A novel glucosylation enzyme: molecular cloning, expression, and characterization of Trichoderma viride JCM22452 alpha-amylase and enzymatic synthesis of some flavonoid monoglucosides and oligoglucosides.

It was found that commercial cellulase preparations from Trichoderma viride showed transglucosylation activity toward (+)-catechin and (-)-epigallocatechin gallate (EGCG) using dextrin as a glucosyl donor. To isolate the enzyme exhibiting transglucosylation activity toward (+)-catechin and EGCG, the present study isolated the cDNA encoding the T. viride JCM22452 alpha-amylase homologue (TRa2), which showed high amino acid sequence identity to functionally uncharacterized alpha-amylase homologues from other ascomycetes, which also produced some (+)-catechin and EGCG glucosides. TRa2 was able to glucosylate a wide range of natural flavonoids, particularly (+)-catechin and EGCG, and to hydrolyze maltooligosaccharides (k(cat)/K(m) for maltotriose, maltotetraose, maltopentaose, maltohexaose, and maltoheptaose were 1.98, 45.2, 58.3, 97.4, and 92.6 s(-1) mM(-1), respectively) except maltose but could not transfer the monoglucosyl residue to maltooligosaccharides. By (1)H NMR and (13)C NMR, the structures of several novel glucosides obtained by commercial cellulase preparations from T. viride and TRa2 were determined as (+)-catechin 5-O-alpha-D-glucopyranoside, (+)-catechin 5-O-alpha-D-maltoside, (+)-catechin 4'-O-alpha-D-maltoside, EGCG 5-O-alpha-D-glucopyranoside, and EGCG 7-O-alpha-D-maltoside. One of these glucosides, EGCG 5-O-alpha-D-glucopyranoside, showed higher heat stability and solubility and lower astringency and astringent stimulation than its aglycon, suggesting that EGCG glucosides are functionally superior to EGCG as food additives.

Improvement of the fatty acid composition of an oil-producing filamentous fungus, Mortierella alpina 1S-4, through RNA interference with delta12-desaturase gene expression.

An oleaginous fungus, Mortierella alpina 1S-4, is used commercially for arachidonic acid production. Delta12-Desaturase, which desaturates oleic acid (18:1n-9) to linoleic acid (18:2n-6), is a key enzyme in the arachidonic acid biosynthetic pathway. To determine if RNA interference (RNAi) by double-stranded RNA occurs in M. alpina 1S-4, we silenced the Delta12-desaturase gene. The silenced strains accumulate 18:2n-9, 20:2n-9, and Mead acid (20:3n-9), which are not detected in either the control strain or wild type strain 1S-4. The fatty acid composition of stable transformants was similar to that of Delta12-desaturation-defective mutants previously identified. Thus, RNAi occurs in M. alpina and could be used to alter the types and relative amounts of fatty acids produced by commercial strains of this fungus without mutagenesis or other permanent changes in the genetic background of the producing strains.

Molecular evidence that the rate-limiting step for the biosynthesis of arachidonic acid in Mortierella alpina is at the level of an elongase.

The oil-producing fungus Mortierella alpina 1S-4 is an industrial strain for arachidonic acid (AA) production. To determine its physiological properties and to clarify the biosynthetic pathways for PUFA, heterologous and homologous gene expression systems were established in this fungus. The first trial was performed with an enhanced green fluorescent protein gene to assess the transformation efficiency for heterologous gene expression. As a result, strong fluorescence was observed in the spores of the obtained transformant, suggesting that the foreign gene was inherited by the spores. The next trial was performed with a homologous PUFA elongase (GLELOp) gene, this enzyme having been reported to catalyze the elongation of GLA (18:3n-6) to dihomo-gamma-linolenic acid (20:3n-6), and to be the rate-limiting step of AA production. The FA composition of the transformant was different from that of the host strain: The GLA content was decreased whereas that of AA was increased. These data support the hypothesis that the GLELOp enzyme plays an important role in PUFA synthesis, and may indicate how to control PUFA biosynthesis.

Transformation of oil-producing fungus, Mortierella alpina 1S-4, using Zeocin, and application to arachidonic acid production.

