Single-cell transcriptomics reveals heterogeneity and intercellular crosstalk in human intervertebral disc degeneration.

The complexity of the human intervertebral disc (IVD) has hindered the elucidation of the microenvironment and mechanisms underlying IVD degeneration (IVDD). Here we determined the landscapes of nucleus pulposus (NP), annulus fibrosus (AF), and immunocytes in human IVD by scRNA-seq. Six NP subclusters and seven AF subclusters were identified, whose functional differences and distribution during different stages of degeneration (Pfirrmann I-V) were investigated. We found MCAM+ progenitor in AF, as well as CD24+ progenitor and MKI67+ progenitor in NP, forming a lineage trajectory from CD24+/MKI67+ progenitors to EffectorNP_⅓ during IVDD. There is a significant increase in monocyte/macrophage (Mφ) in degenerated IVDs (p = 0.044), with Mφ-SPP1 exclusively found in IVDD but not healthy IVDs. Further analyses of the intercellular crosstalk network revealed interactions between major subpopulations and changes in the microenvironment during IVDD. Our results elucidated the unique characteristics of IVDD, thereby shedding light on therapeutic strategies.

A comprehensive evaluation of stable expression "hot spot" in the ScltI gene of Chinese hamster ovary cells.

The Chinese hamster ovary (CHO) cell is the most widely used biopharmaceutical expression system, but its long-term expression is unstable. This issue can be effectively addressed by site-specific integration of exogenous genes into the genome. Therefore, exogenous protein sites with stable expression in the CHO cell genome must be identified. CRISPR/Cas9 technology was used in this study to integrate various exogenous genes into the ScltI site as a "hot spot" at the CHO-K1 cell genome NW_003614095.1, and the stability and adaptability of exogenous genes expressed at the site were investigated. Flow cytometry sorting technology was used to obtain positive monoclonal cell lines that expressed either intracellular protein green fluorescent protein (EGFP) or secretory protein human serum albumin (HSA). For 60 passages, the positive monoclonal cell lines' cell growth cycles and exogenous protein expression were both observed. The results demonstrated that integrating the gene encoding exogenous proteins into the ScltI site had no effect on cell growth. The fluorescence intensity of EGFP was similar after 60 passages, and the expression of HSA increased slightly. Additionally, the super-monomeric protein VWF hydrolase (ADAMTS13) (190 kDa), human coagulation factor VII (FVII) (55 kDa), and interferon α2b (12 kDa) were integrated into the ScltI site for expression. In conclusion, the site located in the first exon of the ScltI gene within the CHO-K1 cell genome NW_003614095.1 is an ideal "hot spot" for the stable expression of various exogenous proteins. KEY POINTS: • The site-specific integration strategy of an exogenous gene in CHO cells was established for the ScltI site. • The genes for EGFP and HSA were site-directed integrated and stably expressed at the ScltI site. • The ScltI site fulfills the expression of exogenous proteins of different molecular weight sizes (15-190 kDa).

Dietary Goji Shapes the Gut Microbiota to Prevent the Liver Injury Induced by Acute Alcohol Intake.

Diet is a major driver of the structure and function of the gut microbiota, which influences the host physiology. Alcohol abuse can induce liver disease and gut microbiota dysbiosis. Here, we aim to elucidate whether the well-known traditional health food Goji berry targets gut microbiota to prevent liver injury induced by acute alcohol intake. The results showed that Goji supplementation for 14 days alleviated acute liver injury as indicated by lowering serum aspartate aminotransferase, alanine aminotransferase, pro-inflammatory cytokines, as well as lipopolysaccharide content in the liver tissue. Goji maintained the integrity of the epithelial barrier and increased the levels of butyric acid in cecum contents. Furthermore, we established the causal relationship between gut microbiota and liver protection effects of Goji with the help of antibiotics treatment and fecal microbiota transplantation (FMT) experiments. Both Goji and FMT-Goji increased glutathione (GSH) in the liver and selectively enriched the butyric acid-producing gut bacterium Akkermansia and Ruminococcaceae by using 16S rRNA gene sequencing. Metabolomics analysis of cecum samples revealed that Goji and its trained microbiota could regulate retinoyl ß-glucuronide, vanillic acid, and increase the level of glutamate and pyroglutamic acid, which are involved in GSH metabolism. Our study highlights the communication among Goji, gut microbiota, and liver homeostasis.

