Cell wall and cell membrane defects contribute to reduced cell growth at 28 °C in Phaa rhodozyma mutant strain MK19

Background: Phaa 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 ecient 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 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. 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. Signicant accumulation of astaxanthin and ergosterol indicated a 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.

Pha a rhodozyma (sexual form: Xanthophyllomyces dendrorhous), a carotenoid-synthesizing yeast having astaxanthin as the main pigment, is the most promising and economical natural source of astaxanthin and has great industrial potential for astaxanthin fermentation [13,14]. P. rhodozyma was initially isolated from exudates of trees in isolated locations in Japan and the West Coast of North America. Because its native environment is fairly cold, P. rhodozyma is a psychrophilic (low temperature preferring) species. Both cell growth and astaxanthin biosynthesis in P. rhodozyma are inhibited by high temperatures [15]. The optimal temperature range for these processes is 17-21 °C, and this fact presents an obstacle to industrial production of astaxanthin [15].
Astaxanthin and ergosterol are isoprenoid compounds involved in the conserved mevalonate (MVA) pathway in P. rhodozyma [16]. All isoprenoid compounds are based on the C5 isoprene unit. In the initial step of the MVA pathway, two molecules of acetyl-CoA (which is also a substrate of the fatty acid synthetic pathway) undergo condensation to yield acetoacetyl-CoA, which is then converted to MVA, catalyzed by the hmgs and hmgr gene products. MVA is then transferred to farnesyl pyrophosphate (FPP) through a series of condensation steps catalyzed by mvk, mpd, idi, and fps-encoded enzymes. This process is collectively termed the MVA pathway because of the importance of the metabolite MVA. FPP undergoes reduction catalyzed by two speci c enzymes, encoded by sqs [17] and crtE, giving rise respectively to ergosterol and astaxanthin.
Formation of phytoene from geranylgeranyl diphosphate (GGPP) is catalyzed by phytoene-β-carotene synthase (encoded by pbs), a bifunctional enzyme that also displays lycopene cyclase function in P. rhodozyma [18,19]. Lycopene is synthesized from phytoene through four subsequent dehydration steps catalyzed by the crtI gene product [20]. β-carotene is generated by introducing ring structures at both ends of lycopene, also catalyzed by phytoene-β-carotene synthase [19]. Finally, β-carotene is hydroxylated and oxidized to astaxanthin by astaxanthin synthase, which is encoded by the single gene ast [21,22].
Numerous studies during the past decade, by our lab and others, have addressed the molecular regulatory mechanisms of cell growth and astaxanthin synthesis in P. rhodozyma [23][24][25][26][27][28]. However, these mechanisms remain poorly understood; in particular, the regulatory effects of temperature have not been investigated.
In our previous studies, an astaxanthin-overproducing mutant strain of P. rhodozyma termed MK19 that grows well at moderate temperature (25 °C) was generated by 1-methyl-3-nitro-1-nitrosoguanidine (NTG) and Co60 mutagenesis, and its properties were evaluated [23,24,25,26]. In the present study, we examined the alterations in cell growth and synthesis of isoprenoids, cell wall, fatty acids, etc. that occur in MK19 during 28 °C stress, a temperature at which wild-type (WT) P. rhodozyma strain JCM9042 is unable to grow. The molecular effect of high temperature (28 °C) on regulation of cell growth and astaxanthin synthesis was investigated through comparison of transcriptional pro ling response to 21 and 28 °C conditions in WT and MK19.
The obtained transcriptome and metabolic data provide new insights into genetic and physiological traits and tolerance mechanisms of P. rhodozyma, and also potential bioprocesses for optimization of industrial-scale cell growth and astaxanthin synthesis.

Results
Mutant strain MK19 grew at 28 °C, Astaxanthin content was enhanced signi cantly at 28 °C in MK19 In our 2010 study, P. rhodozyma WT strain JCM9042 grew optimally in the temperature range 17-21 °C, more slowly at 25 °C [23], and did not grow at 28 °C. Moderate-temperature mutant MK19 grew as well at 25 °C as it did at 21 °C [23]. In the present study, growth of MK19 were inhibited but not eliminated at temperatures >25 °C, and biomass production reached 6 g/L for 100 h culture at 28 °C (Fig. 1A). The temperature resulting in complete suppression of growth for MK19 was higher than that for WT.
