Increased Astaxanthin Production Research Articles

Utilization of silicon dioxide nanoparticles and silicon salts to enhance astaxanthin production in Haematococcus Pluvialis

Haematococcus pluvialis is a type of microalgae that is commercially important as it is the primary source of natural astaxanthin - a potent antioxidant used in nutraceuticals, cosmetics, food, and aquaculture industries. Various nanoparticles and chemicals have been used to stimulate the growth of H. pluvialis to increase astaxanthin production. In this study, silicon nanoparticles were synthesized from tetraethyl orthosilicate (TEOS) and characterized to ensure the pure synthesis of uniform nanocrystals of silicon dioxide. The microalgae were cultivated in optimal Haematococcus medium (OHM) under specific conditions, including a temperature of 25 °C, a pH of 7, and a light intensity of 50 μmol. s−1.m−2 for 12 h of light and 12 h of darkness. To study the amount of viability and astaxanthin production, the microalgae were exposed to different concentrations of silicon, sodium silicate, calcium silicate, and potassium silicate. The highest astaxanthin production (194 mg/g) was obtained after stimulation at 200 μg/ml of silicon compared to the control after 15 days by high-performance liquid chromatography (HPLC) technique. Therefore, it can be concluded that certain concentrations of silicon and silicon salts can be considered a suitable stimulus to produce astaxanthin in the H. pluvialis microalgae. This study highlights the potential of using silicon nanoparticles as a stimulant for astaxanthin production in H. pluvialis, which could have significant implications for the nutraceuticals, cosmetics, food, and aquaculture industries.

Genetic engineering of Haematococcus pluvialis microalgae for the enhancement of astaxanthin production: A review

Haematococcus pluvialis (H. pluvialis) is a significant natural source of astaxanthin, garnering interest in the pharmaceutical and nutraceutical industries. However, astaxanthin production from H. pluvialis is constrained by factors such as a lengthy cultivation period and thick cell walls. Recent research has explored different strategies, such as optimising cultivation conditions, to enhance astaxanthin biosynthesis. This review paper aims to summarise the recent advancement of metabolic and genetic engineering in astaxanthin biosynthesis from H. pluvialis. The review would provide a comprehensive analysis of the molecular components and mechanism involved in the biosynthesis pathway of astaxanthin in H. pluvialis, revealing the specific genes responsible for governing its biosynthesis. Numerous metabolic methodologies are investigated, including manipulating light intensity, salinity, nutrient deficiency, and temperature to enhance microalgae biomass and astaxanthin accumulation. Genetic engineering strategies have recently been studied to manipulate specific genes (e.g. bkt, CrtR-b, pds) to increase astaxanthin production. However, the limitation of genetic engineering is still unclear due to its mechanism of astaxanthin esterification and the transport of secondary β-carotenoids from the chloroplast to the cytosol. This lack of understanding has posed a challenge to maximise astaxanthin production through genetic engineering. This review also provides recent insights and future research directions for genetic engineering by providing a holistic approach to the complex interplay of genetics, metabolism, and biotechnological strategies to maximise astaxanthin production.

Improvement of Astaxanthin Production in Aurantiochytrium limacinum by Overexpression of the Beta-Carotene Hydroxylase Gene.

Aurantiochytrium limacinum is a heterotrophic eukaryotic microorganism that can accumulate high levels of commercial products such as astaxanthin and docosahexaenoic acid. Due to its rapid growth and relatively simple extraction method, A. limacinum is considered a promising astaxanthin resource to replace the conventional microalgal production. However, the astaxanthin biosynthetic process in A. limacinum remains incompletely understood, especially in those catalysed by β-carotene hydroxylase (CrtZ) and ketolase. In this study, we overexpressed a crtZ candidate gene to increase astaxanthin production and expand our understanding of the conversion from beta-carotene to astaxanthin. The resultant transformant AlcrtZ#10 cultivated for 5days showed a significant increase in astaxanthin production per culture (2.8-fold) and per cell (4.5-fold) compared with that of the wild-type strain. Strikingly, longer light exposure increased astaxanthin production and decreased the beta-carotene content in the wild-type strain, suggesting that light exposure duration is important for astaxanthin production in A. limacinum. Among several predicted intermediates, furthermore, the cantaxanthin produced from β-carotene by ketolase activity were enhanced in the transformant AlcrtZ#10. Although the further investigation is needed, this result suggested that the main route of astaxanthin was via cantaxanthin. Thus, our findings will be valuable not only for its application, but also for understanding the astaxanthin biosynthetic process in A. limacinum.

