, PDGF, and ICAM-1. Expression of PDGF-BB and PCNA were examined by western blot. The activation of MAPK signaling pathway was detected by qRT-PCR and western blot. Intracellular Ca2+ density was detected by fluorescent probes. CCK8 assay showed high concentration of CSB inhibited cell viability. Cell proliferation and angiogenesis were inhibited by CSB. ET-1, ICAM-1, VCAM-1, and E-selectin upregulated in CSB groups. CSB enhanced apoptosis of HUVECs. CD11b, ICAM-1 increased and PDGF reduced in CSB groups. The expression level and phosphorylation level of MEK, ERK, JUN, and p38 in MAPK pathway elevated in CSB groups. The expression of PCNA and PDGF-BB was suppressed by CSB. Intracellular Ca2+ intensity was increased by CSB. Abuse of CSB injured HUVECs and caused coagulation disorders.

The complete mitochondrial genome of Cuminum cyminum (Apiales: Apiaceae) and phylogenetic analysis.

Cumin (Cuminum cyminum L). belongs to the family Apiaceae and the order Apiales, which is a widely grown spice and medicinal plant in Xinjiang province, China. In the current study, whole genome sequencing of C. cyminum was performed using the Illumina HiSeq 4000 platform, and the complete mitogenome sequence was assembled and annotated. We found that the single circular mitogenome of C. cyminum was 246,721 bp in length, and has about 45.5% GC content. It comprised 73 genes in the coding region (35 protein-coding genes, 18 tRNA genes, 3 rRNA genes, and 15 open-reading frames) and a non-coding region. Phylogenetic analysis indicated that C. cyminum is closely related to Daucus carota and the subtribes Daucinae. The mitogenome of C. cyminum revealed its phylogenetic relationships with other species in the Apiaceae family, which would further help in understanding its evolution.

Metabolomic analysis of the sera of patients with the long-term inhalation of caffeine-sodium benzoate using LC-MS.

The present study aimed to systematically assess the potential biomarkers in the serum samples of patients with long-term inhalation of caffeine-sodium benzoate (CSB). LC-MS was applied to analyze the metabolic profiles of serum samples of patients with the long-term intake of CSB (n = 35) and other volunteers with no intake of CSB treated as the control group (n = 35). The raw data of metabolic profiles were analyzed via principal component analysis, partial least squares analysis, and orthogonal partial least squares analysis. MBRole 2.0 online tools were used to analyze the Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis of different metabolites. The serum metabolic profiles showed several metabolites with large variations, including 2-propyl-2,4-pentadienoic acid, 24-hydroxycholesterol, 3-O-sulfogalactosylceramide (d18:1/24:1(15Z)), 3-O-sulfogalactosylceramide (d18:1/12:0), 3-O-sulfogalactosylceramide (d18:1/14:0), 3a,7a-dihydroxy-5b-cholestan-26-al, 3a,7a-dihydroxy-5b-cholestane, 7a,25-dihydroxycholesterol, bilirubin, and dehydroepiandrosterone sulfate. The Kyoto Encyclopedia of Genes and Genomes pathways involved in metabolism included 'choline metabolism in cancer' and 'glycerophospholipid metabolism'. In conclusion, the present study provides a basis with which to explore the molecular-specific mechanisms concerning the effects of the long-term inhalation of CSB on human physical and mental health.

Construction and application of an efficient dual-base editing platform for Bacillus subtilis evolution employing programmable base conversion.

