Generation of citric acid-hyperproducers independent of methanol effect by high-level expression of cexA encoding citrate exporter in Aspergillus tubingensis.

Methanol reportedly stimulates citric acid (CA) production by Aspergillus niger and A. tubingensis; however, the underlying mechanisms remain unclear. Here, we elucidated the molecular functions of the citrate exporter gene cexA in relation to CA production by A. tubingensis WU-2223L. Methanol addition to the medium containing glucose as a carbon source markedly increased CA production by strain WU-2223L by 3.38-fold, resulting in a maximum yield of 65.5 g/L, with enhanced cexA expression. Conversely, the cexA-complementing strain with the constitutive expression promoter Ptef1 (strain LhC-1) produced 68.3 or 66.7 g/L of CA when cultivated without or with methanol, respectively. Additionally, strain LhC-2 harboring two copies of the cexA expression cassette produced 80.7 g/L of CA without methanol addition. Overall, we showed that cexA is a target gene for methanol in CA hyperproduction by A. tubingensis WU-2223L. Based on these findings, methanol-independent CA-hyperproducing strains, LhC-1 and LhC-2, were successfully generated.

Specific and sensitive loop-mediated isothermal amplification (LAMP) method for Madurella strains, eumycetoma filamentous fungi causative agent.

BACKGROUND: Filamentous fungi of the genus Madurella are the primary causative agents of mycetoma, a disease observed in tropical and subtropical regions. Since early diagnostics based on a morphological approach are difficult and have many shortcomings, a molecular diagnostic method suitable for rural settings is required. In this study, we developed the loop-mediated isothermal amplification (LAMP) method to present a foundational technique of the diagnosis of Madurella spp. (M. mycetomatis, M. pseudomycetomatis, M. tropicana, and M. fahalii), the common causative organisms of eumycetoma. PRINCIPAL FINDINGS: We successfully designed a primer pair targeting the rDNAs of three Madurella spp. excluding M. fahalii, and detected up to 100 fg of genomic DNA extracted from isolates of M. mycetomatis and 1 pg of M. pseudomycetomatis and M. tropicana, within one hour. Second, a primer pair specific to M. mycetomatis, the most common causative species, or M. fahalii, a drug-resistant species, was constructed, and the detection limit of both primer pairs was 1 pg. The designed primers accurately distinguished 16 strains of the genus Madurella from various fungal species known to cause mycetomas. CONCLUSION: In summary, we established the first model of a LAMP detection method that rapidly and sensitively detects and identifies Madurella isolates for clinical diagnostics. Moreover, the combined designed primer sets could identify mycetoma-causing strains simultaneously.

Draft Genome Sequence of Aspergillus lacticoffeatus WU-2020, a Citric Acid Producer Suitable for Solid Culture That Belongs To Aspergillus Section Nigri.

Aspergillus lacticoffeatus WU-2020 is a citric acid hyperproducer that is suitable for solid culture. Here, we present a high-quality draft of its genome sequence (35.9 Mb), which consists of 11 scaffolds and contains 11,490 genes. We also present the mitochondrial genome, which is 31.3 kb in length.

Selective and high-yield production of ethyl α-d-glucopyranoside by the α-glucosyl transfer enzyme of Xanthomonas campestris WU-9701 and glucose isomerase.

Ethyl α-d-glucopyranoside (α-EG) is detected in sake (Japanese rice wine), that has moisturizing and skin conditioning effects. The production of α-EG by fermentation or enzymatic synthesis to date generates unwanted by-products such as maltooligosaccharides and/or organic acids. In this study, we employed a reaction involving selective α-glucosylation of ethanol by the α-glucosyl transfer enzyme (XgtA) of Xanthomonas campestris WU-9701. Under standard conditions, when 0.80 M ethanol and 1.2 M maltose were used as substrates with XgtA (2.5 units) and incubated in 30 mM HEPES-NaOH buffer (pH 8.0) at 45°C, only one form of ethyl glucopyranoside was selectively obtained as a product. The isolated product was identified as ethyl α-d-glucopyranoside by 1H NMR, 1H-1H COSY, and NOESY analyses. In the reaction mixture, other glucosylated products such as maltotriose and ethylmaltoside were not detected. Under optimum conditions, 180 mM (37.5 g/L) α-EG was produced in one batch production for 80 h. Further, the reaction rate of α-EG production decreased with an increase in glucose, especially more than 500 mM. In contrast, the addition of glucose isomerase decreased the concentration of glucose and was useful for maintaining a glucose concentration of less than 500 mM in the reaction mixture. Thus, owing to the enzymatic reaction with XgtA and glucose isomerase, as much as 260 mM (54.1 g/L) α-EG was produced in one batch production for 100 h. Altogether, this study reports the highest concentration of α-EG produced by enzymatic reaction.

Rapid and marker-free gene replacement in citric acid-producing Aspergillus tubingensis (A. niger) WU-2223L by the CRISPR/Cas9 system-based genome editing technique using DNA fragments encoding sgRNAs.