Metabolic pathway assembly using docking domains from type I cis-AT polyketide synthases

Engineered metabolic pathways in microbial cell factories often have no natural organization and have challenging flux imbalances, leading to low biocatalytic efficiency. Modular polyketide synthases (PKSs) are multienzyme complexes that synthesize polyketide products via an assembly line thiotemplate mechanism. Here, we develop a strategy named mimic PKS enzyme assembly line (mPKSeal) that assembles key cascade enzymes to enhance biocatalytic efficiency and increase target production by recruiting cascade enzymes tagged with docking domains from type I cis-AT PKS. We apply this strategy to the astaxanthin biosynthetic pathway in engineered Escherichia coli for multienzyme assembly to increase astaxanthin production by 2.4-fold. The docking pairs, from the same PKSs or those from different cis-AT PKSs evidently belonging to distinct classes, are effective enzyme assembly tools for increasing astaxanthin production. This study addresses the challenge of cascade catalytic efficiency and highlights the potential for engineering enzyme assembly.

Improving astaxanthin production in Escherichia coli by co-utilizing CrtZ enzymes with different substrate preference

BackgroundThe bifunctional enzyme β-carotene hydroxylase (CrtZ) catalyzes the hydroxylation of carotenoid β-ionone rings at the 3, 3’ position regardless of the presence of keto group at 4, 4’ position, which is an important step in the synthesis of astaxanthin. The level and substrate preference of CrtZ may have great effect on the amount of astaxanthin and the accumulation of intermediates.ResultsIn this study, the substrate preference of PCcrtZ from Paracoccus sp. PC1 and PAcrtZ from Pantoea Agglomerans were certified and were combined utilization for increase astaxanthin production. Firstly, PCcrtZ from Paracoccus sp. PC1 and PAcrtZ from P. Agglomerans were expressed in platform strains CAR032 (β-carotene producing strain) and Can004 (canthaxanthin producing strain) separately to identify their substrate preference for carotenoids with keto groups at 4,4’ position or not. The results showed that PCcrtZ led to a lower zeaxanthin yield in CAR032 compared to that of PAcrtZ. On the contrary, higher astaxanthin production was obtained in Can004 by PCcrtZ than that of PAcrtZ. This demonstrated that PCCrtZ has higher canthaxanthin to astaxanthin conversion ability than PACrtZ, while PACrtZ prefer using β-carotene as substrate. Finally, Ast010, which has two copies of PAcrtZ and one copy of PCcrtZ produced 1.82 g/L of astaxanthin after 70 h of fed-batch fermentation.ConclusionsCombined utilization of crtZ genes, which have β-carotene and canthaxanthin substrate preference respectively, can greatly enhance the production of astaxanthin and increase the ratio of astaxanthin among total carotenoids.

Gibberellic acid-induced fatty acid metabolism and ABC transporters promote astaxanthin production in Phaffia rhodozyma.

