Takasumi Hattori

Enzymatic Kolbe―Schmitt reaction to form salicylic acid from phenol: Enzymatic characterization and gene identification of a novel enzyme, Trichosporon moniliiforme salicylic acid decarboxylase

Salicylic acid decarboxylase (Sdc) can produce salicylic acid from phenol; it was found in the yeast Trichosporon moniliiforme WU-0401 and was for the first time enzymatically characterized, with the sdc gene heterologously expressed. Sdc catalyzed both reactions: decarboxylation of salicylic acid to phenol and the carboxylation of phenol to form salicylic acid without any byproducts. Both reactions were detected without the addition of any cofactors and occurred even in the presence of oxygen, suggesting that this Sdc is reversible, nonoxidative, and oxygen insensitive. Therefore, it is readily applicable in the selective production of salicylic acid from phenol, the enzymatic Kolbe-Schmitt reaction. The deduced amino acid sequence of the gene, sdc, encoding Sdc comprises 350 amino acid residues corresponding to a 40-kDa protein. The recombinant Escherichia coli BL21(DE3) expressing sdc converted phenol to salicylic acid with a 27% (mol/mol) yield at 30 degrees C for 9h.

Regulation of Alternative Oxidase at the Transcription Stage in Aspergillus niger Under the Conditions of Citric Acid Production

The citric acid-producing fungus Aspergillusniger WU-2223L possesses a cyanide-insensitive respiratory pathway catalyzed by alternative oxidase. The regulation of the alternative oxidase under the conditions of citric acid production was determined from the transcription level of the alternative oxidase gene (aox1). PCR and Southern blot analyses revealed that there is only one copy of aox1 on the chromosome of WU-2223L and no homologous gene of aox1. To confirm the regulation stage of alternative oxidase, alternative oxidase activities and aox1 transcription levels were measured under several cultivation conditions, including those for citric acid production. On each cultivation day, the changes in the specific activity of the alternative oxidase were found to be comparable to those in the transcription level of aox1. These results indicate that the activity of the alternative oxidase encoded by aox1 is regulated at the transcription stage under the conditions tested for A. niger WU-2223L.

Expression of alternative oxidase gene (aox1) at the stage of single-cell conidium in citric acid-producing Aspergillus niger

Mycelia of citric acid-producing Aspergillus niger WU-2223L show cyanide-insensitive respiration catalyzed by alternative oxidase. In this study, the constitutive expression of alternative oxidase gene (aox1) even at the stage of single-cell conidium in A. niger WU-2223L was found using the visual expression analysis system of aox1 with green fluorescent protein under microscopy observation.

3.13 – Citric Acid

Citric acid is one of the most widely used organic acids, and its annual worldwide production reached 1.6 million ton during 2009. It is used as an acidulant and preservative in the food industry, and also as a complexing agent in the pharmaceutical and cosmetic industries. Citric acid is also used as a complexing and chelating agent in metal treatment, as a water softener for detergents, and as a buffering agent in toiletry and pharmaceutical industries. Citric acid is exclusively produced by fermentation with the filamentous fungus Aspergillus niger. The industrial production is performed using carbohydrates or agro-industrial residues as substrates by three different types of processes: submerged, surface, and solid fermentations. This article provides a current review of advances in citric acid production by A. niger and application of citric acid in various fields.

Gene Identification and Functional Analysis of Methylcitrate Synthase in Citric Acid-Producing Aspergillus niger WU-2223L

Methylcitrate synthase (EC 2.3.3.5; MCS) is a key enzyme of the methylcitric acid cycle localized in the mitochondria of eukaryotic cells and related to propionic acid metabolism. In this study, cloning of the gene mcsA encoding MCS and heterologous expression of it in Escherichia coli were performed for functional analysis of the MCS of citric acid-producing Aspergillus niger WU-2223L. Only one copy of mcsA (1,495 bp) exists in the A. niger WU-2223L chromosome. It encodes a 51-kDa polypeptide consisting of 465 amino acids containing mitochondrial targeting signal peptides. Purified recombinant MCS showed not only MCS activity (27.6 U/mg) but also citrate synthase (EC 2.3.3.1; CS) activity (26.8 U/mg). For functional analysis of MCS, mcsA disruptant strain DMCS-1, derived from A. niger WU-2223L, was constructed. Although A. niger WU-2223L showed growth on propionate as sole carbon source, DMCS-1 showed no growth. These results suggest that MCS is an essential enzyme in propionic acid metabolism, and that the methylcitric acid cycle operates functionally in A. niger WU-2223L. To determine whether MCS makes a contribution to citric acid production, citric acid production tests on DMCS-1 were performed. The amount of citric acid produced from glucose consumed by DMCS-1 in citric acid production medium over 12 d of cultivation was on the same level to that by WU-2223L. Thus it was found that MCS made no contribution to citric acid production from glucose in A. niger WU-2223L, although MCS showed CS activity.

