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Lab on a Chip Aug 2023Key to our ability to increase recombinant protein production through secretion is a better understanding of the pathways that interact to translate, process and export...
Key to our ability to increase recombinant protein production through secretion is a better understanding of the pathways that interact to translate, process and export mature proteins to the surrounding environment, including the supporting cellular machinery that supplies necessary energy and building blocks. By combining droplet microfluidic screening with large-scale CRISPR libraries that perturb the expression of the majority of coding and non-coding genes in , we identified 345 genes for which an increase or decrease in gene expression resulted in increased secretion of α-amylase. Our results show that modulating the expression of genes involved in the trafficking of vesicles, endosome to Golgi transport, the phagophore assembly site, the cell cycle and energy supply improve α-amylase secretion. Besides protein-coding genes, we also find multiple long non-coding RNAs enriched in the vicinity of genes associated with endosomal, Golgi and vacuolar processes. We validated our results by overexpressing or deleting selected genes, which resulted in significant improvements in α-amylase secretion. The advantages, in terms of precision and speed, inherent to CRISPR based perturbations, enables iterative testing of new strains for increased protein secretion.
Topics: Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Amylases; Microfluidics; alpha-Amylases
PubMed: 37483015
DOI: 10.1039/d3lc00111c -
FEMS Yeast Research Jul 2022The rapid expansion of the application of pharmaceutical proteins and industrial enzymes requires robust microbial workhorses for high protein production. The budding...
The rapid expansion of the application of pharmaceutical proteins and industrial enzymes requires robust microbial workhorses for high protein production. The budding yeast Saccharomyces cerevisiae is an attractive cell factory due to its ability to perform eukaryotic post-translational modifications and to secrete proteins. Many strategies have been used to engineer yeast platform strains for higher protein secretion capacity. Herein, we investigated a line of strains that have previously been selected after UV random mutagenesis for improved α-amylase secretion. A total of 42 amino acid altering point mutations identified in this strain line were reintroduced into the parental strain AAC to study their individual effects on protein secretion. These point mutations included missense mutations (amino acid substitution), nonsense mutations (stop codon generation), and frameshift mutations. For comparison, single gene deletions for the corresponding target genes were also performed in this study. A total of 11 point mutations and seven gene deletions were found to effectively improve α-amylase secretion. These targets were involved in several bioprocesses, including cellular stresses, protein degradation, transportation, mRNA processing and export, DNA replication, and repair, which indicates that the improved protein secretion capacity in the evolved strains is the result of the interaction of multiple intracellular processes. Our findings will contribute to the construction of novel cell factories for recombinant protein secretion.
Topics: Amylases; CRISPR-Cas Systems; Point Mutation; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; alpha-Amylases
PubMed: 35776981
DOI: 10.1093/femsyr/foac033 -
Scientific Reports Mar 2017α-Amylases are glycoside hydrolase enzymes that act on the α(1→4) glycosidic linkages in glycogen, starch, and related α-glucans, and are ubiquitously present in...
α-Amylases are glycoside hydrolase enzymes that act on the α(1→4) glycosidic linkages in glycogen, starch, and related α-glucans, and are ubiquitously present in Nature. Most α-amylases have been classified in glycoside hydrolase family 13 with a typical (β/α)-barrel containing two aspartic acid and one glutamic acid residue that play an essential role in catalysis. An atypical α-amylase (BmaN1) with only two of the three invariant catalytic residues present was isolated from Bacillus megaterium strain NL3, a bacterial isolate from a sea anemone of Kakaban landlocked marine lake, Derawan Island, Indonesia. In BmaN1 the third residue, the aspartic acid that acts as the transition state stabilizer, was replaced by a histidine. Three-dimensional structure modeling of the BmaN1 amino acid sequence confirmed the aberrant catalytic triad. Glucose and maltose were found as products of the action of the novel α-amylase on soluble starch, demonstrating that it is active in spite of the peculiar catalytic triad. This novel BmaN1 α-amylase is part of a group of α-amylases that all have this atypical catalytic triad, consisting of aspartic acid, glutamic acid and histidine. Phylogenetic analysis showed that this group of α-amylases comprises a new subfamily of the glycoside hydrolase family 13.