The arachidonic acid-producing fungus Mortierella alpina 1S-4, an industrial strain, was endowed with Zeocin resistance by integration of the Zeocin-resistance gene at the rDNA locus of genomic DNA. Plasmid DNA was introduced into spores by microprojectile bombardment. Twenty mg/ml Zeocin completely inhibited the germination of M. alpina 1S-4 spores, and decreased the growth rate of fungal filaments to some extent. It was suggested that preincubation period and temperature had a great influence on transformation efficiency. Four out of 26 isolated transformants were selected. Molecular analysis of these stable transformants showed that the plasmid DNA was integrated into the rDNA locus of the genomic DNA. We expect that this system will be applied for useful oil production by gene manipulation of M. alpina 1S-4 and its derivative mutants. On the basis of the fundamental transformation system, we also tried to overexpress a homologous polyunsaturated fatty acid elongase gene, which has been reported to be included in the rate-limiting step for arachidonic acid production, thereby leading to increased arachidonic acid production.

Establishment of an overall transformation system for an oil-producing filamentous fungus, Mortierella alpina 1S-4.

Oil-producing fungus Mortierella alpina 1S-4 is an industrial strain. To determine its physiological properties and to clarify the biosynthetic pathways for polyunsaturated fatty acids, a transformation system for this fungus was established using a derivative of it, i.e., a ura5- mutant lacking orotate phosphoribosyl transferase (OPRTase, EC.2.4.2.10) activity. Transformation with a vector containing the homologous ura5 gene as a marker was successfully performed using microprojectile bombardment, other methods frequently used for transformation, such as the protoplasting, lithium acetate, or electroporation methods, not giving satisfactory results. As a result, two types of transformants were obtained: a few stable transformants overexpressing the ura5 gene, and many unstable transformants showing OPRTase activity comparable to that of the wild-type strain. The results of quantitative PCR indicated that the stable transformants could retain the ura5 genes originating from the transformation vector regardless of the culture conditions. On the other hand, unstable transformants easily lost the marker gene under uracil-containing conditions, as expected. In this paper, we report that an overall transformation system for this fungus was successfully established, and propose how to select useful transformants as experimental and industrial strains.

Cloning and sequencing of the ura3 and ura5 genes, and isolation and characterization of uracil auxotrophs of the fungus Mortierella alpina 1S-4.

The oil-producing fungus Mortierella alpina 1S-4 is an industrial strain. In order to prepare host strains for a transformation system for this fungus, six uracil auxotrophs were obtained by means of random mutation with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). When the activities of orotate phosphoribosyl transferase (OPRTase, EC 2.4.2.10) and orotidine-5'-phosphate decarboxylase (OMPdecase, EC 4.1.1.23) were examined in the mutants and wild strain, OPRTase activity was found to be completely absent in all mutants, on the other hand, OMPdecase activity was intact. The genomic DNA and cDNA of the ura5 gene encoding OPRTase and the ura3 gene encoding OMPdecase were cloned and sequenced. The Ura5p deduced amino acid sequence of this fungus showed highest similarity to that of Vibrio cholerae classed among prokaryote. Furthermore, the mutational points in the ura5 genes of two selected mutants were identified; a base-replacement and a base-insertion.

Deciphering the molecular basis of the broad substrate specificity of alpha-glucosidase from Bacillus sp. SAM1606.

The alpha-glucosidase of Bacillus sp. strain SAM1606 is a member of glycosyl hydrolase family 13, and shows an extraordinarily broad substrate specificity and is one of very few alpha-glucosidases that can efficiently hydrolyze the alpha-1,1-glucosidic linkage of alpha,alpha'-trehalose (trehalose). Phylogenetic analysis of family-13 enzymes suggests that SAM1606 alpha-glucosidase may be evolutionally derived from an alpha-1,6-specific ancestor, oligo-1,6-glucosidase (O16G). Indeed, replacement of Pro(273*) and Thr(342*) of B. cereus O16G by glycine and asparagine (the corresponding residues in the SAM1606 enzyme), respectively, was found to cause 192-fold enhancement of the relative catalytic efficiency for trehalose, suggesting that O16G may easily "evolved" into an enzyme with an extended substrate specificity by substitution of a limited number of amino acids, including that at position 273* (an asterisk indicates the amino-acid numbering of the SAM1606 sequence). To probe the role of the amino acid at position 273* of alpha-glucosidase in determination of the substrate specificity, the amino acid at position 273 of SAM1606 alpha-glucosidase was replaced by all other naturally occurring amino acids, and the resultant mutants were kinetically characterized. The results showed that substitution of bulky residues (e.g., isoleucine and methionine) for glycine at this position resulted in large increases in the K(m) values for trehalose and maltose, whereas the affinity to isomaltose was only minimally affected by such an amino-acid substitution at this position. Three-dimensional structural models of the enzyme-substrate complexes of the wild-type and mutant SAM1606 alpha-glucosidases were built to explore the mechanism responsible for these observations. It is proposed that substitution by glycine at position 273* could eliminate steric hindrance around subsite +1 that originally occurred in parental O16G and is, at least in part, responsible for the acquired broad substrate specificity of SAM1606 alpha-glucosidase.