For WT, astaxanthin synthesis was reduced and cell coloration nearly eliminated at temperatures >25 °C [23]. For MK19, in contrast, astaxanthin content at 28 °C was >2000 μg/g, which was ~2-fold higher than content at 21 or 25 °C (Fig. 1B). Astaxanthin volume yield at 28 °C was low because of limitation of biomass. For MK19, astaxanthin synthesis tolerated a temperature of 28 °C as well as it did 25 °C. These ndings suggest that cell growth and astaxanthin synthesis in P. rhodozyma are controlled by temperature through independent mechanisms.

Transcriptional pro ling of MK19 under 28 °C stress
We used RNA-Seq to investigate genomic transcription changes in MK19 during response to 28 °C and the regulatory network activated by 28 °C stress. Heat shock response (HSR) functions as a molecular chaperone to protect thermally damaged proteins from aggregation, to unfold aggregated proteins, and to refold damaged proteins or target them for e cient degradation. For validation of RNA-Seq data, we performed RT-PCR analysis of 30 genes involved in heat shock-related response, MVA synthetic pathway, and astaxanthin synthetic pathway. For each of these genes, expression was strongly correlated (correlation coe cient 0.86) with RNA-Seq data. Twelve heat shock-related genes coding heat shock proteins (HSPs) such as HSP70, HSP30, HSP60, HSP90, HSP78, and HSP104 were notably upregulated under 28 °C treatment (Table 1). These ndings suggest that HSR, which induces a battery of cytoprotective genes that encode HSPs, is an adaptive mechanism in MK19. Initial functional classi cation of these differentially expressed genes, using Gene Ontology (GO) and KEGG enrichment, showed that the "purine metabolism" and "pyrimidine metabolism" subsets contained the highest number of genes differentially expressed during MK19 exposure to 28 °C stress. In the "purine" subset, 24 out of 25 differentially expressed genes showed signi cant upregulation. In the "pyrimidine" subset, 16 out of 20 differentially expressed genes were upregulated under 28 °C treatment. Several subsets of genes involved in rRNA and amino acid metabolic processing were also upregulated under 28 °C treatment. Protein content were >2-fold higher under 28°C treatment than under 21°C treatment throughout the culture period ( Fig. 2C), while the RNA synthesis was suppressed at 28°C (Fig.   2B).
The genes downregulated under 28 °C treatment belonged mostly to the "base excision repair" and "fatty acid synthesis" subsets. Seven out of 8 differentially expressed genes in the "base excision repair" subset had extremely low mRNA content at 28 °C, and DNA content was >3-fold lower at 28 °C than at 21 °C ( Fig.  2A). These ndings indicate that the low biomass of MK19 at 28 °C was due to strong suppression of DNA and RNA metabolism. Ergosterol synthesis is a branch pathway in carotenoid synthesis. Changes in sterol composition are associated with enhanced thermotolerance in yeast [29]. WT and MK19 did not show notable differences in ergosterol content at 21 vs. 25 °C. However, astaxanthin content in WT was lower at 25 than at 21 °C [23]. Regulation of terpenoids and sterols by temperature is evidently based on different mechanisms; only terpenoid synthesis was inhibited speci cally by 25 °C in WT P. rhodozyma. In contrast, ergosterol content was signi cantly different at 28 °C in comparison with 21 °C; that in MK19 was nearly 2-fold higher at 28 °C than at 25 or 21 °C (Fig. 4B). Astaxanthin synthesis was also higher at 28 °C in MK19 (Fig.  1B). Ergosterol is an important structural enhancement (strengthening) component of cell membranes.
Promotion of ergosterol synthesis in MK19 compensated the reducing of fatty acids and mitigates inhibition of cell growth and helps modulate adaptive response to 28 °C stress. High temperature apparently fosters sterol and terpenoid metabolic uxes simultaneously in MK19.
According to RNA-Seq analysis, genes involved in f.a. synthetic pathway had very low mRNA content at 28 °C. mRNA content at 28 °C for acc1(comp13834_c0), the rst key regulatory gene in f.a. synthetic pathway [26], was ~25% that at 21 °C. Low transcription of acc1 in MK19 at 28 °C accounts for the low f.a. content and to some degree the low biomass at this temperature. Besides acc1, levels of fas1 (comp13900_c0), fas2 (comp13599_c0), f.a.-2 hydroxylase (comp11956_c0), and f.a. desaturase (comp12194_c0) were reduced at 28 °C (Table 1, No. [13][14][15][16]. Metabolic and mRNA data, taken together, indicate that f.a. synthetic pathway was hindered by 28 °C stress, and that growth of P. rhodozyma at 28°C requires an adequate amount of f.a. Therefore, modi cation of acc, fas1, and fas2 expression in future studies could potentially enhance cell growth at 28 °C. sqs is the rst key regulatory gene in ergosterol synthetic pathway [26]. sqs and other genes in this pathway showed no notable change in mRNA content.
