Here we describe a complex enzymatic approach to the efficient transformation of abundant waste chitin, a byproduct of the food industry, into valuable chitooligomers with a degree of polymerization (DP) ranging from 6 to 11. This method involves a three-step process: initial hydrolysis of chitin using engineered variants of a novel fungal chitinase from Talaromyces flavus to generate low-DP chitooligomers, followed by an extension to the desired DP using the high-yielding Y445N variant of β-N-acetylhexosaminidase from Aspergillus oryzae, achieving yields of up to 57%. Subsequently, enzymatic deacetylation of chitooligomers with DP 6 and 7 was accomplished using peptidoglycan deacetylase from Bacillus subtilis BsPdaC. The innovative enzymatic procedure demonstrates a sustainable and feasible route for converting waste chitin into unavailable bioactive chitooligomers potentially applicable as natural pesticides in ecological and sustainable agriculture.
- Klíčová slova
- chitin, chitinase, chitooligomer, peptidoglycan deacetylase, β-N-acetylhexosaminidase,
- MeSH
- Aspergillus oryzae * enzymologie genetika metabolismus MeSH
- Bacillus subtilis genetika enzymologie chemie metabolismus MeSH
- bakteriální proteiny genetika metabolismus chemie MeSH
- biokatalýza MeSH
- chitin * metabolismus chemie MeSH
- chitinasy * metabolismus genetika chemie MeSH
- fungální proteiny * metabolismus genetika chemie MeSH
- hydrolýza MeSH
- oligosacharidy * metabolismus chemie MeSH
- Talaromyces * enzymologie genetika chemie metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- bakteriální proteiny MeSH
- chitin * MeSH
- chitinasy * MeSH
- fungální proteiny * MeSH
- oligosacharidy * MeSH
CRISPR/Cas technology is a powerful tool for genome engineering in Aspergillus oryzae as an industrially important filamentous fungus. Previous study has reported the application of the CRISPR/Cpf1 system based on the Cpf1 (LbCpf1) from Lachnospiraceae bacterium in A. oryzae. However, multiplex gene editing have not been investigated using this system. Here, we presented a new CRISPR/Cpf1 multiplex gene editing system in A. oryzae, which contains the Cpf1 nuclease (FnCpf1) from Francisella tularensis subsp. novicida U112 and CRISPR-RNA expression cassette. The crRNA cassette consisted of direct repeats and guide sequences driven by the A. oryzae U6 promoter and U6 terminator. Using the constructed FnCpf1 gene editing system, the wA and pyrG genes were mutated successfully. Furthermore, simultaneous editing of wA and pyrG genes in A. oryzae was performed using two guide sequences targeting these gene loci in a single crRNA array. This promising CRISPR/Cpf1 genome-editing system provides a powerful tool for genetically engineering A. oryzae.
- Klíčová slova
- Aspergillus oryzae, CRISPR/Cpf1 system, Filamentous fungi, Multiplex gene editing,
- MeSH
- Aspergillus oryzae * genetika MeSH
- editace genu MeSH
- Francisella * MeSH
- vodící RNA, systémy CRISPR-Cas MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- vodící RNA, systémy CRISPR-Cas MeSH
S1 nuclease from Aspergillus oryzae is a single-strand-specific nuclease from the S1/P1 family that is utilized in biochemistry and biotechnology. S1 nuclease is active on both RNA and DNA but with differing catalytic efficiencies. This study clarifies its catalytic properties using a thorough comparison of differences in the binding of RNA and DNA in the active site of S1 nuclease based on X-ray structures, including two newly solved complexes of S1 nuclease with the products of RNA cleavage at atomic resolution. Conclusions derived from this comparison are valid for the whole S1/P1 nuclease family. For proper model building and refinement, multiple lattice-translocation defects present in the measured diffraction data needed to be solved. Two different approaches were tested and compared. Correction of the measured intensities proved to be superior to the use of the dislocation model of asymmetric units with partial occupancy of individual chains. As the crystals suffered from multiple lattice translocations, equations for their correction were derived de novo. The presented approach to the correction of multiple lattice-translocation defects may help to solve similar problems in the field of protein X-ray crystallography.