The functionally evolved bacterial chassis is of great importance to manufacture a group of assorted high value-added chemicals, from small molecules to biologically active macromolecules. However, the current evolution frameworks are less efficienct in generating in vivo genomic diversification because of insufficient tunability, rendering limited evolution spacing for chassis. Here, an engineered genomic diversification platform (CRISPR-ABE8e-CDA-nCas9) leveraging a programmable dual-deaminases base editor was fabricated for rapidly evolving bacterial chassis. The dual-base editor was constructed by reprogramming the CRISPR array, nCas9, and cytidine and adenosine deaminase, enabling single or multiple base conversion at the genomic scale by simultaneous C-to-T and A-to-G conversion in vivo. Employing titration of the Cas-deaminase fusion protein, the platform enabled editing any pre-defined genomic loci with tunable conversion efficiency and editable window, generating a repertoire of mutants with highly diversified genomic sequences. Leveraging the genomic diversification platform, we successfully evolved the nisin-resistant capability of Bacillus subtilis through directed evolution of the subunit of lantibiotic ATP-binding cassette. Therefore, our work provides a portable and programmable genomic diversification platform, which is promising to expedite the fabrication of high-performance and robust bacterial chassis used in the development of biomanufacturing and biopharmaceuticals.

The complete mitochondrial genome of Ophiocordyceps gracilis and its comparison with related species.

In this study, the complete mitochondrial genome of O. gracilis was sequenced and assembled before being compared with related species. As the second largest mitogenome reported in the family Ophiocordycipitaceae, the mitogenome of O. gracilis (voucher OG201301) is a circular DNA molecule of 134,288 bp that contains numerous introns and longer intergenomic regions. UCA was detected as anticodon in tRNA-Sec of O. gracilis, while comparative mitogenome analysis of nine Ophiocordycipitaceae fungi indicated that the order and contents of PCGs and rRNA genes were considerably conserved and could descend from a common ancestor in Ophiocordycipitaceae. In addition, the expansion of mitochondrial organization, introns, gene length, and order of O. gracilis were determined to be similar to those of O. sinensis, which indicated common mechanisms underlying adaptive evolution in O. gracilis and O. sinensis. Based on the mitochondrial gene dataset (15 PCGs and 2 RNA genes), a close genetic relationship between O. gracilis and O. sinensis was revealed through phylogenetic analysis. This study is the first to investigate the molecular evolution, phylogenetic pattern, and genetic structure characteristics of mitogenome in O. gracilis. Based on the obtained results, the mitogenome of O. gracilis can increase understanding of the genetic diversity and evolution of cordycipitoid fungi.

JFK Is a Hypoxia-Inducible Gene That Functions to Promote Breast Carcinogenesis.

Many carcinomas feature hypoxia, a condition has long been associated with tumor progression and poor prognosis, as well as resistance to chemoradiotherapy. Here, we report that the F-box protein JFK promotes mammary tumor initiation and progression in MMTV-PyMT murine model of spontaneous breast cancer. We find that JFK is inducible under hypoxic conditions, in which hypoxia-inducible factor HIF-1α binds to and transcriptionally activates JFK in breast cancer cells. Consistently, analysis of public clinical datasets reveals that the mRNA level of JFK is positively correlated with that of HIF-1α in breast cancer. We show that JFK deficiency leads to a decrease in HIF-1α-induced glycolysis in breast cancer and sensitizes hypoxic breast cancer cells to ionizing radiation and chemotherapeutic treatment. These results indicate that JFK is an important player in hypoxic response, supporting the pursuit of JFK as a potential therapeutic target for breast cancer intervention.

Development of a base editor for protein evolution via in situ mutation in vivo.

Protein evolution has significantly enhanced the development of life science. However, it is difficult to achieve in vitro evolution of some special proteins because of difficulties with heterologous expression, purification, and function detection. To achieve protein evolution via in situ mutation in vivo, we developed a base editor by fusing nCas with a cytidine deaminase in Bacillus subtilis through genome integration. The base editor introduced a cytidine-to-thymidine mutation of approximately 100% across a 5 nt editable window, which was much higher than those of other base editors. The editable window was expanded to 8 nt by extending the length of sgRNA, and conversion efficiency could be regulated by changing culture conditions, which was suitable for constructing a mutant protein library efficiently in vivo. As proof-of-concept, the Sec-translocase complex and bacitracin-resistance-related protein BceB were successfully evolved in vivo using the base editor. A Sec mutant with 3.6-fold translocation efficiency and the BceB mutants with different sensitivity to bacitracin were obtained. As the construction of the base editor does not rely on any additional or host-dependent factors, such base editors (BEs) may be readily constructed and applicable to a wide range of bacteria for protein evolution via in situ mutation.

