Gene manipulation tools for an arachidonic-producing filamentous fungus, Mortierella alpina 1S-4, have not been sufficiently developed. In this study, Agrobacterium tumefaciens-mediated transformation (ATMT) was investigated for M. alpina 1S-4 transformation, using the uracil-auxotrophic mutant (ura5 ؊ strain) of M. alpina 1S-4 as a host strain and the homologous ura5 gene as a selectable marker gene. Furthermore, the gene for 3-desaturase, catalyzing the conversion of n-6 fatty acid to n-3 fatty acid, was overexpressed in M. alpina 1S-4 by employing the ATMT system. As a result, we revealed that the frequency of transformation surpassed 400 transformants/10 8 spores, most of the integrated T-DNA appeared as a single copy at a random position in chromosomal DNA, and most of the transformants (60 to 80%) showed mitotic stability. Moreover, the accumulation of n-3 fatty acid in transformants was observed under the conditions of optimal 3-desaturase gene expression. In particular, eicosapentaenoic acid (20:5n-3), an end product of n-3 fatty acids synthesized in M. alpina 1S-4, reached a maximum of 40% of total fatty acids. In conclusion, the ATMT system was found to be effective and suitable for the industrial strain Mortierella alpina 1S-4 and will be a useful tool for basic mutagenesis research and for industrial breeding of this strain.
Chiral amino acids are important intermediates for the pharmaceutical industry.W eh ave developed an ovel one-pot enzymatic method for damino acid synthesisb yt he dynamic kinetic resolution of N-succinyl-dl-amino acids using d-succinylase (DSA) and N-succinylaminoa cid racemase (NSAR, EC 4.2.1.113). TheD SA from Cupriavidus sp.P 4-10-C,w hich hydrolyzes N-succinyl-d-amino acids enantioselectively to theirc orresponding damino acids,w as identifiedf or the first time by screenings oil microorganisms.S ubsequently,t he DSA gene was cloned ando verexpressed in Escherichia coli.D SA was shown to comprise two subunits with molecular masses of 26 kDa and 60 kDa.Additionally,t he NSAR gene from Geobacillus stearothermphilus NCA1503, which racemizes N-succinylamino acids,w as also cloneda nd overexpressed in E. coli.T he highly purified DSA and NSAR prepared from each recombinant E. coli were characterized and used for d-amino acid synthesis. Ao nepot enzymatic method converted1 00 mM N-succinyl-dl-phenylalaninet od-phenylalanine in 91.1% conversion with 86.7% ee.T his novele nzymatic method may be useful for the industrial production of many d-aminoacids.
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