Dataset Information

Enhanced Expression of Pullulanase in Bacillus subtilis by New Strong Promoters Mined From Transcriptome Data, Both Alone and in Combination.

ABSTRACT: Pullulanase plays an important role as a starch hydrolysis enzyme in the production of bio-fuels and animal feed, and in the food industry. Compared to the methods currently used for pullulanase production, synthesis by Bacillus subtilis would be safer and easier. However, the current yield of pullulanase from B. subtilis is low to meet industrial requirements. Therefore, it is necessary to improve the yield of pullulanase by B. subtilis. In this study, we mined 10 highly active promoters from B. subtilis based on transcriptome and bioinformatic data. Individual promoters and combinations of promoters were used to improve the yield of pullulanase in B. subtilis BS001. Four recombinant strains with new promoters (Phag, PtufA, PsodA, and PfusA) had higher enzyme activity than the control (PamyE). The strain containing PsodA+fusA (163 U/mL) and the strain containing PsodA+fusA+amyE (336 U/mL) had the highest activity among the analyzed dual- and triple-promoter construct stains in shake flask, which were 2.29 and 4.73 times higher than that of the strain with PamyE, respectively. Moreover, the activity of the strain containing PsodA+fusA+amyE showed a maximum activity of 1,555 U/mL, which was 21.9 times higher than that of the flask-grown PamyE strain in a 50-liter fermenter. Our work showed that these four strong promoters mined from transcriptome data and their combinations could reliably increase the yield of pullulanase in quantities suitable for industrial applications.

SUBMITTER: Meng F

PROVIDER: S-EPMC6224515 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Enhanced Expression of Pullulanase in Bacillus subtilis by New Strong Promoters Mined From Transcriptome Data, Both Alone and in Combination.

Meng Fanqiang F Zhu Xiaoyu X Nie Ting T Lu Fengxia F Bie Xiaomei X Lu Yingjian Y Trouth Frances F Lu Zhaoxin Z

Frontiers in microbiology 20181102

Pullulanase plays an important role as a starch hydrolysis enzyme in the production of bio-fuels and animal feed, and in the food industry. Compared to the methods currently used for pullulanase production, synthesis by Bacillus subtilis would be safer and easier. However, the current yield of pullulanase from B. subtilis is low to meet industrial requirements. Therefore, it is necessary to improve the yield of pullulanase by B. subtilis. In this study, we mined 10 highly ac ...[more]

PMID: 30450090

Similar Datasets

Project description:The physiological functions of two amylolytic enzymes, a maltogenic amylase (MAase) encoded by yvdF and a debranching enzyme (pullulanase) encoded by amyX, in the carbohydrate metabolism of Bacillus subtilis 168 were investigated using yvdF, amyX, and yvdF amyX mutant strains. An immunolocalization study revealed that YvdF was distributed on both sides of the cytoplasmic membrane and in the periplasm during vegetative growth but in the cytoplasm of prespores. Small carbohydrates such as maltoheptaose and beta-cyclodextrin (beta-CD) taken up by wild-type B. subtilis cells via two distinct transporters, the Mdx and Cyc ABC transporters, respectively, were hydrolyzed immediately to form smaller or linear maltodextrins. On the other hand, the yvdF mutant exhibited limited degradation of the substrates, indicating that, in the wild type, maltodextrins and beta-CD were hydrolyzed by MAase while being taken up by the bacterium. With glycogen and branched beta-CDs as substrates, pullulanase showed high-level specificity for the hydrolysis of the outer side chains of glycogen with three to five glucosyl residues. To investigate the roles of MAase and pullulanase in glycogen utilization, the following glycogen-overproducing strains were constructed: a glg mutant with a wild-type background, yvdF glg and amyX glg mutants, and a glg mutant with a double mutant (DM) background. The amyX glg and glg DM strains accumulated significantly larger amounts of glycogen than the glg mutant, while the yvdF glg strain accumulated an intermediate amount. Glycogen samples from the amyX glg and glg DM strains exhibited average molecular masses two and three times larger, respectively, than that of glycogen from the glg mutant. The results suggested that glycogen breakdown may be a sequential process that involves pullulanase and MAase, whereby pullulanase hydrolyzes the alpha-1,6-glycosidic linkage at the branch point to release a linear maltooligosaccharide that is then hydrolyzed into maltose and maltotriose by MAase.