- Klíčová slova
- Aspergillus oryzae, S1 nuclease, complexes, lattice-translocation defects, nucleosides, nucleotides,
- MeSH
- Aspergillus oryzae * genetika metabolismus MeSH
- DNA MeSH
- endonukleasy chemie MeSH
- katalytická doména MeSH
- RNA * metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- DNA MeSH
- endonukleasy MeSH
- RNA * MeSH
Kojic acid is an industrially important secondary metabolite produced by Aspergillus oryzae. The construction of genetic materials for kojic acid related genes is important for understanding the mechanism of kojic acid synthesis in A. oryzae. However, multigene simultaneous knockout mutants for kojic acid synthesis genes remain limited because A. oryzae is multinuclear and good selectable markers are scarce. Here, we firstly successfully obtained single mutants of kojA, kojR, and kojT by our previously constructed CRISPR/Cas9 system in A. oryzae, which demonstrated the feasibility of the targeting sgRNAs for kojA, kojR, and kojT. Then, the AMA1-based genome-editing system for multiplex gene editing was developed in A. oryzae. In the multiplex gene-editing system, two guide RNA expression cassettes were ligated in tandem and driven by two U6 promoters in the AMA1-based autonomously replicating plasmid with the Cas9-expression cassette. Moreover, the multiplex gene-editing technique was applied to target the kojic acid synthesis genes kojA, kojR, and kojT, and the double and triple mutants within kojA, kojR, and kojT were obtained successfully. Additionally, the selectable marker pyrG was knocked out in the single and triple mutants of kojA, kojR, and kojT to obtain the auxotrophic strains, which can facilitate to introduce a target gene into the single and triple mutants of kojA, kojR, and kojT for investigating their relationship. The multiplex gene-editing system and release of these materials provide a foundation for further kojic acid research and utilization.
- MeSH
- Aspergillus oryzae * genetika metabolismus MeSH
- CRISPR-Cas systémy MeSH
- fungální proteiny genetika metabolismus MeSH
- pyrony metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- fungální proteiny MeSH
- kojic acid MeSH Prohlížeč
- pyrony MeSH
Fructosyltransferase (FTase) catalyzes the transfer of a fructosyl group to a sucrose molecule or a fructooligosaccharide (FOS) when a FOS with a longer chain is formed. Production of FTase by two Aspergillus species and its mixture was exploited using solid-state fermentation (SSF) and employing agave sap as substrate. The maximum FTase activity (1.59 U/mL) by Aspergillus oryzae was obtained after 24 h, using a temperature of 30 °C, with an inoculum of 2 × 107 spores/mL. The nucleotide sequence coding for the fructosyltransferase showed 1494 bp and encodes for a protein of 498 amino acids. The hypothetical molecular tertiary structure of Aspergillus oryzae BM-DIA FTase showed the presence of structural domains, such as a five-bladed beta-propeller domain characteristic of GH (glycoside hydrolase) and C terminal, which forms a beta-sandwich module. This study contributes to the knowledge of stability, compatibility, and genetic expression of Aspergillus oryzae BM-DIA under SSF bioprocess conditions for industrial production of fructosyltransferase.
- Klíčová slova
- Agave sap, Amino acids, Fructooligosaccharides, Nucleotide sequencing,
- MeSH
- Aspergillus oryzae * enzymologie genetika MeSH
- fermentace * MeSH
- hexosyltransferasy * biosyntéza chemie MeSH
- nukleotidy chemie MeSH
- proteiny chemie MeSH
- průmyslová mikrobiologie * metody MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- hexosyltransferasy * MeSH
- inulosucrase MeSH Prohlížeč
- nukleotidy MeSH
- proteiny MeSH
Aspergillus oryzae G15 was cultured on Czapek yeast extract agar medium containing different concentrations of copper and lead to investigate the mechanisms sustaining metal tolerance. The effects of heavy metals on biomass, metal accumulation, metallothionein (MT), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) were evaluated. Cu and Pb treatment remarkably delayed sclerotial maturation and inhibited mycelial growth, indicating the toxic effects of the metals. Cu decreased sclerotial biomass, whereas Pb led to an increase in sclerotial biomass. G15 bioadsorbed most Cu and Pb ions on the cell surface, revealing the involvement of the extracellular mechanism. Cu treatment significantly elevated MT level in mycelia, and Pb treatment at concentrations of 50-100 mg/L also caused an increase in MT content in mycelia. Both metals significantly increased MDA level in sclerotia. The variations in MT and MDA levels revealed the appearance of heavy metal-induced oxidative stress. The activities of SOD, CAT, and POD varied with heavy metal concentrations, which demonstrated that tolerance of G15 to Cu and Pb was associated with an efficient antioxidant defense system. In sum, the santioxidative detoxification system allowed the strain to survive in high concentrations of Cu and Pb. G15 depended mostly on sclerotial differentiation to defend against Pb stress.