Astaxanthin is an important natural antioxidant with various biological functions; however, the production of astaxanthin does not meet the requirements for industrialization. The aim of the present study was to identify an inducer that increases astaxanthin yield and to evaluate the regulatory mechanism of the induction of astaxanthin synthesis in Phaffia rhodozyma. The effects of indole-3-acetic acid (IAA), jasmonic acid (JA) and gibberellic acid (GA) on astaxanthin synthesis were studied by fermentation kinetics analysis. Then, combined transcriptomics and metabolomics approaches were used to analyse differential metabolites and expressed genes involved in astaxanthin synthesis induced by GA. The results indicated that GA significantly increased astaxanthin production; however, IAA and JA had no significant effect on astaxanthin synthesis. The induction by GA significantly enhanced fatty acid metabolism and ABC transporters, increased the expression of fatty acid desaturase and ABC transporter genes, and elevated the contents of unsaturated fatty acids. These results suggested that fatty acid saturation plays an important role in astaxanthin accumulation and that ABC transporters may be the efflux pumps for astaxanthin. The present study reveals metabolic mechanism of GA-induced astaxanthin synthesis and proposes a new strategy of transporter engineering to improve astaxanthin production.

Biotechnology applied to Haematococcus pluvialis Fotow: challenges and prospects for the enhancement of astaxanthin accumulation

Among natural sources, the green alga Haematococcus pluvialis is the major producer of the potent antioxidant pigment astaxanthin, a high-value compound whose demand is still not sufficiently covered by the current industrial production. Despite the availability of low-cost synthetic astaxanthin, natural astaxanthin is more potent and accepted for human consumption and food additive uses. This review highlights the use of different biotechnological approaches aiming to increase astaxanthin production yields and discusses the advantages and drawbacks of traditional alternatives widely used on other microorganisms. These traditional approaches span from the easy to perform but not devoid of problems random mutagenesis, to advanced methods like microalgae genetic engineering, which has great potential for enhancement, despite being highly restricted in several countries by genetically modified organism legislation. In addition, we propose the underexplored approach of artificial polyploidization for the obtention of strains with increased cell size, which have the advantage of being considered as non-genetically modified organisms that do not require modification of the current industrial production procedures.

Grey relational analysis to explore the culture factors of Haematococcus pluvialis

Astaxanthin has superior antioxidant capacity, and Haematococcus pluvialis is recognized as the most powerful organism to accumulate natural astaxanthin in nature. In artificial culture, environmental factors can affect the yield of astaxanthin. In order to reduce blindness in the cultivation of Haematococcus pluvialis and effectively increase astaxanthin production, grey relational analysis was used to calculate the correlation between the culture factors and the growth condition of Haematococcus pluvialis, and advantage analysis was performed to investigate the influence degree of different culture factors on the growth and astaxanthin accumulation of Haematococcus pluvialis. The results showed that the effects of light intensity and temperature were greatest during the proliferative culture stage, while the effects of temperature and light duration were greatest during the astaxanthin induction stage. The application of grey relational analysis also provides a convenient reference tool for the design of research to improve the yield of astaxanthin induced by Haematococcus pluvialis, and other culture factors affecting astaxanthin yield can be analyzed and compared using this method, providing more ideas for optimizing culture methods and developing culture devices.

Combinatorial expression of different β-carotene hydroxylases and ketolases in Escherichia coli for increased astaxanthin production.

In natural produced bacteria, β-carotene hydroxylase (CrtZ) and β-carotene ketolase (CrtW) convert β-carotene into astaxanthin. To increase astaxanthin production in heterologous strain, simple and effective strategies based on the co-expression of CrtZ and CrtW were applied in E. coli. First, nine artificial operons containing crtZ and crtW genes from different sources were constructed and, respectively, introduced into E. coli ZF237T, a β-carotene producing host. Among the nine resulting strains, five accumulated detectable amounts of astaxanthin ranging from 0.49 to 8.07mg/L. Subsequently, the protein fusion CrtZ to CrtW using optimized peptide linkers further increased the astaxanthin production. Strains expressing fusion proteins with CrtZ rather than CrtW attached to the N-terminus accumulated much more astaxanthin. The astaxanthin production of the best strain ZF237T/CrtZAs-(GS)1-WBs was 127.6% and 40.2% higher than that of strains ZF237T/crtZAsWBs and ZF237T/crtZBsWPs, respectively. The strategies depicted here also will be useful for the heterologous production of other natural products.

