Xanthophyllomyces dendrorhous, a Versatile Platform for the Production of Carotenoids and Other Acetyl-CoA-Derived Compounds.

Xanthophyllomyces dendrorhous (with Phaffia rhodozyma as its anamorphic state) is a basidiomycetous, moderately psychrophilic, red yeast belonging to the Cystofilobasidiales. Its red pigmentation is caused by the accumulation of astaxanthin, which is a unique feature among fungi. The present chapter reviews astaxanthin biosynthesis and acetyl-CoA metabolism in X. dendrorhous and describes the construction of a versatile platform for the production of carotenoids, such as astaxanthin, and other acetyl-CoA-derived compounds including fatty acids by using this fungus.

Engineering of the carotenoid pathway in Xanthophyllomyces dendrorhous leading to the synthesis of zeaxanthin.

Zeaxanthin is an essential nutrient for prevention of macular degeneration. However, it is limited in our diet. For the production of zeaxanthin, we have engineered zeaxanthin synthesis into a carotenoid mutant of Xanthophyllomyces dendrorhous which is blocked in astaxanthin synthesis and accumulates ß-carotene instead. Two strategies were followed to reach high-yield zeaxanthin synthesis. Total carotenoid synthesis was increased by over-expression of genes HMGR, crtE, and crtYB encoding for limiting enzymes in the pathway leading to and into carotenoid biosynthesis. Then bacterial genes crtZ were used to extend the pathway from ß-carotene to zeaxanthin in this mutant. The increase of total carotenoids and the formation of zeaxanthin is dependent on the number of gene copies of crtYB and crtZ integrated into the X. dendrorhous upon transformation. The highest zeaxanthin content around 500 µg/g dw was reached by shaking flask cultures after codon optimization of crtZ for Xanthophyllomyces. Stabilization of carotenoid and zeaxanthin formation in the final transformant in the absence of selection agents was achieved after passing through a sexual cycle and germination of basidiospores. The values for the transformant before and after stabilization were very similar resembling about 70 % of total carotenoids and corresponding to a conversion rate of 80 % for hydroxylation of ß-carotene to zeaxanthin. The stabilized transformant allowed experimental small-scale fermentation yielding X. dendrorhous cells with a zeaxanthin content similar to the shaking flask cultures. Our result demonstrates the potential of X. dendrorhous for its development as a zeaxanthin producer and its suitability for large-scale fermentation.

Metabolic engineering of astaxanthin biosynthesis in maize endosperm and characterization of a prototype high oil hybrid.

Maize was genetically engineered for the biosynthesis of the high value carotenoid astaxanthin in the kernel endosperm. Introduction of a ß-carotene hydroxylase and a ß-carotene ketolase into a white maize genetic background extended the carotenoid pathway to astaxanthin. Simultaneously, phytoene synthase, the controlling enzyme of carotenogenesis, was over-expressed for enhanced carotenoid production and lycopene ε-cyclase was knocked-down to direct more precursors into the ß-branch of the extended ketocarotenoid pathway which ends with astaxanthin. This astaxanthin-accumulating transgenic line was crossed into a high oil- maize genotype in order to increase the storage capacity for lipophilic astaxanthin. The high oil astaxanthin hybrid was compared to its astaxanthin producing parent. We report an in depth metabolomic and proteomic analysis which revealed major up- or down- regulation of genes involved in primary metabolism. Specifically, amino acid biosynthesis and the citric acid cycle which compete with the synthesis or utilization of pyruvate and glyceraldehyde 3-phosphate, the precursors for carotenogenesis, were down-regulated. Nevertheless, principal component analysis demonstrated that this compositional change is within the range of the two wild type parents used to generate the high oil producing astaxanthin hybrid.

Engineered maize as a source of astaxanthin: processing and application as fish feed.