Altered profiles of fecal bile acids correlate with gut microbiota and inflammatory responses in patients with ulcerative colitis.

BACKGROUND: Gut microbiota and its metabolites may be involved in the pathogenesis of inflammatory bowel disease. Several clinical studies have recently shown that patients with ulcerative colitis (UC) have altered profiles of fecal bile acids (BAs). It was observed that BA receptors Takeda G-protein-coupled receptor 5 (TGR5) and vitamin D receptor (VDR) participate in intestinal inflammatory responses by regulating NF-ĸB signaling. We hypothesized that altered profiles of fecal BAs might be correlated with gut microbiota and inflammatory responses in patients with UC. AIM: To investigate the changes in fecal BAs and analyze the relationship of BAs with gut microbiota and inflammation in patients with UC. METHODS: The present study used 16S rDNA sequencing technology to detect the differences in the intestinal flora between UC patients and healthy controls (HCs). Fecal BAs were measured by targeted metabolomics approaches. Mucosal TGR5 and VDR expression was analyzed using immunohistochemistry, and serum inflammatory cytokine levels were detected by ELISA. RESULTS: Thirty-two UC patients and twenty-three HCs were enrolled in this study. It was found that the diversity of gut microbiota in UC patients was reduced compared with that in HCs. Firmicutes, Clostridium IV, Butyricicoccus, Clostridium XlVa, Faecalibacterium, and Roseburia were significantly decreased in patients with UC (P = 3.75E-05, P = 8.28E-07, P = 0.0002, P = 0.003, P = 0.0003, and P = 0.0004, respectively). Proteobacteria, Escherichia, Enterococcus, Klebsiella, and Streptococcus were significantly enriched in the UC group (P = 2.99E-09, P = 3.63E-05, P = 8.59E-05, P = 0.003, and P = 0.016, respectively). The concentrations of fecal secondary BAs, such as lithocholic acid, deoxycholic acid, glycodeoxycholic acid, glycolithocholic acid, and taurolithocholate, in UC patients were significantly lower than those in HCs (P = 8.1E-08, P = 1.2E-07, P = 3.5E-04, P = 1.9E-03, and P = 1.8E-02, respectively) and were positively correlated with Butyricicoccus, Roseburia, Clostridium IV, Faecalibacterium, and Clostridium XlVb (P < 0.01). The concentrations of primary BAs, such as taurocholic acid, cholic acid, taurochenodeoxycholate, and glycochenodeoxycholate, in UC patients were significantly higher than those in HCs (P = 5.3E-03, P = 4E-02, P = 0.042, and P = 0.045, respectively) and were positively related to Enterococcus, Klebsiella, Streptococcus, Lactobacillus, and pro-inflammatory cytokines (P < 0.01). The expression of TGR5 was significantly elevated in UC patients (0.019 ± 0.013 vs 0.006 ± 0.003, P = 0.0003). VDR expression in colonic mucosal specimens was significantly decreased in UC patients (0.011 ± 0.007 vs 0.016 ± 0.004, P = 0.033). CONCLUSION: Fecal BA profiles are closely related to the gut microbiota and serum inflammatory cytokines. Dysregulation of the gut microbiota and altered constitution of fecal BAs may participate in regulating inflammatory responses via the BA receptors TGR5 and VDR.

[Effects of substrate on growth and lipid accumulation of Tribonema sp. FACHB-1786].