Discovery of novel selective PI3Kγ inhibitors through combining machine learning-based virtual screening with multiple protein structures and bio-evaluation.

Introduction: Phosphoinositide 3-kinase gamma (PI3Kγ) has been regarded as a promising drug target for the treatment of various diseases, and the diverse physiological roles of class I PI3K isoforms (α, ß, δ, and γ) highlight the importance of isoform selectivity in the development of PI3Kγ inhibitors. However, the high structural conservation among the PI3K family makes it a big challenge to develop selective PI3Kγ inhibitors. Objectives: A novel machine learning-based virtual screening with multiple PI3Kγ protein structures was developed to discover novel PI3Kγ inhibitors. Methods: A large chemical database was screened using the virtual screening model, the top-ranked compounds were then subjected to a series of bio-evaluations, which led to the discovery of JN-KI3. The selective inhibition mechanism of JN-KI3 against PI3Kγ was uncovered by a theoretical study. Results: 49 hits were identified through virtual screening, and the cell-free enzymatic studies found that JN-KI3 selectively inhibited PI3Kγ at a concentration as low as 3,873 nM but had no inhibitory effect on Class IA PI3Ks, leading to the selective cytotoxicity on hematologic cancer cells. Meanwhile, JN-KI3 potently blocked the PI3K signaling, finally led to distinct apoptosis of hematologic cell lines at a low concentration. Lastly, the key residues of PI3Kγ and the structural characteristics of JN-KI3, which both would influence γ isoform-selective inhibition, were highlighted by systematic theoretical studies. Conclusion: The developed virtual screening model strongly manifests the robustness to find novel PI3Kγ inhibitors. JN-KI3 displays a specific cytotoxicity on hematologic tumor cells, and significantly promotes apoptosis associated with the inhibition of the PI3K signaling, which depicts PI3Kγ as a potential target for the hematologic tumor therapy. The theoretical results reveal that those key residues interacting with JN-KI3 are less common compared to most of the reported PI3Kγ inhibitors, indicating that JN-KI3 has novel structural characteristics as a selective PIK3γ inhibitor.

Decreased Adiponectin Levels Are a Risk Factor for Cognitive Decline in Spinal Cord Injury.

OBJECTIVE: Spinal cord injury (SCI) has become popular in recent years, and cognitive decline is a common complication. Adiponectin is a common protein hormone involved in the course of many diseases, but its relationship with SCI has not yet been elucidated. The purpose of our prospective study is to explore whether adiponectin can be used as a biomarker of cognitive decline in SCI. METHODS: A total of 64 healthy volunteers and 92 patients with acute SCI were recruited by us. Serum adiponectin levels, demographic data (age and gender), lifestyle (smoking and drinking), medical history (diabetes and hypertension), and clinical baseline data (low-density lipoprotein, high-density lipoprotein, and fasting blood glucose) were recorded. Three months after enrollment, we used the Montreal Cognitive Assessment (MoCA) to evaluate cognitive function. Based on a quarter of the serum adiponectin levels, SCI patients were divided into 4 groups, and the differences in their MoCA scores were compared. In addition, we used multivariate linear regression to predict the risk factors of the MoCA score. RESULTS: The serum adiponectin level (6.1 ± 1.1 µg/ml) of SCI patients was significantly lower than that of the healthy control group (6.7 ± 0.9 µg/ml), and there was a significant difference between the two (p < 0.001). The group with higher serum adiponectin levels after 3 months of spinal cord injury had higher MoCA scores. Multivariate regression analysis showed that serum adiponectin level is a protective factor for cognitive function after SCI (ß = 0.210, p = 0.043). CONCLUSIONS: Serum adiponectin levels can be used as an independent predictor of cognitive function in patients with acute SCI.

Genome-wide analysis of cell-Free DNA methylation profiling with MeDIP-seq identified potential biomarkers for colorectal cancer.

