Mucoromycota fungi are powerful cell factories widely applicable in developing modern biorefineries [1
]. Mucoromycota fungi can accumulate a wide range of high-value metabolites, among which lipids and polyphosphates have gained much interest in recent years [3
]. Oleaginous Mucoromycota can accumulate lipids with a similar composition as plant and animal oils, in amounts higher than 20% of their dry cell biomass [4
In order to optimize the production of Mucoromycota lipids and polyphosphate and maximize biomass yield, it is crucial to understand the role of different growth medium components on fungal growth and metabolic activity [5
]. In a recent study, we investigated the effect of metal and phosphate ions on the growth, lipid accumulation, and cell chemistry of Mucor circinelloides
]. We showed that calcium (Ca) starvation enhanced lipid accumulation in M. circinelloides,
while increased Ca availability positively affected polyphosphate accumulation.
Calcium is a unique universal signaling element in prokaryotic and eukaryotic cells. Calcium signaling is an evolutionary conserved process, which, in fungal cells, regulates multiple cell functions ranging from growth [8
], hyphae development, sporulation, and chitin synthesis [11
] to intracellular pH signaling [12
], stress tolerance, and virulence [13
]. The level of Ca2+
in the cytosol is important for signaling and regulation of the above-mentioned processes. In fungal cells, calcium is mainly stored in vacuoles, which can contain approximately 95% of the cellular Ca [14
]. For supporting Ca signaling, cells maintain cytosolic Ca at a low concentration. There are different protein transporters managing the level of Ca ions in cytosol and mediating entry or exit from vacuoles. In eukaryotic cells, Ca is required at the endoplasmic reticulum (ER), where it provides the correct function of protein folding and secretory machinery [15
Since polyphosphate and lipid accumulation are associated with ER, calcium could be directly or indirectly involved in their accumulation. It has been reported that calcium and several other cations neutralize the negative charge of polyphosphate in fungal cells [16
]. Thus, it can be hypothesized that with a higher availability of calcium ions in the medium, more efficient neutralization of the negatively charged polyphosphate occurs and, subsequently, a higher amount of phosphorus can be stored intracellularly in the form of polyphosphate [7
]. Moreover, it has been reported that calcium starvation enhances lipid accumulation in oleaginous algae [18
] and mammalian adipocyte cells [19
]. Currently, there are several hypotheses on the mechanisms behind Ca-deficiency-induced lipid accumulation in oleaginous microorganisms. The first hypothesis is related to the study by Cifuentes et al. [20
]. It is based on mediation of antilipolytic pathways through a calcium-sensing receptor (CaSR) triggered by the low cellular availability of Ca ions. This results in enhanced lipid accumulation in cells. Due to the evolutionary conservation of lipolytic pathways and Ca signaling [21
], it has been suggested that Ca deficiency can mediate similar antilipolytic pathways in oleaginous microorganisms [7
]. The second hypothesis was suggested by Wang et al. [19
], and it is based on the importance of calcium ions in the basal sensitivity of the sterol-sensing mechanism of the sterol response element binding protein (SREBP) pathway. Wang et al. discovered that a reduction in the Ca concentration in ER changes the distribution of intracellular sterol/cholesterol, resulting in the enhancement of SREBP activation and triggering the synthesis of neutral lipids. Sterol response element binding proteins (SREBPs) are transcription factors that are synthesized on ER and are considered as ER-associated integral membrane proteins [19
]. SREBPs were reported for eukaryotic cells, including mammalian and fungal cells [22
In order to investigate whether the role of calcium ions in lipid and polyphosphate accumulation is conserved for different Mucoromycota fungi, in this study, six Mucoromycota strains were grown in the presence or absence of Ca ions at three different phosphorus concentrations and, thus, different pH conditions. Relatively high phosphate concentrations were used to buffer the growth media and provide conditions for polyphosphate accumulation. Nitrogen limitation was used to trigger the lipid accumulation in oleaginous fungi.
To the authors’ knowledge, this study is among the first studies assessing the role of Ca ions on polyphosphate and lipid accumulation in Mucoromycota fungi under different phosphorus concentrations.
