CRISPR/Cas9 genome editing for comparative genetic analysis related to soy sauce brewing in Aspergillus sojae industrial strains.

Aspergillus sojae has traditionally been used in soy sauce brewing. Genetic modification techniques have been established in A. sojae, but it is difficult to apply them to various industrial strains. Although we have previously developed a CRISPR/Cpf1 system for genetic modification of A. sojae, another genome editing system was required for versatile modification. In addition, repetitive genetic modification using the CRISPR system has not been established in A. sojae. In this study, we demonstrated mutagenesis, gene deletion/integration, and large deletion of a chromosomal region in A. sojae using the CRISPR/Cas9 system. We also successfully performed repetitive genetic modification using a method that involved forced recycling of genome-editing plasmids. Moreover, we demonstrated that the effects of genetic modification related to soy sauce brewing differed among A. sojae industrial strains. These results showed that our technique of using the CRISPR/Cas9 system is a powerful tool for genetic modification in A. sojae.

Comparison of taste characteristics between koji mold-ripened cheese and Camembert cheese using an electronic tongue system.

Koji mold, classified in the genus Aspergillus, is used to produce traditional Japanese fermented foods such as miso, soy sauce, and sake. In recent years, the application of koji mold to cheese ripening has attracted attention, and cheese surface-ripened with koji mold (koji cheese) has been studied. In this study, to evaluate the taste characteristics of koji cheese, an electronic tongue system was employed to measure the taste values of cheese samples ripened using 5 strains of koji mold in comparison with commercial Camembert cheese. All koji cheese samples exhibited lower sourness and greater bitterness, astringency, saltiness, and umami richness than the Camembert cheese samples. The intensity of each taste characteristic differed depending on the koji mold strain. These results indicate that koji cheese has a different taste value than conventional mold-ripened cheese. Furthermore, the results also indicate that various taste characteristics can be achieved by selecting different koji molds.

Structural basis for proteolytic processing of Aspergillus sojae α-glucosidase L with strong transglucosylation activity.

An α-glucosidase from Aspergillus sojae, AsojAgdL, exhibits strong transglucosylation activity to produce α-1,6-glucosidic linkages. The most remarkable structural feature of AsojAgdL is that residues 457-560 of AsojAgdL (designated the NC sequence) is not conserved in other glycoside hydrolase family 31 enzymes, and part of this NC sequence is proteolytically cleaved during its maturation. In this study, the enzyme was expressed in Pichia pastoris, and electrophoretic analysis indicated that the recombinant enzyme, rAsojAgdL, consisted of two polypeptide chains, as observed in the case of the enzyme produced in an Aspergillus strain. The crystal structure of rAsojAgdL was determined in complex with the substrate analog trehalose. Electron density corresponding to residues 496-515 of the NC sequence was not seen, and there were no α-helices or ß-strands except for a short α-helix in the structures of residues 457-495 and residues 516-560, both of which belong to the NC sequence. The residues 457-495 and the residues 516-560 both formed extra components of the catalytic domain. The residues 457-495 constituted the entrance of the catalytic pocket of rAsojAgdL, and Gly467, Asp468, Pro469, and Pro470 in the NC sequence were located within 4 Å of Trp400, a key residue involved in binding of the substrate. The results suggest that the proteolytic processing of the NC sequence is related to the formation of the catalytic pocket of AsojAgdL.

Adjunctive application of solid-state culture products and its freeze-dried powder from Aspergillus sojae for semi-hard cheese.

BACKGROUND: Species belonging to the genus Aspergillus have been used in traditional Japanese fermented foods. Aspergillus sojae is a species responsible for strong proteolytic activity. Freeze-drying treatments followed by physical disruption enables the pulverization of the mycelia of A. sojae RIB 1045 grown in whey protein-base solid media. Intracellular proteases were extracted using this protocol to compare extracellular protease activity in terms of the reaction's pH dependence in the presence or absence of inhibitors. RESULT: With different sensitivities to inhibitors, intracellular and extracellular proteases showed the strongest activity under acidic conditions, which were considered suitable for cheese application. The raw culture product (CP) and its freeze-dried product (FDP) were mixed with cheese curds, prepared according to Gouda-type cheese-making methods, and were allowed to ripen for 3 months. Chemical analysis of the products showed 13.3% water-soluble nitrogen (WSN) in the control, which had received noncultured media, whereas 20.0% and 21.1% WSN was found in the CP and FDP experimental cheeses, respectively. Although these adjuncts significantly increased WSN, an insignificant difference was found between CP and FDP. Free fatty acids in all experimental cheeses were similar, showing that CP and FDP caused no rancid defects. CONCLUSION: The introduction of freeze-drying treatments accompanied by cell disruption resulted in a negligible effect in terms of WSN. However, the application of A. sojae can be beneficial when it comes to increasing the level of WSN compared with A. oryzae, as shown in our previous study. © 2020 Society of Chemical Industry.

