RESUMO
The mutation sites on the Delta12 desaturase gene in Mortierella alpina Delta12 desaturase-defective mutants SR88, TM912, and Mut48 accumulating Mead acid were identified. Each mutation resulted in an amino acid replacement (H116Y and P166L) in the Delta12 desaturase gene from SR88 and Mut48, respectively.
Assuntos
Ácidos Graxos Dessaturases/genética , Mortierella/enzimologia , Mutação , Sequência de Bases , Primers do DNARESUMO
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.
Assuntos
Ácidos Graxos Dessaturases/metabolismo , Ácidos Graxos Insaturados/análise , Mortierella/química , Interferência de RNA , Ácido Araquidônico/biossíntese , Meios de Cultura , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Insaturados/biossíntese , Inativação Gênica , Microbiologia Industrial/métodos , Mortierella/genética , Mortierella/crescimento & desenvolvimento , Mortierella/metabolismo , RNA Fúngico/metabolismo , Transformação GenéticaRESUMO
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.
