The formation of protoplasts from Mucor species

Abstract

Methods were developed for the reproducible production of high yields of stable protoplasts from mesophilic and thermophilic strains of Mucor. This is a pre-requisite for the genetic analysis and manipulation of Mucor by cell fusion or transformation and is of special importance in these species since conventional genetic analysis by recombination studies is not feasible. The cell wall lyric enzyme used was the concentrated culture fluid of Streptomyces sp no. 6 grown on chitosan and chitin. Using germlings ofM. circinelloides f. lusitanicus the optimum conditions for protoplast formation were determined as: 0.5-1.0 rag. ml -~ streptozyme ( 1.2 units chitosanase, ml") plus 2.0 rag-ml ' novozym 234 or l%(v/v) heticase in 0.5 M-sorbitol, 0.01 M-sodium phosphate buffer, pH 6.5 for 3-4 hours at 23 ~ Treatment of 107 ml-' germlings gave yields of up to 3x 107. ml -' protoplasts with regeneration and fusion frequencies of up to 40% and 1.7%, respectively. With the thermophilic strains growth at low temperature (25 ~ was critical for the subsequent formation of stable protoplasts. Synchronous growth and germination was obtained at this temperature by a short pre-incubation (1-3 hours) of the sporangiospores at 40 ~

Biochemical events leading to the diversion of carbon into storage lipids in the oleaginous fungi Mucor circinelloides and Mortierella alpina

“…circinelloides was grown in 1 litre stirred bottles, as described above. Harvested biomass was washed twice with spheroplasting buffer (50 mM Tris\HCl, pH 6n5, containing 1n2 M sorbitol) then suspended in the same buffer containing chitinase\chitosanase preparation (Vanheeswijck, 1984) at 2 mg ml − " and incubated for 3 h at 30 mC. The treated biomass was collected by centrifugation (10 000 g for 10 min at 4 mC) and the spheroplasts formed were disrupted by resuspension in 50 mM Tris\HCl (pH 7n5) and incubated at 20 mC for 1 h. The disrupted cells were fractionated by centrifugation at 5000 g for 10 min and then the supernatant centrifuged at 16 000 g for 20 min.…”

Isolation and Use of a Homologous Histone H4 Promoter and a Ribosomal DNA Region in a Transformation Vector for the Oil-Producing FungusMortierella alpina

“…strain no. 6 (16) was obtained from the National Collection of Industrial Bacteria, Aberdeen, United Kingdom, and grown in a chitin-chitosan-containing minimal medium to produce "streptozyme" as described previously (34).…”

“…strain no. 6 (34). Approximately 10 g (wet weight) of mycelium in 15 ml of protoplasting buffer (1 M sorbitol-10 mM NaH 2 PO 4 -Na 2 HPO 4 [pH 6.5]) was incubated with 4 ml of filter-sterilized "streptozyme" in 20 mM NaH 2 PO 4 -Na 2 HPO 4 , pH 6.5, for 1 to 2 h with gentle shaking (60 to 80 rpm) at 25°C.…”

High frequency transformation of Mucor with recombinant plasmid DNA

“…Accompanying papers describe the production of stable protoplasts (19) and the isolation of a number ofphenotypically different auxotrophic mutants (31) in mesophilic and thermophilic Mucor species. The establishment of a transformation system and thereby introduction of recombinant DNA technology into the genetic study of Mucor was considered desirable for three reasons: (i) Formal genetic analysis of mutants by recombination is not feasible in these fungi due to the difficulties in utilising their sexual cycle.…”

“…The germlings were harvested by filtration through nylon cloth (Mon0dur, 22 lam) washed extensively with 0.01 M-sodium phosphate buffer pH 6.5, then incubated in the same buffer containing 0.5 M-sorbitol, 1.5 mg ml" Novozym 234 and 0.5 mg. m]" streptozyme (19), for 2-3 hours at 23 ~ A more detailed description of the formation of protoplasts is given in an accompanying paper (19). The protoplasts and any undigested cells remaining were pelleted by centrifugation (400 g, 5 min, room temperature) washed twice with 0.5 M-sorbitol, once with 0.5 M-sorbitol in MOPS buffer (10 mM-MOPS pH 6.3, 50 mMCaCI2), then resuspended in the same solution to give a final volume of 2 ml.…”