Astaxanthin from a transgenic maize line was evaluated as feed supplement source conferring effective pigmentation of rainbow trout flesh. An extraction procedure using ethanol together with the addition of vegetal oil was established. This resulted in an oily astaxanthin preparation which was not sufficiently concentrated for direct application to the feed. Therefore, a concentration process involving multiple phase partitioning steps was implemented to remove 90 % of the oil. The resulting astaxanthin raw material contained non-esterified astaxanthin with 12 % 4-keto zeaxanthin and 2 % zeaxanthin as additional carotenoids. Isomeric analysis confirmed the exclusive presence of the 3S, 3'S astaxanthin enantiomer. The geometrical isomers were 89 % all-E, 8 % 13-Z and 3 % 9-Z. The incorporation of the oily astaxanthin preparation into trout feed was performed to deliver 7 mg/kg astaxanthin in the final feed formulation for the first 3.5 weeks and 72 mg/kg for the final 3.5 weeks of the feeding trial. The resulting pigmentation of the trout fillets was determined by hue values with a colour meter and further confirmed by astaxanthin quantification. Pigmentation properties of the maize-produced natural astaxanthin incorporated to 3.5 µg/g dw in the trout fillet resembles that of chemically synthesized astaxanthin. By comparing the relative carotenoid compositions in feed, flesh and feces, a preferential uptake of zeaxanthin and 4-keto zeaxanthin over astaxanthin was observed.

Control of light-dependent keto carotenoid biosynthesis in Nostoc 7120 by the transcription factor NtcA.

In Nostoc PCC 7120, two different ketolases, CrtW and CrtO are involved in the formation of keto carotenoids from ß-carotene. In contrast to other cyanobacteria, CrtW catalyzes the formation of monoketo echinenone whereas CrtO is the only enzyme for the synthesis of diketo canthaxanthin. This is the major photo protective carotenoid in this cyanobacterium. Under high-light conditions, basic canthaxanthin formation was transcriptionally up-regulated. Upon transfer to high light, the transcript levels of all investigated carotenogenic genes including those coding for phytoene synthase, phytoene desaturase and both ketolases were increased. These transcription changes proceeded via binding of the transcription factor NtcA to the promoter regions of the carotenogenic genes. The binding was absolutely dependent on the presence of reductants and oxo-glutarate. Light-stimulated transcript formation was inhibited by DCMU. Therefore, photosynthetic electron transport is proposed as the sensor for high-light and a changing redox state as a signal for NtcA binding.

Multiple transformation with the crtYB gene of the limiting enzyme increased carotenoid synthesis and generated novel derivatives in Xanthophyllomyces dendrorhous.

Xanthophyllomces dendrorhous (in asexual state named as Phaffia rhodozyma) is a fungus which produces astaxanthin, a high value carotenoid used in aquafarming. Genetic pathway engineering is one of several steps to increase the astaxanthin yield. The limiting enzyme of the carotenoid pathway is phytoene synthase. Integration plasmids were constructed for transformation with up to three copies of the crtYB gene. Upon stepwise transformation, the copy numbers of crtYB was continuously increased leading to an almost saturated level of phytoene synthase as indicated by total carotenoid content. Several carotenoid intermediates accumulated which were absent in the wild type. Some of them are substrates and intermediates of astaxanthin synthase. They could be further converted into astaxanthin by additional transformation with the astaxanthin synthase gene. However, three intermediates exhibited an unusual optical absorbance spectrum not found before. These novel keto carotenoid were identified by HPLC co-chromatography with reference compounds generated in Escherichia coli and one of them 3-HO-4-keto-7',8'-dihydro-ß-carotene additionally by NMR spectroscopy. The others were 4-keto-ß-zeacarotene and 4-keto-7',8'-dihydro-ß-carotene. A biosynthesis pathway with their origin from neurosporene and the reason for their synthesis especially in our transformants has been discussed.

Genetic engineering of the complete carotenoid pathway towards enhanced astaxanthin formation in Xanthophyllomyces dendrorhous starting from a high-yield mutant.