Strains belonging to Aspergillus section Nigri, including Aspergillus niger, are used for industrial production of citric acid from carbohydrates such as molasses and starch. The objective of this study was to construct the genome editing system that could enable rapid and efficient gene replacement in citric acid-producing fungi for genetic breeding. Using the citric acid-hyperproducer A. tubingensis (formerly A. niger) WU-2223L as a model strain, we developed a CRISPR/Cas9 system-based genome editing technique involving co-transformation of Cas9 and the DNA fragment encoding single guide RNA (sgRNA). Using this system, ATP-sulfurylase gene (sC) knock-out strain derived from WU-2223L was generated; the knock-out efficiency was 29 transformants when 5 µg Cas9 was added to 5 × 105 protoplasts. In the gene replacement method based on this system, a DNA fragment encoding sgRNAs that target both the gene of interest and marker gene was used, and replacement of nitrate reductase gene (niaD) using sC gene as a marker gene was attempted. More than 90% of the sC-knock-out transformants exhibited replaced niaD, indicating efficient gene replacement. Moreover, one-step marker rescue of the sC marker gene was accomplished by excising the knock-in donor via intramolecular homologous recombination, enabling marker-free genome editing and drastically shortening the gene replacement period by circumventing the transformation procedure to recover the sC gene. Thus, we succeeded in constructing a CRISPR/Cas9 system-based rapid and marker-free gene replacement system for the citric acid-hyperproducer strain WU-2223L.

Constitutive production of aconitate isomerase by Pseudomonas sp. WU-0701 in relation to trans-aconitic acid assimilation.

Aconitic acid, an unsaturated tricarboxylic acid, is used in the chemical industry as raw materials for organic synthesis, especially as a specific substrate for a flavoring agent. trans-Aconitic acid (tAA) is a trans-isomer of cis-aconitic acid and detected in some plants and bacteria. However, biosynthetic route and metabolism of tAA in relation to assimilation have been unknown. Aconitate isomerase (AI; EC 5.3.3.7) catalyzes the reversible isomerization between cis-aconitic acid and tAA. Pseudomonas sp. WU-0701 was isolated as a bacterium assimilating tAA as sole carbon source, and characterization and gene identification of AI were already reported. Here, we describe that Pseudomonas sp. WU-0701 exhibited growth in each synthetic medium containing glucose, citric acid, isocitric acid, or tAA as sole carbon source. AI was intracellularly detected all the time during the cultivation of the strain WU-0701 cells, irrespective of the carbon sources; AI activity was detected even in the glucose-grown cells. Through the subcellular fractionation experiments, AI was detected in the periplasmic fraction. This is the first report indicating that a bacterium belonging to the genus Pseudomonas is constitutive for the AI production.

Draft Genome Sequence of Aspergillus tubingensis WU-2223L, a Citric Acid-Producing Filamentous Fungus Belonging to Aspergillus Section Nigri.

Aspergillus tubingensis WU-2223L, belonging to the section Nigri, is a hyperproducer of citric acid. Here, we present the high-quality draft (35 Mb) and mitochondrial (32.4 kb) genome sequences of this strain, which consisted of 16 scaffolds in total. The draft and mitochondrial genome sequences comprised 11,493 and 15 genes, respectively.

Overexpression of the gene encoding alternative oxidase for enhanced glucose consumption in oxalic acid producing Aspergillus niger expressing oxaloacetate hydrolase gene.

The filamentous fungus Aspergillus niger is a well-known hyper-producer of organic acids such as citric acid and oxalic acid. This fungus possesses the cyanide (CN)-insensitive respiration pathway consisting of alternative oxidase (EC 1.10.3.11; AOX), in addition to the cytochrome pathway. Since this CN-insensitive respiration pathway reoxidizes NADH without ATP production, it contributes to continuous glycolysis in A. niger. In this study, to show the availability of aoxA gene encoding AOX as a tool for metabolic engineering, we generated efficient oxalic acid (OA)-producers by genetic engineering of A. niger using aoxA gene. The OA-producing strain EOAH-1, generated by overexpression of the oxaloacetate hydrolase (EC 3.7.1.1; OAH) gene oahA in A. niger WU-2223L, produced 28 g/L OA from 30 g/L glucose during the 9-day cultivation period. Moreover, the strain EAOXOAH-1, generated by overexpression of both aoxA and oahA genes in strain WU-2223L, produced 28 g/L OA during the 7-day cultivation period. Strain EAOXOAH-1 showed higher glucose consumption rate than EOAH-1 did, indicating that overexpression of aoxA contributed to the acceleration of glucose consumption, and that the OA production period was shortened by 2 days. Thus, we clearly show that AOX gene must be an effective tool in metabolic engineering for efficient organic acids production from carbohydrates.

Decrease of citric acid produced by Aspergillus niger through disruption of the gene encoding a putative mitochondrial citrate-oxoglutarate shuttle protein.

The transporter that exports citric acid (CA) generated in mitochondria to the cytosol is an important component of the CA production machinery of Aspergillus niger. In this report, we cloned and identified the gene cocA, encoding a 33.7-kDa putative mitochondrial citrate-oxoglutarate shuttle protein of the CA hyper-producer A. niger WU-2223L. The amount of CA produced by a representative cocA disruptant (35 g/L) was significantly lower than that produced by strain WU-2223L (63 g/L) after culture for 12 days under CA production conditions, and the phenotype of the cocA disruptant differed in part from that of strain WU-2223L. A cocA disruptant complemented with cocA exhibited the same phenotypes as those of strain WU-2223L. This report is the first to show that cocA and its protein product clearly contribute to substantial CA production by A. niger, and provides a significant insight into microbial organic acid production by fermentation. Abbreviations: CA: citric acid; CD medium: Czapek-Dox medium; CS: citrate synthase; CTP: citrate transport protein; HR: homologous recombination; MCF: mitochondrial carrier family; RT-PCR: reverse-transcription PCR; TCA: tricarboxylic acid.