High-level expression, purification, and enzymatic characterization of a recombinant Aspergillus sojae alkaline protease in Pichia pastoris.

An alkaline protease (Ap) was cloned from Aspergillus sojae GIM3.33 via RT-PCR technique. A truncated Ap without the signal peptide was successfully expressed in the Pichia pastoris KM71 strain. The following describes the optimal process conditions for the recombinant engineering of a strain expressing a recombinant Ap (rAp) in a triangular flask: inoculum concentration OD600 value 20.0 in 40 mL working volume (in 500 mL flasks), methanol addition (1.0%; volume ratio), 0.02% biotin solution (60 µL), and YNB primary concentration (13.0 g/L). Under these conditions, the protease activity of rAp in the fermentation broth reached 400.4 ±â€¯40.5 U/mL after induction for three days. The rAp was isolated and purified, and its enzymatic characteristics were tested. Its optimal pH was 10.0, and it remained stable in a pH range of 7.0-10.0. Its optimal temperature was 45 °C and it retained >50% activity at 40 °C for 60 min. The rAp activity was significantly inhibited by PMSF, Zn2+ and Fe2+ and the rAp had a broad substrate specificity for natural proteins and synthetic peptide substrates, and preferred substrates at P1 position with large hydrophobic side-chain groups. Compared to Papain (8.7%) and Alcalase (12.2%), the degree of hydrolysis of rAp to soy protein isolate was 16.5%; therefore, rAp was a good candidate for the processing of food industry byproducts.

Development of enzyme technology for Aspergillus oryzae, A. sojae, and A. luchuensis, the national microorganisms of Japan.

This paper describes the modern enzymology in Japanese bioindustries. The invention of Takadiastase by Jokiti Takamine in 1894 has revolutionized the world of industrial enzyme production by fermentation. In 1949, a new γ-amylase (glucan 1,4-α-glucosidase, EC 3.2.1.3) from A. luchuensis (formerly designated as A. awamori), was found by Kitahara. RNase T1 (guanyloribonuclease, EC 3.1.27.3) was discovered by Sato and Egami. Ando discovered Aspergillus nuclease S1 (single-stranded nucleate endonuclease, EC 3.1.30.1). Aspergillopepsin I (EC 3.4.23.18) from A. tubingensis (formerly designated as A. saitoi) activates trypsinogen to trypsin. Shintani et al. demonstrated Asp76 of aspergillopepsin I as the binding site for the basic substrate, trypsinogen. The new oligosaccharide moieties Man10GlcNAc2 and Man11GlcNAc2 were identified with α-1,2-mannosidase (EC 3.2.1.113) from A. tubingensis. A yeast mutant compatible of producing Man5GlcNAc2 human compatible sugar chains on glycoproteins was constructed. The acid activation of protyrosinase from A. oryzae at pH 3.0 was resolved. The hyper-protein production system of glucoamylase was established in a submerged culture.

Effect of physicochemical parameters on the polygalacturonase of an Aspergillus sojae mutant using wheat bran, an agro-industrial waste, via solid-state fermentation.

BACKGROUND: Polygalacturonases (PGs) are valuable enzymes of the food industry; therefore it is of great importance to discover new and GRAS PG-producing microbial strains. In this study, PG enzyme produced from a high PG activity producer mutant Aspergillus sojae using wheat bran at the flask scale under pre-optimized conditions of solid-state fermentation (SSF) was biochemically characterized. RESULTS: The crude PG enzyme showed optimum activity in the pH range 4.0-5.0 and was stable in the pH range 3.0-7.0. The optimum temperature for the PG was 40 °C and it retained 99% of its activity at 50 °C. The mutant A. sojae PG could preserve more than 50% of its stability between 25 and 50 °C, both for 30 and 60 min, and was found to be stable in the presence of most of the tested compounds and metal ions. The inactivation energy (Ed ) was determined as 125.3 kJ mol(-1) . The enthalpy (ΔH*), free energy (ΔG*) and entropy (ΔS*) of inactivation were found to be stable with increasing temperature. CONCLUSION: The mutant A. sojae PG could be suitable for the clarification (depectinization) of orange and grape juices and wine. © 2015 Society of Chemical Industry.