In contrast to mRNA content, ergosterol content of MK19 was 2-fold higher at 28 °C than at 21 or 25 °C. Increased content of ergosterol may compensate in part for loss of f.a., and promote survival of MK19 at 28 °C.
Another relevant factor is the competition among carotenoids, ergosterol, f.a., and other macromolecules for acetyl-CoA and FPP. When f.a. synthesis was suppressed by high temperature in MK19, a large amount of the intermediate acetyl-CoA was likely accumulated and transferred to isoprenoid biosynthetic pathway through upstream MVA pathway, with the result that astaxanthin and ergosterol content were 2fold higher at 28 °C than at lower temperatures. This observation is consistent with the conclusion from our 2011 study that strengthening of MVA pathway in MK19 is a promising metabolic engineering approach for enhancement of astaxanthin production [24]. In the present study, carotenoid content was inversely correlated with f.a. biosynthesis.
Suppression of cell wall metabolites contributes to reduced cell growth at 28 °C The fungal cell wall plays an essential role in maintenance of cell shape, integrity, and function. It contacts and interacts with the extracellular environment, and can trigger various physiological processes to adapt to changing circumstances. WSC1, a stress circumstance sensory protein located in cell membrane, is used as a probe for cell wall functioning in fungi. Increasing evidence indicates that defects in wsc1 and other wsc family genes in yeast contribute to increased sensitivity to temperature or other stress factors, and may lead to cell lysis [30,31]. In the present study, none of the wsc genes showed mRNA increase. Under 28 °C treatment of MK19, 8 out of 12 Wsc domain-containing proteins showed extremely low mRNA, one (comp11733_c0) showed 3.5-fold downregulation, and others showed 8-to 32fold downregulation ( Table 1, No. [18][19][20][21][22][23][24][25][26][27][28][29]. These ndings suggest that temperature sensitivity in MK19 is related to low mRNA level of wsc genes, and that high temperature suppresses cell growth through its effect on cell wall synthesis. MK19 cell wall structure varied considerably as a function of temperature. Total cell wall thickness was 0.46±0.11 μm at 21 °C and 0.38±0.07 μm at 28 °C. In particular, thickness of the mannan layer at 28 °C (0.15±0.04 μm) was only about half that at 21 °C (0.26 ±0.08 μm). Thickness of the chitin/ glucan layer increased 0.12 μm at 28 °C ( Fig. 6; Table 2). A recent study by H.A. Kang's group suggests that accumulation of mannan in cell wall enhances stress resistance [31]. In MK19 cell wall outer layer, mannose component was notably reduced at 28 °C (Fig. 6, Fig. 7; Table 2), resulting in disruption of cell wall integrity, and inhibition of cell growth. In contrast, 28 °C treatment resulted in increased expression of genes associated with mannan component biogenesis; i.e., the genes encoding α-1,3mannosyltransferase (comp11864_c1), α-1,2-mannosyltransferase (comp12286_c0), and α-1,6mannosyltransferase (comp11585_c0). MK19 glucan levels were higher at 28 °C, consistent with previous ndings that higher β-glucan levels are associated with greater stress resistance in yeast strains [32]. In our study, higher glucan level promoted MK19 survival at 28 °C. In MK19 at 28 °C, mRNAs of most glucan biosynthesis-related genes were downregulated; these included β-1,3-glucanosyltransferase (comp13450_c0), β-1,3-glucanase (comp11848-co, comp11848-c1), and endo-1,3-α-glucanase agn1 (comp12961_c0, comp10598_c0). Yeast cells sometimes deposit more chitin in lateral walls to compensate for compromised cell integrity [32]. In our study, genes that encode enzymes involved in chitin synthesis were upregulated; these include chitin deacetylase (comp13745), chitin synthase CHS1 (comp12056, comp13627), chitin synthase CHS2 (comp12623, comp13980), and chitin synthase CHS2, 1,8 (comp14086). Regulatory patterns for glucans and those for mannan were quite different.