Project description:Porcine circovirus type 2 (PCV2) is one of the most serious pathogens in pig herds worldwide. The Capsid protein (Cap), a structural protein of PCV2, is involved in the host's immune response; it induces neutralizing-antibody production and has good immunogenicity. The main PCV2 subtype currently prevalent in the Chinese pig herd is PCV2d. In this study, We constructed a recombinant Bacillus subtilis (B. subtilis) capable of secreting Cap protein, named pHT43-Cap/B. subtilis; we concentrated the supernatant of the recombinant bacteria and observed virus-like particles (VLPs) of PCV2d formed by Cap protein under transmission electron microscopy, named PCV2d-VLPs. The immunocompetence of the pHT43-Cap/B. subtilis and PCV2d-VLPs were then assessed by oral administration and by intramuscular injection into mice, respectively. The results showed that the levels of PCV2d-Cap protein-specific IgG in the serum and of PCV2d-Cap protein-specific sIgA in the small intestinal fluid of pHT43-Cap/B. subtilis immunized mice were elevated compared to the control group, both of them highly significant (p < 0.01), and the corresponding serum-specific IgG antibodies were effective in neutralizing PCV2d virulence. The virus load in the liver of the immunized mice was significantly lower than that in the control group (p < 0.01), as was the virus load in the spleen and lungs of the immunized mice (p < 0.05). In addition, the serum levels of PCV2d-Cap-specific IgG in mice immunized with PCV2d-VLPs by intramuscular injection were significantly elevated compared to the control group (p < 0.05), and the viral load in all tissues was significantly lower in immunized mice (p < 0.05). In conclusion, the recombinant bacterium pHT43-Cap/B. subtilis can induce effective mucosal and humoral immunity in mice, PCV2d-VLPs can induce humoral immunity in mice, and both vaccines have good immunogenicity; these results provide a theoretical and material basis for the development of a new vaccine against PCV2d.

Project description:BackgroundBacillus subtilis encounters a wide range of environmental pH. The bacteria maintain cytoplasmic pH within a narrow range. Response to acid stress is a poorly understood function of external pH and of permeant acids that conduct protons into the cytoplasm.Methods and principal findingsCytoplasmic acidification and the benzoate transcriptome were observed in Bacillus subtilis. Cytoplasmic pH was measured with 4-s time resolution using GFPmut3b fluorimetry. Rapid external acidification (pH 7.5 to 6.0) acidified the B. subtilis cytoplasm, followed by partial recovery. Benzoate addition up to 60 mM at external pH 7 depressed cytoplasmic pH but left a transmembrane Delta pH permitting growth; this robust adaptation to benzoate exceeds that seen in E. coli. Cytoplasmic pH was depressed by 0.3 units during growth with 30 mM benzoate. The transcriptome of benzoate-adapted cells was determined by comparing 4,095 gene expression indices following growth at pH 7, +/- 30 mM benzoate. 164 ORFs showed > or = 2-fold up-regulation by benzoate (30 mM benzoate/0 mM), and 102 ORFs showed > or = 2-fold down-regulation. 42% of benzoate-dependent genes are regulated up or down, respectively, at pH 6 versus pH 7; they are candidates for cytoplasmic pH response. Acid-stress genes up-regulated by benzoate included drug resistance genes (yhbI, yhcA, yuxJ, ywoGH); an oligopeptide transporter (opp); glycine catabolism (gcvPA-PB); acetate degradation (acsA); dehydrogenases (ald, fdhD, serA, yrhEFG, yjgCD); the TCA cycle (citZ, icd, mdh, sucD); and oxidative stress (OYE-family yqjM, ohrB). Base-stress genes down-regulated by benzoate included malate metabolism (maeN), sporulation control (spo0M, spo0E), and the SigW alkali shock regulon. Cytoplasmic pH could mediate alkali-shock induction of SigW.ConclusionsB. subtilis maintains partial pH homeostasis during growth, and withstands high concentrations of permeant acid stress, higher than for gram-negative neutralophile E. coli. The benzoate adaptation transcriptome substantially overlaps that of external acid, contributing to a cytoplasmic pH transcriptome.

Dataset Information

Enhanced Expression of Pullulanase in Bacillus subtilis by New Strong Promoters Mined From Transcriptome Data, Both Alone and in Combination.

Publications

Enhanced Expression of Pullulanase in <i>Bacillus subtilis</i> by New Strong Promoters Mined From Transcriptome Data, Both Alone and in Combination.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets