Preparation of przewalskinic acid A from salvianolic acid B using a crude enzyme from an Aspergillus oryzae strain

Przewalskinic acid A is a rare, water-soluble, and highly biologically active ingredient found, thus far, only in the Salvia przewalskii Maxim herb; however, the content in S. przewalskii herb is very low. In order to obtain useful quantities of przewalskinic acid A, the biotransformatin of salvianolic acid B from Salvia miltiorrhiza root (danshen in Chinese) into przewalskinic acid A was studied using a crude enzyme produced from Aspergillus oryzae D30s strain. The crude enzyme from the A. oryzae strain hydrolyzed salvianolic acid B into przewalskinic acid A and danshensu. The preparation afforded 31.3 g przewalskinic acid A (91.0 % purity) and 13.1 g danshensu (95 % purity) from 75 g salvianolic acid B. The preparation of przewalskinic acid A was therefore very successful with a yield of over 86 %, but the yield of danshensu was only 33 %. The product przewalskinic acid A was identified using ultra-performance liquid chromatography–mass spectrometry (UPLC–MS) and NMR.     

Overexpression of Aspergillus tubingensis faeA in protease-deficient Aspergillus niger enables ferulic acid production from plant material

The production of ferulic acid esterase involved in the release of ferulic acid side groups from xylan was investigated in strains of Aspergillus tubingensis, Aspergillus carneus, Aspergillus niger and Rhizopus oryzae. The highest activity on triticale bran as sole carbon source was observed with the A. tubingensis T8.4 strain, which produced a type A ferulic acid esterase active against methyl p-coumarate, methyl ferulate and methyl sinapate. The activity of the A. tubingensis ferulic acid esterase (AtFAEA) was inhibited twofold by glucose and induced twofold in the presence of maize bran. An initial accumulation of endoglucanase was followed by the production of endoxylanase, suggesting a combined action with ferulic acid esterase on maize bran. A genomic copy of the A. tubingensis faeA gene was cloned and expressed in A. niger D15#26 under the control of the A. niger gpd promoter. The recombinant strain has reduced protease activity and does not acidify the media, therefore promoting high-level expression of recombinant enzymes. It produced 13.5 U/ml FAEA after 5 days on autoclaved maize bran as sole carbon source, which was threefold higher than for the A. tubingensis donor strain. The recombinant AtFAEA was able to extract 50 % of the available ferulic acid from non-pretreated maize bran, making this enzyme suitable for the biological production of ferulic acid from lignocellulosic plant material.     

Effect of bongkrekic acid on growth and metabolism of filamentous fungi

The antifungal activity of bongkrekic acid against 17 tested molds was determined. Bongkrekic acid prevented spore germination and mycelial proliferation of Aspergillus niger, Rhizopus oryzae and Penicillium italicum. The action of bongkrekic acid was fungicidal. Under these conditions, the incorporation of ¹⁴C-leucine and ¹⁴C-uracil into the perchloric acid insoluble material of germinating A. niger conidia was significantly reduced by bongkrekic acid. Respiratory activity of resting spores was not affected by bongkrekic acid. Respiratory activity of germinated spores was inhibited by bongkrekic acid to the extent of 30 to 60% of controls for A. niger, R. oryzae and P. italicum. It has been concluded that operation of adenine nucleotide translocation in mitochondria of tested fungi is obligatory both for normal spore germination and fungal growth.     

Occurrence and metabolism of sphagnum acid in the cell walls of bryophytes

Sphagnum acid was detected in all 30 Sphagnum species investigated. The content declines in older stem segments. Investigations have so far failed to detect this cinnamic acid derivative outside the Sphagnales. In all the Sphagnum species analysed, a second, conspicuous substance was detected, apparently identical with a degradation product of sphagnum acid produced by enzymatic reaction with peroxidase in vitro. A casual correlation between the sphagnum acid content and peroxidase activity in vivo is discussed. Glyphosate (0.5 mM) inhibits the synthesis of sphagnum acid and shikimate accumulates. Exogenously supplied phenylalanine is able to produce up to 65% reversal of the glyphosate-mediated inhibition of sphagnum acid synthesis. A mixed effect of glyphosate was found on amino acid levels. The content of sphagnum acid is also reduced by daily application of 0.1 mM l-α-aminooxy-β-phenylpropionic acid.     

