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Researchers have created a bioengineered form of cytochrome P450 BM-3 from Bacillus megaterium that has higher activity against polycyclic aromatic hydrocarbons than the native enzyme. The mutant enzyme could be used to degrade these compounds in polluted soil.

A purine nucleosidase isolated from Ochrobactrum anthropi appears to be homologous to nucleosidases from protozoan parasites. It has a molecular weight of 170,000, has four identical subunits, and catalyzes the N-riboside hydrolysis of purine nucleosides.

Conjugated linoleic acid can be produced by Lactobacillus acidophilus from linoleic acid. Animal studies have shown that conjugated linoleic acid has substantial health benefits, and the cis-9, trans-11-octadecadienoic acid isomer appears to be the most important isomer from a health standpoint.

An amidase, named 'half-amidase,' that catalyzes the second step of cyclic imide transformation has been reported. It is involved in the half-amide hydrolysis that is part of the bacterial metabolism of cyclic imides, for which it is highly specific.

Monovalent cation-activated levodione reductase of Corynebacterium aquaticum M-13, not previously reported, is discussed. The reductase was isolated from a cell extract of the soil isolate C. aquaticum. The enzyme required certain cofactors, but catalyzed reversible oxidoreduction between actinol and levodione, activated by monovalent cations. The enzyme is in the short-chain alcohol dehydrogenase/reductase family.

The isolation and characterization of the NADH-cytochrome b5 reductases (CbR) gene from Mortierella alpina 1S-4 and the purification of the CbR expressed in Aspergillus oryzae are described. It was found that the purified CbR used NADH but not NADPH as an electron donor. When NADH was utilized as the electron donor, DCPIP was observed to be reduced by the purified CbR. Results provide support for the hypothesis that the cDNA cloned is definitely a CbR gene.

A study of the cyclic-imide-hydrolyzing activity of a prokaryotic cyclic-ureide-hydrolyzing enzyme, D-hydantoinase, revealed that its structure, physico-chemical properties and NH2-terminal amino acid sequence similar to other reported bacterial D-hydantoinases. It showed the highest catalytic efficiency toard dihydropyrimidines and was induced by dihydropyrimidines. These imply that the cyclic-imide-hydrolyzing activity of D-hydantoinase might likewise be a general property of bacterial D-hydantoinases identical to dihydropyrimidinase.

Site-directed mutagenesis was used to investigate the cloning, sequencing and overexpression in Escherichia coli cells of the low-specificity L-threonine aldolase (L-TA) gene from the Pseudomonas strain NCIMB 10558, purification and characterization of the recombinant enzyme and the identification of the active-site lysine residue of the enzyme. The amino acid sequence displayed less than 20% identity with those of low-specificity L-TA from Saccharomyces cerevisiae, L-allo-threonine aldolase from Aeromonas jandaei and other hypothetical proteins from other microorganisms.

A novel nucleoside oxidase was purified from Flavobacterium meningosepticum. The nucleoside oxidase catalyzes the oxidation of nucleosides to its carboxylic acid congener and forms hydrogen peroxide in the process. This enzyme is induced with the presence of nucleosides indicating that it is involved in nucleoside catabolism in F. meningosepticum. The physiological function of this enzyme is yet to be elucidated.

A novel Mortierella alpina mutant (M209-7) derived from the delta12 desaturase-defective Mut48 strain produced greater amounts of 5,811-eicosatrienoic acid (mead acid) compared to its parent strain. The M209-7 strain was similar to Mut48 in morphology, glucose consumption, total fatty acid composition and growth. Furthermore, optimization of cultivation temperature and glucose feeding enhanced the synthesis of mead acid by the mutant Mortierella alpina M209-7 strain.

The cDNA encoding aldehyde reductase (ALR) in the red yeast Sporobolomyces salmonicolor has been cloned using oligonucleotide probes. The ALR gene contains an open reading frame of 323 residues with two introns in the noncoding sequence and four introns in the coding sequence. The encoded polypeptide has a molecular mass of 35,232 Da and its amino acid sequence is similar to that of other aldo-keto reductases, especially in the N-terminal region. An Escherichia coli strain expressing the ALR gene produces an enzyme structurally and catalytically similar to the yeast ALR.