Improved Astaxanthin Production with Corynebacterium glutamicum by Application of a Membrane Fusion Protein.

Astaxanthin is one of the strongest natural antioxidants and a red pigment occurring in nature. This C40 carotenoid is used in a broad range of applications such as a colorant in the feed industry, an antioxidant in cosmetics or as a supplement in human nutrition. Natural astaxanthin is on the rise and, hence, alternative production systems are needed. The natural carotenoid producer Corynebacterium glutamicum is a potent host for industrial fermentations, such as million-ton scale amino acid production. In C. glutamicum, astaxanthin production was established through heterologous overproduction of the cytosolic lycopene cyclase CrtY and the membrane-bound β-carotene hydroxylase and ketolase, CrtZ and CrtW, in previous studies. In this work, further metabolic engineering strategies revealed that the potential of this GRAS organism for astaxanthin production is not fully exploited yet. It was shown that the construction of a fusion protein comprising the membrane-bound β-carotene hydroxylase and ketolase (CrtZ~W) significantly increased astaxanthin production under high glucose concentration. An evaluation of used carbon sources indicated that a combination of glucose and acetate facilitated astaxanthin production. Moreover, additional overproduction of cytosolic carotenogenic enzymes increased the production of this high value compound. Taken together, a seven-fold improvement of astaxanthin production was achieved with 3.1 mg/g CDW of astaxanthin.

A novel approach to enhance astaxanthin production in Haematococcus lacustris using a microstructure-based culture platform

The green alga Haematococcus lacustris is a freshwater unicellular microalga belonging to Chlorophyceae. It is a natural source of astaxanthin, a secondary metabolite commonly used as an antioxidant and anti-inflammatory agent. Due to the expansive applications of astaxanthin, efforts to increase production are underway. In this study, a novel microfabricated culture platform was developed to physically enhance intracellular stress levels of H. lacustris. The microfabricated culture device increased the level of reactive oxygen species (ROS) in the cells through physical stress, thereby inducing the production of astaxanthin. Consequently, employing the device to stress H. lacustris cells increased astaxanthin production (per cell) by up to 204%. Our novel findings in this study suggest that the microfabricated culture platform can endow mechanical stress on H. lacustris, and could serve as an attractive alternate strategy for increased astaxanthin production.

Metabolic engineering of Escherichia coli for high-level astaxanthin production with high productivity

Astaxanthin is a reddish keto-carotenoid classified as a xanthophyll found in various microbes and marine organisms. As a powerful antioxidant having up to 100 times more potency than other carotenoids such as β-carotene, lutein, and lycopene, astaxanthin is a versatile compound utilized in animal feed, food pigment, health promotion and cosmetic industry. Here, we report development of metabolically engineered Escherichia coli capable of producing astaxanthin to a high concentration with high productivity. First, the heterologous crt genes (crtE, crtY, crtI, crtB, and crtZ) from Pantoea ananatis and the truncated BKT gene (trCrBKT) from Chlamydomonas reinhardtii were introduced to construct the astaxanthin biosynthetic pathway. Then, eight different fusion tags were examined by attaching them to the N- or C-terminus of the trCrBKT membrane protein to allow stable expression and to efficiently guide trCrBKT to the E. coli membrane. When the signal peptide of OmpF and TrxA were tagged to the N-terminus and C-terminus of trCrBKT, respectively, astaxanthin production reached 12.90 mg/L (equivalent to 3.84 mg/gDCW), which was 2.08-fold higher than that obtained without tagging. Upon optimization of culture conditions, this engineered strain WLGB-RPP harboring pAX15 produced 332.23 mg/L (5.38 mg/gDCW) of astaxanthin with the productivity of 3.79 mg/L/h by fed-batch fermentation. In order to further increase astaxanthin production, in silico flux variability scanning based on enforced objective flux (FVSEOF) was performed to identify gene overexpression targets. The engineered strain WLGB-RPP (pAX15, pTrc-ispDF) which simultaneously overexpressing the ispD and ispF genes identified by FVSEOF produced astaxanthin to a higher concentration of 377.10 mg/L (6.26 mg/gDCW) with a productivity of 9.20 mg/L/h upon induction with 1 mM IPTG. When cells were induced with 0.5 mM IPTG to reduce the metabolic burden, astaxanthin concentration further increased to 432.82 mg/L (7.12 mg/gDCW) with a productivity of 9.62 mg/L/h. To more stably maintain plasmid during the fed-batch fermentation of WLGB-RPP (pAX15, pTrc-ispDF), the post-segregational killing hok/sok system was introduced. This strain produced 385.04 mg/L (6.98 mg/gDCW) of astaxanthin with a productivity of 7.86 mg/L/h upon induction with 0.5 mM IPTG. The strategies reported here will be useful for the enhanced production of astaxanthin and related carotenoid products by engineered E. coli strains.

