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Cloning and sequencing of carRA and carB genes from Blakeslea. Applications for carotenoid production

Cloning and characterization of the carB and carRA genes from the beta-carotene overproducing strain B. trispora F-744 (GenBank AY176662). An 8788 bp DNA region contained two complete ORFs read in opposite directions corresponding to the carB and carRA genes. Two introns were predicted in carB and one in carRA. Intron splicing in carRA was proved by cDNA sequence. General transcription signals were detected at the 5-flanking region (TATA and CAAT boxes) and 3-flanking region (AATAAA polyadenylation signal) as were poorly conserved APE-like elements (consensus sequence GAANNTTGCC) involved in gene regulation in response to light. Three copies of the binding site (TTCTTTGTTY) for the transcription factor Ste11 (a �TR box�) were found with minor changes in the carB-carRA promoter region of B. trispora. A conserved sequence (GCXTGTATXTTATAXAAAXAAXAA) including the TATA-box is present upstream of the translation start codon of carB (position -78) and carRA (position -68) genes. RNA secondary structures that can act as potential transcriptional terminators were downstream of carB and carRA genes.

Deduced amino acid sequences encoded by the carB and carRA genes of B. trispora F-744. The CarB deduced protein is similar in size, sequence, and signature pattern to other fungal phytoene dehydrogenases, with amino acid sequence similarity to phytoene dehydrogenases from M. circinelloides (81%), P. blakesleeanus (72%), Xanthophyllomyces dendrorhous (50%), N. crassa (49%), Fusarium fujikuroi (48%), and Cercospora nicotianae (47%). A consensus dinucleotide binding motif for the FAD superfamily was located at the N-terminus (IVVIGAGIGGTATAARLAREGFRVTVVEKNDFSGGRCSFIHHDGHRFDQG), and a bacterial-type phytoene dehydrogenase signature sequence, corresponding to a carotenoid-binding domain, was found at the C-terminus (NLFFVGASTHPGTGVPIVLAG). The deduced CarB protein has a highly hydrophobic region at the C-terminal end that is characteristic of membrane-associated proteins. The carRA deduced protein was similar to fungal proteins with lycopene cyclase/phytoene synthase activity, and had high similarity to lycopene cyclases/phytoene synthases from M. circinelloides (67%), P. blakesleeanus (55%), X. dendrorhous (31%), F. fujikuroi (29%), and N. crassa (28%). The CarRA protein had two different domains, as described for P. blakesleeanus, M. circinelloides, and X. dendrorhous: a hydrophobic transmembrane domain (R), located near to the 5'-end encoding lycopene cyclase (240 residues), and a hydrophilic domain, P, downstream that encodes phytoene synthase (362 residues). A putative protease cleavage site AQAILH (residues 241-246) that could split the polypeptide between residues 243 and 244 is present at the boundary between the two domains.

The wild type carB and carRA genes from B. trispora (GenBank AY176663). The B. trispora wild type (NRRL2457) and mutant (F-744 beta-carotene overproducing) strains both had the same hybridization pattern in Southern blots, suggesting that carB and carRA are present at a single copy per genome. The carB and carRA mutant and wild type genes differ from each other by two nucleotides per gene (four total differences). The A1791C mutation in carB substitutes an arginine for the wild-type serine at position 528 (S528R). An A1958G transition mutation occurred in the carB stop codon, changing the wild-type TAA to TAG, but not affecting the protein sequence. The T497C mutation in the carRA gene changes the wild-type proline at position 143 to a serine (P143S), while the C568T mutation does not change the amino acid encoded (glycine at 166). Transcripts of the carB (1.8 kb) and carRA (1.9 kb) genes were the expected size and present at a constant level during the fermentation.

Expression of the carB and carRA genes of B. trispora F-744 in M. circinelloides. Several transformants for each strain/plasmid combinations were grown on YNB plates. The mycelium of the MS8/pLeu4 and MS23/pEPM9 control transformants remained as white as the parental strains, while MS8/pALBT119 and MS23/pALBT120 had a weak yellowish coloration, suggesting the accumulation of carotenoids other than phytoene. Southern analyses of digested genomic DNA with probes from carRA and carB genes are consistent with autonomous replication, the absence of rearrangements, and the lack of hybridization with the endogenous carRP gene of M. circinelloides. The carotenoids accumulated by wild-type strains of M. circinelloides, mutants and transformants were analyzed by HPLC.

At present, beta-carotene is produced commercially by chemical synthesis. We are now using a new industrial process with B. trispora to produce beta-carotene. Although neither gene number nor sequence in B. trispora can yet be altered by transformation, the carB and carRA sequences described here can be used to guide the development of overproducing strains that are altered either in these genes themselves or in other genes that affect their expression.

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