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Pichia pastoris has been widely exploited for the heterologous expression of proteins in both industry and academia. Recently, it has been shown to be a potentially good chassis host for the production of high-value chemicals and pharmaceuticals. Effective synthetic biology tools for genetic engineering are essential for industrial and biotechnological research in this yeast. Here, we describe a novel and efficient genome editing method mediated by the CRISPR-Cpf1 system, which could facilitate the deletion of large DNA fragments and integration of multiplexed gene fragments. The CRISPR-Cpf1 system exhibited a precise and high editing efficiency for single-gene disruption (99 ± 0.8%), duplex genome editing (65 ± 2.5% to 80 ± 3%), and triplex genome editing (30 ± 2.5%). In addition, the deletion of large DNA fragments of 20kb and one-step integration of multiple genes were first achieved using the developed CRISPR-Cpf1 system. Taken together, this study provides an efficient and simple gene editing tool for P. pastoris. The novel multiloci gene integration method mediated by CRISPR-Cpf1 may accelerate the ability to engineer this methylotrophic yeast for metabolic engineering and genome evolution in both biotechnological and biomedical applications.

Whole-cell biocatalysts have a wide range of applications in many fields. However, the transport of substrates is tricky when applying whole-cell biocatalysts for industrial production. In this research, P. pastoris whole-cell biocatalysts were constructed for rebaudioside A synthesis. Sucrose synthase was expressed intracellularly while UDP-glycosyltransferase was displayed on the cell wall surface simultaneously. As an alternative method, a fermentation process is applied to relieve the substrate transport-limitation of P. pastoris whole-cell biocatalysts. This fermentation process was much simpler, more energy-saving, and greener than additional operating after collecting cells to improve the catalytic ability of whole-cell biocatalysts. Compared with the general fermentation process, the protein production capacity of cells did not decrease. Meanwhile, the activity of whole-cell biocatalysts was increased to 262%, which indicates that the permeability and space resistance were improved to relieve the transport-limitations. Furthermore, the induction time was reduced from 60 h to 36 h. The fermentation process offered significant advantages over traditional permeabilizing reagent treatment and ultrasonication treatment based on the high efficiency and simplicity.

Ready-to-eat (RTE) foods have been considered to be reservoirs of antibiotic resistance bacteria, which constitute direct threat to human health, but the potential microbiological risks of RTE foods remain largely unexplored. In this study, the metagenomic approach was employed to characterize the comprehensive profiles of bacterial community and antibiotic resistance gene (ARG) in 18 RTE food samples (8 RTE meat, 7 RTE vegetables and 3 RTE fruit) in southern China. In total, the most abundant phyla in RTE foods were Proteobacteria, Firmicutes, Cyanobacteria, Bacteroidetes and Actinobacteria. 204 ARG subtypes belonging to 18 ARG types were detected with an abundance range between 2.81 × 10−5 and 7.7 × 10−1 copy of ARG per copy of 16S rRNA gene. Multidrug-resistant genes were the most predominant ARG type in the RTE foods. Chloramphenicol, macrolide-lincosamide-streptogramin, multidrug resistance, aminoglycoside, bacitracin, tetracycline and β-lactam resistance genes were dominant, which were also associated with antibiotics used extensively in human medicine or veterinary medicine/promoters. Variation partitioning analysis indicated that the join effect of bacterial community and mobile genetic elements (MGEs) played an important role in the resistome alteration. This study further deepens the comprehensive understanding of antibiotic resistome and the correlations among the antibiotic resistome, microbiota, and MGEs in the RTE foods.

To build a stronger Pichia pastoris PCAT1 promoter and to identify putative transcriptional factor binding sites (TFBSs) on PCAT1 that affect the activity of the promoter. A synthetic library of PCAT1 was generated by deleting or duplicating putative TFBS motifs in the promoter sequence. CSRE, MIG1, RAP1 and HAP2/3/4 were found to have important effects on PCAT1 activity. The PCAT1 variant P4 with a putative binding site of RAP1 on the promoter sequence showed a stronger activity compared with that of the wild-type PCAT1 and PAOX1, which is the strongest natural P. pastoris promoter that has been reported. This inference was confirmed with EGFP (enhanced green fluorescent protein) and Candida Antarctica lipase B as the reporters. The role of the transcriptional regulator RAP1 may be important in PCAT1 methanol induction. A stronger PCAT1 variant can be constructed by the duplication of the putative binding site of RAP1 on the PCAT1 promoter sequence. This PCAT1 variant has potential value for heterologous protein production, metabolic engineering, and synthetic biology.

Translational regulation plays an important role in protein synthesis. Our goal was to screen translation-related factors to improve heterologous protein expression in Pichia pastoris. Twenty-eight translation-related factors were overexpressed in P. pastoris GS115 expressing enhanced green fluorescent protein (eGFP). The results showed that overexpression of Bcy1, the regulatory subunit of protein kinase A (PKA), significantly increased both eGFP expression and cell biomass by 20% under methanol induction for 120 h. Additionally, overexpression of Bcy1 elevated the growth rate by 18% and increased production of the industrial enzyme Phytase (Phy) by 26%. Transcriptome analysis indicated that the overall expression of ribosomal protein genes was significantly downregulated and that postdiauxic shift genes and stress response element genes were upregulated. Bcy1 regulates ribosome protein genes, postdiauxic shift genes and stress response element genes, leading to improved cell growth and heterologous protein expression. This study provides a convenient and universal factor for heterologous protein production.

Sodium dodecyl sulfate (SDS), a representative anionic surfactant, is a commonly used reagent in studies of the cell membrane and cell wall. However, the mechanisms through which SDS affects cellular functions have not yet been fully examined. Thus, to gain further insights into the cellular functions and responses to SDS, we tested a haploid library of Saccharomyces cerevisiae single-gene deletion mutants to identify genes required for tolerance to SDS. After two rounds of screening, we found 730 sensitive and 77 resistant mutants. Among the sensitive mutants, mitochondrial gene expression; the mitogen-activated protein kinase signaling pathway; the metabolic pathways involved in glycoprotein, lipid, purine metabolic process, oxidative phosphorylation, cellular amino acid biosynthesis and pentose phosphate pathway were found to be enriched. Additionally, we identified a set of transcription factors related to SDS responses. Among the resistant mutants, disruption of ribosome biogenesis and translation alleviated SDS-induced cytotoxicity. Collectively, our results provided new insights into the mechanisms through which SDS regulates the cell membrane or cell wall.