Improved biomass and protein production in solid-state cultures of an Aspergillus sojae strain harboring the Vitreoscilla hemoglobin.

The biotechnological value of Aspergillus sojae ATCC 20235 (A. sojae) for production of pectinases in solid-state fermentation (SSF) has been demonstrated recently. However, a common drawback of fungal solid-state cultures is the poor diffusion of oxygen into the fungi that limits its growth and biological productivity. The bacterial Vitreoscilla hemoglobin (VHb) has favored the metabolism and productivities of various bacterial and yeast strains besides alleviating hypoxic conditions of its native host, but the use of VHb in filamentous fungi still remains poor explored. Based on the known effects of VHb, this study assessed its applicability to improve A. sojae performance in SSF. The VHb gene (vgb) under control of the constitutive Aspergillus nidulants gpdA promoter was introduced into the genome of A. sojae by Agrobacterium-mediated transformation. Successful fungal transformants were identified by fluorescence microscopy and polymerase chain reaction (PCR) analyses. In solid-state cultures, the content of protease, exo-polygalacturonase (exo-PG), and exo-polymethylgalacturonase (exo-PMG) of the transformed fungus (A. sojae vgb+) improved were 26, 60, and 44 % higher, respectively, in comparison to its parental strain (A. sojae wt). Similarly, biomass content was also 1.3 times higher in the transformant strain. No significant difference was observed in endo-polygalacturonase (endo-PG) content between both fungal strains, suggesting dissimilar effects of VHb towards different enzymatic productions. Overall, our results show that biomass, protease, and exo-pectinase content of A. sojae in SSF can be improved by transformation with VHb.

A novel pectin-degrading enzyme complex from Aspergillus sojae ATCC 20235 mutants.

BACKGROUND: In the food industry, the use of pectinase preparations with high pectin esterase (PE) activity leads to the release of methanol, which is strictly regulated in food products. Herein, a pectin-degrading enzyme (PDE) complex exhibiting low PE activity of three Aspergillus sojae ATCC 20235 mutants (M3, DH56 and Guserbiot 2.230) was investigated. Production of exo-/endo-polygalacturonase (PG), exo-polymethylgalacturonase (PMG) and pectin lyase (PL) by mutant M3 and A. sojae using two different carbon sources was evaluated in solid-state fermentation. Finally, experimental preparations obtained from the mutants and commercial pectinases standardized to the same potency were screened for PDEs. RESULTS: Mutant M3 grown on sugar beet was found to be the best producer of exo-PG, endo-PG, exo-PMG and PL, with maximum yields of 1111, 449, 130 and 123 U g(-1), respectively. All experimental preparations exhibited low PE activity, at least 21.5 times less than commercial pectinases, and higher endo-PG (40 U mL(-1)). CONCLUSION: Mutant M3 was the best PDE producer using sugar beet. Mutant strains presented a PDE complex featuring high endo-PG and very low PE activities. This novel complex with low de-esterifying activity can be exploited in the food industry to degrade pectin without releasing methanol.

Ultraviolet-enhanced detoxification of chromate and protection of Brassica napus by Aspergillus sojae SH 20.

The pervasive issue of heavy metal contamination in agricultural lands poses significant concerns and has wide-ranging implications for ecosystems. However, an encouraging solution lies in exploiting the potential of fungal endophytes to alleviate these detrimental effects. This study emphasized on improving the growth-promoting and chromium-alleviating capabilities of fungal endophytes, particularly Aspergillus sojae strain SH20, through ultraviolet (UV) irradiation. Following UV treatment, SH20 exhibited significantly enhanced growth-promoting and chromium-alleviating capabilities in comparison to its non-irradiated counterpart. Distinctly, the UV-treated SH20 strain demonstrated an improved ability to accumulate and reduce toxic chromate in the soil, effectively addressing the growth constraints imposed by elevated chromium levels in Brassica napus L. The UV-irradiated SH20 variant boosted shoot length up to 3 times that of the control. Similarly, this fungal strain displayed a remarkable increase in the total fresh weight of the seedlings, recording nearly 17 times greater than the control. The isolate treated with UV light reduced the absorption of chromium by about 3 times in the roots, helping the young plants to grow well even when exposed to chromate stress. A drop in root colonization by the UV-treated strain further resulted in reduced chromate absorption by the roots. Also, the strain showed great skill in boosting the host's antioxidant defenses by reducing the buildup of harmful reactive oxygen species (ROS), increasing the removal of ROS, and improving the plant's antioxidant levels, including phenols and flavonoids. When the host plants were exposed to 25 ppm of Cr stress, the UV-irradiated variant SH 20 stimulated the production of flavonoids (246 µg/ml) and phenols (952 µg/ml) in comparison to the control (with 220 µg/ml of flavonoids and 919 µg/ml of phenols). In conclusion, this report highlights how exposing the A. sojae strain SH20 to UV light has the potential to enhance its abilities to promote growth and bioremediate. This suggests a promising solution for addressing heavy metal contamination in agricultural lands.