Filamentous microalga Tribonema sp. has the advantages of highly resistance to zooplankton-predation, easy harvesting, and high cellular lipid content, in particular large amounts of palmitoleic acid (PA) and eicosapentaenoic acid (EPA). Therefore, Tribonema sp. is considered as a promising biomass feedstock to produce biodiesel and high-value products. In this work, we studied the effect of different concentrations of nitrogen (NaNO3: 255-3 060 mg/L), phosphorus (K2HPO4: 4-240 mg/L), iron ((NH4)3FeC12H10O14: 0.6-12 mg/L) and magnesium (MgSO4: 7.5-450 mg/L) on the biomass, lipid content, and fatty acid composition of Tribonema sp. FACHB-1786, aiming at enhancing cell lipid productivity. The growth of Tribonema sp. had a positive correlation with the concentration of magnesium, and the maximum biomass of Tribonema sp. (under the condition of 450 mg/L MgSO4) was 8.09 g/L, much greater than those reported in previous studies using the same and other Tribonema species under autotrophic conditions. Different nitrogen concentrations exerted no significant effect on algal growth (P > 0.05), but a higher nitrogen concentration resulted in a greater amount of lipid in the cells. The maximum volumetric productivities of total lipids (319. 6 mg/(L·d)), palmitoleic acid (135.7 mg/(L·d)), and eicosapentaenoic acid (24.2 mg/(L·d)) of Tribonema sp. were obtained when the concentrations of NaNO3, K2HPO4, (NH4)3FeC12H10O14, and MgSO4 were 765 mg/L, 80 mg/L, 6 mg/L, and 75 mg/L, respectively. This study will provide a reference for substrate optimization for Tribonema sp. growth and lipid production.

The effectiveness and safety of curcumin as a complementary therapy in inflammatory bowel disease: A protocol of systematic review and meta-analysis.

BACKGROUND: Inflammatory bowel diseases (IBD), which include Crohn disease and ulcerative colitis, affect several million individuals worldwide. Curcumin as a complementary therapy has been used to cure the IBD, yet the efficacy and safety of curcumin remains to be assessed. In this study, we aim to draw up a protocol for systematic review to evaluate the efficacy and safety of curcumin for IBD. METHODS: We will search the following electronic databases from inception to September 31, 2020: PubMed, Cochrane Library, EMBASE, Web of Science, Medline, the China National Knowledge Infrastructure Database, Wan Fang Database, the Chinese Scientific Journal Database, and Chinese Biomedical Literature Database. Clinical trial registrations, potential gray literatures, relevant conference abstracts and reference list of identified studies will also be searched. Relevant randomized controlled clinical trials were enrolled and analyzed. The literature selection, data extraction, and quality assessment will be completed by 2 independent authors. Either the fixed-effects or random-effects model will be used for data synthesis based on the heterogeneity test. Clinical remission will be evaluated as the primary outcome. Clinical response, endoscopic remission, inflammatory markers and adverse events will be assessed as the secondary outcomes. The RevManV.5.3.5 will be used for Meta-analysis. Subgroup analyses of doses, delivery way, frequency of treatment and the degree of IBD severity or different forms of IBD were also conducted. RESULTS: This study will provide a synthesis of current evidence of curcumin for IBD from several aspects, such as clinical remission, clinical response, endoscopic remission, inflammatory markers, and adverse events. CONCLUSION: The conclusion of our study will provide updated evidence to judge whether curcumin is an effective solution to IBD patients. INPLASY REGISTRATION NUMBER: INPLASY202090065.

Design and Construction of Portable CRISPR-Cpf1-Mediated Genome Editing in Bacillus subtilis 168 Oriented Toward Multiple Utilities.

