Albert J. J. van Ooyen

High-Level Production of Beta-Carotene in Saccharomyces cerevisiae by Successive Transformation with Carotenogenic Genes from Xanthophyllomyces dendrorhous

ABSTRACT To determine whether Saccharomyces cerevisiae can serve as a host for efficient carotenoid and especially β-carotene production, carotenogenic genes from the carotenoid-producing yeast Xanthophyllomyces dendrorhous were introduced and overexpressed in S. cerevisiae. Because overexpression of these genes from an episomal expression vector resulted in unstable strains, the genes were integrated into genomic DNA to yield stable, carotenoid-producing S. cerevisiae cells. Furthermore, carotenoid production levels were higher in strains containing integrated carotenogenic genes. Overexpression of crtYB (which encodes a bifunctional phytoene synthase and lycopene cyclase) and crtI (phytoene desaturase) from X. dendrorhous was sufficient to enable carotenoid production. Carotenoid production levels were increased by additional overexpression of a homologous geranylgeranyl diphosphate (GGPP) synthase from S. cerevisiae that is encoded by BTS1. Combined overexpression of crtE (heterologous GGPP synthase) from X. dendrorhous with crtYB and crtI and introduction of an additional copy of a truncated 3-hydroxy-3-methylglutaryl-coenzyme A reductase gene (tHMG1) into carotenoid-producing cells resulted in a successive increase in carotenoid production levels. The strains mentioned produced high levels of intermediates of the carotenogenic pathway and comparable low levels of the preferred end product β-carotene, as determined by high-performance liquid chromatography. We finally succeeded in constructing an S. cerevisiae strain capable of producing high levels of β-carotene, up to 5.9 mg/g (dry weight), which was accomplished by the introduction of an additional copy of crtI and tHMG1 into carotenoid-producing yeast cells. This transformant is promising for further development toward the biotechnological production of β-carotene by S. cerevisiae.

Cloning and characterization of the astaxanthin biosynthetic gene encoding phytoene desaturase of Xanthophyllomyces dendrorhous.

The first carotenoid biosynthetic gene from the basidiomycetous yeast Xanthophyllomyces dendrorhous was isolated by heterologous complementation in Escherichia coli. The isolated gene, denominated as crtI, was found to encode for phytoene desaturase. The coding region is interrupted by 11 introns. The deduced amino acid sequence showed significant homology with its bacterial and eukaryotic counterparts, especially those of fungal origin. A plasmid containing the geranylgeranyl diphosphate synthase and phytoene synthase encoding genes from Erwinia uredovora was introduced in E. coli together with the phytoene desaturase encoding cDNA from X. dendrorhous. As a result, lycopene accumulation was observed in these transformants. We conclude that in X. dendrorhous the four desaturase steps, by which phytoene is converted into lycopene, are carried out by a single gene product.

Metabolic engineering of the carotenoid biosynthetic pathway in the yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma)

ABSTRACT The crtYB locus was used as an integrative platform for the construction of specific carotenoid biosynthetic mutants in the astaxanthin-producing yeast Xanthophyllomyces dendrorhous. The crtYB gene of X. dendrorhous, encoding a chimeric carotenoid biosynthetic enzyme, could be inactivated by both single and double crossover events, resulting in non-carotenoid-producing transformants. In addition, the crtYB gene, linked to either its homologous or a glyceraldehyde-3-phosphate dehydrogenase promoter, was overexpressed in the wild type and a β-carotene-accumulating mutant of X. dendrorhous. In several transformants containing multiple copies of the crtYB gene, the total carotenoid content was higher than in the control strain. This increase was mainly due to an increase of the β-carotene and echinone content, whereas the total content of astaxanthin was unaffected or even lower. Overexpression of the phytoene synthase-encoding gene (crtI) had a large impact on the ratio between mono- and bicyclic carotenoids. Furthermore, we showed that in metabolic engineered X. dendrorhous strains, the competition between the enzymes phytoene desaturase and lycopene cyclase for lycopene governs the metabolic flux either via β-carotene to astaxanthin or via 3,4-didehydrolycopene to 3-hydroxy-3′-4′-didehydro-β-ψ-caroten-4-one (HDCO). The monocylic carotenoid torulene and HDCO, normally produced as minority carotenoids, were the main carotenoids produced in these strains.

STRUCTURAL AND PHYLOGENETIC ANALYSIS OF THE ACTIN GENE FROM THE YEAST PHAFFIA RHODOZYMA

The gene coding for actin from Phaffia rhodozyma was cloned and sequenced. The Phaffia actin gene contains four intervening sequences and the predicted protein consists of 375 amino acids. The structural features of the Phaffia actin introns were studied and compared with actin introns from seven fungi and yeasts with ascomycetous and basidiomycetous affinity. It was shown that the architecture of the Phaffia introns most resembles that of the basidiomycete Filobasidiella neoformans (perfect stage of Cryptococcus neoformans), whereas least resemblance occurs with the ascomycetous yeasts. Based on the intron structure, the ascomycetous yeasts can be accommodated in one group in that their splice site sequences are very similar and show less homology with the other fungi investigated, including Phaffia. It was demonstrated that the Phaffia actin introns cannot be spliced in Saccharomyces cerevisiae, which shows that the differences found in intron structure are significant. Alignment of the Phaffia actin gene with the actin sequences from the yeasts and fungi investigated showed a high level of homology both on the DNA level and on the protein level. Based on these alignments Phaffia showed highest homology with F. neoformans and both organisms were accommodated in the same cluster. In addition, the actin gene comparisons also supported the distant relationship of Phaffia with the ascomycetous yeasts. These results supported the usefulness of actin sequences for phylogenetic studies. The sequence presented here has been submitted to the EMBL data library under Accession Number X89898.