A monophenol oxidase activity in extracts of sorghum

A p-hydroxycinnamic acid oxidase activity was present in enzyme preparations from first internodes of Sorghum vulgare variety Wheatland milo when incubated in phosphate buffer at pH 7.5. This preparation had no classical polyphenolase activity but had both peroxidase and catalase activities. Since horseradish preparations catalyzed the same reaction, the oxidation probably is another example of a peroxidase-oxidase reaction. A second substrate was p-hydroxyphenylpyruvic acid. Ferulic acid was slightly active at low concentrations and inhibitory at higher ones. Diphenols such as caffeic and chlorogenic acids were inactive and inhibitory to p-hydroxycinnamic acid oxidation. A variety of monophenols such as tyrosine and cinnamic acid were inactive. An active substrate must have a free monophenolic group and para to this a C₃ side chain with a double bond and probably a free terminal acid group. A sulfhydryl reducing agent at the 5 millimolar level such as mercaptoethanol, reduced glutathione, or dithiothreitol was obligatory. Products were varied and were found in both the ethyl acetate-soluble and insoluble fractions after acidification of the incubation mixtures. With internode extracts, about 1 micromole of O₂ was consumed per micromole of p-hydroxycinnamic acid that disappeared in the presence of mercaptoethanol. Tetrahydrafolic acid plus mercaptoethanol were required for a second step oxidation or a parallel reaction; about 2 micromoles of O₂ were consumed per micromole of p-hydroxycinnamic acid that disappeared. Potassium cyanide, diethyldithiocarbamate, ascorbic acid, and ethylenediaminetetraacetate were inhibitory. A similar mercaptoethanol-dependent monophenol oxidase was present in preparations from green shoots that also contained a classical polyphenolase activity. The activity was present in both soluble and particulate (500 to 100,000 gravity) fractions of internodes. Preliminary studies were made of enzyme complexes in the particulate fractions capable of converting phenylalanine and tyrosine to the level of ferulic acid when the above p-hydroxycinnamic acid oxidase was blocked with ascorbic acid. The ratelimiting step was the hydroxylation of p-hydroxycinnamic acid.     

Sporogenic Effect of Polyunsaturated Fatty Acids on Development of Aspergillus spp.

Aspergillus spp. are frequently occurring seed-colonizing fungi that complete their disease cycles through the development of asexual spores, which function as inocula, and through the formation of cleistothecia and sclerotia. We found that development of all three of these structures in Aspergillus nidulans, Aspergillus flavus, and Aspergillus parasiticus is affected by linoleic acid and light. The specific morphological effects of linoleic acid include induction of precocious and increased asexual spore development in A. flavus and A. parasiticus strains and altered sclerotium production in some A. flavus strains in which sclerotium production decreases in the light but increases in the dark. In A. nidulans, both asexual spore production and sexual spore production were altered by linoleic acid. Spore development was induced in all three species by hydroperoxylinoleic acids, which are linoleic acid derivatives that are produced during fungal colonization of seeds. The sporogenic effects of these linoleic compounds on A. nidulans are similar to the sporogenic effects of A. nidulans psi factor, an endogenous mixture of hydroxylinoleic acid moieties. Light treatments also significantly increased asexual spore production in all three species. The sporogenic effects of light, linoleic acid, and linoleic acid derivatives on A. nidulans required an intact veA gene. The sporogenic effects of light and linoleic acid on Aspergillus spp., as well as members of other fungal genera, suggest that these factors may be significant environmental signals for fungal development.     

Isolation and Characterization of Esters of Indole-3-Acetic Acid from the Liquid Endosperm of the Horse Chestnut (Aesculus species)

Esters of indole-3-acetic acid were extracted and purified from the liquid endosperm of immature fruits of various species of the horse chestnut (Aesculus parviflora, A. baumanni, A.pavia rubra, and A. pavia humulis). The liquid endosperm contained, at least 12 chromatographically distinct esters. One of these compounds was purified and characterized as an ester of indole-3-acetic acid and myo-inositol. A second compound was found to be an ester of indole-3-acetic acid and the disaccharide rutinose (glucosyl-rhamnose). A third compound was partially characterized as an ester of indole-3-acetic acid and a desoxyaminohexose.     