Enhanced Production of Astaxanthin by Metabolically Engineered Non-mevalonate Pathway in Escherichia coli

Astaxanthin is one of the major carotenoids used in pigment has a great economical value in pharmaceutical markets, feeding, nutraceutical and food industries. This study was to increase the production of astaxanthin by co-expression with transformed Escherichia coli using six genes involved in the non-mevalonate pathway. Involved in the non-mevalonate biosynthetic pathway of the strain Kocuria gwangalliensis were cloned dxs, ispC, ispD, ispE, ispF, ispG, ispH and idi genes in order to increase astaxanthin production from the transformed E. coli. And co-expression with the genes to compared the amount of astaxanthin production. This engineered E. coli, containing both the non-mevalonate pathway gene and the astaxanthin biosynthesis gene cluster, produced astaxanthin at 1,100 μg/g DCW (dry cell weight), resulting in approximately three times the production of astaxanthin.

Enhancement of astaxanthin production in Xanthophyllomyces dendrorhous by efficient method for the complete deletion of genes.

BackgroundRed yeast, Xanthophyllomyces dendrorhous is the only yeast known to produce astaxanthin, an anti-oxidant isoprenoid (carotenoid) widely used in the aquaculture, food, pharmaceutical and cosmetic industries. The potential of this microorganism as a platform cell factory for isoprenoid production has been recognized because of high flux through its native terpene pathway. Recently, we developed a multiple gene expression system in X. dendrorhous and enhanced the mevalonate synthetic pathway to increase astaxanthin production. In contrast, the mevalonate synthetic pathway is suppressed by ergosterol through feedback inhibition. Therefore, releasing the mevalonate synthetic pathway from this inhibition through the deletion of genes involved in ergosterol synthesis is a promising strategy to improve isoprenoid production. An efficient method for deleting diploid genes in X. dendrorhous, however, has not yet been developed.ResultsXanthophyllomyces dendrorhous was cultivated under gradually increasing concentrations of antibiotics following the introduction of antibiotic resistant genes to be replaced with target genes. Using this method, double CYP61 genes encoding C-22 sterol desaturases relating to ergosterol biosynthesis were deleted sequentially. This double CYP61 deleted strain showed decreased ergosterol biosynthesis compared with the parental strain and single CYP61 disrupted strain. Additionally, this double deletion of CYP61 genes showed increased astaxanthin production compared with the parental strain and the single CYP61 knockout strain. Finally, astaxanthin production was enhanced by 1.4-fold compared with the parental strain, although astaxanthin production was not affected in the single CYP61 knockout strain.ConclusionsIn this study, we developed a system to completely delete target diploid genes in X. dendrorhous. Using this method, we deleted diploid CYP61 genes involved in the synthesis of ergosterol that inhibits the pathway for mevalonate, which is a common substrate for isoprenoid biosynthesis. The resulting decrease in ergosterol biosynthesis increased astaxanthin production. The efficient method for deleting diploid genes developed in this study has the potential to improve industrial production of various isoprenoids in X. dendrorhous.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0556-x) contains supplementary material, which is available to authorized users.