Bacillus subtilis is an important Gram-positive bacterium for industrial biotechnology, which has been widely used to produce diverse high-value added chemicals and industrially and pharmaceutically relevant proteins. Robust and versatile toolkits for genome editing in B. subtilis are highly demanding to design higher version chassis. Although the Streptococcus pyogenes (Sp) CRISPR-Cas9 has been extensively adapted for genome engineering of multiple bacteria, it has many defects, such as higher molecular weight which leads to higher carrier load, low deletion efficiency and complexity of sgRNA construction for multiplex genome editing. Here, we designed a CRISPR-Cpf1-based toolkit employing a type V Cas protein, Cpf1 from Francisella novicida. Using this platform, we precisely deleted single gene and gene cluster in B. subtilis with high editing efficiency, such as sacA, ganA, ligD & ligV, and bac operon. Especially, an extremely large gene cluster of 38 kb in B. subtilis genome was accurately deleted from the genome without introducing any unexpected mutations. Meanwhile, the synthetic platform was further upgraded to a version for multiplex genome editing, upon which two genes sacA and aprE were precisely and efficiently deleted using only one plasmid harboring two targeting sequences. In addition, we successfully inserted foreign genes into the genome of the chassis using the CRISPR-Cpf1 platform. Our work highlighted the availability of CRISPR-Cpf1 to gene manipulation in B. subtilis, including the flexible deletion of a single gene and multiple genes or a gene cluster, and gene knock-in. The designed genome-editing platform was easily and broadly applicable to other microorganisms. The novel platforms we constructed in this study provide a promising tool for efficient genome editing in diverse bacteria.

Efficient Overproduction of Active Nitrile Hydratase by Coupling Expression Induction and Enzyme Maturation via Programming a Controllable Cobalt-Responsive Gene Circuit.

A robust and portable expression system is of great importance in enzyme production, metabolic engineering, and synthetic biology, which maximizes the performance of the engineered system. In this study, a tailor-made cobalt-induced expression system (CIES) was developed for low-cost and eco-friendly nitrile hydratase (NHase) production. First, the strong promoter Pveg from Bacillus subtilis, the Ni(II)/Co(II) responsive repressor RcnR, and its operator were reorganized to construct a CIES. In this system, the expression of reporter green fluorescent protein (GFP) was specifically triggered by Co(II) over a broad range of concentration. The performance of the cobalt-induced system was evolved to version 2.0 (CIES 2.0) for adaptation to different concentrations of Co(II) through programming a homeostasis system that rebalances cobalt efflux and influx with RcnA and NiCoT, respectively. Harnessing these synthetic platforms, the induced expression of NHase was coupled with enzyme maturation by Co(II) in a synchronizable manner without requiring additional inducers, which is a unique feature relative to other induced systems for production of NHase. The yield of NHase was 111.2 ± 17.9 U/ml using CIES and 114.9 ± 1.4 U/ml using CIES 2.0, which has a producing capability equivalent to that of commonly used isopropyl thiogalactoside (IPTG)-induced systems. In a scale-up system using a 5-L fermenter, the yielded enzymatic activity reached 542.2 ± 42.8 U/ml, suggesting that the designer platform for NHase is readily applied to the industry. The design of CIES in this study not only provided a low-cost and eco-friendly platform to overproduce NHase but also proposed a promising pipeline for development of synthetic platforms for expression of metalloenzymes.

[Study of mycelial polysaccharide from Paraisaria dubia of Ophiocordyceps gracilis asexual].

In order to provide a foundation for the development and application of Ophiocordyceps gracilis and increase the new resources of cordyceps,an asexual Paraisaria dubia was isolated from an O. gracilis fruit body. After 10 days of liquid fermentation,white globular mycelium and clear transparent fermentation were produced. The mycelium was extracted by hot water and precipitated with ethanol to obtain intracellular crude polysaccharide. The protein was deproteinized to obtain deproteinized polysaccharide. The intracellular pure polysaccharide was purified by Sepharose 4 B column chromatography and were analyzed by UV,IR,1 H-NMR,and13 CNMR data,as well as GC and HPLC. The results showed that the intracellular polysaccharide of P. dubia was composed of glucose,galactose and mannose with a molar ratio of 25. 54 ∶2 ∶1. It was a ß-configuration glycosylic bond,containing pyranoside. The initial connection of polysaccharide was ß(1→2)(1→4)(1→6) connection. This experiment provides a theoretical basis for the development and application of P. dubia.