Cloning of beta-primeverosidase from tea leaves, a key enzyme in tea aroma formation.

A beta-primeverosidase from tea (Camellia sinensis) plants is a unique disaccharide-specific glycosidase, which hydrolyzes aroma precursors of beta-primeverosides (6-O-beta-D-xylopyranosyl-beta-D-glucopyranosides) to liberate various aroma compounds, and the enzyme is deeply concerned with the floral aroma formation in oolong tea and black tea during the manufacturing process. The beta-primeverosidase was purified from fresh leaves of a cultivar for green tea (C. sinensis var sinensis cv Yabukita), and its partial amino acid sequences were determined. The beta-primeverosidase cDNA has been isolated from a cDNA library of cv Yabukita using degenerate oligonucleotide primers. The cDNA insert encodes a polypeptide consisting of an N-terminal signal peptide of 28 amino acid residues and a 479-amino acid mature protein. The beta-primeverosidase protein sequence was 50% to 60% identical to beta-glucosidases from various plants and was classified in a family 1 glycosyl hydrolase. The mature form of the beta-primeverosidase expressed in Escherichia coli was able to hydrolyze beta-primeverosides to liberate a primeverose unit and aglycons, but did not act on 2-phenylethyl beta-D-glucopyranoside. These results indicate that the beta-primeverosidase selectively recognizes the beta-primeverosides as substrates and specifically hydrolyzes the beta-glycosidic bond between the disaccharide and the aglycons. The stereochemistry for enzymatic hydrolysis of 2-phenylethyl beta-primeveroside by the beta-primeverosidase was followed by (1)H-nuclear magnetic resonance spectroscopy, revealing that the enzyme hydrolyzes the beta-primeveroside by a retaining mechanism. The roles of the beta-primeverosidase in the defense mechanism in tea plants and the floral aroma formation during tea manufacturing process are also discussed.

Deletion and insertion of a 192-residue peptide in the active-site domain of glycosyl hydrolase family-2 beta-galactosidases.

The monomeric multimetal-binding beta-galactosidase of Saccharopolyspora rectivirgula (srbg), a glycosyl hydrolase family-2 enzyme, has a unique sequence consisting of 192 amino acid residues with no similarity to known proteins. This 192-residue sequence (termed the "iota [iota] sequence") appears to be inserted into a sequence homologous to the active-site domain of the Escherichia coli lacZ enzyme (lacZbg). To assess the effects of the t sequence at specific sites of beta-galactosidase on the catalytic functioning and molecular properties of beta-galactosidase, deletion or insertion mutants of beta-galactosidases were constructed, expressed in LacZ- E. coli strains, and characterized: srbgdelta in which the iota sequence was deleted from srbg, and lacZbgI, in which the 192-residue iota sequence was inserted into the corresponding position (between Asp591 and Phe592) in the active-site domain of lacZbg. srbgdelta was a catalytically inactive, dimeric protein which retained multimetal-binding characteristics, suggesting that the iota sequence is very important for maintaining the structure necessary for the catalytic functioning and the monomeric structure of srbg but is not responsible for the unique metal ion requirements of srbg. On the other hand, lacZbgI existed as a mixture of a monomer, a tetramer, and higher multimers. The monomeric species was inactive, whereas the tetramer and other multimers were catalytically active (V(max )K(m) value, 25% of that of lacZbg) and highly specific for beta-D-galactoside. The tetrameric lacZbgI was activated by Mg2+ and Mn2+ with lowered metal affinities, and the stoichiometry of metal binding was unchanged from that of lacZbg. These results, along with the published stereo structure of lacZbg, suggest that, in lacZbgI, the inserted 192-residue iota peptide could fold independently of the lacZbg domains into a "sub-domain," lying distant from the active site and subunit interfaces.