The yeast Xanthophyllomyces dendrorhous is one of the rare organisms which can synthesize the commercially interesting carotenoid astaxanthin. However, astaxanthin yield in wild-type and also in classical mutants is still too low for an attractive bioprocess. Therefore, we combined classical mutagenesis with genetic engineering of the complete pathway covering improved precursor supply for carotenogenesis, enhanced metabolite flow into the pathway, and efficient conversion of intermediates into the desired end product astaxanthin. We also constructed new transformation plasmids for the stepwise expression of the genes of 3-hydroxymethyl-3-glutaryl coenzyme A reductase, geranylgeranyl pyrophosphate synthase, phytoene synthase/lycopene cyclase, and astaxanthin synthase. Starting from two mutants with a 15-fold higher astaxanthin, we obtained transformants with an additional 6-fold increase in the final step of pathway engineering. Thus, a maximum astaxanthin content of almost 9 mg per g dry weight was reached in shaking cultures. Under optimized fermenter conditions, astaxanthin production with these engineered transformants should be comparable to Haematococcus pluvialis, the leading commercial producer of natural astaxanthin.

Catalytic properties and reaction mechanism of the CrtO carotenoid ketolase from the cyanobacterium Synechocystis sp. PCC 6803.

CrtW and CrtO are two distinct non-homologous ß-carotene ketolases catalyzing the formation of echinenone and canthaxanthin. CrtO belongs to the CrtI family which comprises carotene desaturases and carotenoid oxidases. The CrtO protein from Synechocystis sp. PCC 6803 has been heterologously expressed, extracted and purified. Substrate specificity has been determined in vitro. The enzyme from Synechocystis is basically a mono ketolase. Nevertheless, small amounts of diketo canthaxanthin can be formed. The poor diketolation reaction could be explained by the low relative turnover numbers for the mono keto echinenone. Also other carotenoids with an unsubstituted ß-ionone ring were utilized with low conversion rates by CrtO regardless of the substitutions at the other end of the molecule. The CrtO ketolase was independent of oxygen and utilized an oxidized quinone as co-factor. In common to CrtI-type desaturases, the first catalytic step involved hydride transfer to the quinone. The stabilization reaction of the resulting carbo cation was a reaction with OH(-) forming a hydroxy group. Finally, the keto group resulted from two subsequent hydroxylations at the same C-atom and water elimination. This reaction mechanism was confirmed by in vitro conversion of the postulated hydroxy intermediates and by their enrichment and identification as trace intermediates during ketolation.

Contribution of two ζ-carotene desaturases to the poly-cis desaturation pathway in the cyanobacterium Nostoc PCC 7120.

The presence of two completely unrelated ζ-carotene desaturases CrtQa and CrtQb in some Nostoc strains is unique. CrtQb is the ζ-carotene desaturase, which was acquired by almost all cyanobacteria. The additional CrtQa can be regarded as an evolutionary relict of the CrtI desaturase present in non-photosynthetic bacteria. By reconstruction of the carotene desaturation pathway, we showed that both enzymes from Nostoc PCC 7120 were active. However, they differed in their preferred utilization of ζ-carotene Z isomers. CrtQa converted ζ-carotene isomers that were poorly metabolized by CrtQb. In this respect, CrtQa complemented the reactions of CrtQb, which is an advantage avoiding dead ends in the poly-cis desaturation pathway. In addition to ζ-carotene desaturation, CrtQa still possesses the Z to E isomerase function of the ancestral desaturase CrtI. Biochemical characterization showed that CrtQb is an enzyme with one molecule of tightly bound FAD and acts as a dehydrogenase transferring hydrogen to oxidized plastoquinone.

Biosynthesis of fucoxanthin and diadinoxanthin and function of initial pathway genes in Phaeodactylum tricornutum.

The biosynthesis pathway to diadinoxanthin and fucoxanthin was elucidated in Phaeodactylum tricornutum by a combined approach involving metabolite analysis identification of gene function. For the initial steps leading to ß-carotene, putative genes were selected from the genomic database and the function of several of them identified by genetic pathway complementation in Escherichia coli. They included genes encoding a phytoene synthase, a phytoene desaturase, a ζ-carotene desaturase, and a lycopene ß-cyclase. Intermediates of the pathway beyond ß-carotene, present in trace amounts, were separated by TLC and identified as violaxanthin and neoxanthin in the enriched fraction. Neoxanthin is a branching point for the synthesis of both diadinoxanthin and fucoxanthin and the mechanisms for their formation were proposed. A single isomerization of one of the allenic double bounds in neoxanthin yields diadinoxanhin. Two reactions, hydroxylation at C8 in combination with a keto-enol tautomerization and acetylation of the 3'-HO group results in the formation of fucoxanthin.