Lycopene is a highly valued carotenoid with wide applications in various industries. The market demand for lycopene promotes research in metabolic engineering of heterologous hosts for lycopene. In this study, Pichia pastoris strain GS115 was genetically engineered to produce lycopene by integrating the heterologous lycopene biosynthesis genes from Corynebacterium glutamicum ATCC13032. The resulting strain, L1, produced 0.115 mg/g cell dry weight (DCW) lycopene. Through optimization by promoter selection, improving the precursor supply and expanding the Geranylgeranyl diphosphate (GGPP) pool, ultimately, the lycopene yield of the final optimal strain was 6.146 mg/g DCW with shake flask fermentation and 9.319 mg/g DCW (0.714 g/L) in a 3 L fermenter. The lycopene yield in this study is the highest yield of lycopene in P. pastoris reported to date, which demonstrated the potential of P. pastoris in lycopene synthesis and as a candidate host organism for the synthesis of other high value-added terpenoids. P. pastoris strain GS115 was genetically engineered to produce lycopene and the lycopene yield was improved step by step.

Background The methylotrophic yeast Pichia pastoris is well-known for the production of a broad spectrum of functional types of heterologous proteins including enzymes, antigens, engineered antibody fragments, and next gen protein scaffolds and many transcription factors are utilized to address the burden caused by the high expression of heterologous proteins. In this article, a novel P. pastoris transcription factor currently annotated as Fhl1p, an activator of ribosome biosynthesis processing, was investigated for promoting the expression of the recombinant proteins. Results The function of Fhl1p of P. pastoris for improving the expression of recombinant proteins was verified in strains expressing phytase, pectinase and mRFP, showing that the productivity was increased by 20–35%. RNA-Seq was used to study the Fhl1p regulation mechanism in detail, confirming Fhl1p involved in the regulation of rRNA processing genes, ribosomal small/large subunit biogenesis genes, Golgi vesicle transport genes, etc., which contributed to boosting the expression of foreign proteins. The overexpressed Fhl1p strain exhibited increases in the polysome and monosome levels, showing improved translation activities. Conclusion This study illustrated that the transcription factor Fhl1p could effectively enhance recombinant protein expression in P. pastoris. Furthermore, we provided the evidence that overexpressed Fhl1p was related to more active translation state.

The purpose of this article is to study the underlying cause of the induction of autophagy in Pichia pastoris cells grown in amino acid-rich methanol medium during methanol adaptation. Autophagy was induced in P. pastoris GS115 when cells were grown in amino acid-rich methanol medium. Transcriptome analysis revealed that genes involved in amino acid biosynthesis were upregulated. The deletion of Gcw13, a GPI-anchored protein that plays a role in the endocytosis of the general amino acid permease Gap1, resulted in the inhibition of autophagy, the activation of TORC1 and an increase in the uptake of glutamine and asparagine in methanol-grown cells. Our results demonstrated that the autophagy induced in P. pastoris cells grown in amino acid-rich methanol medium was nitrogen source independent and may be due to a Gcw13-dependent decrease in amino acid uptake during methanol adaptation.

We screened a haploid library of Saccharomyces cerevisiae single-gene deletion mutants to identify nonessential genes associated with increased sensitivity to or resistance against the cell wall antagonist calcofluor white. Through a genome-wide screen, we isolated 537 strains that had an altered growth rate relative to wild type, of which 485 showed increased sensitivity and 52 showed increased resistance to calcofluor white. The MAPK signaling pathway, N-glycan biosynthesis, endocytosis, vacuole acidification, autophagy, and the sulfur relay system were identified as being associated with calcofluor white sensitivity. Resistance genes were mainly involved in chitin metabolism and the RIM101 pathway or encoded several components of the ESCRT complexes or related to cysteine and methionine metabolism and RNA degradation. Further investigation indicated a clear global response network that S. cerevisiae relies on in the presence of the cell wall antagonist calcofluor white, which may help us to understand fungal cell wall remodeling and the mechanisms of toxicity of calcofluor white with respect to eukaryotic cells.

Background Translational regulation played an important role in the correct folding of heterologous proteins to form bioactive conformations during biogenesis. Translational pausing coordinates protein translation and co-translational folding. Decelerating translation elongation speed has been shown to improve the soluble protein yield when expressing heterologous proteins in industrial expression hosts. However, rational redesign of translational pausing via synonymous mutations may not be feasible in many cases. Our goal was to develop a general and convenient strategy to improve heterologous protein synthesis in Pichia pastoris without mutating the expressed genes. Results Here, a large-scale deletion library of ribosomal protein (RP) genes was constructed for heterologous protein expression in Pichia pastoris, and 59% (16/27) RP deletants have significantly increased heterologous protein yield. This is due to the delay of 60S subunit assembly by deleting non-essential ribosomal protein genes or 60S subunit processing factors, thus globally decreased the translation elongation speed and improved the co-translational folding, without perturbing the relative transcription level and translation initiation. Conclusion Global decrease in the translation elongation speed by RP deletion enhanced co-translational folding efficiency of nascent chains and decreased protein aggregates to improve heterologous protein yield. A potential expression platform for efficient pharmaceutical proteins and industrial enzymes production was provided without synonymous mutation.

The carotenoid, α-carotene, is very beneficial for human health and wellness, but microbial production of this compound is notoriously difficult, due to the asymmetric rings on either end of its terpenoid backbone. Here, we report for the first time the efficient production of α-carotene in the industrial bacterium Corynebaterium glutamicum by using a combined pathway engineering approach including evaluation of the performance of different cyclases and analysis of key metabolic intermediates to determine flux bottlenecks in the carotenoid biosynthesis pathway. A multi-copy chromosomal integration method was pivotal in achieving stable expression of the cyclases. In fed-batch fermentation, 1,054 mg/L of α-carotene was produced by the best strain, which is the highest reported titer achieved in microbial fermentation. The success of increased α-carotene production suggests that the multi-copy chromosomal integration method can be a useful metabolic engineering tool for overexpression of key enzymes in C. glutamicum and other bacterium as well.

