scholarly journals Comparative Analysis of Structural Variations Due to Genome Shuffling of Bacillus Subtilis VS15 for Improved Cellulase Production

2020 ◽  
Vol 21 (4) ◽  
pp. 1299 ◽  
Author(s):  
Soujanya Lakshmi Ega ◽  
Gene Drendel ◽  
Steve Petrovski ◽  
Eleonora Egidi ◽  
Ashley E. Franks ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Potassium Dichromate ◽  
Cellulase Activity ◽  
Genome Shuffling ◽  
Cellulase Production ◽  
Wild Type ◽  
Structural Variations ◽  
Renewable Biomass ◽  
Ethyl Methyl ◽  
A Genome

Cellulose is one of the most abundant and renewable biomass products used for the production of bioethanol. Cellulose can be efficiently hydrolyzed by Bacillus subtilis VS15, a strain isolate obtained from decomposing logs. A genome shuffling approach was implemented to improve the cellulase activity of Bacillus subtilis VS15. Mutant strains were created using ethyl methyl sulfonate (EMS), N-Methyl-N′ nitro-N-nitrosoguanidine (NTG), and ultraviolet light (UV) followed by recursive protoplast fusion. After two rounds of shuffling, the mutants Gb2, Gc8, and Gd7 were produced that had an increase in cellulase activity of 128%, 148%, and 167%, respectively, in comparison to the wild type VS15. The genetic diversity of the shuffled strain Gd7 and wild type VS15 was compared at whole genome level. Genomic-level comparisons identified a set of eight genes, consisting of cellulase and regulatory genes, of interest for further analyses. Various genes were identified with insertions and deletions that may be involved in improved celluase production in Gd7. Strain Gd7 maintained the capability of hydrolyzing wheatbran to glucose and converting glucose to ethanol by fermentation with Saccharomyces cerevisiae of the wild type VS17. This ability was further confirmed by the acidified potassium dichromate (K2Cr2O7) method.

scholarly journals Wild type Bacillus subtilis treated with ethyl methyl sulphonate produced catabolite insensitive mutants with improved cellulase production

2021 ◽  
Author(s):  
Oladipo Olaniyi
Keyword(s):  
Bacillus Subtilis ◽  
Carbon Source ◽  
Enzyme Production ◽  
Cellulase Activity ◽  
Cellulase Production ◽  
Minimal Salt Medium ◽  
Salt Medium ◽  
Production Medium ◽  
Wild Type ◽  
Ethyl Methyl

Abstract The goal of this present investigation was to mutagenize Bacillus subtilis with Ethyl Methyl Sulphonate (EMS), screen the mutants for cellulase production and evaluate the influence of different glucose concentrations on their cellulase production potentials. The wild type B. subtilis was treated with 20, 40, 60 and 80 µl of EMS and the mutants generated were screened for cellulase production in minimal salt medium containing carboxylmethylcellulose (CMC) as the carbon source. Quantitatively, cellulase activity and protein contents were determined by dinitrosalicylic acid and Lowry methods respectively. Seven mutants were developed from each of the EMS concentration bringing the total to twenty-eight from all the concentrations. Approximately 14 and 57% of the mutants developed from 40 and 60µl of EMS had higher cellulase activities than the wild type, while none of the mutants developed from 20 and 80 µl of EMS had better activities than the wild type. The supplementation of 0.2, 0.5, 1.0 and 1.5% glucose in enzyme production medium caused approximately 100, 14, 29 and 14% cellulase repression respectively in the mutants developed from 60µl EMS. Mutants MSSS02 and MSSS05 were considered as catabolite insensitive mutants because their cellulase production were enhanced in comparison to wild type.

scholarly journals Heterotrimeric G-Protein Signaling Is Required for Cellulose Degradation in Neurospora crassa

mBio ◽  
2020 ◽  
Vol 11 (6) ◽  
Author(s):  
Logan A. Collier ◽  
Arit Ghosh ◽  
Katherine A. Borkovich
Keyword(s):  
G Protein ◽  
Cellulose Degradation ◽  
Cellulase Activity ◽  
Cellulase Production ◽  
Camp Signaling ◽  
G Protein Signaling ◽  
Protein Signaling ◽  
Wild Type ◽  
Posttranscriptional Control ◽  
Heterotrimeric G Protein Signaling