A novel delta 5-desaturase defective mutant, S14, obtained from an arachidonic-acid generating fungus, Mortierella alpina, 1S-4, generates dihomo-gamma-linolenic acid (DGLA). Under favorable conditions, more than twograms of DGLA per liter of culture broth is generated by delta 5-desaturase-defective mutant. This is possible when it is developed for seven days in a 5-litre jar fermentor at 28 degrees Celsius under submerged batch culture conditions.

A delta 5-desaturase-defective mutant, termed Mut44, of the fungus Mortierella alpina 1S-4 was found to exhibit high mycelial dihomo-gamma-linolenic acid (DHGA) activity. The mutant grew in a simple medium with significant DHGA productivity. Various factors affecting the DHGA production were the glucose and yeast extract concentrations and growth temperature. There were no problems, however, with the inhibitors present in oils. Most of the DHGA was present in the triglyceride fraction among lipid classes.

The overexpression of the diacylglycerol diphosphate phosphatase (DPP1) gene has promoted the production of (E,E,E)-geranylgeraniol (GGOH). The construction of diploid prototrophic strain coexpressing the hydroxymethylglutaryl-coenzyme A reductase (HMG1), geranylgeranyl diphosphate synthase (BTS1)-DPP1 and BTS1-ERG20 genes from multicopy integration vectors is described.

Agrobacterium tumefaciens-mediated transformation (ATMT) is examined for Mortierella alpina 1S-4 transformation by using the uracil-auxotrophic mutant of Mortierella alpina 1S-4 as a host strain and the homologous ura5 gene as a selectable marker gene. The ATMT system is effective and suitable for the industrial strain Mortierella alpina 1S-4 and is a useful tool for basic mutagenesis research and for industrial breeding of this strain.

Flavin mononucleotide (FMN) and riboflavin are identified as the extracellular electron shuttles that are produced by a range of Fe(III)-respiring bacteria such as Shewanella species. The implications of flavin secretion by Shewanella cells living at redox boundaries and their role in extracellular electron transfer are analyzed.

The molecular characterization of the gene encoding sterol [DELTA]7 reductase from the fungus Mortierella alpina 1S-4 is presented. The molecular characterization of Mo[DELTA]7SR gene has helped in studying the steroid production by microbial fermentation, investigation of structure-function relationships in [DELTA]7SR and the elucidation of evolution of sterol reductases in various organisms.

The delta1-desturase gene was silenced in order to determine if RNA interference (RNAi) by double-stranded RNA occurs in Mortierella alpina IS-4. 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.

Results demonstrate that 2-deoxyribose 5-phosphate is produced by deoxyriboaldolase encoding gene of Klebsiella pneumoniae, which is overexpressed in Escherichia coli, in the presence of ATP using glucose and acetaldehyde. Data indicate that under the optimal conditions, 100 millimolar 2-deoxyribose 5-phosphate is produced, which is further transformed to 2'-deoxyribonucleoside.

Research demonstrates the application of Saccharomyces cerevisiae old yellow enzyme for the production of actinol, a precursor for the synthesis of several useful compounds. Actinol is produced stoichiometrically by the stereoselective hydrogenation of theketoisophorone followed by the stereoselective reduction of the carbonyl group at the C-4 position of (6R)-levodione.

Molecular cloning of the gene encoding sterol Delta7 reductase from the filamentous fungus Mortierella alpina 1S-4, which accumulates cholesta-5,24-dienol (desmosterol) as the main sterol, revealed that the open reading frame of this gene, designated MoDelta7SR, consists of 1,404 bp and codes for 468 amino acids with a molecular weight of 53,965. The predicted amino acid sequence of MoDelta7SR showed highest homology of 51% with that of sterol Delta7 reductase (EC 1.3.1.21) from Xenopus laevis (African clawed frog). Heterologous expression of the MoDelta7SR gene in yeast Saccharomyces cerevisiae revealed that MoDelta7SR converts ergosta-5,7-dienol to ergosta-5-enol (campesterol) by the activity of Delta7 reductase. In addition, with gene silencing of MoDelta7SR gene by RNA interference, the transformant accumulated cholesta-5,7,24-trienol up to 10% of the total sterols with a decrease in desmosterol. Cholesta-5,7,24-trienol is not detected in the control strain. This indicates that MoDelta7SR is involved in desmosterol biosynthesis in M. alpina 1S-4. This study is the first report on characterization of sterol Delta7 reductase from a microorganism.

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.