Search

Background Phaffia rhodozyma has many desirable properties for astaxanthin production, including rapid heterotrophic metabolism and high cell densities in fermenter culture. The low optimal temperature range (17–21 °C) for cell growth and astaxanthin synthesis in this species presents an obstacle to efficient industrial-scale astaxanthin production. The inhibition mechanism of cell growth at > 21 °C in P. rhodozyma have not been investigated. Results MK19, a mutant P. rhodozyma strain grows well at moderate temperatures, its cell growth was also inhibited at 28 °C, but such inhibition was mitigated, and low biomass 6 g/L was obtained after 100 h culture. Transcriptome analysis indicated that low biomass at 28 °C resulted from strong suppression of DNA and RNA synthesis in MK19. Growth inhibition at 28 °C was due to cell membrane damage with a characteristic of low mRNA content of fatty acid (f.a.) pathway transcripts (acc, fas1, fas2), and consequent low f.a. content. Thinning of cell wall and low mannose content (leading to loss of cell wall integrity) also contributed to reduced cell growth at 28 °C in MK19. Levels of astaxanthin and ergosterol, two end-products of isoprenoid biosynthesis (a shunt pathway of f.a. biosynthesis), reached 2000 µg/g and 7500 µg/g respectively; ~2-fold higher than levels at 21 or 25 °C. Abundance of ergosterol, an important cell membrane component, compensated for lack of f.a., making possible the biomass production of 6 g/L for MK19 at 28 °C. Conclusions Inhibition of growth of P. rhodozyma at 28 °C results from blocking of DNA, RNA, f.a., and cell wall biosynthesis. In MK19, abundant ergosterol made possible biomass production 6 g/L at 28 °C. Significant accumulation of astaxanthin and ergosterol indicated an active MVA pathway in MK19 at 28 °C. Strengthening of the MVA pathway can be a feasible metabolic engineering approach for enhancement of astaxanthin synthesis in P. rhodozyma. The present findings provide useful mechanistic insights regarding adaptation of P. rhodozyma to 28 °C, and improved understanding of feasible metabolic engineering techniques for industrial scale astaxanthin production by this economically important yeast species.

Background: Phaffia rhodozyma has many desirable properties for astaxanthin production, including rapid heterotrophic metabolism and high cell densities in fermenter culture. The low optimal temperature range (17-21 °C) for cell growth and astaxanthin synthesis in this species presents an obstacle to efficient industrial-scale astaxanthin production. The inhibition mechanism of cell growth at > 21 °C in P. rhodozyma have not been investigated.Results: MK19, a mutant P. rhodozyma strain grows well at moderate temperatures, its cell growth was also inhibited at 28 °C, but such inhibition was mitigated, and low biomass 6 g/L was obtained after 100 h culture. Transcriptome analysis indicated that low biomass at 28 °C resulted from strong suppression of DNA and RNA synthesis in MK19. Growth inhibition at 28 °C was due to cell membrane damage with a characteristic of low mRNA content of fatty acid (f.a.) pathway transcripts (acc, fas1, fas2), and consequent low f.a. content. Thinning of cell wall and low mannose content (leading to loss of cell wall integrity) also contributed to reduced cell growth at 28 °C in MK19. Levels of astaxanthin and ergosterol, two end-products of isoprenoid biosynthesis (a shunt pathway of f.a. biosynthesis), reached 2000 μg/g and 7500 μg/g respectively; ~2-fold higher than levels at 21 or 25 °C. Abundance of ergosterol, an important cell membrane component, compensated for lack of f.a., making possible the biomass production of 6 g/L for MK19 at 28 °C.Conclusion: Inhibition of growth of P. rhodozyma at 28 °C results from blocking of DNA, RNA, f.a., and cell wall biosynthesis. In MK19, abundant ergosterol made possible biomass production 6 g/L at 28 °C. Significant accumulation of astaxanthin and ergosterol indicated an active MVA pathway in MK19 at 28 °C. Strengthening of the MVA pathway can be a feasible metabolic engineering approach for enhancement of astaxanthin synthesis in P. rhodozyma. The present findings provide useful mechanistic insights regarding adaptation of P. rhodozyma to 28 °C, and improved understanding of feasible metabolic engineering techniques for industrial scale astaxanthin production by this economically important yeast species.

