Mutant strain MK19 grew at 28 °C, Astaxanthin content was enhanced significantly 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, viability and 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 findings suggest that cell growth and astaxanthin synthesis in P. rhodozyma are controlled by temperature through independent mechanisms.
Transcriptional profiling 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 efficient 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 coefficient 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 findings suggest that HSR, which induces a battery of cytoprotective genes that encode HSPs, is an adaptive mechanism in MK19.
Table 1
Fold expression changes of HSP, f.a., etc. related genes at 28 vs. 21 °C, based on RT-PCR and RNA-Seq analyses.
| Gene | Function | RT-PCR: fold-change 28/21 °C | RNA-Seq: fold-change 28/21 °C |
1 | comp12727_c0 | HSP | 1.75 | 1.93 |
2 | comp13502_c0 | HSP homolog pss1 | 16.16 | 3.87 |
3 | comp12521_c0 | DnaJ family | 1.25 | -1.29 |
4 | comp11731_c2 | HSP | 2.25 | 8.65 |
5 | comp11698_c0 | stress-induced protein STI1 | 2.63 | 2.33 |
6 | comp12783_c0 | HSP104 | 2.58 | 4.35 |
7 | comp12308_c0 | HSP90 | 12.06 | 3.60 |
8 | comp11869_c0 | HSP60 | 2.77 | 2.71 |
9 | comp12396_c0 | HSP78 | 2.97 | 4.50 |
10 | comp10325_c0 | HSP SSB | 2.45 | 2.23 |
11 | comp11696_c0 | hsp70-like protein | 3.74 | 3.65 |
12 | comp12989_c0 | HSP70 | 5.66 | N.D. |
13 | comp13599_c0 | FAS2 | 0.29 | N.D. |
14 | comp13900_c0 | FAS1 | 0.40 | N.D. |
15 | comp13834_c0 | ACC1, acetyl-CoA carboxylase | 0.25 | N.D. |
16 | comp11956_c0 | f.a.-2 hydroxylase | 0.48 | N.D. |
17 | comp12194_c0 | f.a. desaturase | 0.32 | N.D. |
18 | comp10129_c0 | WSC domain-containing protein | 0.03 | N.D. |
19 | comp10153_c0 | WSC | 1.80 | N.D. |
20 | comp10598_c1 | WSC domain-containing protein | 0.18 | N.D. |
21 | comp10909_c0 | related to WSC2 glucoamylase III | 0.77 | N.D. |
22 | comp10944_c0 | WSC domain-containing protein | 0.03 | N.D. |
23 | comp11101_c1 | WSC | 1.52 | N.D. |
24 | comp11381_c0 | WSC domain-containing protein | 0.02 | N.D. |
25 | comp11733_c0 | WSC | 0.30 | N.D. |
26 | comp12382_c1 | WSC-domain-containing protein | 0.04 | N.D. |
27 | comp12404_c0 | WSC | 0.15 | N.D. |
28 | comp12789_c0 | WSC | 0.82 | N.D. |
29 | comp12838_c0 | WSC | 0.11 | N.D. |
30 | comp11864_c1 | alpha-1,3-mannosyltransferase CMT1 | 2.82 | N.D. |
31 | comp12286_c0 | glycosyltransferase family 22 protein | 3.88 | N.D. |
32 | comp12082_c1 | alpha-1,6-mannosyltransferase | 0.53 | N.D. |
33 | comp11585_c0 | glycosyltransferase family 22 protein | 1.95 | N.D. |
34 | comp13450_c0 | 1,3-beta-glucanosyltransferase | 0.45 | N.D. |
35 | comp11848_c1 | endo-1,3(4)-beta-glucanase | 0.27 | N.D. |
36 | comp11848_c2 | endo-1,3(4)-beta-glucanase | 0.31 | N.D. |
37 | comp11853_c0 | endo-1,3(4)-beta-glucanase | 2.89 | N.D. |
38 | comp12961_c0 | glucan endo-1,3-alpha-glucosidase agn1 | 0.33 | N.D. |
39 | comp10598_c0 | glycoside hydrolase family 71 protein | 0.14 | N.D. |
40 | comp13745_c1 | chitin deacetylase | 4.50 | N.D. |
41 | comp14086_c0 | glycosyltransferase family 2 protein | 2.41 | N.D. |
42 | comp12056_c0 | chitin synthase 1 | 1.75 | N.D. |
43 | comp12623_c0 | glycosyltransferase family 2 protein | 1.99 | N.D. |
44 | comp13627_c0 | glycosyltransferase family 2 protein | 2.2 | N.D. |
45 | comp13980_c0 | chitin synthase 6 | 2.2 | N.D. |
N.D.: not detected. |
No. 1–12: HSP genes. No. 13–17: f.a. biosynthetic pathway genes. No. 18–29: WSC genes. No. 30–33: mannan biosynthetic pathway genes. No. 34–39: glucan biosynthetic pathway genes. No. 40–45: chitin biosynthetic pathway genes. |
Initial functional classification 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. is “purine metabolism” and “pyrimidine metabolism”. In the “purine” subset, 24 out of 25 differentially expressed genes showed significant 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. Both RNA and protein content were > 2-fold higher under 28 °C treatment than under 21 °C treatment throughout the culture period (Fig. 2B, C).