ABSTRACT The filamentous fungus Neurospora crassa decomposes lignocellulosic biomass to generate soluble sugars as carbon sources. In this study, we investigated a role for heterotrimeric G-protein signaling in cellulose degradation. Loss of the Gα subunit genes gna-1 and gna-3, the Gβ subunit genes gnb-1 and cpc-2, the Gγ gene gng-1, or the gene for downstream effector adenylyl cyclase (cr-1) resulted in loss of detectable cellulase activity. This defect was also observed in strains expressing a constitutively active version of gna-3 (gna-3Q208L). We found that GNA-1 levels are greatly reduced in Δgna-3, Δgnb-1, and Δgng-1 strains, likely contributing to cellulase defects in these genetic backgrounds. The observation that gna-3Q208L Δgnb-1 strains exhibit cellulase activity, despite greatly reduced levels of GNA-1 protein, is consistent with positive control of cellulase production by GNA-3 that is manifested in the absence of gnb-1. Expression patterns for five cellulase genes showed that Δgna-1, Δgnb-1, and Δgna-3 mutants produce less cellulase mRNA than the wild type, consistent with transcriptional regulation. Δcpc-2 mutants had wild-type levels of cellulase transcripts, suggesting posttranscriptional control. In contrast, results for Δcr-1 mutants support both transcriptional and posttranscriptional control of cellulase activity by cAMP signaling. Cellulase activity defects in Δgna-3 mutants were fully remediated by cAMP supplementation, consistent with GNA-3 operating upstream of cAMP signaling. In contrast, cAMP addition only partially corrected cellulase activity defects in Δgna-1 and Δgnb-1 mutants, suggesting participation of GNA-1 and GNB-1 in additional cAMP-independent pathways that control cellulase activity. IMPORTANCE Filamentous fungi are critical for the recycling of plant litter in the biosphere by degrading lignocellulosic biomass into simpler compounds for metabolism. Both saprophytic and pathogenic fungi utilize plant cell wall-degrading enzymes to liberate carbon for metabolism. Several studies have demonstrated a role for cellulase enzymes during infection of economically relevant crops by fungal pathogens. Especially in developing countries, severe plant disease means loss of entire crops, sometimes leading to starvation. In this study, we demonstrate that G-protein signaling is a key component of cellulase production. Therefore, understanding the role of G-protein signaling in the regulation of the unique metabolism of cellulose by these organisms can inform innovations in strain engineering of industrially relevant species for biofuel production and in combatting food shortages caused by plant pathogens.

scholarly journals Identification of a Pathogenicity Locus, rpfA, in Erwinia carotovora subsp. carotovora That Encodes a Two-Component Sensor-Regulator Protein

1997 ◽  
Vol 10 (3) ◽  
pp. 407-415 ◽  
Author(s):  
Reid D. Frederick ◽  
Jiliang Chiu ◽  
Jeffrey L. Bennetzen ◽  
Autar K. Handa
Keyword(s):  
Xanthomonas Campestris ◽  
Genetic Locus ◽  
Cellulase Activity ◽  
Pectate Lyase ◽  
Erwinia Carotovora ◽  
Cellulase Production ◽  
Extracellular Protease ◽  
Wild Type ◽  
Reading Frame ◽  
Two Component

A mutant of Erwinia carotovora subsp. carotovora, AH2552, created by a Mud1 insertion was found to be reduced in plant pathogenicity and deficient in extracellular protease and cellulase activity, although it produced normal levels of pectate lyase and polygalacturonase. A cosmid clone, pEC462, was isolated from a wild-type E. carotovora subsp. carotovora DNA library that concomitantly restored pathogenicity and protease and cellulase activities of AH2552 to wild-type levels when present in trans. The genetic locus that was disrupted in AH2552 by insertion of Mud1 has been designated rpfA, for regulator of pathogenicity factors. Sequencing of the rpfA region identified an open reading frame of 2,787 bp, and the predicted 929-amino acid polypeptide shared high identity with several two-component sensor-regulator proteins: BarA from Escherichia coli, ApdA from Pseudomonas fluorescens, PheN from P. tolaasii, RepA from P. viridiflava, LemA from P. syringae pv. syringae, and RpfC from Xanthomonas campestris pv. campestris. The RpfA locus described in this study encodes a putative sensor kinase protein that is involved in both extracellular protease and cellulase production and the pathogenicity of E. carotovora subsp. carotovora on potato tubers.

scholarly journals Enhanced production of cellulase by Escherichia coli engineered with UV-mutated cellulase gene from Aspergillus niger UVMT-I

BioResources ◽  
2019 ◽  
Vol 14 (4) ◽  
pp. 9054-9063
Author(s):  
Qurat-ul-ain Daud ◽  
Zara Hamid ◽  
Tayyaba Sadiq ◽  
Asif Abbas ◽  
Shahzad Hussain Shah ◽  
...  
Keyword(s):  
Aspergillus Niger ◽  
Specific Activity ◽  
Cellulase Activity ◽  
Pcr Amplification ◽  
Type A ◽  
Cellulase Production ◽  
Cellulase Gene ◽  
Wild Type ◽  
E Coli ◽  
Enhanced Production