The Molecular Identification and Antifungal Susceptibility of Clinical Isolates of Aspergillus Section Flavi from Three French Hospitals.

(1) Background: Aspergillus flavus is a cosmopolitan mold with medical, veterinary, and agronomic concerns. Its morphological similarity to other cryptic species of the Flavi section requires molecular identification techniques that are not routinely performed. For clinical isolates of Aspergillus section Flavi, we present the molecular identification, susceptibility to six antifungal agents, and clinical context of source patients. (2) Methods: One hundred forty fungal clinical isolates were included in the study. These isolates, recovered over a 15-year period (2001-2015), were identified based on their morphological characteristics as belonging to section Flavi. After the subculture, sequencing of a part of the ß-tubulin and calmodulin genes was performed, and resistance to azole antifungals was screened on agar plates containing itraconazole and voriconazole. Minimum inhibitory concentrations were determined for 120 isolates by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) broth microdilution method. (3) Results: Partial ß-tubulin and calmodulin sequences analysis showed that 138/140 isolates were A. flavus sensu stricto, 1 isolate was A. parasiticus/sojae, and 1 was A. nomiae. Many of the isolates came from samples collected in the context of respiratory tract colonization. Among probable or proven aspergillosis, respiratory infections were the most frequent, followed by ENT infections. Antifungal susceptibility testing was available for isolates (n = 120, all A. flavus ss) from one hospital. The MIC range (geometric mean MIC) in mg/L was 0.5-8 (0.77), 0.5-8 (1.03), 0.125-2 (0.25), 0.03-2 (0.22), 0.25-8 (1.91), and 0.03-0.125 (0.061) for voriconazole, isavuconazole, itraconazole, posaconazole, amphotericin B, and caspofungin, respectively. Two (1.67%) isolates showed resistance to isavuconazole according to current EUCAST breakpoints with MICs at 8 mg/L for isavuconazole and voriconazole. One of these two isolates was also resistant to itraconazole with MIC at 2 mg/L. (4) Conclusions: The present characterization of a large collection of Aspergillus belonging to the Flavi section confirmed that A. flavus ss is the predominant species. It is mainly implicated in respiratory and ENT infections. The emergence of resistance highlights the need to perform susceptibility tests on section Flavi isolates.

Are Current Aspergillus sojae Strains Originated from a Native Aflatoxigenic Aspergillus Species Population Also Present in California?

Aspergillus sojae has long been considered a domesticated strain of Aspergillus parasiticus. This study delineated relationships among the two species and an Aspergillus PWE36 isolate. Of 25 examined clustered aflatoxin genes of PWE36, 20 gene sequences were identical to those of A. sojae, but all had variations to those of A. parasiticus. Additionally, PWE36 developmental genes of conidiation and sclerotial formation, overall, shared higher degrees of nucleotide sequence identity with A. sojae genes than with A. parasiticus genes. Examination of defective cyclopiazonic acid gene clusters revealed that the PWE36 deletion pattern was identical only to those of A. sojae. Using A. sojae SMF134 genome sequence as a reference, visualization of locally collinear blocks indicated that PWE36 shared higher genome sequence homologies with A. sojae than with A. parasiticus. Phylogenetic inference based on genome-wide single nucleotide polymorphisms (SNPs) and total SNP counts showed that A. sojae strains formed a monophyletic clade and were clonal. Two (Argentinian and Ugandan) A. parasiticus isolates but not including an Ethiopian isolate formed a monophyletic clade, which showed that A. parasiticus population is genetically diverse and distant to A. sojae. PWE36 and A. sojae shared a most recent common ancestor (MRCA). The estimated divergence time for PWE36 and A. sojae was about 0.4 mya. Unlike Aspergillus oryzae, another koji mold that includes genetically diverse populations, the findings that current A. sojae strains formed a monophyletic group and shared the MRCA with PWE36 allow A. sojae to be continuously treated as a species for food safety reasons.