Background The yeast Phaffia rhodozyma has many desirable properties for astaxanthin production, including rapid heterotrophic metabolism and high cell densities in fermenter culture. The optimal temperature range for cell growth and astaxanthin synthesis in this species is low (17–21 °C), which presents an obstacle to efficient industrial-scale astaxanthin production. The molecular regulatory mechanisms of cell growth and astaxanthin synthesis in P. rhodozyma remain poorly understood, and the regulatory effects of temperature have not been investigated. Results MK19, a mutant P. rhodozyma strain that grows well at moderate temperatures, was generated from wild-type (WT) strain JCM9042, which is unable to grow at 28 °C. MK19 cell growth was also inhibited at 28 °C, but such inhibition was mitigated, and biomass production reached 6 g/L after 100 h culture. Transcriptome analysis indicated that low viability at 28 °C resulted from strong suppression of DNA synthesis. Growth inhibition at 28 °C was due to low mRNA content of fatty acid (f.a.) pathway genes (acc, fas1, fas2), and consequent low f.a. content. Thinning of cell wall and low mannose content (leading to loss of cell wall integrity) also contributed to reduced cell growth at 28 °C. In MK19 cultured for 48 h at 28 °C, levels of astaxanthin and ergosterol, two end-products of isoprenoid biosynthesis (a shunt pathway of f.a. biosynthesis), reached 2000 µg/g and 7500 µg/g respectively; ~2-fold higher than levels at 21 or 25 °C. Abundance of ergosterol, an important cell membrane component, compensated for lack of f.a., making possible the biomass production of 6 g/L for MK19 at 28 °C. Downregulation of crtI mRNA, whose product catalyzes four dehydrogenation steps in the phytoene-to-lycopene process, contributed to inhibition of astaxanthin synthesis in WT at 25 °C. High crtI mRNA content in MK19 (> 10-fold higher than that in WT) counteracted the inhibitory effect of high temperature on crtI transcription, making possible the efficient synthesis of lycopene which was subsequently converted to astaxanthin. Conclusion Inhibition of growth of WT P. rhodozyma at 28 °C results from blocking of DNA, f.a., and cell wall biosynthesis. Growth at this temperature could be increased by enhancement of such biosynthesis. In moderate-temperature strain MK19, precursor molecules accumulated from f.a. pathway are diverted to astaxanthin and ergosterol biosynthesis, making possible biomass production 6 g/L at 28 °C. Strengthening of the MVA pathway to make available more precursors from f.a. pathway is a feasible metabolic engineering approach for enhancement of astaxanthin synthesis in MK19 or other moderate-temperature producer strains. The present findings provide useful mechanistic insights regarding adaptation of P. rhodozyma to 28 °C, and improved understanding of transcription regulatory networks and feasible metabolic engineering techniques for industrial scale astaxanthin production by this economically important yeast species.

