scholarly journals Development and Characterization of Acidic-pH Tolerant Mutants of Zymomonas mobilis through Adaptation and Next Generation Sequencing based Genome Resequencing and RNA-Seq

2020 ◽  
Author(s):  
Qing Yang ◽  
Yongfu Yang ◽  
Xia Wang ◽  
Yunhao Chen ◽  
Wei Shen ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Zymomonas Mobilis ◽  
Acidic Condition ◽  
Acidic Ph ◽  
Rna Seq ◽  
Genome Resequencing ◽  
Ph Tolerance ◽  
Mutant Strains ◽  
Ph Conditions ◽  
Generation Sequencing

Abstract Background: Acid pretreatment is a common strategy used to break down lignocellulosic biomass as substrate for biochemical production, which however generates inhibitory compounds and results in acidic pH condition. Although the natural ethanologenic bacterium Zymomonas mobilis can grow in a broad pH range, cell growth and ethanol fermentation are still affected at acidic pH conditions below pH 4.0.Results: In this study, adaptive laboratory evolution (ALE) strategy was applied to adapt Z. mobilis under acidic pH condition. Two mutant strains named 3.6M and 3.5M with enhanced acidic-pH tolerance were selected and confirmed. Mutant strains 3.6M and 3.5M exhibited 50~130% enhancement on growth rate, 4~9 h reduction on fermentation time, and 20~63% improvement on ethanol productivity than wild-type ZM4 at pH 3.8. Next-generation sequencing (NGS)-based whole genome resequencing (WGR) and RNA-Seq technologies were applied to unravel the acidic pH tolerance mechanism of mutant strains. WGR result indicated that mutations in four genes ZMO0421 (Ala67Thr), ZMO0712 (Gly539Asp), ZMO1432 (Pro480Leu), and ZMO1733 (Thr7Lys) with non-synonymous amino acid changes might be related with the acidic-pH tolerance. Additionally, RNA-Seq result showed that the upregulation of genes involved in glycolysis and the downregulation of mobility related genes would help generate and redistribute cellular energy to help resist acidic pH while keep normal biological processes in Z. mobilis . Moreover, genes involved in RND efflux pump, ATP-binding cassette (ABC) transporter, proton consumption, and alkaline metabolite production were significantly upregulated in mutants under acidic condition compared with ZM4, which could help maintain the pH homeostasis in mutant strains for low acidic-pH resistance. Furthermore, our results also demonstrated that genes related to branch amino acid biosynthesis from threonine to isoleucine were significantly upregulated in mutant 3.6M under acidic condition compared with ZM4, and genes encoding F 1 F 0 ATPase to pump excess protons out of cells were upregulated in mutant 3.6M under pH 3.8 compared with pH 6.2. These differences could help mutant 3.6M manage acidic condition better than ZM4. A few gene targets were then selected for genetics study to confirm their role on acidic pH tolerance, and our results demonstrated that the expression of two operons in the shuttle plasmids could help Z. mobilis tolerate acidic pH condition, which are ZMO0956-ZMO0958 encoding cytochrome bc1 complex and ZMO1428-ZMO1432 encoding RND efflux pump.Conclusion: Two acidic-pH tolerant mutants 3.6M and 3.5M obtained through this study especially 3.6M can be used as candidate strains for commercial bioethanol production under acidic fermentation conditions, and molecular mechanism of acidic pH tolerance of Z. mobilis \was further proposed, which can facilitate future research on rational design of synthetic microorganisms with enhanced tolerance against acidic pH conditions. In addition, the strategy developed in this study combining approaches of ALE, genome resequencing, RNA-Seq and classical genetics study for mutant evolution and characterization can be applied in other industrial microorganisms.

scholarly journals Development and Characterization of Acidic-pH Tolerant Mutants of Zymomonas mobilis through Adaptation and Next Generation Sequencing based Genome Resequencing and RNA-Seq

2020 ◽  
Author(s):  
Qing Yang ◽  
Yongfu Yang ◽  
Yin Tang ◽  
Xia Wang ◽  
Yunhao Chen ◽  
...  
Keyword(s):  
Efflux Pump ◽  
Acidic Ph ◽  
Rna Seq ◽  
Genome Resequencing ◽  
Ph Tolerance ◽  
Mutant Strains ◽  
Rnd Efflux Pump ◽  
Ph Conditions ◽  
Generation Sequencing ◽  
Better Than