BACKGROUND: Colorectal cancer is the most common malignancy and the third leading cause of cancer-related death worldwide. This study aimed to identify potential diagnostic biomarkers for colorectal cancer by genome-wide plasma cell-free DNA (cfDNA) methylation analysis. METHODS: Peripheral blood from colorectal cancer patients and healthy controls was collected for cfDNA extraction. Genome-wide cfDNA methylation profiling, especially differential methylation profiling between colorectal cancer patients and healthy controls, was performed by methylated DNA immunoprecipitation coupled with high-throughput sequencing (MeDIP-seq). Logistic regression models were established, and the accuracy of this diagnostic model for colorectal cancer was verified using tissue-sourced data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) due to the lack of cfDNA methylation data in public datasets. RESULTS: Compared with the control group, 939 differentially methylated regions (DMRs) located in promoter regions were found in colorectal cancer patients; 16 of these DMRs were hypermethylated, and the remaining 923 were hypomethylated. In addition, these hypermethylated genes, mainly PRDM14, RALYL, ELMOD1, and TMEM132E, were validated and confirmed in colorectal cancer by using publicly available DNA methylation data. CONCLUSIONS: MeDIP-seq can be used as an optimal approach for analyzing cfDNA methylomes, and 12 probes of four differentially methylated genes identified by MeDIP-seq (PRDM14, RALYL, ELMOD1, and TMEM132E) could serve as potential biomarkers for clinical application in patients with colorectal cancer.

Expression of ANGPTL4 in Nucleus Pulposus Tissues Is Associated with Intervertebral Disc Degeneration.

OBJECTIVE: Angiopoietin-like protein 4 (ANGPTL4), encoding a glycosylated secreted protein, has been reported to be closely related to many kinds of diseases, including diabetes, tumor, and some musculoskeletal pathologies, such as rheumatoid arthritis, osteoarthritis, and osteoporosis. The aim of the current study is to investigate the role of ANGPTL4 in intervertebral disc degeneration and analyze the association of ANGPTL4 expression with Pfirrmann grades. METHODS: A total of 162 nucleus pulposus tissues were collected from lumbar intervertebral disc herniation patients undergoing interforaminal endoscopic surgery. Real-time quantitative PCR and western blot were performed to determine the mRNA and protein expression of ANGPTL4 in nucleus pulposus samples. Statistical analysis was performed to analyze the association of ANGPTL4 expression with Pfirrmann grades. RESULTS: Based on the clinical data of 162 patients, results showed that Pfirrmann grades were significantly associated with patients' age (r = 0.162, P = 0.047) and were not significantly associated with patients' gender (P > 0.05). RT-qPCR and western blot results showed that the mRNA (r = 0.287, P < 0.05) and protein (r = 0.356, P < 0.05) expressions of ANGPTL4 were both closely associated with Pfirrmann grades. The expression of ANGPTL4 was remarkably increased in the groups of high IVDD Pfirrmann grades. CONCLUSION: The results demonstrated that ANGPTL4 expression was positively associated with the Pfirrmann grades and the severity of intervertebral disc degeneration. ANGPTL4 may be served as a candidate biomarker for intervertebral disc degeneration.

Discovery of potential inhibitors targeting the kinase domain of polynucleotide kinase/phosphatase (PNKP): Homology modeling, virtual screening based on multiple conformations, and molecular dynamics simulation.

In recent years, the level of interest has been increased in developing the DNA-repair inhibitors, to enhance the cytotoxic effects in the treatment of cancers. Polynucleotide kinase/phosphatase (PNKP) is a critical human DNA repair enzyme that repairs DNA strand breaks by catalyzing the restoration of 5'-phosphate and 3'-hydroxyl termini that are required for subsequent processing by DNA ligases and polymerases. PNKP is the only protein that repairs the 3'-hydroxyl group and 5'-phosphate group, which depicts PNKP as a potential therapeutic target. Besides, PNKP is the only DNA-repair enzyme that contains the 5'-kinase activity, therefore, targeting this kinase domain would motivate the development of novel PNKP-specific inhibitors. However, there are neither crystal structures of human PNKP nor the kinase inhibitors reported so far. Thus, in this present study, a sequential molecular docking-based virtual screening with multiple PNKP conformations integrating homology modeling, molecular dynamics simulation, and binding free energy calculation was developed to discover novel PNKP kinase inhibitors, and the top-scored molecule was finally submitted to molecular dynamics simulation to reveal the binding mechanism between the inhibitor and PNKP. Taken together, the current study could provide some guidance for the molecular docking based-virtual screening of novel PNKP kinase inhibitors.

A multi-conformational virtual screening approach based on machine learning targeting PI3Kγ.

Nowadays, more and more attention has been attracted to develop selective PI3Kγ inhibitors, but the unique structural features of PI3Kγ protein make it a very big challenge. In the present study, a virtual screening strategy based on machine learning with multiple PI3Kγ protein structures was developed to screen novel PI3Kγ inhibitors. First, six mainstream docking programs were chosen to evaluate their scoring power and screening power; CDOCKER and Glide show satisfactory reliability and accuracy against the PI3Kγ system. Next, virtual screening integrating multiple PI3Kγ protein structures was demonstrated to significantly improve the screening enrichment rate comparing to that with an individual protein structure. Last, a multi-conformational Naïve Bayesian Classification model with the optimal docking programs was constructed, and it performed a true capability in the screening of PI3Kγ inhibitors. Taken together, the current study could provide some guidance for the docking-based virtual screening to discover novel PI3Kγ inhibitors.

Cyclic Mechanical Stretch Ameliorates the Degeneration of Nucleus Pulposus Cells through Promoting the ITGA2/PI3K/AKT Signaling Pathway.

BACKGROUND: Intervertebral disc degeneration (IVDD) is one of the major causes of low back pain and motor deficiency. Nucleus pulposus (NP) degeneration plays a key role in the process of IVDD. The mechanical and biological interactions involved in NP degeneration have not been elucidated. The present study is aimed at investigating the effect and mechanism of cyclic mechanical stretch in regulating the function and degeneration of NP cells. METHODS: NP cells were subjected to cyclic tensile stress (10% deformation) of 0.1 Hz for 8640 cycles. Cell proliferation was conducted through the MTT assay. The cell cycle and apoptosis were detected by flow cytometry. A gene expression profile chip was used to analyze the differentially expressed genes between the tensile stress group and the control group. Enrichment analysis of Gene Ontology (GO) annotation and signaling pathways were analyzed. Western blot and RNA interference were carried out to investigate the role of the ITGA2/PI3K/AKT pathway in the effect of cyclic mechanical stretch on NP cells. RESULTS: NP cells exhibited a greater (P < 0.05) growth rate in the tensile stress group compared to the control group. Cyclic mechanical stress significantly promoted the cell cycle transition of NP cells from the S phase to the G2/M phase. A fewer proportion of apoptotic cells were found in the tensile stress group (P < 0.05), indicating that cyclic mechanical stretch inhibits NP cell apoptosis. Microarray analysis revealed 689 significant differentially expressed genes between the two groups (P < 0.05), of which 333 genes were upregulated and another 356 genes were downregulated. Cyclic mechanical stretch altered the expression of 31 genes involved in the ITGA2/PI3K/AKT pathway and remarkably promoted this pathway in NP cells. Downregulation of ITGA2 and AKT further demonstrated that the PI3K/AKT pathway was responsible for the proliferation and COL2A1 expression of NP cells upon cyclic mechanical stretch. CONCLUSIONS: Cyclic mechanical stretch promoted the proliferation and cell cycle and reversely inhibited the apoptosis of NP cells. Cyclic mechanical stretch promoted COL2A1 expression and ameliorated the degeneration of NP cells via regulation of the ITGA2/PI3K/AKT signaling pathway. Our results may provide a potential target and a possibility of IVDD disease treatment by ameliorating the degenerative changes.

Integrated molecular modeling techniques to reveal selective mechanisms of inhibitors to PI3Kδ with marketed Idelalisib.

Phosphatidylinositol-3-kinase (PI3K) is important for cell proliferation, differentiation, and apoptosis, and the diverse physiological roles of different PI3K isoforms have highlighted the significance of the development of PI3Kδ inhibitors. A large number of PI3Kδ inhibitors have been reported after the FDA approval of Idelalisib, but the clinical use of Idelalisib was limited because of its serious side effects. Therefore, great efforts have been made on the development of PI3Kδ inhibitors with higher selectivity and lower toxicity, but there is no new PI3Kδ inhibitor coming into the market so far. Even so, as the first listed PI3K inhibitor, Idelalisib could be used as an effective tool to investigate the selective inhibition mechanism of PI3Kδ. Thus, in this study, a modeling strategy integrated 3D-QSAR, pharmacophore model, and molecular dynamics simulation was employed to reveal the key chemical characteristics of Idelalisib analogs and the binding pattern between the inhibitors and PI3Kδ. First, the CoMFA model with high statistical significance was built to reveal the general structure-activity relationships. And then, a reliable pharmacophore model with a robust discrimination capability was constructed to expound the main chemical characteristics of the PI3Kδ inhibitors. Finally, molecular dynamics simulation was conducted to explore the binding modes and some key residues refer to δ-selective binding were highlighted with binding-free energy calculation. In summary, these models and results would provide some effective help for the discovery or the rational design of novel PI3Kδ inhibitors.

[Rational design of the C-terminal Loop region of leucine dehydrogenase and cascade biosynthesis L-2-aminobutyric acid].

Leucine dehydrogenase (LDH) is the key rate-limiting enzyme in the production of L-2-aminobutyric acid (L-2-ABA). In this study, we modified the C-terminal Loop region of this enzyme to improve the specific enzyme activity and stability for efficient synthesis of L-2-ABA. Using molecular dynamics simulation of LDH, we analyzed the change of root mean square fluctuation (RMSF), rationally designed the Loop region with greatly fluctuated RMSF, and obtained a mutant EsLDHD2 with a specific enzyme activity 23.2% higher than that of the wild type. Since the rate of the threonine deaminase-catalyzed reaction converting L-threonine into 2-ketobutyrate was so fast, the multi-enzyme cascade catalysis system became unbalanced. Therefore, the LDH and the formate dehydrogenase were double copied in a new construct E. coli BL21/pACYCDuet-RM. Compared with E. coli BL21/pACYCDuet-RO, the molar conversion rate of L-2-ABA increased by 74.6%. The whole cell biotransformation conditions were optimized and the optimal pH, temperature and substrate concentration were 7.5, 35 °C and 80 g/L, respectively. Under these conditions, the molar conversion rate was higher than 99%. Finally, 80 g and 40 g L-threonine were consecutively fed into a 1 L reaction mixture under the optimal conversion conditions, producing 97.9 g L-2-ABA. Thus, this strategy provides a green and efficient synthesis of L-2-ABA, and has great industrial application potential.

[Engineering the C4 pathway of Corynebacterium glutamicum for efficient production of 5-aminolevulinic acid].

5-aminolevulinic acid (5-ALA) plays an important role in the fields of medicine and agriculture. 5-ALA can be produced by engineered Escherichia coli and Corynebacterium glutamicum. We systematically engineered the C4 metabolic pathway of C. glutamicum to further improve its ability to produce 5-ALA. Firstly, the hemA gene encoding 5-ALA synthase (ALAS) from Rhodobacter capsulatus and Rhodopseudomonas palustris were heterologously expressed in C. glutamicum, respectively. The RphemA gene of R. palustris which showed relatively high enzyme activity was selected. Screening of the optimal ribosome binding site sequence RBS5 significantly increased the activity of RphemA. The ALAS activity of the recombinant strain reached (221.87±3.10) U/mg and 5-ALA production increased by 14.3%. Subsequently, knocking out genes encoding α-ketoglutarate dehydrogenase inhibitor protein (odhI) and succinate dehydrogenase (sdhA) increased the flux of succinyl CoA towards the production of 5-ALA. Moreover, inhibiting the expression of hemB by means of sRNA reduced the degradation of 5-ALA, while overexpressing the cysteine/O-acetylserine transporter eamA increased the output efficiency of intracellular 5-ALA. Shake flask fermentation using the engineered strain C. glutamicum 13032/∆odhI/∆sdhA-sRNAhemB- RBS5RphemA-eamA resulted in a yield of 11.90 g/L, which was 57% higher than that of the original strain. Fed-batch fermentation using the engineered strain in a 5 L fermenter produced 25.05 g/L of 5-ALA within 48 h, which is the highest reported-to-date yield of 5-ALA from glucose.

Effectiveness of a community-based integrated strategy to control soil-transmitted helminthiasis and clonorchiasis in the People's Republic of China.

