A single gene for lycopene cyclase, phytoene synthase, and regulation of carotene biosynthesis in Phycomyces

Abstract

Previous complementation and mapping of mutations that change the usual yellow color of the Zygomycete Phycomyces blakesleeanus to white or red led to the definition of two structural genes for carotene biosynthesis. We have cloned one of these genes, carRA, by taking advantage of its close linkage to the other, carB, responsible for phytoene dehydrogenase. The sequences of the wild type and six mutants have been established, compared with sequences in other organisms, and correlated with the mutant phenotypes. The carRA and carB coding sequences are separated by 1,381 untranslated nucleotides and are divergently transcribed. Gene carRA contains separate domains for two enzymes, lycopene cyclase and phytoene synthase, and regulates the overall activity of the pathway and its response to physical and chemical stimuli from the environment. The lycopene cyclase domain of carRA derived from a duplication of a gene from a common ancestor of fungi and Brevibacterium linens; the phytoene synthase domain is similar to the phytoene and squalene synthases of many organisms; but the regulatory functions appear to be specific to Phycomyces.

Figure 1

Genes, enzymes, and chemical reactions for carotene biosynthesis in Phycomyces. Two molecules of geranylgeranyl pyrophosphate are converted to one molecule of β-carotene by phytoene synthase (diamonds), four copies of phytoene dehydrogenase (squares), and two copies of lycopene cyclase (circles). The scheme on the left represents the genes that code for these enzymes. The chemical changes are marked with arrowheads.

Figure 2

Structural genes for carotene biosynthesis in Phycomyces. (A) A DNA region that contains genes carB and carRA. Probes B and R were used to screen a lambda library and a partial plasmid library and isolate the 14-kb and the 9-kb fragments, respectively. Sequencing reactions are indicated by the thin arrows S1 through S13. The gray segment represents the DNA sequence published by Ruiz-Hidalgo et al. (19). (B) A 6494-bp DNA sequence that contains genes carB and carRA. Hatched segments represent introns and white segments represent intergenic sequences. Thin arrows represent genomic sequencing reactions, and thicker arrows below represent cDNA sequencing reactions. The target sites of restriction endonucleases are: B, BglII; Bm, BamHI; C, ClaI; E, EcoRI; H, HindIII; K, KpnI; P, PstI; S, SacI; Sp, SpeI; V, EcoRV; X, XhoI; and Xb, XbaI.

Figure 3

Hybridization of probe R (Fig. 2A) with Phycomyces genomic DNA fragments cut with several restriction enzymes (abbreviated as in Fig. 2) and separated by electrophoresis. The two pictures differ in the stringency of the test (washed at 65°C, Left, and 42°C, Right).

Figure 4

Hydrophobicity index of the predicted CarRA protein sequence. The vertical dotted line marks the location of the putative protease cleavage site AHAIV. The sites of the mutations in six color mutants are indicated below.

Figure 5

Alignment of the protein sequences Pb, Mc, Nc, and Xd predicted from the sequences of genes carRA from Phycomyces blakesleeanus, al-2 (ref. ; GenBank accession no. L27652) from Neurospora crassa, crtYB (ref. ; accession no. AJ133646) from Xanthophyllomyces dendrorhous (syn. Rhodomyces dendrorhous and Phaffia rhodozyma), and carRP (ref. ; accession no. AJ250827) from Mucor circinelloides. The line marks the putative cleavage site in the Phycomyces sequence at the approximate limit of the lycopene cyclase and phytoene synthase domains. The black circles locate the mutations in six Phycomyces color mutants. The arrowheads indicate the presence of introns in the gene sequence.

Figure 6

Alignment of two parts of the lycopene cyclase domain of the CarRA protein from Phycomyces and proteins CrtYc and CrtYd from Brevibacterium linens (ref. ; GenBank accession no. AF139916). CarRA(1) and CarRA(2) represent the 133-aa sequence that precedes the intron location and the next 128 aa, respectively. The black circles locate missense mutations in red mutants of Phycomyces.