Abstract Background: Acid pretreatment is a common strategy used to break down the hemicellulose component of the lignocellulosic biomass to release pentoses, and a subsequent enzymatic hydrolysis step is usually applied to release hexoses from the cellulose. The hydrolysate after pretreatment and enzymatic hydrolysis containing both hexoses and pentoses can then be used as substrates for biochemical production. However, the acid-pretreated liquor can also be directly used as the substrate for microbial fermentation, which has an acidic pH and contains inhibitory compounds generated during pretreatment. Although the natural ethanologenic bacterium Zymomonas mobilis can grow in a broad range of pH 3.5~7.5, cell growth and ethanol fermentation are still affected under acidic-pH conditions below pH 4.0. Results: In this study, adaptive laboratory evolution (ALE) strategy was applied to adapt Z. mobilis under acidic-pH conditions. Two mutant strains named 3.6M and 3.5M with enhanced acidic-pH tolerance were selected and confirmed, of which 3.5M grew better than ZM4 but worse than 3.6M in acidic-pH conditions that is served as a reference strain between 3.6M and ZM4 to help unravel the acidic-pH tolerance mechanism. Mutant strains 3.5M and 3.6M exhibited 50~130% enhancement on growth rate, 4~9 h reduction on fermentation time to consume glucose, and 20~63% improvement on ethanol productivity than wild-type ZM4 at pH 3.8. Next-generation sequencing (NGS)-based whole genome resequencing (WGR) and RNA-Seq technologies were applied to unravel the acidic-pH tolerance mechanism of mutant strains. WGR result indicated that compared to wild-type ZM4, 3.5M and 3.6M have seven and five single nucleotide polymorphisms (SNPs) respectively, among which four are shared in common. Additionally, RNA-Seq result showed that the upregulation of genes involved in glycolysis and the downregulation of flagellar and mobility related genes would help generate and redistribute cellular energy to resist acidic pH while keeping normal biological processes in Z. mobilis. Moreover, genes involved in RND efflux pump, ATP-binding cassette (ABC) transporter, proton consumption, and alkaline metabolite production were significantly upregulated in mutants under the acidic-pH condition compared with ZM4, which could help maintain the pH homeostasis in mutant strains for acidic-pH resistance. Furthermore, our results demonstrated that in mutant 3.6M, genes encoding F1F0 ATPase to pump excess protons out of cells were upregulated under pH 3.8 compared to pH 6.2. This difference might help mutant 3.6M manage acidic conditions better than ZM4 and 3.5M. A few gene targets were then selected for genetics study to explore their role on acidic-pH tolerance, and our results demonstrated that the expression of two operons in the shuttle plasmids, ZMO0956-ZMO0958 encoding cytochrome bc1 complex and ZMO1428-ZMO1432 encoding RND efflux pump, could help Z. mobilis tolerate acidic-pH conditions. Conclusion: An acidic-pH tolerant mutant 3.6M obtained through this study can be used for commercial bioethanol production under acidic fermentation conditions. In addition, the molecular mechanism of acidic-pH tolerance of Z. mobilis was further proposed, which can facilitate future research on rational design of synthetic microorganisms with enhanced tolerance against acidic-pH conditions. Moreover, the strategy developed in this study combining approaches of ALE, genome resequencing, RNA-Seq, and classical genetics study for mutant evolution and characterization can be applied in other industrial microorganisms.

scholarly journals Erratum: Multi-platform assessment of transcriptome profiling using RNA-seq in the ABRF next-generation sequencing study

2014 ◽  
Vol 32 (11) ◽  
pp. 1166-1166 ◽  
Author(s):  
Sheng Li ◽  
Scott W Tighe ◽  
Charles M Nicolet ◽  
Deborah Grove ◽  
Shawn Levy ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Transcriptome Profiling ◽  
Rna Seq ◽  
Next Generation ◽  
Generation Sequencing

scholarly journals Megakaryocytes differentially sort mRNAs for matrix metalloproteinases and their inhibitors into platelets: a mechanism for regulating synthetic events

Blood ◽  
2011 ◽  
Vol 118 (7) ◽  
pp. 1903-1911 ◽  
Author(s):  
Luca Cecchetti ◽  
Neal D. Tolley ◽  
Noemi Michetti ◽  
Loredana Bury ◽  
Andrew S. Weyrich ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Matrix Metalloproteinases ◽  
Mrna Expression ◽  
Vascular Remodeling ◽  
Differentially Express ◽  
Expression Patterns ◽  
Critical Role ◽  
Rna Seq ◽  
Next Generation ◽  
Generation Sequencing

Abstract Megakaryocytes transfer a diverse and functional transcriptome to platelets during the final stages of thrombopoiesis. In platelets, these transcripts reflect the expression of their corresponding proteins and, in some cases, serve as a template for translation. It is not known, however, if megakaryocytes differentially sort mRNAs into platelets. Given their critical role in vascular remodeling and inflammation, we determined whether megakaryocytes selectively dispense transcripts for matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) into platelets. Next-generation sequencing (RNA-Seq) revealed that megakaryocytes express mRNA for 10 of the 24 human MMP family members. mRNA for all of these MMPs are present in platelets with the exception of MMP-2, 14, and 15. Megakaryocytes and platelets also express mRNA for TIMPs 1-3, but not TIMP-4. mRNA expression patterns predicted the presence and, in most cases, the abundance of each corresponding protein. Nonetheless, exceptions were observed: MMP-2 protein is present in platelets but not its transcript. In contrast, quiescent platelets express TIMP-2 mRNA but only traces of TIMP-2 protein. In response to activating signals, however, platelets synthesize significant amounts of TIMP-2 protein. These results demonstrate that megakaryocytes differentially express mRNAs for MMPs and TIMPs and selectively transfer a subset of these into platelets. Among the platelet messages, TIMP-2 serves as a template for signal-dependent translation.

Immune functional portraits of head and neck cancer using next generation sequencing.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. 6561-6561
Author(s):  
Susan Raju Paul ◽  
Nikita Kotlov ◽  
Viktor Svekolkin ◽  
Felix Frenkel ◽  
Nava Almog ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Head And Neck ◽  
Functional State ◽  
Caspase 8 ◽  
Rna Seq ◽  
Type B ◽  
Tp53 Mutations ◽  
Immune Infiltration ◽  
Type C ◽  
Generation Sequencing

6561 Background: The addition of biomarkers as companion diagnostics and Next Generation Sequencing (NGS) have dramatically increased therapeutic efficacy and have aided precision medicine development. The unique genomic profile and tumor microenvironment (TME) composition of each patient can be ascertained through NGS. Using TCGA and Geo datasets, we characterized head and neck cancers (HNC) according to the cellular and functional state of their TME and conducted a pilot validation study using prospectively collected HNC tumors. Methods: To stratify the TME of HNC tumors into molecular functional portraits, we analyzed the sequencing data of 1,486 HNC tumor samples and 143 controls (normal, oral leukoplakia) from TCGA and GEO data sets. For the prospective pilot study, resected tissue from oropharyngeal carcinomas independent of HPV status were processed for whole exome (WES) and RNA-seq (n = 6; HPV-positive = 1). Results: To characterize the cellular composition and functional state of HNC tumors and their TMEs, we created 26 separate molecular signatures related to functional processes such as immune checkpoint inhibition, immune infiltration, immunosuppression, and stromal activities represented by angiogenesis and mesenchymal stromal cells. Unsupervised clustering of these signatures delineated tumors into 4 types: immune infiltration with increased stromal signatures (type A), immune infiltration with decreased stromal signatures (type B), no immune infiltration with increased stromal signature (type C), and no immune infiltration and decreased stromal signatures (type D). Most HPV-positive tumors were type B (p = 1e-27) and associated with increased survival compared to the HPV-negative tumors (types C and D; p = 3e-05). Type B HPV-positive tumors had reduced FAT1 and TP53 mutations, whereas type B HPV-negative tumors had increased caspase 8 mutations/loss. In the validation cohort, actionable mutations were found in PI3KCA and TSC2 in types A and B HPV-negative tumors. Moreover, while the HPV-positive tumor was classified as type C, we identified a caspase 8 homozygous deletion and absence of FAT1 and TP53 mutations, supporting the TCGA and GEO analysis. Conclusions: Exome and transcriptome analyses with cellular deconvolution from bulk RNA-seq enrich tumor characterization by including major TME components, providing a comprehensive biomarker profile for precision therapy and clinical decision making. Our prospective analysis identified TME parameters comparable with the large datasets and revealed targetable genomic alterations.

scholarly journals Connectivity Mapping for Candidate Therapeutics Identification Using Next Generation Sequencing RNA-Seq Data

PLoS ONE ◽  
2013 ◽  
Vol 8 (6) ◽  
pp. e66902 ◽  
Author(s):  
Darragh G. McArt ◽  
Philip D. Dunne ◽  
Jaine K. Blayney ◽  
Manuel Salto-Tellez ◽  
Sandra Van Schaeybroeck ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Rna Seq ◽  
Next Generation ◽  
Connectivity Mapping ◽  
Generation Sequencing

scholarly journals Next Generation Sequencing Identifies DNA Methylation Patterns Indicative of Disease Progression in Ph+ CML

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4526-4526
Author(s):  
Gerwin Heller ◽  
Thais Topakian ◽  
Corinna Altenberger ◽  
Sabine Cerny-Reiterer ◽  
Barbara Ziegler ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Disease Progression ◽  
Transcriptional Start Site ◽  
Start Site ◽  
Rna Seq ◽  
Next Generation ◽  
Neoplastic Cells ◽  
Number Of Genes ◽  
Methylation Patterns ◽  
Generation Sequencing

Abstract Ph+ chronic myeloid leukemia (CML) is a stem cell malignancy characterized by the BCR-ABL1 oncoprotein and leukemic expansion of myeloid progenitor cells. In the chronic phase (CP) of CML, clonal cells undergo myeloid differentiation and respond well to BCR-ABL1 inhibitors. In the accelerated phase (AP) and blast phase (BP) of CML, however, neoplastic cells are immature and often resistant against most BCR-ABL1-targeting drugs which is a challenge in clinical hematology. So far, little is known about molecular mechanisms underlying disease progression in CML. Methylation of CG sites around the transcriptional start site of various cancer-related genes including diverse tumor suppressor genes (referred to as methylation) is a frequently occurring epigenetic feature in neoplastic cells resulting in silencing of these genes. Although methylation is considered a critical factor in the pathogenesis of various malignant diseases including myeloid neoplasms, no comprehensive studies on the impact of methylation in the pathogenesis of CML have been conducted so far. We hypothesized that methylation may be an important mechanism regulating the transcriptional gene activity in CML cells during disease progression. Therefore, we investigated the methylome and the transcriptome of neoplastic cells in patients with CML in various phases of the disease (CP, n=15; AP, n=5; BP, n=7). Genome-wide methylation and gene expression patterns were analysed by next generation sequencing approaches using bone marrow (BM) or peripheral blood (PB) mononuclear cells (MNC) obtained from patients with CML and BM or PB MNC from control individuals. Methylation was analysed by reduced representation bisulfite sequencing (RRBS), and mRNA expression was determined by RNA-sequencing (RNA-seq). By comparing the methylome of patients who were initially diagnosed with CP-CML and who relapsed several months later (AP-CML, n=1; BC-CML, n=3), we identified a large number of genes which were methylated around their transcriptional start site in leukemic cells in patients at the time of progression compared to the time of CP-CML (range in the 4 patients: 423-1209 genes, adjusted p<0.05). These methylated genes were found to be less methylated or not methylated in BM or PB MNC of control individuals. When the methylome of all patients in all cohorts was examined and compared, more genes were found to be methylated in AP-CML and BC-CML compared to CP-CML (CP-CML, n=200; AP-CML, n=311; BC-CML, n=570). In addition, we identified several genes that were less methylated or not methylated in AP-CML and BC-CML cells compared to cells in CP-CML samples (range: 16-541 genes). Moreover, we analysed and compared the transcriptome of CP-CML, AP-CML and BC-CML samples and identified a large number of genes whose expression is downregulated in AP-CML and BC-CML samples compared to CP-CML (range: 187-382 genes). By correlating RRBS results and RNA-seq data, we found that expression of >100 of the methylated genes is downregulated in AP-CML/BC-CML compared to CP-CML suggesting that these genes may be regulated by methylation. Expression of the majority of these genes was detected in BM or PB MNC of control individuals. In silico pathway analyses and gene network analyses revealed that some methylated genes are involved in the regulation of apoptosis (e.g. CYP1B1, ZBTB16), negative regulation of cell proliferation (e.g. BTG3, VSX2) or regulation of Wnt signalling (e.g. SFRP1). Currently, a large number of CML patients are analysed gene-specifically for methylation by methylation-sensitive high resolution melting PCR and for expression of selected genes by RT-PCR in order to define prognostic patterns in CML. In summary, our results demonstrate that methylation changes are frequent events accompanying disease progression in patients with CML. These results may contribute to the identification of clinically relevant methylation patterns in CML and thus may improve prognostication. In addition, our data may reveal new epigenetic targets of therapy and may help to develop new treatment strategies for high risk or relapsing patients with CML. Disclosures Valent: Novartis: Honoraria; Pfizer: Honoraria; Ariad: Honoraria; BMS: Honoraria.

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