Heterologous Expression of the Phycomyces blakesleeanus Phytoene Dehydrogenase Gene ( carB ) in Mucor circinelloides

Abstract

A phytoene dehydrogenase-deficient mutant of Mucor circinelloides accumulating only phytoene was transformed with the gene encoding the corresponding enzyme (carB gene) of Phycomyces blakesleeanus. Carotenoids derived from phytoene were detected in the transformants showing that the P. blakesleeanus carB gene complements the M. circinelloides carB mutation. These newly formed carotenoids accumulated in low quantities, indicating that functional complementation was poor. carB mRNA molecules correctly transcribed were detected in the transformants, but they represented a small proportion of the total population of carB-derived mRNAs, mostly constituted by truncated transcripts and by transcripts longer than the transcript that is functional in Phycomyces. These results showed that the P. blakesleeanus carB gene was expressed in M. circinelloides and suggested that the poor complementation observed was owing, at least in part, to the lack of specificity in the recognition of the transcription initiation and termination signals of the P. blakesleeanus carB gene by the M. circinelloides transcriptional machinery.

Genetic damage following introduction of DNA in Phycomyces

“…Self-replicative transformation seems to prevail, in contrast with the predominance of integrative transformation in other fungi (Fincham, 1989). The best results are obtained with Mucor circinelloides, an obscure organism until it was found that it is transformable at high rates, about 1 transformed colony per ng DNA and 10 6 spheroplasts, and that many of its gene sequences support self replication of plasmids (Benito et al, , 1995Heeswijck and Roncero, 1984;Heeswijck, 1986;Iturriaga et al, 1992;Ruiz-Hidalgo et al, 1999;Velayos et al, 1998). The transformed phenotype is much less frequent and very unstable in Absidia glauca (Burmester, 1995;Burmester et al, 1992;W€ o ostemeyer et al, 1987), Parasitella simplex (Burmester, 1992), Rhizopus niveus (Takaya et al, 1996), Rhizopus oryzae (Skory, 2002), and Phycomyces blakesleeanus (see below).…”

Blue-light regulation of phytoene dehydrogenase ( carB ) gene expression in Mucor circinelloides

“…In M. circinelloides the carotenogenic pathway appears to be rather similar to that in P. blakesleeanus, except for the negative endproduct regulation (Ruiz-Hidalgo et al 1995;Velayos et al 1997). A very ecient transformation system has been developed for this fungus (Van Heeswijck and Roncero 1984), and genes from P. blakesleeanus have been successfully expressed in M. circinelloides (Iturriaga et al 1992;Benito et al 1995;Ruiz-Hidalgo et al 1999).…”

The crtS gene of Xanthophyllomyces dendrorhous encodes a novel cytochrome-P450 hydroxylase involved in the conversion of β-carotene into astaxanthin and other xanthophylls

“…Plasmid pALMC11 was constructed, to express the crtS gene in M. circinelloides MS12 (leuA1, pyrG4), by inserting into pLeu4 (Roncero et al, 1989) the cDNA from the crtS gene (1.6 kb NcoI-NotI) expressed under the control of the carRA promoter from Blakeslea trispora (611 pb NcoI; Rodríguez-Sáiz et al, 2004). M. circinelloides MS12 was transformed as previously described (Ruiz-Hidalgo et al, 1999) with a mix of 1.5 mg/ml Caylase (Cayla, Toulouse, France) and 4-12 U/ml of Streptozyme for protoplast preparation. Several transformants of each plasmid, prototrophic for leucine, were selected on YNB minimal medium (Lampila et al, 1985) supplemented with uridine and osmotically stabilized with 0.55 M sorbitol.…”