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Feasibility study of on-site solid-state enzyme production by Aspergillus oryzae.

ABSTRACT: Background:The development of biorefinery systems that use lignocellulosic biomass as a renewable carbon source to produce fuels and chemicals is attracting increasing attention. The process cost of enzymatic saccharification of biomass is a major challenge for commercialization. To decrease this cost, researchers have proposed on-site solid-state fermentation (SSF). This study investigated the feasibility of using Aspergillus oryzae as a host microorganism for SSF recombinant enzyme production with ammonia-treated rice straw as model biomass. Eight A. oryzae strains were tested, all of which are used in the food industry. We evaluated the effects of acetic acid, a fermentation inhibitor. We also developed a platform strain for targeted recombinant enzyme production by gene engineering technologies. Results:The SSF validation test showed variation in the visibility of mycelium growth and secreted protein in all eight A. oryzae strains. The strains used to produce shoyu and miso grew better under test conditions. The ammonia-treated rice straw contained noticeable amounts of acetic acid. This acetic acid enhanced the protein production by A. oryzae in a liquid-state fermentation test. The newly developed platform strain successfully secreted three foreign saccharifying enzymes. Conclusions:A. oryzae is a promising candidate as a host microorganism for on-site SSF recombinant enzyme production, which bodes well for the future development of a more cost-efficient saccharifying enzyme production system.

SUBMITTER: Shinkawa S

PROVIDER: S-EPMC7045521 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Feasibility study of on-site solid-state enzyme production by <i>Aspergillus oryzae</i>.

Shinkawa Satoru S Mitsuzawa Shigenobu S

Biotechnology for biofuels 20200226

<h4>Background</h4>The development of biorefinery systems that use lignocellulosic biomass as a renewable carbon source to produce fuels and chemicals is attracting increasing attention. The process cost of enzymatic saccharification of biomass is a major challenge for commercialization. To decrease this cost, researchers have proposed on-site solid-state fermentation (SSF). This study investigated the feasibility of using <i>Aspergillus oryzae</i> as a host microorganism for SSF recombinant enz ...[more]

PMID: 32127918

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Project description:Sake (rice wine) produced by multiple parallel fermentation (MPF) involving Aspergillus oryzae (strain RW) and Saccharomyces cerevisiae under solid-state cultivation conditions contained 3.5 mM agmatine, while that produced from enzymatically saccharified rice syrup by S. cerevisiae contained <0.01 mM agmatine. Agmatine was also produced in ethanol-free rice syrup prepared with A. oryzae under solid-state cultivation (3.1 mM) but not under submerged cultivation, demonstrating that A. oryzae in solid-state culture produces agmatine. The effect of cultivation conditions on agmatine production was examined. Agmatine production was boosted at 30°C and reached the highest level (6.3 mM) at pH 5.3. The addition of l-lactic, succinic, and citric acids reduced the initial culture pHs to 3.0, 3.5, and 3.2, respectively, resulting in a further increase in agmatine accumulation (8.2, 8.7, and 8.3 mM, respectively). Homogenate from a solid-state culture exhibited a maximum l-arginine decarboxylase (ADC) activity (74 pmol · min-1 · μg-1) at pH 3.0 at 30°C; homogenate from a submerged culture exhibited an extremely low activity (<0.3 pmol · min-1 · μg-1) under all conditions tested. These observations indicated that efficient agmatine production in ethanol-free rice syrup is achieved by an unidentified low-pH-dependent ADC induced during solid-state cultivation of A. oryzae, even though A. oryzae lacks ADC orthologs and instead possesses four ornithine decarboxylases (ODC1 to ODC4). Recombinant ODC1 and ODC2 exhibited no ADC activity at acidic pH (pH < 4.0), suggesting that other decarboxylases or an unidentified ADC is involved in agmatine production.IMPORTANCE It has been speculated that, in general, fungi do not synthesize agmatine from l-arginine because they do not possess genes encoding arginine decarboxylase. Numerous preclinical studies have shown that agmatine exerts pleiotropic effects on various molecular targets, leading to an improved quality of life. In the present study, we first demonstrated that l-arginine was a feasible substrate for agmatine production by the fungus Aspergillus oryzae RW. We observed that the productivity of agmatine by A. oryzae RW was elevated at low pH only during solid-state cultivation. A. oryzae is utilized in the production of various Asian fermented foods. The saccharification conditions optimized in the current study could be employed not only in the production of an agmatine-containing ethanol-free rice syrup but also in the production of many types of fermented foods, such as soy sauce (shoyu), rice vinegar, etc., as well as for use as novel therapeutic agents and nutraceuticals.

