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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 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.

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.

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.

Molecular cloning of the gene encoding sterol Δ7 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 MoΔ7SR, consists of 1,404 bp and codes for 468 amino acids with a molecular weight of 53,965. The predicted amino acid sequence of MoΔ7SR showed highest homology of 51% with that of sterol Δ7 reductase (EC 1.3.1.21) from Xenopus laevis (African clawed frog). Heterologous expression of the MoΔ7SR gene in yeast Saccharomyces cerevisiae revealed that MoΔ7SR converts ergosta-5,7-dienol to ergosta-5-enol (campesterol) by the activity of Δ7 reductase. In addition, with gene silencing of MoΔ7SR 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 MoΔ7SR is involved in desmosterol biosynthesis in M. alpina IS-4. This study is the first report on characterization of sterol Δ7 reductase from a microorganism. [ABSTRACT FROM AUTHOR]

An oleaginous fungus, Mortierella alpina 1S-4, is used commercially for arachidonic acid production. £12-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 £12-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 £12-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. [ABSTRACT FROM AUTHOR]

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.