Topics: Bacillus megaterium; Bacterial Proteins; Catalysis; Starch; alpha-Amylases
PubMed: 28287181
DOI: 10.1038/srep44230 -
BMC Biotechnology Jun 2017Alpha amylase hydrolyzes α-bonds of polysaccharides such as starch and produces malto-oligosaccharides. Its starch saccharification applications make it an essential...
BACKGROUND
Alpha amylase hydrolyzes α-bonds of polysaccharides such as starch and produces malto-oligosaccharides. Its starch saccharification applications make it an essential enzyme in the textile, food and brewing industries. Commercially available α-amylase is mostly produced from Bacillus or Aspergillus. A hyper-thermostable and Ca independent α-amylase from Pyrococcus furiosus (PFA) expressed in E.coli forms insoluble inclusion bodies and thus is not feasible for industrial applications.
RESULTS
We expressed PFA in Nicotiana tabacum and found that plant-produced PFA forms functional aggregates with an accumulation level up to 3.4 g/kg FW (fresh weight) in field conditions. The aggregates are functional without requiring refolding and therefore have potential to be applied as homogenized plant tissue without extraction or purification. PFA can also be extracted from plant tissue upon dissolution in a mild reducing buffer containing SDS. Like the enzyme produced in P. furiosus and in E. coli, plant produced PFA preserves hyper-thermophilicity and hyper-thermostability and has a long shelf life when stored in lyophilized leaf tissue. With tobacco's large biomass and high yield, hyper-thermostable α-amylase was produced at a scale of 42 kg per hectare.
CONCLUSIONS
Tobacco may be a suitable bioreactor for industrial production of active hyperthermostable alpha amylase.
Topics: Cloning, Molecular; Enzyme Activation; Enzyme Stability; Plants, Genetically Modified; Protein Aggregates; Pyrococcus furiosus; Substrate Specificity; Temperature; Nicotiana; alpha-Amylases
PubMed: 28629346
DOI: 10.1186/s12896-017-0372-3 -
Scientific Reports Mar 2019Gene duplication is a source of genetic materials and evolutionary changes, and has been associated with gene family expansion. Functional divergence of duplicated genes...
Gene duplication is a source of genetic materials and evolutionary changes, and has been associated with gene family expansion. Functional divergence of duplicated genes is strongly directed by natural selections such as organism diversification and novel feature acquisition. We show that, plant α-amylase gene family (AMY) is comprised of six subfamilies (AMY1-AMY6) that fell into two ancient phylogenetic lineages (AMY3 and AMY4). Both AMY1 and AMY2 are grass-specific and share a single-copy ancestor, which is derived from grass AMY3 genes that have undergone massive tandem and whole-genome duplications during evolution. Ancestral features of AMY4 and AMY5/AMY6 genes have been retained among four green algal sequences (Chrein_08.g362450, Vocart_0021s0194, Dusali_0430s00012 and Monegl_16464), suggesting a gene duplication event following Chlorophyceae diversification. The observed horizontal gene transfers between plant and bacterial AMYs, and chromosomal locations of AMY3 and AMY4 genes in the most ancestral green body (C. reinhardtii), provide evidences for the monophyletic origin of plant AMYs. Despite subfamily-specific sequence divergence driven by natural selections, the active site and SBS1 are well-conserved across different AMY isoforms. The differentiated electrostatic potentials and hydrogen bands-forming residue polymorphisms, further imply variable digestive abilities for a broad substrates in particular tissues or subcellular localizations.
Topics: Evolution, Molecular; Gene Duplication; Gene Expression; Gene Ontology; Genes, Duplicate; Isoenzymes; Molecular Sequence Annotation; Multigene Family; Phylogeny; Plant Proteins; Selection, Genetic; Viridiplantae; alpha-Amylases
PubMed: 30894656
DOI: 10.1038/s41598-019-41420-w -
PloS One 2015Alpha-amylase is a very important enzyme in the starch conversion process. Most of the α-amylases are calcium-dependent and exhibit poor performance in the simultaneous...