Soil-transmitted helminthiases (STHs) are caused by a group of intestinal nematode infections due to poor hygiene and environments, and clonorchiasis is a food-borne trematode (FBT) infection caused by ingestion of raw freshwater fish. Both are endemic in the People's Republic of China. To explore a suitable control strategy, integrated interventions were applied between 2007 and 2009 in ten pilot counties (eight for the STHs and two for clonorchiasis). Drug administration was used for treatment and complementary efforts to improve the situation based on health education, provision of clean water and sanitation were carried out. Significant achievements were gained as reflected by a drastic decrease in prevalence these infections were demonstrated. The overall prevalence of STHs and clonorchiasis decreased from 35.9% to 7.8% and from 41.4% to 7.0%, respectively. The reduction of prevalence and high cost-effectiveness were documented supporting large-scale application of this integrated intervention in China and elsewhere.

Reduction of charge variants by CHO cell culture process optimization.

Over the past decade, global interest in the development of therapeutic monoclonal antibodies (mAbs) has risen rapidly. As therapeutic agents, antibodies have shown marked efficacy in combatting a range of cancers and immune diseases with high target specificity and low toxicity (Carla Lucia et al. in PLoS ONE 6:e24071, 2011; Donaghy in MAbs 8:659-671, 2016; Nasiri et al. in J Cell Physiol 9:6441-6457, 2018; Teo et al. in Cancer Immunol Immunother 61:2295-2309, 2012). Recent advances in cell culture technology, such as high-throughput clone screening, have facilitated antibody production at concentrations exceeding 10 g/L (Chen et al. in BMC Immunol 19:35, 2018; Huang et al. in Biotechnol Prog 26:1400-1410, 2010; Lu et al. in Biotechnol Bioeng 110:191-205, 2013; Singh et al. in Biotechnol Bioeng 113:698-716, 2016). As titers have improved, the industry has begun to focus on the adjustment of target antibody quality profiles to improve efficacy. Cell lines, culture media, and culture conditions impact protein quality (Van Beers and Bardor in Biotechnol J 7:1473-1484, 2012). Optimization of critical quality attributes (CQAs), such as charge variants, can be achieved through bioprocess development and is the preferred approach as changes to the cell line or growth media used is considered unfavorable by regulatory bodies (Gawlitzek et al. in Biotechnol Bioeng 103:1164-1175, 2009; Jordan et al. in Cytotechnology 65:31-40, 2013; Pan et al. in Cytotechnology 69:39-56, 2016). In this study, the effect of process control and ion supplementation on charge variants of mAbs produced by Chinese hamster ovary (CHO) cells was investigated. Results of this study demonstrated that the concentration of Zn2+, duration of culturing, and temperature affect charge variants of a given mAb. Under the optimum conditions of 3L bioreactors, the most significant was that Zn2 + and temperature shift could further improve the quality of antibody. The main peak increased by 12%, and the acid peak decreased by 16%. At the same time, there was no significant loss of titer. This study provided supporting evidence for methods to improve charge variants arising during mAb production.

Biocatalytic synthesis of lactosucrose using a recombinant thermostable ß-fructofuranosidase from Arthrobacter sp. 10138.

As a prebiotics, lactosucrose plays an important role in maintaining human gastrointestinal homeostasis. In this study, a thermostable enzyme from Arthrobacter sp. 10138 was screened from six ß-fructofuranosidase-producing strains for the lactosucrose production and the coding gene was heterologously expressed in Escherichia coli for efficient expression. Recombinant ß-fructofuranosidase was purified and biochemically characterized by MALDI-TOFMS spectrometry. The transfructosylation product by this recombinant enzyme was determined to be lactosucrose rather than other oligosaccharides or polysaccharides by HPLC and LC-MS. Efficient extracellular secretion of ß-fructofuranosidase was achieved by the optimization of signal peptide and induction conditions. It was found that with the signal peptide torT, the highest extracellular activity reached 111.01 U/mL, which was 38.4-fold higher than that with the OmpA signal peptide. Under the optimal conditions (pH 6.0, temperature 50°C, enzyme amount 40 µg/ml, sucrose 150 g/L and lactose 150 g/L), 109 g/L lactosucrose was produced with a molar conversion ratio of 49.3%. Here the thermostable ß-fructofuranosidase from Arthrobacter sp. 10138 can be used for efficient synthesis of lactosucrose, and this provides a good startpoint for the industrial production of lactosucrose in the future.