Transgenic multivitamin corn through biofortification of endosperm with three vitamins representing three distinct metabolic pathways.

Vitamin deficiency affects up to 50% of the world's population, disproportionately impacting on developing countries where populations endure monotonous, cereal-rich diets. Transgenic plants offer an effective way to increase the vitamin content of staple crops, but thus far it has only been possible to enhance individual vitamins. We created elite inbred South African transgenic corn plants in which the levels of 3 vitamins were increased specifically in the endosperm through the simultaneous modification of 3 separate metabolic pathways. The transgenic kernels contained 169-fold the normal amount of beta-carotene, 6-fold the normal amount of ascorbate, and double the normal amount of folate. Levels of engineered vitamins remained stable at least through to the T3 homozygous generation. This achievement, which vastly exceeds any realized thus far by conventional breeding alone, opens the way for the development of nutritionally complete cereals to benefit the world's poorest people.

Engineering of geranylgeranyl pyrophosphate synthase levels and physiological conditions for enhanced carotenoid and astaxanthin synthesis in Xanthophyllomyces dendrorhous.

The basidiomycetous yeast, Xanthophyllomyces dendrorhous, is one of the very few organisms which can be used for biological production of the carotenoid astaxanthin. crtE cDNA has been cloned from this fungus for engineering of the terpenoid pathway. The function of its gene product as a geranylgeranyl pyrophosphate synthase was established. X. dendrorhous was transformed with the crtE cDNA to divert metabolite flow from the sterol pathway towards carotenoid biosynthesis. Transformants were obtained with increased levels of geranylgeranyl pyrophosphate synthase leading to higher carotenoid levels including astaxanthin. Physiological conditions for maximum carotenoid synthesis for wild type and the CrtE transformant were dim light and extra air supply of the shaking culture. These conditions and the transformation with crtE had additive effects and resulted in an 8-fold higher astaxanthin formation as compared to the initial wild type culture without illumination and extra air supply yielding 451 µg/g dry wt within 4 days of growth.

Combinatorial genetic transformation generates a library of metabolic phenotypes for the carotenoid pathway in maize.

Combinatorial nuclear transformation is a novel method for the rapid production of multiplex-transgenic plants, which we have used to dissect and modify a complex metabolic pathway. To demonstrate the principle, we transferred 5 carotenogenic genes controlled by different endosperm-specific promoters into a white maize variety deficient for endosperm carotenoid synthesis. We recovered a diverse population of transgenic plants expressing different enzyme combinations and showing distinct metabolic phenotypes that allowed us to identify and complement rate-limiting steps in the pathway and to demonstrate competition between beta-carotene hydroxylase and bacterial beta-carotene ketolase for substrates in 4 sequential steps of the extended pathway. Importantly, this process allowed us to generate plants with extraordinary levels of beta-carotene and other carotenoids, including complex mixtures of hydroxycarotenoids and ketocarotenoids. Combinatorial transformation is a versatile approach that could be used to modify any metabolic pathway and pathways controlling other biochemical, physiological, or developmental processes.

Cloning and functional characterization of the maize carotenoid isomerase and ß-carotene hydroxylase genes and their regulation during endosperm maturation.

In order to gain further insight into the partly-characterized carotenoid biosynthetic pathway in corn (Zea mays L.), we cloned cDNAs encoding the enzymes carotenoid isomerase (CRTISO) and ß-carotene hydroxylase (BCH) using endosperm mRNA isolated from inbred line B73. For both enzymes, two distinct cDNAs were identified mapping to different chromosomes. The two crtiso cDNAs (Zmcrtiso1 and Zmcrtiso2) mapped to unlinked genes each containing 12 introns, a feature conserved among all crtiso genes studied thus far. ZmCRTISO1 was able to convert tetra-cis prolycopene to all-trans lycopene but could not isomerize the 15-cis double bond of 9,15,9'-tri-cis-ζ-carotene. ZmCRTISO2 is inactivated by a premature termination codon in B73 corn, but importantly the mutation is absent in other corn cultivars and the active enzyme showed the same activity as ZmCRTISO1. The two bch cDNAs (Zmbch1 and Zmbch2) mapped to unlinked genes each coding sequences containing five introns. ZmBCH1 was able to convert ß-carotene into ß-cryptoxanthin and zeaxanthin, but ZmBCH2 was able to form ß-cryptoxanthin alone and had a lower overall activity than ZmBCH1. All four genes were expressed during endosperm development, with mRNA levels rising in line with carotenoid accumulation (especially zeaxanthin and lutein) until 25 DAP. Thereafter, expression declined for three of the genes, with only Zmcrtiso2 mRNA levels maintained by 30 DAP. We discuss the impact of paralogs with different expression profiles and functions on the regulation of carotenoid synthesis in corn.