Genome mining of astaxanthin biosynthetic genes from Sphingomonas sp. ATCC 55669 for heterologous overproduction in Escherichia coli.

As a highly valued keto‐carotenoid, astaxanthin is widely used in nutritional supplements and pharmaceuticals. Therefore, the demand for biosynthetic astaxanthin and improved efficiency of astaxanthin biosynthesis has driven the investigation of metabolic engineering of native astaxanthin producers and heterologous hosts. However, microbial resources for astaxanthin are limited. In this study, we found that the α‐Proteobacterium Sphingomonas sp. ATCC 55669 could produce astaxanthin naturally. We used whole‐genome sequencing to identify the astaxanthin biosynthetic pathway using a combined PacBio‐Illumina approach. The putative astaxanthin biosynthetic pathway in Sphingomonas sp. ATCC 55669 was predicted. For further confirmation, a high‐efficiency targeted engineering carotenoid synthesis platform was constructed in E. coli for identifying the functional roles of candidate genes. All genes involved in astaxanthin biosynthesis showed discrete distributions on the chromosome. Moreover, the overexpression of exogenous E. coli idi in Sphingomonas sp. ATCC 55669 increased astaxanthin production by 5.4‐fold. This study described a new astaxanthin producer and provided more biosynthesis components for bioengineering of astaxanthin in the future.

Overexpression of a bifunctional enzyme, CrtS, enhances astaxanthin synthesis through two pathways in Phaffia rhodozyma.

BackgroundA moderate-temperature, astaxanthin-overproducing mutant strain (termed MK19) of Phaffia rhodozyma was generated in our laboratory. The intracellular astaxanthin content of MK19 was 17-fold higher than that of wild-type. The TLC profile of MK19 showed a band for an unknown carotenoid pigment between those of β-carotene and astaxanthin. In the present study, we attempted to identify the unknown pigment and to enhance astaxanthin synthesis in MK19 by overexpression of the crtS gene that encodes astaxanthin synthase (CrtS).ResultsA crtS-overexpressing strain was constructed without antibiotic marker. A recombinant plasmid with lower copy numbers was shown to be stable in MK19. In the positive recombinant strain (termed CSR19), maximal astaxanthin yield was 33.5% higher than MK19, and the proportion of astaxanthin as a percentage of total carotenoids was 84%. The unknown carotenoid was identified as 3-hydroxy-3′,4′-didehydro-β,Ψ-carotene-4-one (HDCO) by HPLC, mass spectrometry, and NMR spectroscopy. CrtS was found to be a bifunctional enzyme that helped convert HDCO to astaxanthin. Enhancement of crtS transcriptional level increased transcription levels of related genes (crtE, crtYB, crtI) in the astaxanthin synthesis pathway. A scheme of carotenoid biosynthesis in P. rhodozyma involving alternative bicyclic and monocyclic pathways is proposed.ConclusionsCrtS overexpression leads to up-regulation of synthesis-related genes and increased astaxanthin production. The transformant CSR19 is a stable, secure strain suitable for feed additive production. The present findings help clarify the regulatory mechanisms that underlie metabolic fluxes in P. rhodozyma carotenoid biosynthesis pathways.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0279-4) contains supplementary material, which is available to authorized users.

Increase in the astaxanthin synthase gene (crtS) dose by in vivo DNA fragment assembly in Xanthophyllomyces dendrorhous.