Alpha-amylase is a very important enzyme in the starch conversion process. Most of the α-amylases are calcium-dependent and exhibit poor performance in the simultaneous saccharification and fermentation process of industrial bioethanol production that uses starch as feedstock. In this study, an extracellular amylolytic enzyme was purified from the culture broth of newly isolated Talaromyces pinophilus strain 1-95. The purified amylolytic enzyme, with an apparent molecular weight of 58 kDa on SDS-PAGE, hydrolyzed maltopentaose, maltohexaose, and maltoheptaose into mainly maltose and maltotriose and minor amount of glucose, confirming the endo-acting mode of the enzyme, and hence, was named Talaromyces pinophilus α-amylase (TpAA). TpAA was most active at pH 4.0-5.0 (with the temperature held at 37°C) and 55°C (at pH 5.0), and stable within the pH range of 5.0-9.5 (at 4°C) and below 45°C (at pH 5.0). Interestingly, the Ca2+ did not improve its enzymatic activity, optimal temperature, or thermostability of the enzyme, indicating that the TpAA was Ca2+-independent. TpAA displayed higher enzyme activity toward malto-oligosaccharides and dextrin than other previously reported α-amylases. This highly active Ca2+-independent α-amylase may have potential applications in starch-to-ethanol conversion process.
Topics: Amylose; Calcium; Chromatography, High Pressure Liquid; Electrophoresis, Polyacrylamide Gel; Enzyme Stability; Hydrogen-Ion Concentration; Hydrolysis; Ions; Kinetics; Molecular Sequence Data; Molecular Weight; Solubility; Solvents; Substrate Specificity; Talaromyces; Temperature; alpha-Amylases
PubMed: 25811759
DOI: 10.1371/journal.pone.0121531 -
Molecules (Basel, Switzerland) Apr 2019A series of symmetrical salicylaldehyde-bishydrazine azo molecules, -, have been synthesized, characterized by H-NMR and C-NMR, and evaluated for their in vitro...
A series of symmetrical salicylaldehyde-bishydrazine azo molecules, -, have been synthesized, characterized by H-NMR and C-NMR, and evaluated for their in vitro α-glucosidase and α-amylase inhibitory activities. All the synthesized compounds efficiently inhibited both enzymes. Compound was the most potent derivative in the series, and powerfully inhibited both α-glucosidase and α-amylase. The IC of against α-glucosidase was 0.35917 ± 0.0189 µM (standard acarbose IC = 6.109 ± 0.329 µM), and the IC value of against α-amylase was 0.4379 ± 0.0423 µM (standard acarbose IC = 33.178 ± 2.392 µM). The Lineweaver-Burk plot indicated that compound is a competitive inhibitor of α-glucosidase. The binding interactions of the most active analogues were confirmed through molecular docking studies. Docking studies showed that interacts with the residues Trp690, Asp548, Arg425, and Glu426, which form hydrogen bonds to with distances of 2.05, 2.20, 2.10 and 2.18 Å, respectively. All compounds showed high mutagenic and tumorigenic behaviors, and only showed irritant properties. In addition, all the derivatives showed good antioxidant activities. The pharmacokinetic evaluation also revealed promising results.
Topics: Animals; Glycoside Hydrolase Inhibitors; Molecular Docking Simulation; Molecular Structure; Swine; alpha-Amylases; alpha-Glucosidases
PubMed: 30999646
DOI: 10.3390/molecules24081511 -
Scientific Reports Feb 2020Butea monosperma is one of the extensively used plants in traditional system of medicines for many therapeutic purposes. In this study, the antioxidant activity,...
Butea monosperma is one of the extensively used plants in traditional system of medicines for many therapeutic purposes. In this study, the antioxidant activity, α-glucosidase and α-amylase inhibition properties of freeze drying assisted ultrasonicated leaf extracts (hydro-ethanolic) of B. monosperma have been investigated. The findings revealed that 60% ethanolic fraction exhibited high phenolic contents, total flavonoid contents, highest antioxidant activity, and promising α-glucosidase and α-amylase inhibitions. The UHPLC-QTOF-MS/MS analysis indicated the presence of notable metabolites of significant medicinal potential including apigenin, apigenin C-hexoside C-pentoside, apigenin C-hexoside C-hexoside, apigenin-6,8-di-C-pentoside and genistin etc., in B. monosperma leave extract. Docking studies were carried out to determine the possible role of each phytochemical present in leaf extract. Binding affinity data and interaction pattern of all the possible phytochemicals in leaf extract of B. monosperma revealed that they can inhibit α-amylase and α-glucosidase synergistically to prevent hyperglycemia.