In vitro cosmeceutical activity of alcoholic extract from chestnut inner shell fermented with Aspergillus sojae.

Chestnut inner shell was fermented in solid state with Aspergillus sojae, and then extracted using ethanol (95%) to analyze its cosmeceutical activity and phenolic composition. The fermentation significantly increased the antioxidant activity, and in vitro cosmeceutical activities. The ethanol extract showed the higher activities than ethyl acetate and water extracts. DPPH radical scavenging activity of the alcoholic extract was 80.53%, and tyrosinase and elastase inhibition activities were 101.01%, and 76.73%, respectively, after 10 days of fermentation. Kojic acid, a secondary metabolite of A. sojae was produced by the fermentation as a major bioactive component. Gallic acid, ellagic acid, and coumaric acid appeared the major phenolic acids in the alcoholic extract from fermented chestnut inner shell. The alcoholic extract from chestnut inner shell fermented by A. sojae may be used as an effective and bioactive cosmeceutical. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-022-01044-9.

Solid-State Fermented Okara with Aspergillus spp. Improves Lipid Metabolism and High-Fat Diet Induced Obesity.

Okara is a major by-product of soymilk and tofu production. Despite retaining abundant nutrients after the process, okara is often under-utilized. In this study, solid-state fermentation (SSF) of okara was carried out using a koji starter (containing both Aspergillus oryzae and Aspergillus sojae) with the intention of releasing its untapped nutrients. Its effects on lipid metabolism in diet-induced obesity (DIO) were observed. The nutritional profile of fermented okara was elucidated using the following parameters: total phenolic content (TPC), pH, protein content, dietary fiber, amino acid content, and free sugar content. In vivo experiments were conducted using high-fat diets supplemented with unfermented okara and fermented okara over three weeks. Supplementation with fermented okara reduced body weight gain, adipose tissue weight, the serum triglyceride profile, and lipid accumulation in the liver, and altered the mRNA expression levels related to lipid metabolism; however, it did not affect pH and short-chain fatty acid (SCFA) production in this study. In conclusion, high-fat diets supplemented using okara fermented with Aspergillus spp. improved the lipid metabolism in mice, due to their high nutritional value, such as TPC, soy protein, and amino acids, and their synergistic effects without altering the gut microbiota.

Heterologous production of ascofuranone and ilicicolin A in Aspergillus sojae.

Ascofuranone and its precursor, ilicicolin A, are secondary metabolites with various pharmacological activities that are produced by Acremonium egyptiacum. In particular, ascofuranone strongly inhibits trypanosome alternative oxidase and represents a potential drug candidate against African trypanosomiasis. However, difficulties associated with industrial production of ascofuranone by A. egyptiacum, specifically the co-production of ascochlorin, which inhibits mammalian respiratory chain complex III at low concentrations, has precluded its widespread application. Therefore, in this study, ascofuranone biosynthetic genes (ascA-E and H-J) were heterologously expressed in Aspergillus sojae, which produced very low-levels of endogenous secondary metabolites under conventional culture conditions. As a result, although we obtained transformants producing both ilicicolin A and ascofuranone, they were produced only when an adequate concentration of chloride ions was added to the medium. In addition, we succeeded in increasing the production of ilicicolin A, by enhancing the expression of the rate-determining enzyme AscD, using a multi-copy integration system. The heterologous expression approach described here afforded the production of both ascofuranone and ilicicolin A, allowing for their development as therapeutics.

CRISPR/Cpf1-mediated mutagenesis and gene deletion in industrial filamentous fungi Aspergillus oryzae and Aspergillus sojae.

In industrial applications such as fermentation and heterologous protein production, various Aspergillus oryzae and A. sojae strains are used. Although genetic engineering techniques have been developed for these filamentous fungi, applying such classical techniques to many strains is difficult. Therefore, the establishment of innovative technologies applicable to various industrial strains is required. We previously developed a genome editing technology using the CRISPR/Cas9 system for the efficient genetic engineering of A. oryzae; however, this system is limited by its protospacer adjacent motif sequence. In A. sojae, no genetic engineering using genome editing has been developed. In this study, we aimed to develop a genome editing technology using the Cpf1 nuclease for the genetic engineering of A. oryzae and A. sojae. AMA1-based genome editing vectors bearing codon-optimized cpf1 expression cassettes were constructed, and guide RNA expression cassettes were inserted into the Cpf1 genome editing vectors. Using the resultant plasmids, we performed mutagenesis of the AowA and sC genes in A. oryzae and the AswA gene in A. sojae. We deleted these genes by co-introducing the Cpf1 genome editing plasmid and the donor plasmid. Our study demonstrates that the CRISPR/Cpf1 system can be used as an efficient alternative to the CRISPR/Cas9 system to genetically engineer A. oryzae and as a new approach for efficient genetic engineering of A. sojae.