Project description:The production of petroleum-based plastics increased dramatically following industrialization. Because of multifaceted properties such as durability, thermostability, water resistance, and many others, these plastics have become an indispensable part of daily life. However, while improving people's quality of life, indiscriminate use of plastics has caused pollution and raised environmental concerns. To address this situation and reduce environmental risks, microbially produced biopolymers such as poly-3-hydroxyalkanoates can be used to make bioplastics that are completely biodegradable under normal environmental conditions. At the moment, the cost of bioplastic production is high when compared to petroleum-based plastics, so alternate strategies for making the bioplastic process economical are urgently needed. Agricultural waste is abundant around the world and can be efficiently used as a low-cost renewable feedstock after pretreatment and enzymatic hydrolysis. Fungi are well known as primary degraders of lignocellulosic waste, and this property was used in the current study to enzymatically hydrolyze the pretreated paddy straw for the production of reducing sugars, which were then used in the microbial fermentation for the production of PHB. In this study, Aspergillus nidulans was used to advance a low-cost and efficient enzyme hydrolysis system for the generation of reducing sugars from lignocellulosic biomass. For the production of the holocellulosic enzyme complex, the fungus was grown on wheat straw with Reese mineral medium as a wetting agent. After 216 h of solid-state fermentation at 30 °C, pH 6.0, the enzyme extract from A. nidulans demonstrated the highest activity, CMCase 68.58 (± 0.55), FPase 12.0 (± 0.06), Xylanase 27.17 (± 0.83), and β-glucosidase 1.89 (± 0.037). The initial pH, incubation temperature, and time all had a significant impact on final enzyme activity. Enzymatic hydrolysis of pretreated paddy straw produced reducing sugars (8.484 to 30.91 gL-1) that were then used to produce poly(3-hydroxybutyrate) using halophilic bacterial isolates. Burkholderia gladioli 2S4R1 and Bacillus cereus LB7 accumulated 26.80% and 20.47% PHB of the cell dry weight, respectively. This suggests that the holocellulosic enzyme cocktail could play a role in the enzymatic hydrolysis of lignocellulosic materials and the production of PHA from less expensive feedstocks such as agricultural waste.

Project description:Copper ligands of the recombinant tyrosinase from the fungus Aspergillus oryzae expressed in Saccharomyces cerevisiae or Escherichia coli were identified by site-directed mutagenesis. The recombinant protyrosinases expressed in S. cerevisiae were assayed for catalytic activities of mono-oxygenase and L-dopa oxidase at pH 5.5 after acid shock at pH 3.0. Replacements of His-63, His-84, His-93, His-290, His-294, His-332 or His-333 with asparagine resulted in mutant enzymes exhibiting no activities. The site-directed mutant Cys82Ala showed that Cys-82 was also an essential residue for the activity. We obtained homogeneous preparations of activated tyrosinases from mutated thioredoxin fusion gene products expressed in E. coli by acid shock. The copper contents of engineered mutants and wild-type enzyme expressed in E. coli were determined by atomic absorption spectrophotometry. The wild-type enzyme contained 2 g-atoms of copper/mol of the subunit. The His63Asn, His84Asn, His93Asn, His290Asn, His294Asn, His332Asn, His333Asn or Cys82Ala substitution decreased copper binding by approx. 50%, indicating that the mutants contain only approx. 1 g-atom of copper/mol of the subunit. The five mutants His63Asn, His93Asn, His290Asn, His294Asn and Cys82Ala contain only one copper ion, which is fully detectable by EPR. From the correlation of g( parallel) and (Cu)A( parallel), we deduced that the nitrogen or sulphur donors in the copper ligands should be in a square or a distorted tetrahedral geometric environment. In further atomic absorption spectrophotometry experiments, no copper atom was observed in the seven double mutants His63Asn/His290Asn, His63Asn/His294Asn, His63Asn/His332Asn, His63Asn/His333Asn, Cys82Ala/His290Asn, His84Asn/His333Asn and His93Asn/His290Asn. We propose a new structure of active sites of tyrosinase from A. oryzae: the most likely binding sites of tyrosinase for Cu(A) are His-63, His-84 and His-93, with the remaining conserved Cys-82 providing the fourth ligand. Cu(B) liganded by four histidine residues, His-290, His-294, His-332 and His-333, is identified as new binding motif of Cu(B).

Project description:Background:Monoclonal antibodies (mAbs) as biopharmaceuticals take a pivotal role in the current therapeutic applications. Generally mammalian cell lines, such as those derived from Chinese hamster ovaries (CHO), are used to produce the recombinant antibody. However, there are still concerns about the high cost and the risk of pathogenic contamination when using mammalian cells. Aspergillus oryzae, a filamentous fungus recognized as a GRAS (Generally Regarded As Safe) organism, has an ability to secrete a large amount of proteins into the culture supernatant, and thus the fungus has been used as one of the cost-effective microbial hosts for heterologous protein production. Pursuing this strategy the human anti-TNF? antibody adalimumab, one of the world's best-selling antibodies for the treatment of immune-mediated inflammatory diseases including rheumatoid arthritis, was chosen to produce the full length of mAbs by A. oryzae. Generally, N-glycosylation of the antibody affects immune effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) via binding to the Fc receptor (Fc?R) on immune cells. The CRISPR/Cas9 system was used to first delete the Aooch1 gene encoding a key enzyme for the hyper-mannosylation process in fungi to investigate the binding ability of antibody with Fc?RIIIa. Results:Adalimumab was expressed in A. oryzae by the fusion protein system with ?-amylase AmyB. The full-length adalimumab consisting of two heavy and two light chains was successfully produced in the culture supernatants. Among the producing strains, the highest amount of antibody was obtained from the ten-protease deletion strain (39.7 mg/L). Two-step purifications by Protein A and size-exclusion chromatography were applied to obtain the high purity sample for further analysis. The antigen-binding and TNF? neutralizing activities of the adalimumab produced by A. oryzae were comparable with those of a commercial product Humira®. No apparent binding with the Fc?RIIIa was detected with the recombinant adalimumab even by altering the N-glycan structure using the Aooch1 deletion strain, which suggests only a little additional activity of immune effector functions. Conclusion:These results demonstrated an alternative low-cost platform for human antibody production by using A. oryzae, possibly offering a reasonable expenditure for patient's welfare.

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Feasibility study of on-site solid-state enzyme production by Aspergillus oryzae.

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Feasibility study of on-site solid-state enzyme production by <i>Aspergillus oryzae</i>.

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