Carotenoids and carotenogenic genes in Podospora anserina: engineering of the carotenoid composition extends the life span of the mycelium.

Carotenoids have been identified in the fungus Podospora anserina and a parallel pathway to neurosporene and beta-carotene was established. Three genes for the beta-carotene branch have been cloned and their function elucidated. They correspond to the al-1, al-2 and al-3 genes from Neurospora crassa. They were individually and in combinations over-expressed in P. anserina in order to modify the carotenoid composition qualitatively and quantitatively. In the resulting transformants, carotenoid synthesis was up to eightfold increased and several intermediates of the pathway together with special cyclic carotenoids, beta-zeacarotene and 7,8-dihydro-beta-carotene, accumulated. All transformants with an over-expressed al-2 gene (encoding a phytoene synthase and a lycopene cyclase) displayed up to 31% prolonged life span.

Structure, function and biosynthesis of carotenoids in the moderately halophilic bacterium Halobacillus halophilus.

Inhibitor studies and mutant analysis revealed a C(30) pathway via 4,4'-diapophytoene and 4,4'-diaponeurosporene to 4,4'-diaponeursoporene-4-oic acid esters related to staphyloxanthin in Halobacillus halophilus. Six genes may be involved in this biosynthetic pathway and could be found in two adjacent gene clusters. Two genes of this pathway could be functionally assigned by functional pathway complementation as a 4,4'-diapophytoene synthase and a 4,4'-diapophytoene desaturase gene. These genes were organized in two operons together with two putative oxidase genes, a glycosylase and an acyl transferase ortholog. Pigment mutants were obtained by chemical mutagenesis. Carotenoid analysis showed that a white mutant accumulated 4,4'-diapophytoene due to a block in desaturation. In a yellow mutant carotenogenesis was blocked at the stage of 4,4'-diaponeurosporene and in an orange mutant at the stage of 4,4'-diaponeurosporene-4-oic acid. The protective function of these pigments could be demonstrated for H. halophilus after inhibition of carotenoid synthesis by initiation of oxidative stress. A degree of oxidative stress which still allowed 50% growth of carotenogenic cells resulted in the death of the cells devoid of colored carotenoids.

Genetic modification of the carotenoid pathway in the red yeast Xanthophyllomyces dendrorhous: Engineering of a high-yield zeaxanthin strain.

The red yeast Xanthophyllomyces dendrorhous was genetically engineered for high-yield accumulation of the carotenoid zeaxanthin. Initially, an astaxanthin hyper-producing mutant was used to generate a ß-carotene synthesizing transformant by inactivation of the astaxanthin synthase gene. Subsequently, a bacterial ß-carotene hydroxylase gene was genome integrated to establish ß-carotene to zeaxanthin conversion. Crucial for efficient zeaxanthin formation was the rate of this hydroxylation which was related to the number of integrated gene copies. Two strategies were followed to get multiple integrations, either random integration into the ribosomal DNA which resulted in a maximum copy number of 10, or directly integration of a total of 8 copies into both alleles of the astaxanthin synthase gene. Combining both procedures with additional insertion of the gene to enhance expression of the carotenogenesis limiting phytoene synthase, a transformant reaching a high level of zeaxanthin of 5.2 mg/g dw was finally generated. The application of pentose sugars including xylose as substrates for X. dendrorhous which avoids the inhibitory Crab-tree effect of glucose is favorable for carotenogenesis allowing the replacement of glucose by a hydrolysate of the waste product hemicellulose which is rich in xylose demonstrating ithe effectiveness as a sustainable and cost-efficient alternative for high-yield zeaxanthin formation.