Background Glycosylphosphatidylinositol (GPI)-anchored glycoproteins have diverse intrinsic functions in yeasts, and they also have different uses in vitro. In this study, the functions of potential GPI proteins in Pichia pastoris were explored by gene knockout approaches. Results Through an extensive knockout of GPI proteins in P. pastoris, a single-gene deletion library was constructed for 45 predicted GPI proteins. The knockout of proteins may lead to the activation of a cellular response named the ‘compensatory mechanism’, which is characterized by changes in the content and relationship between cell wall polysaccharides and surface proteins. Among the 45 deletion strains, five showed obvious methanol tolerance, four owned high content of cell wall polysaccharides, and four had a high surface hydrophobicity. Some advantages of these strains as production hosts were revealed. Furthermore, the deletion strains with high surface hydrophobicity were used as hosts to display Candida antarctica lipase B (CALB). The strain gcw22Δ/CALB-GCW61 showed excellent fermentation characteristics, including a faster growth rate and higher hydrolytic activity. Conclusions This GPI deletion library has some potential applications for production strains and offers a valuable resource for studying the precise functions of GPI proteins, especially their putative functions.

Pectate lyases play an essential role in textiles, animal feed, and oil extraction industries. Pichia pastoris can be an ideal platform for pectate lyases production, and BspPel (a thermo-alkaline pectate lyase from Bacillus sp. RN1) was overexpressed by combined strategies, reaching 1859 U/mL in a 50 L fermentator. It displayed the highest activity at 80°C, and maintained more than 60% of the activity between 30 and 70°C for 1 h. It showed an optimal pH of 10.0, and exhibited remarkable stability over a wider pH range (3.0-11.0), retaining more than 80.0% of enzyme activity for 4 h. The K m and k cat of BspPel on PGA (polygalacturonic acid) was 2.19 g L-1 and 116.1 s-1, respectively. The activity was significantly enhanced by Ca2+, Mn2+, and Cu2+, and a slight increase was observed with the addition of Ba2+ and Mg2+. Scanning electron microscope was used to show the degumming efficiency of BspPel on ramie fibers. The loss weight was 9.2% when treated with crude enzyme supernatant and 20.8% when treated with the enzyme-chemical method, which was higher than the 14.2% weight loss in the positive control treated with 0.5% (w/v) NaOH alone. In conclusion, BspPel could be a good candidate for the ramie degumming industry.

FKS1 encodes a β-1,3-glucan synthase, which is a key player in cell wall assembly in Saccharomyces cerevisiae. Here we analyzed the global transcriptomic changes in the FKS1 mutant to establish a correlation between the changes in the cell wall of the FKS1 mutant and the molecular mechanism of cell wall maintenance. These transcriptomic profiles showed that there are 1151 differentially expressed genes (DEGs) in the FKS1 mutant. Through KEGG pathway analysis of the DEGs, the MAPK pathway and seven pathways involved in carbon metabolism were significantly enriched. We found that the MAPK pathway is activated for FKS1 mutant survival and the synthesis of cell wall components are reinforced in the FKS1 mutant. Our results confirm that the FKS1 mutant has a β-1,3-glucan defect that affects the cell wall and partly elucidate the molecular mechanism responsible for cell wall synthesis. Our greater understanding of these mechanisms helps to explain how the FKS1 mutant survives, has useful implications for the study of similar pathways in other fungi, and increases the theoretical foundation for the regulation of the cell wall in S. cerevisiae.

Rebaudioside A is an attractive non-nutritive natural sweetener. UDP-glycosyltransferase UGT76G1 from Stevia rebaudiana Bertoni has been found to effectively convert stevioside to rebaudioside A. In this study, sucrose synthase mbSUS from Vigna radiata was first reported to co-operate with glycosyltransferase. Compared with other sucrose synthases, mbSUS was selected for co-expression with UGT76G1 in Pichia pastoris GS115 because of its high efficiency in improving the synthesis of rebaudioside A. After optimizing the gene dose ratio of mbSUS and UGT76G1, which is an effective whole-cell biocatalyst GS115 (U76/mb3:1) was constructed. For the biosynthesis of rebaudioside A with whole-cell biocatalyst GS115 (U76/mb3:1), the reaction parameters were optimized and the limiting conditions were identified. Under optimal conditions, the yield of rebaudioside A reached 261.2 mM in a 1 L batch within 26 h. Based on its the high reaction efficiency, the whole-cell catalyst GS115 (U76/mb3:1) is a powerful biocatalyst with the potential for industrial-scale synthesis.

Pichia pastoris is a widely used heterologous protein production workhorse. However, with its multiple genetic modifications to solve bottlenecks for heterologous protein productivity, P. pastoris lacks selectable markers. Existing selectable marker recycling plasmids have drawbacks (e.g., slow growth and conditional lethality). Here, zeocin-resistance marker recycling vectors were constructed using the Cre/loxP recombination system. The vectors were used to (i) knock in heterologous phytase, xylanase and lipase expression cassettes, (ii) increase the phytase, xylanase and lipase gene copy number to 13, 5, and 5, respectively, with vector introduction and (iii) engineer the secretion pathway by co-overexpressing secretion helper factors (Sly1p and Sec1p) without introducing selectable markers, giving a phytase field of 0.833 g/L. The vectors allow selectable marker recycling and would be a useful tool to engineer P. pastoris for high heterologous protein productivity.

Objectives: Patients with tuberous sclerosis complex (TSC) present multiple cortical tubers in the brain, which are responsible for epilepsy. It is still difficult to localize the epileptogenic tuber. The value of cortico-cortical evoked potentials (CCEPs) was assessed in epileptogenic tuber localization in patients with TSC using stereo-electroencephalography (SEEG). Methods: Patients with TSC who underwent SEEG and CCEP examination in preoperative evaluation during 2014-2017 and reached postoperative seizure freedom at 1-year follow-up were enrolled in this study (n = 11). CCEPs were conducted by stimulating every two adjacent contacts of SEEG electrodes and recording on other contacts of SEEG electrodes in one epileptogenic tuber and its early-stage propagating tuber, and their perituberal cortexes in each patient. The CCEP was defined as positive when N1 and/or N2 wave presented, and then the occurrence rates of positive CCEPs were then compared among different tubers and perituberal regions. Results: Occurrence rates of positive CCEP from epileptogenic tubers to early propagating tubers and epileptogenic tubers to perituberal cortexes were 100%, which were significantly higher than the occurrence rates of CCEP between other locations. The occurrence rates of CCEP from peripheral portions of epileptogenic tubers to peripheral portions of early propagating tubers or perituberal cortexes were 100%, which were significant higher than the occurrence rates of CCEP from peripheral regions of early propagating tubers to peripheral portions of epileptogenic tubers, from the central part of early propagating tuber to central portions of epileptogenic tubers, or from perituberal cortexes to the center part of epileptogenic tubers. Conclusion: Epileptogenic tubers presented much more diffusive connectivity with other tubers and perituberal cortexes than any other connectivity relationships across propagating tubers, and the peripheral region of epileptogenic tubers presented the greatest connectivity with propagating tubers and perituberal cortexes. CCEP can be an effective tool in epileptogenic tuber localization in patients with TSC.

