Figure 1.
Carotenoid biosynthetic pathway of N. crassa.
The gene products responsible for each enzymatic reaction are indicated. NCU06054 is the name annotated for squalene synthetase in the N. crassa genome. Chemical changes from precursor molecules are shaded with different colors. Blue and red arrows indicate the predominant steps from phytoene upon illumination at 8°C or at 30°C, respectively. Squalene synthesis is also depicted in the initial steps. The boxed reaction (diapolycopene synthesis) is proposed from the data.
Figure 2.
Phenotype of the mutant #2666.
A: Left picture: slant cultures from the strain #2666 compared to the wild type and mutant JA26. The strains were grown for three days at 30°C in the dark and four days at 22°C under light. Right picture: flasks of the cultures used for the carotenoid analysis displayed in B and C. The cultures were incubated 48 h in the dark at 30°C and 24 h under illumination at 8°C. B: Absorption spectra of the crude carotenoid samples from the cultures of the wild type and the mutants JA26 and #2666. Peak wavelengths are indicated. C: HPLC profiles of the carotenoid mixtures from the samples shown on graph B. Absorption spectra of major peaks are shown in inner boxes.
Table 1.
Pigmentation and carotenoid content of the wild type and al-2 mutants.
Figure 3.
Spectra of the major carotenoid produced by the wild type strain, the reddish mutant #2666 and five leaky al-2 mutants.
The cultures were incubated 48 h in the dark at 30°C and 24 h under illumination at 8°C. Their carotenoids extracts were concentrated and separated by HPLC and spectra of the major peaks were displayed. Maximal and secondary absorbance peaks for neurosporaxanthin (469 and 497 nm) and apo-4′-lycopenoic acid (478 and 507 nm) are indicated by continuous and dashed grey bars, respectively.
Figure 4.
Detection of a carotenoid-like pigment in al-2 mutants.
A: Absorption spectra of the crude carotenoid samples from the cultures of the mutants #896 and the null mutants Δal-1 and Δal-2 incubated 48 h in the dark at 30°C and 24 h under illumination at 8°C. Relevant peak wavelengths are indicated. The spectrum of the wild type sample (WT) diluted about 150 times is shown for comparison. B: HPLC profiles of the carotenoid mixtures from the samples of the mutants displayed on the left. Absorption spectra of the major peaks are shown in inner boxes.
Figure 5.
Predicted AL-2 truncated polypeptides in the mutants #896, #897, #904, #913 and #914.
A: Scheme of the AL-2 wild type polypeptide and locations of the different mutations indicated in the text. The cyclase and phytoene synthase domains are indicated in orange and blue colors, respectively. Vertical bars: substitutions; upside triangles: small deletions; downside triangle: small insertion. FGSC numbers of the mutants are indicated for each mutation. B: Above: Map of conserved residues upon Clustal comparison of AL-2 from N. crassa (accession number L27652), with the cyclase/PS enzymes CarRA from F. fujikuroi (AJ426417) and P. blakesleeanus (CAB93661), CarRP from M. circinelloides (AJ250827), CrtYB from X. dendrorhous (AAO47570), and Car2 from U. maydis (UM06287). Full bars in the scheme indicate identical positions in the six proteins, 2/3 bars indicate conserved substitutions, and 1/3 bars indicate semi-conserved-substitutions (“*”, “:” and “.” symbols in the Clustal alignment, respectively). Small vertical bars over the scheme indicate amino acids involved in phytoene synthase catalytic domain. The small grey boxes indicate two short amino acid segments associated with substrate-Mg2+ binding. Below, schematic representation of predicted AL-2 truncated proteins. Random amino acid segments of the same reading frame are indicated with the same color at the carboxy ends of the polypeptides. The numbers below indicate predicted protein lengths plus number of random amino acids in the cases of frame-shift mutations.
Figure 6.
Point mutations in the al-2 alleles in the mutants #2666, #4014, #900 and #910.
A: Schematic representation of the mutations on the AL-2 polypeptide. Amino acid changes are indicated by black bars and silent mutations are indicated by green bars. Upper bars: mutations in strain #2666. Lower bars, mutations in strain, #4014. Dashed bar: mutation in strains #900 and #910. B: Comparison of the six polypeptide segments containing amino acid changes, taken from the Clustal analysis of AL-2 with the five fungal cyclase/PS enzymes mentioned in the legend of Figure 4. The location of the six protein segments are shown below the map of conserved residues. Numbers in parentheses indicate amino acids in the AL-2 protein. The sequences representing the mutations from each strain are boxed, and the affected residues are highlighted on blue background. In sequence number 3, the tryptophan affected in the mutant JA26 is indicated on yellow background. C: Proposed molecular events leading to the formation of the mutants #2666 and #4014. The X represents a cross-over in one of the backcrosses. Mutations are indicated with bars, as displayed in panel A. Relevant amino acid changes are boxed. Direct evidence for wild-type sequence is only available for wild type 1.
Figure 7.
In vivo activity of AL-2 alleles in lycopene-producing E. coli strains.
HPLC analyses of the carotenoids accumulated in E. coli cell expressing the genes crtE, crtB and crtI from E. herbicola and the al-2 alleles either from the wild type (pFarbeR-AL2) or mutants #896 and #897 (pFarbeR-896 and -897, respectively). Absorption spectra of the major peaks and carotene identifications are shown in inner boxes.
Figure 8.
Effect of light on transcript levels of genes al-1 and al-2 in the wild type and al-2 mutants.
Real-time RT-PCR analyses of RNA samples isolated from the wild-type strain and the al-2 mutants under investigation, grown in the dark (dark bars) or illuminated for 30 min (light bars). Data represent average and standard deviations of four determinations from two independent experiments. Relative mRNA levels for each gene are referred to the value from illuminated wild type samples in each set of experiments.