Oxidation of Metabolites Highlights the Microbial Interactions and Role of Acetobacter pasteurianus during Cocoa Bean Fermentation

Four cocoa-specific acetic acid bacterium (AAB) strains, namely, Acetobacter pasteurianus 386B, Acetobacter ghanensis LMG 23848^T, Acetobacter fabarum LMG 24244^T, and Acetobacter senegalensis 108B, were analyzed kinetically and metabolically during monoculture laboratory fermentations. A cocoa pulp simulation medium (CPSM) for AAB, containing ethanol, lactic acid, and mannitol, was used. All AAB strains differed in their ethanol and lactic acid oxidation kinetics, whereby only A. pasteurianus 386B performed a fast oxidation of ethanol and lactic acid into acetic acid and acetoin, respectively. Only A. pasteurianus 386B and A. ghanensis LMG 23848^T oxidized mannitol into fructose. Coculture fermentations with A. pasteurianus 386B or A. ghanensis LMG 23848^T and Lactobacillus fermentum 222 in CPSM for lactic acid bacteria (LAB) containing glucose, fructose, and citric acid revealed oxidation of lactic acid produced by the LAB strain into acetic acid and acetoin that was faster in the case of A. pasteurianus 386B. A triculture fermentation with Saccharomyces cerevisiae H5S5K23, L. fermentum 222, and A. pasteurianus 386B, using CPSM for LAB, showed oxidation of ethanol and lactic acid produced by the yeast and LAB strain, respectively, into acetic acid and acetoin. Hence, acetic acid and acetoin are the major end metabolites of cocoa bean fermentation. All data highlight that A. pasteurianus 386B displayed beneficial functional roles to be used as a starter culture, namely, a fast oxidation of ethanol and lactic acid, and that these metabolites play a key role as substrates for A. pasteurianus in its indispensable cross-feeding interactions with yeast and LAB during cocoa bean fermentation.     

In Vitro Fatty Acid Synthesis and Complex Lipid Metabolism in the Cyanobacterium Anabaena variabilis: I. Some Characteristics of Fatty Acid Synthesis

In vitro fatty acid synthesis was examined in crude cell extracts, soluble fractions, and 80% (NH₄)₂SO₄ fractions from Anabaena variabilis M3. Fatty acid synthesis was absolutely dependent upon acyl carrier protein and required NADPH and NADH. Moreover, fatty acid synthesis and elongation occurred in the cytoplasm of the cell. The major fatty acid products were palmitic acid (16:0) and stearic acid (18:0). Of considerable interest, both stearoyl-acyl carrier protein and stearoyl-coenzyme A desaturases were not detected in any of the fractions from A. variabilis. The similarities and differences in fatty acid synthesis between A. variabilis and higher plant tissues are discussed with respect to the endosymbiotic theory of chloroplast evolution.     

Identification of a fatty acid ∆6-desaturase gene from the eicosapentaenoic acid-producing fungus Pythium splendens RBB-5

A new full-length cDNA (PsD6) putatively encoding a ?⁶-desaturase was cloned from the eicosapentaenoic acid-producing fungus Pythium splendens RBB-5. PsD6 contained an open reading frame of 1380 bp encoding a protein of 459 amino acids. The deduced amino acid sequence showed high similarity to those of other ?⁶-desaturases. A recombinant vector expressing PsD6 (pPIC3.5K-D6) was constructed and transformed into Pichia pastoris GS115. The heterologous expressed PsD6 in P. pastoris desaturated linoleic acid to γ-linolenic acid but not desaturated α-linolenic acid to stearidonic acid. The results indicated that PsD6 was a fatty acid ?⁶-desaturase and it had a substrate specificity for linoleic acid.     

Cloning of a cluster of chitinase genes from Aeromonas sp. No. 10S-24

A gene encoding chitinases from Aeromonas sp. No. 10S-24 was cloned into Escherichia coli DH5α using pUC19, and its nucleotides were sequenced. The chitinase gene was clustered in ORFs (open reading frame) 1 to 4, in a 8-kb fragment of DNA. ORF-1 consisted of 1608 bp encoding 535 amino acid residues, and ORF-2 consisted of 1425 bp encoding 474 amino acid residues. ORF-3 was 1617 bp long and encodes a protein consisting of 538 amino acids. ORF-4 encodes 287 amino acids of the N-terminal region. The amino acid sequences of ORF-1 and ORF-3 share sequence homology with chitinase D from Bacillus circulans, and chitinase A and B from Streptomyces lividans. The amino acid sequence of ORF-2 shared sequence homology with chitinase II from Aeromonas sp. No. 10S-24, and chitinase from Saccharopolyspora erythraea. A region of the sequence starting from Ala-28 of the amino acid sequence of ORF-3 coincided with the N-terminal amino acid sequence of chitinase III from Aeromonas sp. No. 10S-24.     