Background Electronic conduction, a new treatment approach for epilepsy, has been confirmed to reduce epileptiform discharge on EEG and convulsive behaviors, particularly epileptic discharge propagation and serious behavioral seizures, in rats with kainic acid (KA)-induced acute temporal lobe epilepsy (TLE). Objective Hippocampal cell apoptosis was examined to confirm the neuroprotective effect of electronic conduction therapy in rats with KA-induced acute TLE. Methods Rats were divided into four groups: control group (right CA3 injection of saline), KA group (right CA3 injection of KA), sham conduction group (KA rats with sham conduction), and conduction group (KA rats with electric conduction). Apoptotic cells were evaluated by flow cytometry, TUNEL staining, and mRNA expression levels of caspase-3, tumor necrosis factor-alpha, and glial fibrillary acidic protein measured by real-time quantitative PCR (qRT-PCR). Results The frequency of convulsive behaviors in the conduction group decreased significantly compared with the KA group and the sham conduction group. Significantly fewer apoptotic cells were detected in rats with conduction based on flow cytometry and TUNEL staining results. The qRT-PCR results indicated that KA-induced up-regulation of hippocampal caspase-3 mRNA expression was reduced 24 hours after KA injection in rats that received conduction treatment. Conclusion Electronic conduction treatment can reduce seizure frequency and hippocampal cell apoptosis in rats with KA-induced acute TLE.

In Pichia pastoris, most of the Glycosylphosphatidylinositol (GPI)-anchored proteins are of unknown function. Gcw13, one of these GPI-anchored proteins, was found to exert an inhibitory effect on the growth of the histidine auxotrophic P. pastoris strain GS115 on methanol as the sole carbon source. To investigate the biological function of Gcw13, RNA sequencing (RNA-Seq) was performed to compare the difference of gene expression between GS115 and GCW13-deletion strain D13. RNA-Seq analysis showed that, in strain D13, the expression of genes involved in the methanol utilization pathway or peroxisome biogenesis was not changed, and a high proportion of genes involved in the biosynthesis of amino acids were down-regulated, whereas GAP1, which encodes a general amino acid permease, was significantly up-regulated. Besides, the intracellular concentrations of various amino acids were significantly higher in D13 than that in GS115. We also observed that deletion of GCW13 resulted in more Gap1 presented on the cell surface and more active uptake of the toxic proline analogue l-azetidine-2-carboxylate acid (AzC). These results suggest that Gcw13 suppresses the expression of GAP1 and facilitates the endocytosis of Gap1 on methanol, resulting in decreasing Gap1-dependent uptake of amino acids in P. pastoris, which might contribute to the poor growth of GS115 on methanol.

To characterize a putative laccase gene, LacMP, of Moniliophthora perniciosa FA553 that had been screened using a genome mining approach, then cloned and expressed in Pichia pastoris.The purified recombinant LacMP was ~57 kDa with a maximum laccase activity of 232 U/l. The optimal pH for oxidation reactions with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), syringaldazine, guaiacol and 2,6-dimethoxyphenol, were 6, 7.5, 6.5, and 6.5, respectively. The laccase activity was optimal at 60, 45, 45, and 55 °C for the four respective substrates. LacMP was stable at pH 6-8 and30 °C. Li(+), K(+), Co(2+), Ni(2+), Mn(2+), and Mg(2+) at 1 mM had no obvious effect on its activity.Given that LacMP has optimal activity under neutral and alkaline oxidation reaction conditions, it could be a potential candidate for industrial biocatalytic applications.

Endoglucanase 1 (EG1) isolated from the straw mushroom has great potential in the textile and paper industries. Improving EG1 expression level will add to its value for industrial applications. In this study, we employed two combined strategies to enhance the expression quantity of EG1, which are increase the copy number of EG1 and enhance the folding and secretion efficiency of EG1 in the endoplasmic reticulum by overexpress HAC1. Multiple plasmids, which contains four copies of EG1, were constructed by isocaudamers, resulted a recombinant strain with EG1 activity up to 39.6 U/mL, 262% higher than that measured in the strain containing only a single copy. A significant increase in activity (151%) was found when eight copies of EG1 was introduced into a different host, compared with a host harboring four copies. Further overexpression of the HAC1 transcription factor in the host harboring eight EG1 copies led to activity of 91.9 U/mL, which is 619% higher than that measured in the original strain. Finally, EG1 activity of 650.1 U/mL was achieved in a 3-L scaled-up fed-batch fermenter and the protein yield was 4.05 g/L. The characteristics of recombinant EG1 were also investigated, the optimal values for enzyme activity were 60 °C and pH 5.0, which yielded a catalytic efficiency of 312.9 mL mg-1min-1 using carboxymethyl cellulose(CMC) as the substrate.

Kinetic resolution of sec-alcohols is a green process with biocatalyst. Candida antarctica lipase B (CALB) displayed on Pichia pastoris cell-surface (Pp-CALB) was characterized in kinetic resolution of sec-alcohols with different structures. The reaction parameters including acyl donors, molar ratio of substrates, solvents and temperatures were examined with 2-octanol as model substrate. 47.4% molar conversion of 2-octanol and 99.7% eep were obtained after a 5h reaction with Pp-CALB, and 90% of its original activity still remained after being reused for 10 cycles. Pp-CALB was then used to several sec-alcohols and it showed great enzymatic activity and enantioselectivity to all tested sec-alcohols, more than 93.1% of eep. The enantioselective characteristics of Pp-CALB catalysed sec-alcohols with different structures were compared with Novozyme 435 which was almost the same. Solvent free system as one way of green chemistry was applied to Pp-CALB and Pp-CALB showed great catalytic activity and enantioselectivity. Pp-CALB was potential biocatalyst of high enzymatic activity and enantioselectivity using in resolution of sec-alcohols.