Background A major obstacle to industrial-scale astaxanthin production by the yeast Phaffia rhodozyma is the strong inhibitory effect of high glucose concentration on astaxanthin synthesis. We investigated, for the first time, the mechanism of the regulatory effect of high glucose (> 100 g/L) at the metabolite and transcription levels. Results Total carotenoid, β-carotene, and astaxanthin contents were greatly reduced in wild-type JCM9042 at high (110 g/L) glucose; in particular, β-carotene content at 24–72 h was only 14–17% of that at low (40 g/L) glucose. The inhibitory effect of high glucose on astaxanthin synthesis appeared to be due mainly to repression of lycopene-to-β-carotene and β-carotene-to-astaxanthin steps in the pathway. Expression of carotenogenic genes crtE, pbs, and ast was also strongly inhibited by high glucose; such inhibition was mediated by creA, a global negative regulator of carotenogenic genes which is strongly induced by glucose. In contrast, astaxanthin-overproducing, glucose metabolic derepression mutant strain MK19 displayed de-inhibition of astaxanthin synthesis at 110 g/L glucose; this de-inhibition was due mainly to deregulation of pbs and ast expression, which in turn resulted from low creA expression. Failure of glucose to induce the genes reg1 and hxk2, which maintain CreA activity, also accounts for the fact that astaxanthin synthesis in MK19 was not repressed at high glucose. Conclusion We conclude that astaxanthin synthesis in MK19 at high glucose is enhanced primarily through derepression of carotenogenic genes (particularly pbs), and that this process is mediated by CreA, Reg1, and Hxk2 in the glucose signaling pathway.

An astaxanthin-overproducing (∼1000 μg g(-1)) strain of Phaffia rhodozyma, termed MK19, was established through 1-methyl-3-nitro-1-nitrosoguanidine and Co60 mutagenesis from wild-type JCM9042 (merely 35-67 μg g(-1)). The total fatty acid content of MK19 was much lower than that of the wild type. Possible causes of the astaxanthin increase were studied at the gene expression level. The expression of the carotenogenic genes crtE, crtI, pbs, and ast, which are responsible for astaxanthin biosynthesis from geranylgeranyl pyrophosphate, was highly induced at the mRNA level, leading to excessive astaxanthin accumulation. In contrast, transcription levels of the genes (hmgs, hmgr, idi, mvk, mpd, fps), responsible for the initial steps in the terpenoid pathway, were essentially the same in wild type and MK19. Although fatty acid and total ergosterol content were reduced by 40-70 mg g(-1) and 760.3 μg g(-1) , respectively, in MK19 as compared with the wild type, but the transcription levels of rate-limiting genes in fatty acid and ergosterol pathways such as acc and sqs were similar. Because fatty acids and ergosterol are two branch pathways of astaxanthin biosynthesis in P. rhodozyma, our findings indicate that enhancement of astaxanthin in MK19 results from decreased fatty acid and ergosterol biosynthesis, leading to precursor accumulation, and transfer to the astaxanthin pathway. Strengthening of the mevalonate pathway is suggested as a promising metabolic engineering approach for further astaxanthin enhancement in MK19.

Aims: The present study was designed to investigate quality of life (QOL) among caregivers of cancer inpatients, and to explore the related factors with the long-term goal to improve QOL of cancer caregivers in China. Methods: A cross-sectional study was performed during the period January–March 2008. Subjects comprised 358 caregivers of cancer inpatients. The questionnaires pertaining to QOL, sociodemographic characteristics, caregiving demands, available social resources, caregiving situation, and caregiver's health status were used. Data were collected by nurses through interviews. Results: The average QOL score of these subjects was 5.26. General linear model analysis showed that caregiver's QOL was significantly associated with, in standardized estimate sequence, spousal relationship, patient's activities of daily living, and chronic disease (no). Overall spousal relationship to patient had the strongest association with caregiver's QOL. Conclusions: Psychological well-being and social concerns of cancer caregivers were disrupted more seriously than spiritual well-being and physical well-being. In addition, spousal relationship, patient's disability, and caregiver's health status could affect the caregiver's QOL.