Heterologous carotenoid production in Saccharomyces cerevisiae induces the pleiotropic drug resistance stress response

To obtain insight into the genome‐wide transcriptional response of heterologous carotenoid production in Saccharomyces cerevisiae, the transcriptome of two different S. cerevisiae strains overexpressing carotenogenic genes from the yeast Xanthophyllomyces dendrorhous grown in carbon‐limited chemostat cultures was analysed. The strains exhibited different absolute carotenoid levels as well as different intermediate profiles. These discrepancies were further sustained by the difference of the transcriptional response exhibited by the two strains. Transcriptome analysis of the strain producing high carotenoid levels resulted in specific induction of genes involved in pleiotropic drug resistance (PDR). These genes encode ABC‐type and major facilitator transporters which are reported to be involved in secretion of toxic compounds out of cells. β‐Carotene was found to be secreted when sunflower oil was added to the medium of S. cerevisiae cells producing high levels of carotenoids, which was not observed when added to X. dendrorhous cells. Deletion of pdr10, one of the induced ABC transporters, decreased the transformation efficiency of a plasmid containing carotenogenic genes. The few transformants that were obtained had decreased growth rates and lower carotenoid production levels compared to a pdr5 deletion and a reference strain transformed with the same genes. Our results suggest that production of high amounts of carotenoids in S. cerevisiae leads to membrane stress, in which Pdr10 might play an important role, and a cellular response to secrete carotenoids out of the cell. Copyright

Characterisation of Aspergillus niger prolyl aminopeptidase

We have cloned a gene (papA) that encodes a prolyl aminopeptidase from Aspergillus niger. Homologous genes are present in the genomes of the Eurotiales A. nidulans, A. fumigatus and Talaromyces emersonii, but the gene is not present in the genome of the yeast Saccharomyces cerevisiae. Cell extracts of strains overexpressing the gene under the control of its own promoter showed a fourfold to sixfold increase in prolyl aminopeptidase activity, but no change in phenylalanine or leucine aminopeptidase activity. The overexpressed enzyme was subsequently purified and characterised. The enzyme specifically removes N-terminal proline and hydroxyproline residues from peptides. It is the first enzyme of its kind from a eukaryotic organism that has been characterised.

Molecular Characterization of the Glyceraldehyde‐3‐phosphate Dehydrogenase Gene of Phaffia rhodozyma

The glyceraldehyde‐3‐phosphate dehydrogenase (GPD; EC1.2.1.12)‐encoding gene (gpd) was isolated from a genomic library of Phaffia rhodozyma CBS 6938. Unlike some other eukaryotic organisms the gpd gene is represented by a single copy in P. rhodozyma. The complete nucleotide sequence of the coding, as well as the flanking non‐coding regions was determined. The nucleotide sequence of gpd predicted six introns and a polypeptide chain of 339 amino acids. The codon usage in the gpd gene of P. rhodozyma was highly biased and was significantly different from the codon usage in other yeasts. Phylogenetic analysis of different yeasts and filamentous asco‐ and basidiomycetes gpd sequences indicated that the gpd gene of P. rhodozyma forms a cluster with the corresponding genes of filamentous basidiomycetes.

Cloning, characterization and heterologous expression of epoxide hydrolase-encoding cDNA sequences from yeasts belonging to the genera Rhodotorula and Rhodosporidium

Epoxide hydrolase-encoding cDNA sequences were isolated from the basidiomycetous yeast species Rhodosporidium toruloides CBS 349, Rhodosporidium toruloides CBS 14 and Rhodotorula araucariae CBS 6031 in order to evaluate the molecular data and potential application of this type of enzymes. The deduced amino acid sequences were similar to those of the known epoxide hydrolases from Rhodotorula glutinis CBS 8761, Xanthophyllomyces dendrorhous CBS 6938 and Aspergillus niger LCP 521, which all correspond to the group of the microsomal epoxide hydrolases. The epoxide hydrolase encoding cDNAs of the Rhodosporidium and Rhodotorula species were expressed in Escherichia coli. The recombinant strains were able to hydrolyze trans-1-phenyl-1,2-epoxypropane with high enantioselectivity.

Genes involved in carotene synthesis and mating in Blakeslea trispora

Mating of Blakeslea trispora and other molds of the order Mucorales requires the interaction of mycelia of opposite sex, (+) and (−), leading to the development of specialized structures and to an enhanced accumulation of β-carotene. Industry obtains β-carotene by co-cultivating appropriate strains of Blakeslea (“mated cultures”). Gene transcription in single and mated cultures was assayed by cDNA-AFLP, a technique to observe the differential expression of subsets of mRNA fragments. Overexpression in mated cultures is about ten times more frequent than underexpression. We obtained and sequenced fragments of 97 candidate genes that appeared to be overexpressed during mating and confirmed four of them by reverse transcription and real-time PCR. Comparisons with gene sequences from other organisms suggest functions in carotene biosynthesis (4 genes), energy metabolism (8), cell wall synthesis (1), transfer of acetyl groups (1), and regulatory processes (10). Sodium acetate inhibited sexual overexpression in about two-thirds of the candidate genes and acted as a signal with broad effects on the metabolism and the morphology of mated cultures. Our work offers new materials for the study of carotene biosynthesis and its regulation and for the improvement of carotene production with Mucorales.