Citrus essential oils (CEOs) are important flavors in the food and confectionary industries. A lipase process was proposed for enhancing the flavor profiles and increasing the proportions of esters in CEOs. The effects of the enzymatic process were explored by detecting the constituents of the CEOs of American sweet orange oil (ASO) and Brazil mandarin oil (BMO) through GC/MS and sensory evaluation by a trained panel, and positive effects were confirmed by both methods. A further eleven kinds of CEOs were treated via the lipase process and increments of 10 – 1170% were achieved in the proportions of esters, which were mostly ethyl esters. Enhancement in fruity odor, especially the top note, was demonstrated by all CEOs after enzymatic processing. All CEOs were tested for antimicrobial activities, and only ASO displayed fairly ideal antimicrobial activities. Meanwhile, modified ASO showed a certain increase in antimicrobial activities. This methodology might be considered a sustainable route for acquiring ‘natural’ essential oils with enhanced flavor profiles and simultaneously enhancing the comprehensive utilization of citrus fruits.

Nuclear receptor subfamily 4 group A member 1 (NR4A1), a downstream target of CREB that is a key regulator of epileptogenesis, has been implicated in a variety of biological processes and was previously identified as a seizure-associated molecule. However, the relationship between NR4A1 and epileptogenesis remains unclear. Here, we showed that NR4A1 protein was predominantly expressed in neurons and up-regulated in patients with epilepsy as well as pilocarpine-induced mouse epileptic models. NR4A1 knockdown by lentivirus transfection (lenti-shNR4A1) alleviated seizure severity and prolonged onset latency in mouse models. Moreover, reciprocal coimmunoprecipitation of NR4A1 and NR2B demonstrated their interaction. Furthermore, the expression of p-NR2B (Tyr1472) in epileptic mice and the expression of NR2B in the postsynaptic density (PSD) were significantly reduced in the lenti-shNR4A1 group, indicating that NR4A1 knockdown partly decreased surface NR2B by promoting NR2B internalization. These results are the first to indicate that the expression of NR4A1 in epileptic brain tissues may provide new insights into the molecular mechanisms underlying epilepsy.

Yeast cell-surface display is an efficient and cost-effective technology to produce recyclable whole-cell biocatalysts for numerous industrial applications. In this study, the optimized lipase 1 gene from Candida rugosa (CRL1) was displayed, for the first time, on the cell wall of Pichia pastoris using a truncated flocculation functional domain of Flo1p (FS) anchor. The hydrolytic activity of displayed CRL1 reached 7517 U/g under the control of the AOX1 promoter. Higher lipase activity, 19,771 U/g (an increase of 163%), was obtained by a five-copy CRL1 construct overexpressing the HAC1 factor. Flow cytometry and quantitative analysis demonstrated the CRL1 was successfully displayed on the yeast surface with high efficiency. Displayed CRL1 was characterized by the optimal temperature and pH at 50℃ and 8.5, respectively; the half-life was both 6 h under the optimal temperature and pH8.0. Displayed CRL1 also exhibited remarkable stability in the presence of different organic solvents, surfactants, and metal ions. A 3-L fed-batch fermentation confirmed the reproducibility of yeast-displayed CRL1 in large-scale production. Taken together, these findings provide a novel recyclable whole-cell biocatalyst, which not only had a high lipase activity but also with superior characteristics over free lipase, indicating great potential for further application.

Earlier studies on fructose laurate ester products have shown that recombinant Pichia pastoris displaying Candida antarctica lipase B (CALB) on the cell surface acts as an efficient whole-cell biocatalyst for sugar ester production from fructose and lauric acid in an organic solvent. The effects of various reaction factors, including solvent composition, substrate molar ratio, enzyme dose, temperature and water activity, on esterification catalyzed by the CALB-displaying P. pastoris whole-cell biocatalyst were examined in the present study. Under the preferred reaction conditions, specifically, 5 mL organic solvent mixture of 2-methyl-2-butanol/DMSO (20% v/v), 2 mmol fructose with a lauric acid to fructose molar ratio of 2:1, 0.3 g whole-cell biocatalyst (1,264 U/g dry cell) with an initial water activity of 0.11, 1.2 g 4A molecular sieve, reaction temperature of 55oC and 200 rpm stirring speed, the fructose mono laurate ester yield was 78% (w/w). The CALBdisplaying P. pastoris whole-cell biocatalyst exhibited good operational stability, with an evident increase, rather than decrease, in relative activity after the continuous recover and reuse cycle. The relative activity of the biocatalyst remained 50% higher than that of the first batch, even following reuse for 15 batches. Our results collectively indicate that the CALB-displaying P. pastoris whole-cell biocatalyst may be potentially utilized in lieu of free or immobilized enzyme to effectively produce non-ionic surfactants such as fatty acid sugar esters, offering the significant advantages of cost-effectiveness, good operational stability and mild reaction conditions.

Glycosylphosphatidylinositol (GPI)-anchored glycoproteins have diverse intrinsic functions in yeasts, and they also have different uses in vitro. The GPI-modified cell wall proteins GCW21, GCW51, and GCW61 of Pichia pastoris were chosen as anchoring proteins to construct co-expression strains in P. pastoris GS115. The hydrolytic activity and the amount of Candida antarctica lipase B (CALB) displayed on cell surface increased significantly following optimization of the fusion gene dosage and combination of the homogeneous or heterogeneous cell wall proteins. Maximum CALB hydrolytic activity was achieved at 4920 U/g dry cell weight in strain GS115/CALB-GCW (51 + 51 + 61 + 61) after 120 h of methanol induction. Changes in structural morphology and the properties of the cell surfaces caused by co-expression of fusion proteins were observed by transmission electron microscopy (TEM) and on plates containing cell-wall-destabilizing reagent. Our results suggested that both the outer and inner cell layers were significantly altered by overexpression of GPI-modified cell wall proteins. Interestingly, quantitative analysis of the inner layer components showed an increase in β-1,3-glucan, but no obvious changes in chitin in the strains overexpressing GPI-modified cell wall proteins.