β-1,3-glucanosyltransferases play essential roles in cell wall biosynthesis in yeast. Kluyveromyces lactis has six putative β-1,3-glucanosyltransferase genes. KlGAS1-1 and KlGAS1-2 are homologs of Saccharomyces cerevisiae gene GAS1. RT-qPCR indicated the transcription level of KlGAS1-1 was significantly reduced while heterologous protein (thermostable xylanase B) secretion was enhanced during medium optimization. To evaluate if these two events were related, and to improve xylanase B secretion in K. lactis, we constructed KlGAS1-1 and KlGAS1-2 single deletion strains and double deletion strain, respectively. KlGAS1-1 gene deletion resulted in the highest xylanase B activity among the three mutants. Only the double deletion strain showed morphology similar to that of the GAS1 deletion mutant in S. cerevisiae. The two single deletion strains differed in terms of cell wall thickness and xylanase B secretion. Transcription levels of β-1,3-glucanosyltransferase genes and genes related to protein secretion and transport were assayed. The β-1,3-glucanosyltransferase genes displayed transcription complementation in the cell wall synthesis process. KlGAS1-1 and KlGAS1-2 affected transcription levels of secretion- and transport-related genes. Differences in protein secretion ratio among the three deletion strains were associated with changes of transcription levels of secretion- and transport-related genes. Our findings indicate that KlGAS1-1 deletion is an effective tool for enhancing industrial-scale heterologous protein secretion in K. lactis.

An astaxanthin-overproducing (∼1000 μg g(-1)) strain of Phaffia rhodozyma, termed MK19, was established through 1-methyl-3-nitro-1-nitrosoguanidine and Co60 mutagenesis from wild-type JCM9042 (merely 35-67 μg g(-1)). The total fatty acid content of MK19 was much lower than that of the wild type. Possible causes of the astaxanthin increase were studied at the gene expression level. The expression of the carotenogenic genes crtE, crtI, pbs, and ast, which are responsible for astaxanthin biosynthesis from geranylgeranyl pyrophosphate, was highly induced at the mRNA level, leading to excessive astaxanthin accumulation. In contrast, transcription levels of the genes (hmgs, hmgr, idi, mvk, mpd, fps), responsible for the initial steps in the terpenoid pathway, were essentially the same in wild type and MK19. Although fatty acid and total ergosterol content were reduced by 40-70 mg g(-1) and 760.3 μg g(-1) , respectively, in MK19 as compared with the wild type, but the transcription levels of rate-limiting genes in fatty acid and ergosterol pathways such as acc and sqs were similar. Because fatty acids and ergosterol are two branch pathways of astaxanthin biosynthesis in P. rhodozyma, our findings indicate that enhancement of astaxanthin in MK19 results from decreased fatty acid and ergosterol biosynthesis, leading to precursor accumulation, and transfer to the astaxanthin pathway. Strengthening of the mevalonate pathway is suggested as a promising metabolic engineering approach for further astaxanthin enhancement in MK19.

A moderate-temperature mutant strain of the yeast Phaffia rhodozyma, termed MK19, was selected by 1-methyl-3-nitro-1-nitrosoguanidine (NTG) and Co60 mutagenesis. MK19 displayed fast cell growth and elevated astaxanthin content at 25 degrees C, whereas optimal temperature for growth and astaxanthin synthesis of wild-type P. rhodozyma was 17-21 degrees C. Optimized astaxanthin yield for MK19 after 4 days culture in shaking flask at 25 degrees C, determined by response surface methodology, was 25.8 mg/l, which was 17-fold higher than that of the wild-type. MK19 was tolerant of high initial concentration of glucose (100 g/l) in optimized medium. Total fatty acid content of MK19 was much lower than that of the wild-type. Acetyl-CoA is a common precursor of fatty acid and terpenoid biosynthesis, and it is possible that decreased fatty acid synthesis results in transfer of acetyl-CoA to the carotenoid biosynthetic pathway. Our results indicate that astaxanthin content is negatively correlated with fatty acid content in P. rhodozyma. Nutrient analysis showed that MK19 cells are enriched in lysine, vitamin E, and other rare nutrients, and have potential application as fish food without nutritional supplementation. This moderate-temperature mutant strain is a promising candidate for economical industrial-scale production.