Expanding the Isoprenoid Building Block Repertoire with an IPP Methyltransferase from Streptomyces monomycini.

Many synthetic biology approaches aim at expanding the product diversity of enzymes or whole biosynthetic pathways. However, the chemical structure space of natural product forming routes is often restricted by the limited cellular availability of different starting intermediates. Although the terpene biosynthesis pathways are highly modular, their starting intermediates are almost exclusively the C5 units IPP and DMAPP. To amplify the possibilities of terpene biosynthesis through the modification of its building blocks, we identified and characterized a SAM-dependent methyltransferase converting IPP into a variety of C6 and C7 prenyl pyrophosphates. Heterologous expression in Escherichia coli not only extended the intracellular prenyl pyrophosphate spectrum with mono- or dimethylated IPP and DMAPP, but also enabled the biosynthesis of C11, C12, C16, and C17 prenyl pyrophosphates. We furthermore demonstrated the general high promiscuity of terpenoid biosynthesis pathways toward uncommon building blocks by the E. coli-based production of polymethylated C41, C42, and C43 carotenoids. Integration of the IPP methyltransferase in terpene synthesis pathways enables an expansion of the terpenoid structure space beyond the borders predetermined by the isoprene rule which indicates a restricted synthesis by condensation of C5 units.

Development of Xanthophyllomyces dendrorhous as a production system for the colorless carotene phytoene.

Phytoene is a colorless carotenoid with increasing economic potential for skin care but with limited availability. The red yeast Xanthophyllomyces dendrorhous which has previously been used as a production platform for carotenoids was engineered as a prototype for the yield of this carotene. Phytoene was accumulated by prevention of its metabolization by desaturation in the carotenoid pathway. In a first step, the phytoene desaturase gene crtI was disrupted by insertion of a hygromycin-resistance gene. Most of the resulting transformants were heterozygote for intact and inactivated crtI. Upon re-cultivation of this orange transformants under selection pressure, white colonies homozygote for disrupted crtI were obtained. In contrast to reddish wild-type, the orange transformants contained colored carotenoids together with phytoene whereas the homozygote transformant synthesized phytoene exclusively. This targeted mutagenesis approach was first tested with the wild type and then applied to a high-yield carotenoid synthesizing X. dendrorhous mutant. In a second step, precursor supply for phytoene synthesis was enhanced by over-expression of the genes HMGR, crtE and crtYB which encode limiting enzymes of the pathway. The combination of this engineering approaches resulted in a phytoene producing X. dendrorhous strain which accumulated 7.5mg/g dw in shaking cultures. Finally, experimental small scale fermenter studies demonstrated continuous growth of this strain during fermentation and stable phytoene production without selection pressure. This fermenter culture contained the highest phytoene content ever reached by any organism with more than 10mg/g dw.

Combinatorial Biosynthesis of Novel Multi-Hydroxy Carotenoids in the Red Yeast Xanthophyllomyces dendrorhous.

The red yeast Xanthophyllomyces dendrorhous is an established platform for the synthesis of carotenoids. It was used for the generation of novel multi oxygenated carotenoid structures. This was achieved by a combinatorial approach starting with the selection of a ß-carotene accumulating mutant, stepwise pathway engineering by integration of three microbial genes into the genome and finally the chemical reduction of the resulting 4,4'-diketo-nostoxanthin (2,3,2',3'-tetrahydroxy-4,4'-diketo-ß-carotene) and 4-keto-nostoxanthin (2,3,2',3'-tetrahydroxy-4-monoketo-ß-carotene). Both keto carotenoids and the resulting 4,4'-dihydroxy-nostoxanthin (2,3,4,2',3',4'-hexahydroxy-ß-carotene) and 4-hydroxy-nostoxanthin (2,3,4,2'3'-pentahydroxy-ß-carotene) were separated by high-performance liquid chromatography (HPLC) and analyzed by mass spectrometry. Their molecular masses and fragmentation patterns allowed the unequivocal identification of all four carotenoids.