Nuclear receptor subfamily 4 group A member 1 (NR4A1), a downstream target of CREB that is a key regulator of epileptogenesis, has been implicated in a variety of biological processes and was previously identified as a seizure-associated molecule. However, the relationship between NR4A1 and epileptogenesis remains unclear. Here, we showed that NR4A1 protein was predominantly expressed in neurons and up-regulated in patients with epilepsy as well as pilocarpine-induced mouse epileptic models. NR4A1 knockdown by lentivirus transfection (lenti-shNR4A1) alleviated seizure severity and prolonged onset latency in mouse models. Moreover, reciprocal coimmunoprecipitation of NR4A1 and NR2B demonstrated their interaction. Furthermore, the expression of p-NR2B (Tyr1472) in epileptic mice and the expression of NR2B in the postsynaptic density (PSD) were significantly reduced in the lenti-shNR4A1 group, indicating that NR4A1 knockdown partly decreased surface NR2B by promoting NR2B internalization. These results are the first to indicate that the expression of NR4A1 in epileptic brain tissues may provide new insights into the molecular mechanisms underlying epilepsy.

By predicting the potential signal peptides from proteins that are naturally secreted by Pichia pastoris, we identified three possible endogenous signal peptides: Scw, Dse and Exg. We compared their capability to mediate the secretion of enhanced green fluorescent protein (EGFP) and Candida antarctica lipase B (CALB) with that of the Saccharomyces cerevisiae α-factor prepro-signal. EGFP entered the secretory pathway of P. pastoris and was efficiently secreted into the culture medium by all three endogenous peptides. Further, these three putative endogenous signal peptides were also effective in secreting CALB. These endogenous signal peptides thus have the potential to mediate the efficient secretion of heterologous proteins in P. pastoris.

Cancer stem cells (CSCs), or tumor initiating cells, are a subpopulation of cancer cells with self-renewal and differentiation properties. However, there has been no direct observation of the properties of gastric CSCs in vitro. Here we describe a vincristine (VCR)-preconditioning approach to obtain cancer stem-like cells (CSLCs) from the gastric cancer cell line SGC7901. The CSLCs displayed mesenchymal characteristics, including the up-regulated mesenchymal markers Snail, Twist, and vimentin, and the down-regulated epithelial marker E-cadherin. Using a Matrigel-based differentiation assay, CSLCs formed 2D tube-like and 3D complex lumen-like structures, which resembled differentiated gastric crypts. The characteristic of cellular differentiation was also found by transmission electron microscopy and up-regulation of gastrointestinal genes CDX2 and SOX2. We further showed that CSLCs could self-renew through significant asymmetric division compared with parent cells by tracing PKH-26, BrdU, and EDU label-retaining cells. In addition, these CSLCs also increased expression of CD44, CD90, and CXCR4 at the mRNA level, which was identified as novel targets. Furthermore, drug sensitivity assays and xenograft experiments demonstrated that the cells developed multi-drug resistance (MDR) and significant tumorigenicity in vivo. In summary, gastric CSCs were identified from VCR-preconditioned SGC7901 cell line, characterized by high tumorigenicity and the capacity for self-renewal and differentiation.

Summary Grid cells and theta oscillations are fundamental components of the brain’s navigation system. Grid cells provide animals [ 1 , 2 ] and humans [ 3 , 4 ] with a spatial map of the environment by exhibiting multiple firing fields arranged in a regular grid of equilateral triangles. This unique firing pattern presumably constitutes the neural basis for path integration [ 5 , 6 , 7 , 8 ] and may also enable navigation in visual and conceptual spaces [ 9 , 10 , 11 , 12 ]. Theta frequency oscillations are a prominent mesoscopic network phenomenon during navigation in both rodents and humans [ 13 , 14 ] and encode movement speed [ 15 , 16 , 17 ], distance traveled [ 18 ], and proximity to spatial boundaries [ 19 ]. Whether theta oscillations may also carry a grid-like signal remains elusive, however. Capitalizing on previous fMRI studies revealing a macroscopic proxy of sum grid cell activity in human entorhinal cortex (EC) [ 20 , 21 , 22 ], we examined intracranial EEG recordings from the EC of epilepsy patients (n = 9) performing a virtual navigation task. We found that the power of theta oscillations (4–8 Hz) exhibits 6-fold rotational modulation by movement direction, reminiscent of grid cell-like representations detected using fMRI. Modulation of theta power was specific to 6-fold rotational symmetry and to the EC. Hexadirectional modulation of theta power by movement direction only emerged during fast movements, stabilized over the course of the experiment, and showed sensitivity to the environmental boundary. Our results suggest that oscillatory power in the theta frequency range carries an imprint of sum grid cell activity potentially enabled by a common grid orientation of neighboring grid cells [ 23 ].

To modify the Pichia pastoris cell surface, two classes of hydrophobins, SC3 from Schizophyllum commune and HFBI from Trichoderma reesei, were separately displayed on the cell wall. There was an observable increase in the hydrophobicity of recombinant strains. Candida antarctica lipase B (CALB) was then co-displayed on the modified cells, generating strains GS115/SC3-61/CALB-51 and GS115/HFBI-61/CALB-51. Interestingly, the hydrolytic and synthetic activities of strain GS115/HFBI-61/CALB-51 increased by 37% and 109%, respectively, but decreased by 26% and 43%, respectively, in strain GS115/SC3-61/CALB-51 compared with the hydrophobin-minus recombinant strain GS115/CALB-GCW51. The amount of glycerol by-product from the transesterification reaction adsorbed on the cell surface was significantly decreased following hydrophobin modification, removing the glycerol barrier and allowing substrates to access the active sites of lipases. Electron micrographs indicated that the cell wall structures of both recombinant strains appeared altered, including changes to the inner glucan layer and outer mannan layer. These results suggest that the display of hydrophobins can change the surface structure and hydrophobic properties of P. pastoris, and affect the catalytic activities of CALB displayed on the surface of P. pastoris cells.