Crystal structure and proposed mechanism of an enantioselective hydroalkoxylation enzyme from Penicillium herquei.

Hydroalkoxylation is a useful and efficient reaction which generates C-O bond and produces cyclic ethers, the common structural elements of natural products. The dedicative enzyme which can catalyze enantioselective hydroalkoxylation named PhnH was recently identified in the herqueinone biosynthetic gene from Penicillium herquei. It catalyzes addition of a phenol to the terminal olefin on substrate to produce a dihydrobenzofuran. Here, the crystal structure of PhnH is reported and the putative substrate-binding pocket is illustrated. Through docking experiment, possible substrate-binding poses are displayed and the catalytic mechanism is therefore proposed. Our findings form the basis for further studies of enantioselective hydroalkoxylation enzymes.

[Heterologous expression of Streptomyces coelicolor trehalose synthase and whole-cell biocatalyst production of trehalose in Escherichia coli].

The trehalose synthase (ScTreS) gene from Streptomyces coelicolor was successfully cloned and heterologously expressed in Escherichia coli BL21(DE3). The protein purified by Ni-NTA affinity column showed an apparent molecular weight (MW) of 62.3 kDa analyzed by SDS-PAGE. The optimum temperature of the enzyme was 35 °C and the optimum pH was 7.0; the enzyme was sensitive to acidic conditions. By homologous modeling and sequence alignment, the enzyme was modified by site-directed mutagenesis. The relative activities of the mutant enzymes K246A and A165T were 1.43 and 1.39 times that of the wild type, an increased conversion rate of 14% and 10% respectively. To optimize the synthesis conditions of trehalose, the mutant strain K246A was cultivated in a 5-L fermentor and used for whole-cell transformation. The results showed that with the substrate maltose concentration of 300 g/L at 35 °C and pH 7.0, the highest conversion rate reached 71.3%, and the yield of trehalose was 213.93 g/L. However, when maltose concentration was increased to 700 g/L, the yield of trehalose can reach 465.98 g/L with a conversion rate of 66%.

Structural insights to heterodimeric cis-prenyltransferases through yeast dehydrodolichyl diphosphate synthase subunit Nus1.

The polyprenoid glycan carriers are produced by cis-prenyltransferases (cis-PTs), which function as heterodimers in metazoa and fungi or homodimers in bacteria, but both are found in plants, protista and archaea. Heterodimeric cis-PTs comprise catalytic and non-catalytic subunits while homodimeric enzymes contain two catalytic subunits. The non-catalytic subunits of cis-PT shows low sequence similarity to known cis-PTs and their structure information is of great interests. Here we report the crystal structure of Nus1, the non-catalytic subunit of cis-PT from Saccharomyces cerevisiae. We also investigate the heterodimer formation and active site conformation by constructing a homology model of Nus1 and its catalytic subunit. Nus1 does not contain an active site, but its C-terminus may participate in catalysis by interacting with the substrates bound to the catalytic subunit. These results provide important basis for further investigation of heterodimeric cis-PTs.

Site-specific integration of light chain and heavy chain genes of antibody into CHO-K1 stable hot spot and detection of antibody and fusion protein expression level.

Expression cell line constructed by random integration method will often meet with unstable expression problem because target genes may be integrated into unstable region of chromatin. Rational cell line construction can overcome this shortcoming by inserting target gene into stable region of chromatin specifically. Here, we successfully got one knock-in cell line where light chain and heavy chain genes of antibody was site specifically integrated into stable hot spot reported before via homologous dependent recombination method mediated by CRISPR/Cas9. The targeting efficiency was around 1.35%. This cell line together with other three pre-established targeting cell lines (targeting with glucagon-like peptide 1 with human serum albumin fusion protein gene, or NGGH) were all undergoing protein expression level detection. In adherent cell mode, the amount of antibody expressed per cell per day were all around 0.006 pg/cell/day over passage 3, 12, 23, 35 and 50 while the amount of NGGH expressed per cell per day of 3 cell lines were all around 1.2 pg/cell/day over passage 3, 12, 23, 35 and 50. In batch mode, the antibody concentration within supernatant were around 2.5 µg/L over passage 1, 25, and 50 while the NGGH fusion protein concentration within supernatant were around 17 mg/L over passage 1, 25, and 50.