Expression of MVA pathway and astaxanthin pathway genes
The MVA pathway includes the early steps of terpenoid synthesis. hmgr and hmgs, the key regulatory genes in the terpenoid pathway in eukaryotes, are subject to feedback control at multiple levels; e.g., transcriptional, translational, and enzyme stability [33]. RNA-Seq analysis of MK19 showed no notable expression changes for genes upstream of MVA pathway at 21 vs. 28 °C. The same was true for RT-PCR analysis, except in the case of idi, the gene that encodes the enzyme isopentenyl diphosphate (IDP) isomerase. idi transcription was induced at 25 and 28 °C in both WT and MK19. For MK19, the maximal increase at 28 °C was ~10-fold higher than at 21 °C (data not shown). hmgs and hmgr expression was not inhibited at 28 °C in MK19, despite the fact that ergosterol content was 2-fold higher at this temperature than at 21 or 25 °C. hmgs expression was enhanced at temperatures >21 °C in both WT and MK19.
Relative expression of carotenoid pathway genes at these three temperatures were compared between WT and MK19. The results (Fig. 5) were consistent with those from RNA-Seq analysis. Expression of pbs and ast did not differ notably between three temperatures in WT and MK19. crtE expression was slightly higher at 28 °C, whereas crtI expression was reduced ~1.5-fold at temperatures >21 °C in both WT and MK19.
The inhibitory effect of high temperature on astaxanthin synthesis was exerted mainly at the four dehydrogenation steps leading from phytoene to lycopene in both WT and MK19. Reduced mRNA content of crtI was an important cause of astaxanthin inhibition at temperatures >25 °C. crtI mRNA level was >10fold higher in MK19 than in WT. This level in MK19 was reduced 1.5-fold at 25 and 28 °C, but was still su cient to counteract the inhibitory effect of higher temperature and lead to e cient transfer in the four from phytoene-to-lycopene dehydrogenation steps. Therefore, metabolic engineering of these steps, e.g., overexpression of crtI, is a feasible and promising method for enhancement of astaxanthin content in WT or other P. rhodozyma strains.

Discussion
The yeast Pha a rhodozyma has great potential for industrial-scale astaxanthin production, and has been a subject of great biotechnological interest for several decades [13,14]. It is a psychrophilic (low temperature preferring) species whose native habitats in Japan and the North American West Coast are isolated and fairly cold. Applicability of WT P. rhodozyma in industrial production is limited because its temperature range for optimal cell growth and astaxanthin biosynthesis is 17-21 °C. We previously established an astaxanthin-overproducing, moderate-temperature mutant strain of P. rhodozyma, termed MK19, by NTG and Co 60 mutagenesis [23,24]. The regulatory effects of temperature on cell growth and astaxanthin synthesis of P. rhodozyma have not been investigated, and our knowledge of the molecular regulatory mechanisms in general is quite fragmentary.
We describe here for the rst time the biosynthetic and regulatory mechanisms of cell growth and astaxanthin production at high temperature (28 °C) in P. rhodozyma. WT strain cannot grow at 28 °C. MK19 biomass production at 28 °C reached 6 g/L, which was 80% less than values at 21 or 25 °C. Our RNA-Seq and metabolic analyses revealed 5 regulatory patterns involving temperature, as follows. (1) Upregulation of genes that encode proteins involved in protein folding and stabilization, including HSPs, rRNA processing, and amino acid synthesis. These genes have high copy numbers, re ecting protein denaturation and misfolding, which may result from 28 °C stress. The high copy numbers help mitigate such stress through increased protein synthesis, and refolding and reactivating functions of denatured proteins. (2)  Engineering of the f.a. biosynthetic pathway could potentially convert MK19 or other P. rhodozyma strains to mesophilic strains having optimal growth temperature ~28 °C. (5) Fungi have a rigid cell wall that plays an important role in cell growth. Cell wall in P. rhodozyma loses integrity at 28 °C because the outer mannose layer becomes thinner, another reason why WT is unable to grow at this temperature.