Enzymatic characterization and gene identification of aconitate isomerase, an enzyme involved in assimilation of trans-aconitic acid, from Pseudomonas sp. WU-0701

trans‐Aconitic acid is an unsaturated organic acid that is present in some plants such as soybean and wheat; however, it remains unclear how trans‐aconitic acid is degraded and/or assimilated by living cells in nature. From soil, we isolated Pseudomonas sp. WU‐0701 assimilating trans‐aconitic acid as a sole carbon source. In the cell‐free extract of Pseudomonas sp. WU‐0701, aconitate isomerase (AI; EC 5.3.3.7) activity was detected. Therefore, it seems likely that strain Pseudomonas sp. WU‐0701 converts trans‐aconitic acid to cis‐aconitic acid with AI, and assimilates this via the tricarboxylic acid cycle. For the characterization of AI from Pseudomonas sp. WU‐0701, we performed purification, determination of enzymatic properties and gene identification of AI. The molecular mass of AI purified from cell‐free extract was estimated to be ~ 25 kDa by both SDS/PAGE and gel filtration analyses, indicating that AI is a monomeric enzyme. The optimal pH and temperature of purified AI for the reaction were 6.0 °C and 37 °C, respectively. The gene ais encoding AI was cloned on the basis of the N‐terminal amino acid sequence of the protein, and Southern blot analysis revealed that only one copy of ais is located on the bacterial genome. The gene ais contains an ORF of 786 bp, encoding a polypeptide of 262 amino acids, including the N‐terminal 22 amino acids as a putative periplasm‐targeting signal peptide. It is noteworthy that the amino acid sequence of AI shows 90% and 74% identity with molybdenum ABC transporter substrate‐binding proteins of Pseudomonas psychrotolerans and Xanthomonas albilineans, respectively. This is the first report on purification to homogeneity, characterization and gene identification of AI.

Overexpression of the NADP+-specific isocitrate dehydrogenase gene (icdA) in citric acid-producing Aspergillus niger WU-2223L

In the tricarboxylic acid (TCA) cycle, NADP+-specific isocitrate dehydrogenase (NADP+-ICDH) catalyzes oxidative decarboxylation of isocitric acid to form α-ketoglutaric acid with NADP+ as a cofactor. We constructed an NADP+-ICDH gene (icdA)-overexpressing strain (OPI-1) using Aspergillus niger WU-2223L as a host and examined the effects of increase in NADP+-ICDH activity on citric acid production. Under citric acid-producing conditions with glucose as the carbon source, the amounts of citric acid produced and glucose consumed by OPI-1 for the 12-d cultivation period decreased by 18.7 and 10.5%, respectively, compared with those by WU-2223L. These results indicate that the amount of citric acid produced by A. niger can be altered with the NADP+-ICDH activity. Therefore, NADP+-ICDH is an important regulator of citric acid production in the TCA cycle of A. niger. Thus, we propose that the icdA gene is a potentially valuable tool for modulating citric acid production by metabolic engineering. Graphical Abstract We revealed that NADP+-specific isocitrate dehydrogenase (NADP+-ICDH) is an important regulator of citric acid production in Aspergillus niger by constructing NADP+-ICDH gene (icdA)-overexpressing strain (OPI-1) using strain WU-2223L as a host.

Phenotypes of gene disruptants in relation to a putative mitochondrial malate–citrate shuttle protein in citric acid-producing Aspergillus niger

The mitochondrial citrate transport protein (CTP) functions as a malate–citrate shuttle catalyzing the exchange of citrate plus a proton for malate between mitochondria and cytosol across the inner mitochondrial membrane in higher eukaryotic organisms. In this study, for functional analysis, we cloned the gene encoding putative CTP (ctpA) of citric acid-producing Aspergillus niger WU-2223L. The gene ctpA encodes a polypeptide consisting 296 amino acids conserved active residues required for citrate transport function. Only in early-log phase, the ctpA disruptant DCTPA-1 showed growth delay, and the amount of citric acid produced by strain DCTPA-1 was smaller than that by parental strain WU-2223L. These results indicate that the CTPA affects growth and thereby citric acid metabolism of A. niger changes, especially in early-log phase, but not citric acid-producing period. This is the first report showing that disruption of ctpA causes changes of phenotypes in relation to citric acid production in A. niger. Graphical abstract Disruption of the gene encoding the citrate transport protein (CTPA, i.e., malate–citrate shuttle protein) affects the phenotype of Aspergillus niger.