Disruption Of The Aicd Protease And M1G1 Gene In Saccharmpis SFibulgera A11 Affects Enzymes Production Trehlse Accumlationm

Saccharomycopsis fibuligera is one species of the teleomorphic ascomycetous genera. It is found to actively accumulate trehalose from starch. This yeast is also found to secrete a large amount of amylases, acid protease andβ-glucosidase. Although S. fibuligera has great potential for application in both fermentation industry and medicine, very little has been known of the genetic system of S. fibuligera.The acid protease gene in S. fibuligera A11 was disrupted by integrating the HPT (hygromycin B phosphotransferase) gene into ORF (Open Reading Frame) of the acid protease gene. The mutant A11-a obtained could grow in the medium containing hygromycin. No clear zone formed by the mutant grown on the plate containing milk protein was observed whereas a big clear zone formed by the strain A11 was detected. The acid protease and amylases activities produced by the mutant within 3 days were 0.89 U/mL and 424.7 U/mL, respectively while those produced by the strain A11 were 13.5 U/mL and 259.9 U/mL, respectively. The amylases preparations produced by the mutant A11-a and the strain A11 kept 88.8% and 45.5% of amylase activity, respectively after they were incubated at 37℃for two days. Trehalose amount accumulated in the mutant cells was 28.3% (w/w) while that accumulated in the cells of S. fibuligera A11 was 23.6 (w/w).Highly thermosensitive and permeable mutants are the mutants from which intracellular contents can be released when they are incubated both in low osmolarity water and at non-permissive temperature (usually 37℃). After mutagenesis by using nitrosoguanidine, a highly thermosensitive and permeable mutant named A11-b was obtained from S. fibuligera A11-a, a trehalose overproducer in which the acid protease gene has been disrupted. Of the total trehalose, 73.8% was released from the mutant cells suspended in distilled water after they had been treated at 37℃overnight. However, only 10.0% of the total trehalose was released from the cells of S. fibuligera A11-a treated under the same conditions. The cell volume of the mutant cells suspended in distilled water and treated at 37℃overnight was much bigger than that of S. fibuligera A11-a treated under the same conditions. The cell growth and trehalose accumulation of the mutant were almost the same as those of S. fibuligera A11-a during the cultivation at the flask level and in a 5-l fermentor. Both could accumulate around 28.0% (w/w) trehalose from cassava starch. After purification, the trehalose crystal from the aqueous extract of the mutant was obtained.The MIG1(Multicopy Inhibitor of Glucose)gene of S. fibuligera A11 was cloned from the genomic DNA using the degenerated primers and inverse PCR. The MIG1 gene (1152 bp accession number: HM450676) encodes a 384-amino acid protein very similar to Mig1s from other fungi. Besides their highly conserved zinc fingers, the Mig1 proteins display short conserved motifs of possible significance in glucose repression. The MIG1 gene in S. fibuligera A11 was disrupted by integrating the HPT gene into ORF of the MIG1 gene. The disruptant A11-c obtained could grow in the media containing hygromycin and 2-deoxy-D-glucose, respectively. Amylases, acid protease andβ-glucosidase production by the disruptant and expression of the genes encodingα-amylase, glucoamylase, acid protease andβ-glucosidase in the disruptant were enhanced. This confirms that Mig1, the transcriptional repressor indeed also regulate expression of many genes and production of many extracellular enzymes in S. fibuligera A11.