Metabolic engineering of Escherichia coli for production of salvianic acid A via an artificial biosynthetic pathway

Salvianic acid A, a valuable derivative from L-tyrosine biosynthetic pathway of the herbal plant Salvia miltiorrhiza, is well known for its antioxidant activities and efficacious therapeutic potential on cardiovascular diseases. Salvianic acid A was traditionally isolated from plant root or synthesized by chemical methods, both of which had low efficiency. Herein, we developed an unprecedented artificial biosynthetic pathway of salvianic acid A in E. coli, enabling its production from glucose directly. In this pathway, 4-hydroxyphenylpyruvate was converted to salvianic acid A via D-lactate dehydrogenase (encoding by d-ldh from Lactobacillus pentosus) and hydroxylase complex (encoding by hpaBC from E. coli). Furthermore, we optimized the pathway by a modular engineering approach and deleting genes involved in the regulatory and competing pathways. The metabolically engineered E. coli strain achieved high productivity of salvianic acid A (7.1 g/L) with a yield of 0.47 mol/mol glucose.     

Biological activity of antheridic acid,an antheridiogen of anemia phyllitidis

The biological activities of synthetic antheridiogen of Anemia phyllitidis, (±)-antheridic acid, and naturally derived antheridic acid with regard to induction of antheridial formation and dark spore germination in A. phyllitidis were closely similar. The activity of (±)-3-epi-antheridic acid was weaker than that of (±)-antheridic acid in inducing these phenomena. (±)-Antheridic acid was active in inducing elongation growth in the dwarf rice bioassay system, although its activity was weaker than that of GA₃. In this bioassay system, (±)-3-epi-antheridic acid showed higher activity than did (±)-antheridic acid.     

On the reaction specificity of the lipoxygenase from tomato fruits

A lipoxygenase was purified 300-fold from a homogenate supernatant of ripe tomato fruits by fractionated ammonium sulfate precipitation and anion exchange fast protein liquid chromatography. The specific linoleate oxygenase activity of the final enzyme preparation was 1300 nkat per mg protein at pH 6.8 and 25°C in the absence of any detergent. The enzyme oxygenated linoleic acid and α-linolenic acid at comparable rates, whereas γ-linolenic acid, arachidonic acid, 11,14-eicosadienoic acid and 11,14,17-eicosatrienoic acid were poor substrates. Linoleic acid was converted to 9(S)-hydroperoxy-10E,12Z-octadecadienoic acid, whereas 5(S)-HpETE, 11(S)-HpETE and 8(S)-HpETE were identified as major oxygenation products from arachidonic acid. The tomato lipoxygenase did not react with either dilinoleyl phosphatidylcholine or the lipid extract from beef heart mitochondria. The possible biological importance of the reaction of tomato lipoxygenase with arachidonic acid is discussed.     

Stereochemistry of 2-aminopimelic acid and related amino acids in three species of asplenium

We previously reported two free D-amino acids, D-2-aminopimelic acid (D-APA) and trans-3,4-dehydro-D-2-aminopimelic acid (D-Δ-APA), from Asplenium unilaterale. In the present work we isolated 4-hydroxy-2-aminopimelic acid (OH-APA) from the same plant and determined it to be the α-L-form. We also investigated the configurations of these amino acids isolated from A. prolongatum and A. wilfordii which are morphologically distinct from A. unilaterale. In A. prolongatum, APA was the D- and OH-APA was the L-isomer. In contrast, APA from A. wilfordii was partially racemized and the degree of racemization was significantly different in plant material collected in July and November, L:D = 3:2 and 3:7, respectively. In A. wilfordii OH-APA was almost pure L- and Δ-APA was mostly the D-isomer.     