High-level heterologous protein production using an attenuated selection marker in Aspergillus sojae.

Filamentous fungi, including Aspergillus sojae, are essential for the industrial production of enzymes. Although multi-copy introduction of a gene encoding the protein of interest is useful for increasing protein production, this method has not been established in the case of filamentous fungi. In this study, we aimed to establish an efficient system for multi-copy chromosomal integration and high-level expression of a heterologous gene in A. sojae using an attenuated selectable marker. Consequently, by truncating the promoter region of selectable markers, we efficiently introduced multiple copies of a heterologous gene and enhanced the rate of high-level protein-production in the strains. Since the multi-copy strains obtained in this study maintained high productivity even in a non-selective medium, this system could be applicable for industrial protein production.

Koji Molds for Japanese Soy Sauce Brewing: Characteristics and Key Enzymes.

Soy sauce is a traditional Japanese condiment produced from the fermentation of soybeans, wheat, and salt by three types of microorganisms, namely koji molds, halophilic lactic acid bacteria, and salt-tolerant yeast. The delicate balance between taste, aroma, and color contributes to the characteristic delicious flavor imparted by soy sauce. In soy sauce brewing, protein and starch of the raw materials are hydrolyzed into amino acids and sugars by enzymes derived from koji molds. These enzymatically hydrolyzed products not only directly contribute to the taste but are further metabolized by lactic acid bacteria and yeasts to most of organic acids and aromatic compounds, resulting in its distinctive flavor and aroma. The color of the soy sauce is also due to the chemical reactions between amino acids and sugars during fermentation. Therefore, koji mold, which produces various enzymes for the breakdown of raw materials, is an essential microorganism in soy sauce production and plays an essential role in fermenting the ingredients. In this review, we describe the manufacturing process of Japanese soy sauce, the characteristics of koji molds that are suitable for soy sauce brewing, and the key enzymes produced by koji molds and their roles in the degradation of materials during soy sauce fermentation, focusing on the production of umami taste in soy sauce brewing.

Whole genome analysis of Aspergillus sojae SMF 134 supports its merits as a starter for soybean fermentation.

Aspergillus sojae is a koji (starter) mold that has been applied for food fermentation in Asia. The whole genome of A. sojae SMF 134, which was isolated from meju (Korean soybean fermented brick), was analyzed at the genomic level to evaluate its potential as a starter for soybean fermentation. The genome size was 40.1 Mbp, which was expected to be composed of eight chromosomes with 13,748 ORFs. Strain SMF 134 had a total of 151 protease genes, among which two more leucine aminopeptidase (lap) genes were found in addition to the previously known lap 1, and three γ-glutamyltranspeptidase (ggt) genes were newly identified. Such genomic characteristics of SMF 134 with many protease and flavor-related (lap and ggt) genes support its merits as a starter for soybean fermentation. In addition, this first complete genome of A. sojae will allow for further genetic studies to better understand the production of various enzymes, including proteases, LAPs, and GGTs, as well as other characteristics as a starter mold for soybean fermentation.

Strain improvement of Aspergillus sojae for increased l-leucine aminopeptidase and protease production.

Conventional random mutagenesis was implemented to improve l-leucine aminopeptidase (LAP) and protease production in Aspergillus sojae. Through successive mutagenesis by ethyl methanesulfonate (EMS), UV, and 1-methyl-2-nitro-1-nitrosoguanidine (NTG), EMS25, EU36, and EUN13 mutants from each mutagenesis process were screened using a newly developed quick and easy screening method. The mutant EUN13 exhibited a 9.6-fold increase in LAP [50.61 ± 4.36 U/g-initial dried substrate (IDS)] and a 3.8-fold increase in protease production (13.36 ± 0.31 U/g-IDS) on solid-state fermentation. This mutant showed more frequent branching and higher lap1 mRNA expression as compared to the parent strain SMF 131, which at least in part contributed to the increased LAP and protease production. The mutant EUN13 can be used as a starter organism for diverse industrial soybean fermentation processes for the production of conventional products such as meju, doenjang, and ganjang as well as for the production of new fermented soybean-based sauces.