Enhanced cellulase production was studied with ultraviolet mutagenesis and the mutated cellulase gene in E. coli DH5α was cloned for production under controlled conditions. Aspergillus niger inoculum was exposed to UV radiation for different time intervals. The UV exposure of 10 min to A. niger yielded 330 μmol/min/mg specific activity. The mRNA of mutant A. niger yielding maximum enzyme activity was isolated and used for the synthesis of cDNA. The cDNA prepared from mRNA was used for the PCR amplification of mutated cellulase gene with primers designed on the basis of a cellulase gene database from A. niger. The amplified cellulase gene was cloned into E. coli DH5α followed by expression in E. coli BL21. The cellulase activity by wild type A. niger, A. niger UVMT-I, and recombinant E. coli was compared by analysis of variance test. The specific activity of cellulase by recombinant E. coli was maximum (441 μmol/min/mg), followed by A. niger UVMT-I (330 μmol/min/mg) and wild type A. niger (96 μmol/min/mg).

scholarly journals Chromosome-level genome assembly of a regenerable maize inbred line A188

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Guifang Lin ◽  
Cheng He ◽  
Jun Zheng ◽  
Dal-Hoe Koo ◽  
Ha Le ◽  
...  
Keyword(s):  
Inbred Line ◽  
Genome Assembly ◽  
Gene Function ◽  
Maize Inbred Line ◽  
Carotenoid Cleavage Dioxygenase ◽  
Structural Variations ◽  
Embryonic Callus ◽  
Network Analyses ◽  
A Genome ◽  
Chromosome Level

Abstract Background The maize inbred line A188 is an attractive model for elucidation of gene function and improvement due to its high embryogenic capacity and many contrasting traits to the first maize reference genome, B73, and other elite lines. The lack of a genome assembly of A188 limits its use as a model for functional studies. Results Here, we present a chromosome-level genome assembly of A188 using long reads and optical maps. Comparison of A188 with B73 using both whole-genome alignments and read depths from sequencing reads identify approximately 1.1 Gb of syntenic sequences as well as extensive structural variation, including a 1.8-Mb duplication containing the Gametophyte factor1 locus for unilateral cross-incompatibility, and six inversions of 0.7 Mb or greater. Increased copy number of carotenoid cleavage dioxygenase 1 (ccd1) in A188 is associated with elevated expression during seed development. High ccd1 expression in seeds together with low expression of yellow endosperm 1 (y1) reduces carotenoid accumulation, accounting for the white seed phenotype of A188. Furthermore, transcriptome and epigenome analyses reveal enhanced expression of defense pathways and altered DNA methylation patterns of the embryonic callus. Conclusions The A188 genome assembly provides a high-resolution sequence for a complex genome species and a foundational resource for analyses of genome variation and gene function in maize. The genome, in comparison to B73, contains extensive intra-species structural variations and other genetic differences. Expression and network analyses identify discrete profiles for embryonic callus and other tissues.

scholarly journals Synergistic Effect of Different Plant Cell Wall–Degrading Enzymes Is Important for Virulence of Fusarium graminearum

2017 ◽  
Vol 30 (11) ◽  
pp. 886-895 ◽  
Author(s):  
Maria Chiara Paccanaro ◽  
Luca Sella ◽  
Carla Castiglioni ◽  
Francesca Giacomello ◽  
Ana Lilia Martínez-Rocha ◽  
...  
Keyword(s):  
Cell Wall ◽  
Fusarium Graminearum ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Xylanase Activity ◽  
Cellulase Activity ◽  
Wild Type ◽  
Cell Wall Degrading Enzymes ◽  
Xylanase Production ◽  
Significant Difference

Endo-polygalacturonases (PGs) and xylanases have been shown to play an important role during pathogenesis of some fungal pathogens of dicot plants, while their role in monocot pathogens is less defined. Pg1 and xyr1 genes of the wheat pathogen Fusarium graminearum encode the main PG and the major regulator of xylanase production, respectively. Single- and double-disrupted mutants for these genes were obtained to assess their contribution to fungal infection. Compared with wild-type strain, the ∆pg mutant showed a nearly abolished PG activity, slight reduced virulence on soybean seedlings, but no significant difference in disease symptoms on wheat spikes; the ∆xyr mutant was strongly reduced in xylanase activity and moderately reduced in cellulase activity but was as virulent as wild type on both soybean and wheat plants. Consequently, the ΔpgΔxyr double mutant was impaired in xylanase, PG, and cellulase activities but, differently from single mutants, was significantly reduced in virulence on both plants. These findings demonstrate that the concurrent presence of PG, xylanase, and cellulase activities is necessary for full virulence. The observation that the uronides released from wheat cell wall after a F. graminearum PG treatment were largely increased by the fungal xylanases suggests that these enzymes act synergistically in deconstructing the plant cell wall.