Cell wall plasticity and composition depend on active regulation of underlying biosynthetic and restructuring processes. The cell wall integrity (CWI) pathway is a central signaling cascade that is highly conserved in fungi [30,31]. CWI pathway is essential for adaptation to a wide variety of cell wall disrupting conditions, including heat stress. Heat shock triggers activation of CWI and HSP pathways, resulting in global transcriptomic changes in various biosynthetic pathways, including cell wall remodeling enzymes and mannan and glucan biosynthesis. Observed differential expression of genes encoding WSC proteins, f.a., and other cell wall components re ects the essential role of CWI and HSP pathways in adaptation to 28 °C stress in MK19. A schematic model of candidate processes contributing to adaptive response of MK19 to 28°C stress, based on RNA-Seq and metabolic analyses, is presented in Fig. 8.
In our 2011 study, astaxanthin content of WT P. rhodozyma was lower at 25 °C than at 21 °C whereas ergosterol content was the same at these two temperatures, indicating that astaxanthin and ergosterol are regulated through independent mechanisms [24]. Synthesis of carotenoids (but not of other isoprenoids) was inhibited at 25 °C. crtI mRNA level at 25 °C, which was ~1.5-fold lower than that at 21°C , accounted for such inhibition in WT.
Despite the transcriptional inhibition of crtI, the high crtI mRNA of MK19 (>10-fold higher than that of WT) was enough and accounted for the high e ciency of astaxanthin synthesis at 25 or 28 °C. Thus, inhibition of astaxanthin synthesis by high temperature in P. rhodozyma appears to occur mainly at the transfer step from phytoene to lycopene. Metabolic engineering of this step (e.g., overexpression of crtI) could be a feasible method for increasing astaxanthin content at temperatures >25 °C in this species.
Astaxanthin and ergosterol content in MK19 were increased >2-fold at 28 °C, in contrast to cell growth, suggesting that isoprenoid biosynthetic pathway (particularly MVA pathway which includes the initial steps of isoprenoid synthesis) was activated at this temperature. There is competition among intermediate compounds of the f.a., astaxanthin, and ergosterol pathways because these macromolecules share common precursors, such as acetyl-CoA and FPP. Astaxanthin content is inversely correlated with f.a. synthesis in P. rhodozyma [23]. F.a. synthesis in MK19 was inhibited at 28 °C, resulting in a probable accumulation of acetyl-CoA and other precursors, enhanced activity of MVA pathway, excessive synthesis of ergosterol, and reduction of cell survival. Precursors from MVA pathway and F. a. synthesis were also diverted to astaxanthin biosynthesis via activated carotenoid pathway in MK19, resulting in increase of astaxanthin content to 2000 μg/g. The present ndings, taken together, clearly indicate that strengthening of the MVA pathway is a feasible and e cient metabolic engineering approach for enhancement of astaxanthin synthesis in MK19 or other moderate-temperature strains.

Conclusion
The inability of WT P. rhodozyma to grow at 28 °C is due to blocking of DNA, RNA, cell membrane, and cell wall biosynthesis through transcriptional regulation. In moderate-temperature mutant strain MK19, excessive accumulation of ergosterol, glucan, andprotein, at this temperature led to biomass production of 6 g/L. Except ergosterol, acetyl-coA from impeded f.a. pathway, was also diverted to astaxanthin biosynthesis via activated carotenoid pathway. Strengthening of the MVA pathway could be a feasible metabolic engineering approach for enhancement of astaxanthin synthesis in MK19 or other moderatetemperature producer strains.

Methods
Strains and culture conditions WT P. rhodozyma strain JCM9042 was from the Institute of Physical and Chemical Research, Japan. MK19, an astaxanthin-overproducing and moderate-temperature mutant strain, was generated and screened by NTG and Co 60 mutagenesis in our laboratory [24]. Both strains were maintained on potato dextrose agar slants at 4 °C.
Seed medium and fermentation medium were prepared as described previously [24]. All experiments were conducted in shaking ask culture in 250 ml asks containing xed liquid volume 25 ml. The DNA, RNA, Protein content was prepared using extraction kit (Omega, US), and determined by Nano drop2000 (Thermo), the fatty acid content was determined the same as Miao et al 2010 [23].
Total RNA puri cation and reverse transcription Cells of WT and MK19 in 1 ml broth were harvested by centrifugation (12000 rpm, 1 min), frozen immediately in liquid nitrogen, and stored at -70 ºC until processing. RNA extraction was performed using  Figure 1 Cell growth (A) and astaxanthin content (B) of P. rhodozyma strain MK19 at three temperatures as indicated.