Topics: Butea; Ethanol; Glycoside Hydrolase Inhibitors; Hypoglycemic Agents; Phytochemicals; Plant Extracts; Plant Leaves; alpha-Amylases; alpha-Glucosidases
PubMed: 32103043
DOI: 10.1038/s41598-020-60076-5 -
BioMed Research International 2018A novel cold-adapted and salt-tolerant -amylase gene (175) from Antarctic sea ice bacterium sp. M175 was successfully cloned and expressed. The open reading frame (ORF)...
A novel cold-adapted and salt-tolerant -amylase gene (175) from Antarctic sea ice bacterium sp. M175 was successfully cloned and expressed. The open reading frame (ORF) of 175 had 1722 bp encoding a protein of 573 amino acids residues. Multiple alignments indicated Amy175 had seven highly conserved sequences and the putative catalytic triad (Asp, Glu, and Asp). It was the first identified member of GH13_36 subfamily which contained QPDLN in the CSR V. The recombinant enzyme (Amy175) was purified to homogeneity with a molecular mass of about 62 kDa on SDS-PAGE. It had a mixed enzyme specificity of -amylase and -glucosidase. Amy175 displayed highest activity at pH 8.0 and 25°C and exhibited extreme salt-resistance with the maximum activity at 1 M NaCl. Amy175 was strongly stimulated by Mg, Ni, K, 1 mM Ca, 1 mM Ba, 1 mM Pb, 1 mM sodium dodecyl sulphate (SDS), and 10% dimethyl sulfoxide (DMSO) but was significantly inhibited by Cu, Mn, Hg, 10 mM -mercaptoethanol (-ME), and 10% Tween 80. Amy175 demonstrated excellent resistance towards all the tested commercial detergents, and wash performance analysis displayed that the addition of Amy175 improved the stain removal efficiency. This study demonstrated that Amy175 would be proposed as a novel -amylase source for industrial application in the future.
Topics: Amino Acid Sequence; Antarctic Regions; Cloning, Molecular; Detergents; Enzyme Stability; Hydrogen-Ion Concentration; Ice Cover; Pseudoalteromonas; alpha-Amylases
PubMed: 30050926
DOI: 10.1155/2018/3258383 -
PloS One 2016We previously reported that Aspergillus oryzae strain S2 had produced two α-amylase isoforms named AmyA and AmyB. The apparent molecular masses revealed by SDS-PAGE...
We previously reported that Aspergillus oryzae strain S2 had produced two α-amylase isoforms named AmyA and AmyB. The apparent molecular masses revealed by SDS-PAGE were 50 and 42 kDa, respectively. Yet AmyB has a higher catalytic efficiency. Based on a monitoring study of the α-amylase production in both the presence and absence of different protease inhibitors, a chymotrypsin proteolysis process was detected in vivo generating AmyB. A. oryzae S2 α-amylase gene was amplified, cloned and sequenced. The sequence analysis revealed nine exons, eight introns and an encoding open reading frame of 1500 bp corresponding to AmyA isoform. The amino-acid sequence analysis revealed aY371 potential chymotrypsin cleaving site, likely to be the AmyB C-Terminal end and two other potential sites at Y359, and F379. A zymogram with a high acrylamide concentration was used. It highlighted two other closed apparent molecular mass α-amylases termed AmyB1 and AmyB2 reaching40 kDa and 43 kDa. These isoforms could be possibly generated fromY359, and F379secondary cut, respectively. The molecular modeling study showed that AmyB preserved the (β/α)8 barrel domain and the domain B but lacked the C-terminal domain C. The contact map analysis and the docking studies strongly suggested a higher activity and substrate binding affinity for AmyB than AmyA which was previously experimentally exhibited. This could be explained by the easy catalytic cleft accessibility.
Topics: Amino Acid Sequence; Aspergillus oryzae; Molecular Docking Simulation; Proteolysis; Sequence Homology, Amino Acid; Substrate Specificity; alpha-Amylases
PubMed: 27101008
DOI: 10.1371/journal.pone.0153868