scholarly journals Role of SpoVA Proteins in Release of Dipicolinic Acid during Germination of Bacillus subtilis Spores Triggered by Dodecylamine or Lysozyme

2006 ◽  
Vol 189 (5) ◽  
pp. 1565-1572 ◽  
Author(s):  
Venkata Ramana Vepachedu ◽  
Peter Setlow
Keyword(s):  
Bacillus Subtilis ◽  
Small Molecules ◽  
Spore Germination ◽  
Dipicolinic Acid ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Ph Optimum ◽  
Inner Membrane ◽  
Temperature Sensitive Mutants

ABSTRACT The release of dipicolinic acid (DPA) during the germination of Bacillus subtilis spores by the cationic surfactant dodecylamine exhibited a pH optimum of ∼9 and a temperature optimum of 60°C. DPA release during dodecylamine germination of B. subtilis spores with fourfold-elevated levels of the SpoVA proteins that have been suggested to be involved in the release of DPA during nutrient germination was about fourfold faster than DPA release during dodecylamine germination of wild-type spores and was inhibited by HgCl2. Spores carrying temperature-sensitive mutants in the spoVA operon were also temperature sensitive in DPA release during dodecylamine germination as well as in lysozyme germination of decoated spores. In addition to DPA, dodecylamine triggered the release of amounts of Ca2+ almost equivalent to those of DPA, and at least one other abundant spore small molecule, glutamic acid, was released in parallel with Ca2+ and DPA. These data indicate that (i) dodecylamine triggers spore germination by opening a channel in the inner membrane for Ca2+-DPA and other small molecules, (ii) this channel is composed at least in part of proteins, and (iii) SpoVA proteins are involved in the release of Ca2+-DPA and other small molecules during spore germination, perhaps by being a part of a channel in the spore's inner membrane.

scholarly journals Complete Genome Sequence of Bacillus subtilis subsp. subtilis Strain ∆6

2016 ◽  
Vol 4 (4) ◽  
Author(s):  
Daniel R. Reuß ◽  
Andrea Thürmer ◽  
Rolf Daniel ◽  
Wim J. Quax ◽  
Jörg Stülke
Keyword(s):  
Bacillus Subtilis ◽  
Genome Sequence ◽  
Complete Genome Sequence ◽  
Complete Genome ◽  
Full Genome Sequence ◽  
Subtilis Strain ◽  
Full Genome ◽  
Biotechnological Applications ◽  
A Genome ◽  
Subtilis Subsp

Bacillus subtilis ∆6 is a genome-reduced strain that was cured from six prophages and AT-rich islands. This strain is of great interest for biotechnological applications. Here, we announce the full-genome sequence of this strain. Interestingly, the conjugative element ICE Bs 1 has most likely undergone self-excision in B. subtilis ∆6.

Tetramer formation of Bacillus subtilis YabJ protein that belongs to YjgF/YER057c/UK114 family

2021 ◽  
Vol 85 (2) ◽  
pp. 297-306
Author(s):  
Zui Fujimoto ◽  
Le Thi Thu Hong ◽  
Naomi Kishine ◽  
Nobuhiro Suzuki ◽  
Keitarou Kimura
Keyword(s):  
Bacillus Subtilis ◽  
Quaternary Structure ◽  
Single Amino Acid ◽  
Wild Type ◽  
Amino Acid Mutation ◽  
X Ray Crystallography ◽  
Ligand Binding Sites ◽  
Potential Ligand ◽  
Single Amino Acid Mutation

ABSTRACT Bacillus subtilis YabJ protein belongs to the highly conserved YjgF/YER057c/UK114 family, which has a homotrimeric quaternary structure. The dominant allele of yabJ gene that is caused by a single amino acid mutation of Ser103Phe enables poly-γ-glutamic acid (γPGA) production of B. subtilis under conditions where the cell-density signal transduction was disturbed by the loss of DegQ function. X-ray crystallography of recombinant proteins revealed that unlike the homotrimeric wild-type YabJ, the mutant YabJ(Ser103Phe) had a homotetrameric quaternary structure, and the structural change appeared to be triggered by an inversion of the fifth β-strand. The YabJ homotetramer has a hole that is highly accessible, penetrating through the tetramer, and 2 surface concaves as potential ligand-binding sites. Western blot analyses revealed that the conformational change was also induced in vivo by the Ser103Phe mutation.

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