Development of a novel strategy for robust synthetic bacterial promoters based on a stepwise evolution targeting the spacer region of the core promoter in Bacillus subtilis.

BACKGROUND: Promoter evolution by synthetic promoter library (SPL) is a powerful approach to development of functional synthetic promoters to synthetic biology. However, it requires much tedious and time-consuming screenings because of the plethora of different variants in SPL. Actually, a large proportion of mutants in the SPL are significantly lower in strength, which contributes only to fabrication of a promoter library with a continuum of strength. Thus, to effectively obtain the evolved synthetic promoter exhibiting higher strength, it is essential to develop novel strategies to construct mutant library targeting the pivotal region rather than the arbitrary region of the template promoter. In this study, a strategy termed stepwise evolution targeting the spacer of core promoter (SETarSCoP) was established in Bacillus subtilis to effectively evolve the strength of bacterial promoter. RESULTS: The native promoter, PsrfA, from B. subtilis, which exhibits higher strength than the strong promoter P43, was set as the parental template. According to the comparison of conservation of the spacer sequences between - 35 box and - 10 box among a set of strong and weak native promoter, it revealed that 7-bp sequence immediately upstream of the - 10 box featured in the regulation of promoter strength. Based on the conservative feature, two rounds of consecutive evolution were performed targeting the hot region of PsrfA. In the first round, a primary promoter mutation library (pPML) was constructed by mutagenesis targeting the 3-bp sequence immediately upstream of the - 10 box of the PsrfA. Subsequently, four evolved mutants from pPML were selected to construction of four secondary promoter mutation libraries (sPMLs) based on mutagenesis of the 4-bp sequence upstream of the first-round target. After the consecutive two-step evolution, the mutant PBH4 was identified and verified to be a highly evolved synthetic promoter. The strength of PBH4 was higher than PsrfA by approximately 3 times. Moreover, PBH4 also exhibited broad suitability for different cargo proteins, such as ß-glucuronidase and nattokinase. The proof-of-principle test showed that SETarSCoP successfully evolved both constitutive and inducible promoters. CONCLUSION: Comparing with the commonly used SPL strategy, SETarSCoP facilitates the evolution process to obtain strength-evolved synthetic bacterial promoter through fabrication and screening of small-scale mutation libraries. This strategy will be a promising method to evolve diverse bacterial promoters to expand the toolbox for synthetic biology.

The complete mitochondrial genome of wood-rotting fungus Xylaria hypoxylon.

Xylaria hypoxylon is a noticeable black fungus, and also habitual to cluster on rotting wood. In this study, the high-quality whole-genome of X. hypoxylon strain SFY20170806 was sequenced on the Illumina sequencing platform. The complete mitochondrial genome of X. hypoxylon was assembled and annotated. The single circular structure of 129,366 bp length is the largest species found in the order Xylariales. The overall GC content is 29.7% and gene composition includes 13 protein-coding genes (PCGs), 30 transfer RNA genes(tRNA), 2 ribosomal RNA genes(rRNA) and 6 open reading frames (ORF). Phylogenetic tree was constructed to validate the evolutionary relationship based on the complete mitogenomes from twelve taxa of four species of Xylariales, four species of Hypocreales, two species of Helotiales, one species of Microascales and X. hypoxylon. Phylogenetic analysis demonstrated that X. hypoxylon has a special evolutionary status and close genetic relationship with Annulohypoxylon stygium.

Exploitation of Bacillus subtilis as a robust workhorse for production of heterologous proteins and beyond.

Bacillus subtilis, belonging to the type species of Bacillus, is a type of soil-derived, low %G+C, endospore-forming Gram-positive bacterium. After the discovery of B. subtilis 168 that displayed natural competence, this bacterium has been intensively considered to be an ideal model organism and a robust host to study several basic mechanisms, such as metabolism, gene regulation, bacterial differentiation, and application for industrial purposes, such as heterologous protein expression and the overproduction of an array of bioactive molecules. Since the first report of heterologous overproduction of recombinant proteins in this strain, the bulk production of a multitude of valuable enzymes, especially industrial enzymes, has been performed on a relatively large scale. Since B. subtilis can non-specifically secrete recombinant proteins using various signal peptides, it has tremendous advantages over Gram-negative bacterial hosts. Along with the report of the complete genome sequence of B. subtilis, a number of genetic tools, including diverse types of plasmids, bacterial promoters, regulatory elements, and signal peptides, have been developed and characterized. These novel genetic elements tremendously accelerated genetic engineering in B. subtilis recombinant systems. In addition, with the development of several complex gene expression systems, B. subtilis has performed a number of more complex functions. This ability enables it to be a substantial chassis in synthetic biology rather than just a workhorse for the overproduction of recombinant proteins. In this review, we review the progress in the development of B. subtilis as a universal platform to overproduce heterologous diverse high-value enzymes. This progress has occurred from the development of biological parts, including the characterization and utilization of native promoters, the fabrication of synthetic promoters and regulatory elements, and the assembly and optimization of genetic systems. Some important industrial enzymes that have been produced in large quantities in this host are also summarized in this review. Furthermore, the ability of B. subtilis to serve as a cellular tool was also briefly recapitulated and reviewed.

Stepwise modifications of genetic parts reinforce the secretory production of nattokinase in Bacillus subtilis.

Nattokinase (NK) is an important serine-protease with direct fibrinolytic activity involving the prevention of cardiovascular disease as an antithrombotic agent. Dozens of studies have focused on the characterization of intrinsic novel promoters and signal peptides to the secretory production of recombinant proteins in Bacillus subtilis. However, intrinsic genetic elements have several drawbacks, which cannot mediate the production of NK to the desired level. In this study, the genetic elements, which were used to overproduce the recombinant secretory NK, were rationally modified in B. subtilis in a stepwise manner. The first step was to select a suitable signal peptide for the highly efficient secretion of NK. By comparison of the secretory levels mediated by two different signal peptides, which were encoded by the genes of a minor extracellular protease epr (SPepr ) and cell-wall associated protease wapA (SPwapA ), respectively, SPwapA was verified as the superior secretory element. Second, P04, which was a synthetic promoter screened from an array of mutants based on the promoter cloned from the operon of a quorum-sensing associated gene srfA (PsrfA ), was paired to SPwapA. The secretory level of NK was obviously augmented by the combination of these two genetic elements. Third, the cis-acting element CodY-binding sequence positioned at the 5'UTR was deleted (yielding P08), and thus the secretory level was significantly elevated. The activity of NK, which was defined as fibrinolytic units (FU), reached to a level of 270 FU ml-1 . Finally, the superior genetic element composed of P08 and SPwapA was utilized to overproduce NK in the host B. subtilis WB800, which was able to produce the secretory NK at 292 FU ml-1 . The strategy established in this study can not only be used to overproduce NK in B. subtilis but also might be a promising pipeline to modify the genetic element for the synthetic secretory system.

The complete mitochondrial genome of a transitional form in secondary endosymbiotic Cryptophyte algae Guillardia theta strain CCMP2712.

The complete mitochondrial genome of Guillardia theta strain CCMP2712 was sequenced, assembled, and annotated in this study. The circular genome is 35,013 bp in size and it contains 36 protein-coding genes (PCGs), 28 transfer RNA genes (tRNA), and 2 ribosomal RNA genes (rRNA). The overall GC contents of the mitochondrial genome are 28.9%. The phylogenetic tree was constructed to validate the taxonomic relationship based on the complete mitogenomes of G. theta strain CCMP2712 through combining with seven Cryptophyta and four Heterokontophyta algae. The complete mitochondrial genome of the Guillardia theta strain CCMP2712 will provide more information for the evolution of secondary endosymbiotic species.