- MeSH
- Aspergillus oryzae účinky léků genetika růst a vývoj metabolismus MeSH
- fungální proteiny genetika metabolismus MeSH
- katalasa genetika metabolismus MeSH
- malondialdehyd metabolismus MeSH
- měď farmakologie MeSH
- mycelium účinky léků genetika růst a vývoj metabolismus MeSH
- olovo farmakologie MeSH
- oxidační stres účinky léků MeSH
- superoxiddismutasa genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- fungální proteiny MeSH
- katalasa MeSH
- malondialdehyd MeSH
- měď MeSH
- olovo MeSH
- superoxiddismutasa MeSH
An expression plasmid containing the agdA gene encoding Aspergillus oryzae ZL-1 α-glucosidase was constructed and expressed in Pichia pastoris X-33. The molar mass of the purified protein was estimated by SDS-PAGE. HPLC analysis showed that the purified enzyme has a transglucosylating activity with maltose as substrate. The main component of the enzyme products was panose, while amounts of isomaltose and isomaltotriose were very low or absent. pH 5.2 and temperature of 37 °C were optimum for enzyme activity.
- MeSH
- alfa-glukosidasy chemie genetika metabolismus MeSH
- Aspergillus oryzae enzymologie genetika MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- exprese genu * MeSH
- genetické vektory MeSH
- glukany metabolismus MeSH
- isomaltosa metabolismus MeSH
- klonování DNA MeSH
- koncentrace vodíkových iontů MeSH
- maltosa metabolismus MeSH
- molekulární sekvence - údaje MeSH
- molekulová hmotnost MeSH
- Pichia enzymologie genetika metabolismus MeSH
- plazmidy MeSH
- rekombinantní proteiny chemie genetika metabolismus MeSH
- sekvence aminokyselin MeSH
- sekvenční seřazení MeSH
- stabilita enzymů MeSH
- teplota MeSH
- trisacharidy metabolismus MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- alfa-glukosidasy MeSH
- glukany MeSH
- isomaltosa MeSH
- isomaltotriose MeSH Prohlížeč
- maltosa MeSH
- panose MeSH Prohlížeč
- rekombinantní proteiny MeSH
- trisacharidy MeSH
Microdochium nivale carbohydrate oxidase was produced by heterologous recombinant expression in Aspergillus oryzae, purified and crystallized. The enzyme crystallizes with varying crystal morphologies depending on the crystallization conditions. Several different crystal forms were obtained using the hanging-drop vapour-diffusion method, two of which were used for diffraction measurements. Hexagon-shaped crystals (form I) diffracted to 2.66 A resolution, with unit-cell parameters a = b = 55.7, c = 610.4 A and apparent space group P6(2)22. Analysis of the data quality showed almost perfect twinning of the crystals. Attempts to solve the structure by molecular replacement did not give satisfactory results. Recently, clusters of rod-shaped crystals (form II) were grown in a solution containing PEG MME 550. These crystals belonged to the monoclinic system C2, with unit-cell parameters a = 132.9, b = 56.6, c = 86.5 A, beta = 95.7 degrees . Data sets were collected to a resolution of 2.4 A. The structure was solved by the molecular-replacement method. Model refinement is currently in progress.
- MeSH
- Aspergillus oryzae genetika MeSH
- časové faktory MeSH
- difrakce rentgenového záření MeSH
- galaktosa metabolismus MeSH
- genetické vektory MeSH
- izoelektrický bod MeSH
- karbohydrátdehydrogenasy chemie genetika izolace a purifikace metabolismus MeSH
- klonování DNA MeSH
- krystalizace MeSH
- krystalografie rentgenová MeSH
- molekulární sekvence - údaje MeSH
- molekulová hmotnost MeSH
- sběr dat MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie aminokyselin MeSH
- statistika jako téma MeSH
- substrátová specifita MeSH
- teplota MeSH
- Xylariales enzymologie genetika MeSH
- xylosa metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- galaktosa MeSH
- karbohydrátdehydrogenasy MeSH
- xylosa MeSH