Phytase is used as an animal feed additive that degrades phytic acid and reduces feeding costs and pollution caused by fecal excretion of phosphorus. Some phytases have been expressed in Pichia pastoris, among which the phytase from Citrobacter amalonaticus CGMCC 1696 had high specific activity (3548 U/mg). Improvement of the phytase expression level will contribute to facilitate its industrial applications. To improve the phytase expression, we use modification of P AOX1 and the α-factor signal peptide, increasing the gene copy number, and overexpressing HAC1 i to enhance folding and secretion of the protein in the endoplasmic reticulum. The genetic stability and fermentation in 10-L scaled-up fed-batch fermenter was performed to prepare for the industrial production. The phytase gene from C. amalonaticus CGMCC 1696 was cloned under the control of the AOX1 promoter (P AOX1 ) and expressed in P. pastoris. The phytase activity achieved was 414 U/mL. Modifications of P AOX1 and the α-factor signal peptide increased the phytase yield by 35 and 12 %, respectively. Next, on increasing the copy number of the Phy gene to six, the phytase yield was 141 % higher than in the strain containing only a single gene copy. Furthermore, on overexpression of HAC1 i (i indicating induced), a gene encoding Hac1p that regulates the unfolded protein response, the phytase yield achieved was 0.75 g/L with an activity of 2119 U/mL, 412 % higher than for the original strain. The plasmids in this high-phytase expression strain were stable during incubation at 30 °C in Yeast Extract Peptone Dextrose (YPD) Medium. In a 10-L scaled-up fed-batch fermenter, the phytase yield achieved was 9.58 g/L with an activity of 35,032 U/mL. The production of a secreted protein will reach its limit at a specific gene copy number where further increases in transcription and translation due to the higher abundance of gene copies will not enhance the secretion process any further. Enhancement of protein folding in the ER can alleviate bottlenecks in the folding and secretion pathways during the overexpression of heterologous proteins in P. pastoris. Using modification of P AOX1 and the α-factor signal peptide, increasing the gene copy number, and overexpressing HAC1 i to enhance folding and secretion of the protein in the endoplasmic reticulum, we have successfully increased the phytase yield 412 % relative to the original strain. In a 10-L fed-batch fermenter, the phytase yield achieved was 9.58 g/L with an activity of 35,032 U/mL. Large-scale production of phytase can be applied towards different biocatalytic and feed additive applications.

N-acetyl glutamate kinase (NAGK) is a key enzyme in the synthesis of L-arginine, and L-arginine-sensitive NAGK typically has hexameric architecture. Defining the relationship between this architecture and L-arginine inhibition can provide a foundation to identify the key amino acids involved in the allosteric regulation network of L-arginine. In the present study, the key amino acids in the N-terminal helix (N-helix) of Corynebacterium glutamicum (Cg) NAGK required for hexamer formation were determined using structural homology modeling and site-directed mutagenesis. It was also verified that hexameric architecture is required for the positive cooperativity of inhibition by L-arginine and for efficient catalysis, but that it is not the determinant of inhibition by L-arginine. Monomeric mutants retained a similar sensitivity to L-arginine as the hexameric form, indicating that monomers contain an independent, sensitive signal transduction network of L-arginine to mediate allosteric regulation. Mutation studies of CgNAGKs also revealed that amino acid residues 18-23 of the N-helix are required for inhibition by L-arginine, and that E19 may be an essential amino acid influencing the apparent affinity of L-arginine. Collectively, these studies may illuminate the basic mechanism of metabolic homeostasis of C. glutamicum.

Laccases have been used for the decolorization and detoxification of synthetic dyes due to their ability to oxidize a wide variety of dyes with water as the sole byproduct. A putative laccase gene (LacTT) from Thermus thermophilus SG0.5JP17-16 was screened using the genome mining approach, and it was highly expressed in Pichia pastoris, yielding a high laccase activity of 6130 U/L in a 10-L fermentor. The LacTT open reading frame encoded a protein of 466 amino acid residues with four putative Cu-binding regions. The optimal pH of the recombinant LacTT was 4.5, 6.0, 7.5 and 8.0 with 2,2'-azino-bis(3-ethylbenzothazoline-6-sulfonic acid) (ABTS), syringaldazine (SGZ), guaiacol, and 2,6-dimethoxyphenol (2,6-DMP) as the substrate, respectively. The optimal temperature of LacTT was 90°C with guaiacol as the substrate. LacTT was highly stable at pH 4.0–11.0 and thermostable at 40°C–90°C, confirming that it is a pH-stable and thermostable laccase. Furthermore, LacTT also exhibited high tolerance to halides such as NaCl, NaBr and NaF, and decolorized 100%, 94%, 94% and 73% of Congo Red, Reactive Black B and Reactive Black WNN, and Remazol Brilliant Blue R, respectively. Interestingly, addition of high concentration of NaCl increased the RBBR decolorization efficiency of LacTT. These results suggest that LacTT is a good candidate for industrial applications such as dyestuff processing and degradation of dyes in textile wastewaters.

Phytase expressed and anchored on the cell surface of Pichia pastoris avoids the expensive and time-consuming steps of protein purification and separation. Furthermore, yeast cells with anchored phytase can be used as a whole-cell biocatalyst. In this study, the phytase gene of Citrobacter amalonaticus was fused with the Pichia pastoris glycosylphosphatidylinositol (GPI)-anchored glycoprotein homologue GCW61. Phytase exposed on the cell surface exhibits a high activity of 6413.5 U/g, with an optimal temperature of 60°C. In contrast to secreted phytase, which has an optimal pH of 5.0, phytase presented on the cell surface is characterized by an optimal pH of 3.0. Moreover, our data demonstrate that phytase anchored on the cell surface exhibits higher pH stability than its secreted counterpart. Interestingly, our in vitro digestion experiments demonstrate that phytase attached to the cell surface is a more efficient enzyme than secreted phytase.

Glycosylphosphatidylinositol (GPI)-anchored glycoproteins have various intrinsic functions in yeasts and different uses in vitro . In the present study, the genome of Pichia pastoris GS115 was screened for potential GPI-modified cell wall proteins. Fifty putative GPI-anchored proteins were selected on the basis of (i) the presence of a C-terminal GPI attachment signal sequence, (ii) the presence of an N-terminal signal sequence for secretion, and (iii) the absence of transmembrane domains in mature protein. The predicted GPI-anchored proteins were fused to an alpha-factor secretion signal as a substitute for their own N-terminal signal peptides and tagged with the chimeric reporters FLAG tag and mature Candida antarctica lipase B (CALB). The expression of fusion proteins on the cell surface of P. pastoris GS115 was determined by whole-cell flow cytometry and immunoblotting analysis of the cell wall extracts obtained by β-1,3-glucanase digestion. CALB displayed on the cell surface of P. pastoris GS115 with the predicted GPI-anchored proteins was examined on the basis of potential hydrolysis of p -nitrophenyl butyrate. Finally, 13 proteins were confirmed to be GPI-modified cell wall proteins in P. pastoris GS115, which can be used to display heterologous proteins on the yeast cell surface.

PURPOSE: To examine the effects of current shunt on rats with temporal lobe epilepsy and neocortex epilepsy. EXPERIMENTAL DESIGN: A kainic acid (KA)-induced model of temporal lobe seizure and a penicillin-induced model of neocortical partial seizure were used in this study. Rats of each model were randomly allocated into two groups: control and model groups. The model group was further divided into the KA or penicillin group, sham conduction group and conduction group. The current shunt was realized through the implantation of a customized conduction electrode. After surgery, electroencephalogram (EEG) was recorded for two hours for each rat under anesthesia. Subsequently, the rats were video monitored for 72 h to detect the occurrence of behavioral seizures upon awakening. The average number and duration of seizures on EEG and the number of behavioral seizures were measured. RESULTS: In KA model, the number of total EEG seizures in conduction group (9.57±2.46) was significantly less than that in sham conduction group (15.13±3.45) (p

The methylotrophic yeast Pichia pastoris is an attractive platform for a plethora of recombinant proteins. There is growing evidence that host cells producing recombinant proteins are exposed to a variety of cellular stresses resulting in the induction of the unfolded protein response (UPR) pathway. At present, there is only limited information about the cellular reactions of the host cells at the level of the proteome, especially with regard to recombinant protein secretion. Here we monitored xylanase A secretion from Bacillus halodurans C-125 (xynA) in P. pastoris, using strains containing different copy numbers of the gene encoding xylanase A and co-overexpressing the gene encoding the UPR-regulating transcription factor HAC1 by applying a quantitative proteomics approach (iTRAQ-LC–MS/MS). Many important cellular processes, including carbon metabolism, stress response and protein folding are affected in the investigated conditions. Notably, the analysis revealed that strong over-expression of xynA can efficiently improve protein production but simultaneously cause an unfolded protein burden with a subsequent induction of the UPR. This limits the further improvement of protein production levels. Remarkably, constitutive expression of the gene encoding HAC1 lessens the unfolded protein burden by attenuating protein synthesis and increasing ER protein folding efficiency which is beneficial for protein secretion. Biological significance Pichia pastoris expression systems have been successfully used for over 20 years in basic research and in the biotechnology industry for the production and secretion of a wide range of recombinant proteins. In particular, secretion of recombinant proteins is still one of the main reasons for using P. pastoris. It has become obvious that many protein products can lead to severe stress on the host cell when being over-expressed, thus limiting the potential yield. Detailed understanding of the physiological responses to such stresses gives rise to engineering of host cells that can better cope with the stress factors. Therefore, the regulatory mechanism of heterologous protein secretion by quantitative mass-spectrometry (MS) proteomics is a growing field and an important endeavor in improving protein annotation. Many important cellular processes, including carbon and amino acid metabolism, stress response and protein folding are affected in the over-expression strains. This data represent a first step towards a systems wide approach to assess the response with recombinant protein induced stress in P. pastoris.

Background The methylotrophic yeast Pichia pastoris is widely used as a bioengineering platform for producing industrial and biopharmaceutical proteins, studying protein expression and secretion mechanisms, and analyzing metabolite synthesis and peroxisome biogenesis. With the development of DNA microarray and mRNA sequence technology, the P. pastoris transcriptome has become a research hotspot due to its powerful capability to identify the transcript structures and gain insights into the transcriptional regulation model of cells under protein production conditions. The study of the P. pastoris transcriptome helps to annotate the P. pastoris transcript structures and provide useful information for further improvement of the production of recombinant proteins. Results We used a massively parallel mRNA sequencing platform (RNA-Seq), based on next-generation sequencing technology, to map and quantify the dynamic transcriptome of P. pastoris at the genome scale under growth conditions with glycerol and methanol as substrates. The results describe the transcription landscape at the whole-genome level and provide annotated transcript structures, including untranslated regions (UTRs), alternative splicing (AS) events, novel transcripts, new exons, alternative upstream initiation codons (uATGs), and upstream open reading frames (uORFs). Internal ribosome entry sites (IRESes) were first identified within the UTRs of genes from P. pastoris, encoding kinases and the proteins involved in the control of growth. We also provide a transcriptional regulation model for P. pastoris grown on different carbon sources. Conclusions We suggest that the IRES-dependent translation initiation mechanism also exists in P. pastoris. Retained introns (RIs) are determined as the main AS event and are produced predominantly by an intron definition (ID) mechanism. Our results describe the metabolic characteristics of P. pastoris with heterologous protein production under methanol induction and provide rich information for further in-depth studies of P. pastoris protein expression and secretion mechanisms.

The imitation of translation, as mediated by internal ribosome entry sites, has not yet been reported in Pichia pastoris. An IRES element from Saccharomyces cerevisiae was demonstrated to direct the translation of a dicistronic mRNA in P. pastoris. The 5′-untranslated region of GPR1 mRNA, termed GPR, was cloned into a dual reporter construct containing an upstream Rhizomucor miehei lipase (RML) and a downstream β-galactosidase gene (lacZ) from Escherichia coli BL21. After being transformed into P. pastoris, the RML gene and lacZ were simultaneously expressed. The possibility of DNA rearrangement, spurious splicing, or cryptic promoter in the GPR sequence were eliminated, indicating that expression of a second ORF was IRES-dependent. These findings strongly suggested that the IRES-dependent translation initiation mechanism is conserved in P. pastoris and provides a useful means to express multiple genes simultaneously.

RELA and FOS are targets of miR-7 in gastric cancer cells and the miR-7/IKKε/RELA reciprocal feedback loop is important for gastric cancer induced by H. pylori infection., MicroRNAs play essential roles in gene expression regulation during carcinogenesis. Here, we investigated the role of miR-7 and the mechanism by which it is dysregulated in gastric cancer (GC). We used genome-wide screenings and identified RELA and FOS as novel targets of miR-7. Overexpression of miR-7 repressed RELA and FOS expression and prevented GC cell proliferation and tumorigenesis. These effects were clinically relevant, as low miR-7 expression was correlated with high RELA and FOS expression and poor survival in GC patients. Intriguingly, we found that miR-7 indirectly regulated RELA activation by targeting the IκB kinase IKKε. Furthermore, IKKε and RELA can repress miR-7 transcription, which forms a feedback circuit between miR-7 and nuclear factor κB (NF-κB) signaling. Additionally, we demonstrate that down-regulation of miR-7 may occur as a result of the aberrant activation of NF-κB signaling by Helicobacter pylori infection. These findings suggest that miR-7 may serve as an important regulator in GC development and progression.