Heterologous production of caffeic acid from tyrosine in Escherichia coli

Caffeic acid is a plant secondary metabolite and its biological synthesis has attracted increased attention due to its beneficial effects on human health. In this study, Escherichia coli was engineered for the production of caffeic acid using tyrosine as the initial precursor of the pathway. The pathway design included tyrosine ammonia lyase (TAL) from Rhodotorula glutinis to convert tyrosine to p-coumaric acid and 4-coumarate 3-hydroxylase (C3H) from Saccharothrix espanaensis or cytochrome P450 CYP199A2 from Rhodopseudomonas palustris to convert p-coumaric acid to caffeic acid. The genes were codon-optimized and different combinations of plasmids were used to improve the titer of caffeic acid. TAL was able to efficiently convert 3 mM of tyrosine to p-coumaric acid with the highest production obtained being 2.62 mM (472 mg/L). CYP199A2 exhibited higher catalytic activity towards p-coumaric acid than C3H. The highest caffeic acid production obtained using TAL and CYP199A2 and TAL and C3H was 1.56 mM (280 mg/L) and 1 mM (180 mg/L), respectively. This is the first study that shows caffeic acid production using CYP199A2 and tyrosine as the initial precursor. This study suggests the possibility of further producing more complex plant secondary metabolites like flavonoids and curcuminoids.     

Oxalic acid production by citric acid-producing Aspergillus niger overexpressing the oxaloacetate hydrolase gene oahA

The filamentous fungus Aspergillus niger is used worldwide in the industrial production of citric acid. However, under specific cultivation conditions, citric acid-producing strains of A. niger accumulate oxalic acid as a by-product. Oxalic acid is used as a chelator, detergent, or tanning agent. Here, we sought to develop oxalic acid hyperproducers using A. niger as a host. To generate oxalic acid hyperproducers by metabolic engineering, transformants overexpressing the oahA gene, encoding oxaloacetate hydrolase (OAH; EC 3.7.1.1), were constructed in citric acid-producing A. niger WU-2223L as a host. The oxalic acid production capacity of this strain was examined by cultivation of EOAH-1 under conditions appropriate for oxalic acid production with 30 g/l glucose as a carbon source. Under all the cultivation conditions tested, the amount of oxalic acid produced by EOAH-1, a representative oahA-overexpressing transformant, exceeded that produced by A. niger WU-2223L. A. niger WU-2223L and EOAH-1 produced 15.6 and 28.9 g/l oxalic acid, respectively, during the 12-day cultivation period. The yield of oxalic acid for EOAH-1 was 64.2 % of the maximum theoretical yield. Our method for oxalic acid production gave the highest yield of any study reported to date. Therefore, we succeeded in generating oxalic acid hyperproducers by overexpressing a single gene, i.e., oahA, in citric acid-producing A. niger as a host.     

Degradation of 1,4-Dioxane and Cyclic Ethers by an Isolated Fungus

By using 1,4-dioxane as the sole source of carbon, a 1,4-dioxane-degrading microorganism was isolated from soil. The fungus, termed strain A, was able to utilize 1,4-dioxane and many kinds of cyclic ethers as the sole source of carbon and was identified as Cordyceps sinensis from its 18S rRNA gene sequence. Ethylene glycol was identified as a degradation product of 1,4-dioxane by the use of deuterated 1,4-dioxane-d₈ and gas chromatography-mass spectrometry analysis. A degradation pathway involving ethylene glycol, glycolic acid, and oxalic acid was proposed, followed by incorporation of the glycolic acid and/or oxalic acid via glyoxylic acid into the tricarboxylic acid cycle.     

Production of Hydrocinnamic Acid by Clostridia

Hydrocinnamic acid was found in acid extracts of spent growth medium from cultures of Clostridium sporogenes. The acid was identified by mass spectrometry and its identity was confirmed by gas chromatography. The acid was produced in relatively large amounts (2 to 3 μmoles/ml of medium) by C. sporogenes, toxigenic types A, B, D, and F of C. botulinum, and some strains of C. bifermentans. Other strains of C. bifermentans and strains of C. sordellii and C. caproicum produced only small amounts (0.1 to 0.4 μmoles/ml) of the acid. The acid was not detected in spent medium from toxigenic types C and E of C. botulinum or from 25 other strains representing eight Clostridium species. Resting cell suspensions exposed to l-phenylalanine produced hydrocinnamic and cinnamic acid; the latter compound probably functions as an intermediate in the metabolism of l-phenylalanine.