BackgroundXanthophyllomyces dendrorhous is a basidiomycetous yeast that is relevant to biotechnology, as it can synthesize the carotenoid astaxanthin. However, the astaxanthin levels produced by wild-type strains are low. Although different approaches for promoting increased astaxanthin production have been attempted, no commercially competitive results have been obtained thus far. A promising alternative to facilitate the production of carotenoids in this yeast involves the use of genetic modification. However, a major limitation is the few available molecular tools to manipulate X. dendrorhous.ResultsIn this work, the DNA assembler methodology that was previously described in Saccharomyces cerevisiae was successfully applied to assemble DNA fragments in vivo and integrate these fragments into the genome of X. dendrorhous by homologous recombination in only one transformation event. Using this method, the gene encoding astaxanthin synthase (crtS) was overexpressed in X. dendrorhous and a higher level of astaxanthin was produced.ConclusionsThis methodology could be used to easily and rapidly overexpress individual genes or combinations of genes simultaneously in X. dendrorhous, eliminating numerous steps involved in conventional cloning methods.

Increased astaxanthin production by a Phaffia rhodozyma mutant grown on date juice from Yucca fillifera

The wild strain and the astaxanthin-overproducing mutant strain 25–2 of Phaffia rhodozyma were analyzed in order to assess their ability to grow and synthesize astaxanthin in a minimal medium composed of g L−1: KH2PO4 2.0; MgSO4 0.5; CaCl2 0.1; urea 1.0 and supplemented with date juice of Yucca fillifera as a carbon source (yuca medium). The highest astaxanthin production (6170 μg L−1) was obtained at 22.5 g L−1 of reducing sugars. The addition of yeast extract to the yuca medium at concentrations of 0.5–3.0 g L−1 inhibited astaxanthin synthesis. The yuca medium supported a higher production of astaxanthin, 2.5-fold more than that observed in the YM medium. Journal of Industrial Microbiology & Biotechnology (2000) 24, 187–190.

Increased astaxanthin production by Phaffia rhodozyma mutants isolated as resistant to diphenylamine

To improve astaxanthin production by Phaffia rhodozyma, resistance to a carotenoid biosynthesis inhibitor was employed to screen higher astaxanthin producers. The wild type strain generated pinkish salmon-colored colonies due to the intracellular astaxanthin accumulation, but the colonies have turned white when grown in the presence of some of carotenoid biosynthesis inhibitors. Among the compounds tested, diphenylamine (DPA) was selected as the most effective inhibitor that did not significantly affect colony numbers and sizes. Upon mutagenesis and screening, DPA-resistant mutants were isolated as salmon-colored colonies on plates containing DPA, and two of the DPA-resistant mutants exhibited a two-fold increase in astaxanthin production over that of the wild type. In the DPA-treated wild type, 15- cis phytoene was identified as the major carotenoid, indicating that the target enzyme of DPA was likely to be phytoene desaturase (PDS). When treated with DPA and/or nicotine, an inhibitor of a lycopene cyclase, lycopene accumulation was similarly enhanced in the resistant mutant, and the degrees of inhibition by DPA were also found analogous to astaxanthin formation in the wild type and in the DPA-resistant mutant. These results indicated that the enhanced carotenogenesis most probably occured at the PDS stage.

Selection and evaluation of astaxanthin-overproducing mutants of Phaffia rhodozyma

Mutagenesis of Phaffia rhodozyma with NTG yielded a mutant with an astaxanthin content of 1688 μg (g dry biomass)(-1), a cell yield coefficient of 0.47 on glucose and a maximum specific growth rate of 0.12 h(-1). Re-mutation of the mutant decreased the cell yield and maximum specific growth rate but increased the astaxanthin content. The use of mannitol or succinate as carbon sources enhanced pigmentation, yielding astaxanthin contents of 1973 μg g(-1) and 1926 μg g(-1), respectively. The use of valine as sole nitrogen source also increased astaxanthin production, but severely decreased the maximum specific growth rate and cell yield coefficient. The optimum pH for growth of P. rhodozyma was between pH 4.5 and 5.5, whereas the astaxanthin content remained constant above pH 3.