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 findings indicate that the low viability of MK19 at 28 °C was due to strong suppression of DNA metabolism.
MK19 cell membrane was damaged at 28 °C
Biological membranes function as permeable or semi-permeable barriers and play key roles in a variety of physiological processes. Maintenance of proper membrane function depends on a precise balance of various lipid species. The biosynthetic pathway of fatty acids (f.a.), essential component of cell membranes, competes with the astaxanthin biosynthetic pathway for the precursor acetyl coenzyme A. Synthesis of f.a., particularly 18:0, 16:0, and 18:1 f.a., was significantly lower under 28 °C treatment than under 21 °C treatment (Fig. 3). These f.a. species declined steadily throughout the culture period, particularly after 48 h. Total f.a. content under 28 °C treatment was ~ 50% of that under 21 °C treatment (Fig. 3A). 18:1 is the most abundant f.a. species in P. rhodozyma (Fig. 3D), and the inhibitory effect of temperature on f.a. synthesis is based mainly on control of 18:1 synthesis. These findings indicate that reduced viability of P. rhodozyma is due in part to deficiency of f.a., mainly 18:1.
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 (Fig. 4A). Regulation of terpenoids and sterols by temperature is evidently based on different mechanisms; only terpenoid synthesis was inhibited specifically by 25 °C in WT P. rhodozyma. In contrast, ergosterol content was significantly 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 mitigates inhibition of cell growth and helps modulate adaptive response to 28 °C stress. High temperature apparently induces sterol and terpenoid metabolic fluxes 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 first 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. 13–16). Metabolic and mRNA data, taken together, indicate that f.a. synthetic pathway was suppressed by 28 °C stress, and that growth of P. rhodozyma at 28 °C requires an adequate amount of f.a. Therefore, modification of acc, fas1, and fas2 expression in future studies could potentially enhance cell growth at 28 °C. sqs is the first 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 accumulated and transferred to isoprenoid biosynthetic pathway through upstream MVA pathway, with the result that astaxanthin and ergosterol content were 2-fold 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 32-fold downregulation (Table 1, No. 18–29). These findings 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; 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-1,3-mannosyltransferase (comp11864_c1), α-1,2-mannosyltransferase (comp12286_c0), and α-1,6-mannosyltransferase (comp11585_c0). MK19 glucan levels were higher at 28 °C, consistent with previous findings 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.
Table 2
Cell wall thickness comparisons for MK19 at 21 and 28 °C, and JCM9042 at 21 °C.
| | Total cell wall (µm) | chitin/ glucan layer (µm) | mannan layer (µm) |
MK19 | 21 °C | 0.46 ± 0.11 | 0.18 ± 0.05 | 0.26 ± 0.08 |
28 °C | 0.38 ± 0.07 | 0.24 ± 0.04 | 0.15 ± 0.04 |
JCM9042 | 21 °C | 0.48 ± 0.13 | 0.16 ± 0.05 | 0.32 ± 0.08 |
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 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. Reduced mRNA content of crtI was an important cause of astaxanthin inhibition at temperatures > 25 °C. crtI mRNA level was > 10-fold higher in MK19 than in WT. This level in MK19 was reduced 1.5-fold at 25 and 28 °C, but was still sufficient to counteract the inhibitory effect of higher temperature and lead to efficient 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.