Calcium is an important second messenger in the transduction of cellular signals and cell growth under stress conditions. Exposure of fungal cells to environmental stress triggers an immediate response in cytoplasmic calcium levels. This process is fundamental for the survival of eukaryotic cells. Through a variety of calcium signal transduction mechanisms, fungal cells can tolerate numerous environmental changes, including pH stress [33
]. There are at least two calcium-based signal transduction pathways regulating the processes necessary for pH adjustment and ion homeostasis in eukaryotic cells [33
]. In this study, fungi were grown in media with ammonium sulphate as a nitrogen source and various phosphorus substrate concentrations combined with different levels of calcium availability. Due to the low buffering capacity of ammonium sulphate and the fact that the uptake of ammonium ions causes an increase in the release of H+
by fungal cells, the variation in phosphorus concentration caused a significant drop in pH from 6.0 to 2.0. The biomass concentration data showed that calcium deficiency negatively affected the adaptation of fungal cells to the different phosphorus/pH conditions. Thus, reduced growth and biomass formation at lower phosphorus/pH levels were observed. A large number of studies on a variety of eukaryotic cell types, including fungal cells, reported interactions between changes in pH and calcium cellular signals, where both cytosolic acidification and alkalization caused increases in cytoplasmic calcium for providing ion homeostasis in the cell [34
]. Therefore, calcium availability is critical for pH stress tolerance, as was shown in our study. While a significant growth-inhibiting effect of calcium deficiency was recorded at high (Pi4) and reference (Pi1) phosphorus substrate concentrations, resulting in pH 5.0 and 3.0, respectively, an increase in biomass concentration was observed when calcium was absent in the media with a low phosphorus level (Pi0.5) and pH 2.0. Such a twisting effect of calcium deficiency could be explained by the higher lipid accumulation under the Ca0-Pi0.5 condition, meaning that the biomass increase under this condition was due to the higher lipid content and not elevated growth rate.
In addition to pH stress tolerance, it has been reported that calcium ions are involved in lipid and phosphorus metabolism of eukaryotic cells [16
]. Thus, the synthesis and accumulation of phosphorus in the form of energy storage compounds such as polyphosphates are linked to the storage of cellular calcium. Polyphosphate granules, also known as acidocalcisomes, are membrane-bound evolutionary conserved organelles found in prokaryotic and eukaryotic cells, whose main function is the accumulation of polyphosphate and cations such as calcium, magnesium, zinc, and sodium [35
]. Calcium, as well as other cations, functions as a neutralizing agent for negatively charged polyphosphates in the formation of acidocalcisomes. Therefore, calcium availability is an important prerequisite for the formation of polyphosphate granules. In our study, calcium deprivation led to a decrease in the total phosphorus content in Mucoromycota fungi. Some exceptions where the total phosphorus content in calcium-deficient conditions was higher than when calcium was present in the media were recorded for M. circinelloides
FRR 5020, M. racemosus
, and U. vinacea
. This could be explained by the possible involvement of other cations present in the media, such as magnesium and zinc, in polyphosphate accumulation. Furthermore, it was reported that for the cells grown under an alkaline pH 7.5, the activities of a microbial polyphosphate synthesis enzyme—polyphosphate kinase (PPK)—and a polyphosphate hydrolysis enzyme—exopolyphosphatase (PPX)—were approximately equal [36
]. In contrast, at slightly acidic pH (5.5), PPK activity increased sixfold, while PPX activity remained unchanged [36
]. This elevation in PPK activity could be responsible for the increased intracellular accumulation of polyphosphate at pH 5.5. This observation is in accordance with our study, where the highest polyphosphate accumulation was observed at pH 5.5.
The positive effect of calcium-starved growth media on lipid accumulation was observed for oleaginous algae [18
], although, to the authors’ knowledge, there is no study reporting the role of calcium in the lipogenesis of oleaginous Mucoromycota fungi. Recently, we reported the first indication on the influence of calcium ions on lipid accumulation in oleaginous M. circinelloides
]. The aim of this study was to investigate whether calcium displayed some general or strain-specific patterns in lipid accumulation in Mucoromycota fungi. In this study, the lipid-triggering effect of calcium deprivation was remarkably pronounced in all fungi depending on the phosphorus substrate/pH conditions. Interestingly, the absence of calcium in the medium with Pi0.5/pH 2.0 showed a general effect of increased lipid accumulation in all fungi except M. circinelloides
FRR 5020. Concerning the effect of pH on lipid accumulation in fungal cells, the reference literature indicates that pH variation in the culture medium affects the lipid composition rather than the total lipid content [37
]. Thus, it is possible that energy was translocated to lipid synthesis rather than polyphosphate synthesis. The response to pH variations is suspected to be strain- and species-specific. Therefore, the variation in the calcium availability effect on lipid accumulation in Mucoromycota fungi observed in this study could be associated with the strain-specific response to pH changes in the culture media associated with the different levels of phosphorus substrate.
The observation of a higher total lipid content in Mucoromycota fungi under calcium deficiency at a low pH of 2.0 could presumably be explained by the activation of the unfolded protein response (UPR). UPR is known as a signal transduction pathway activated in a response to ER stress. ER stress can be mediated by the extremely low pH of the surrounding environment (for example, culture medium) or calcium deficiency, and it results in the disruption of the ER protein-folding capacity [38
]. Disruption of the ER protein-folding capacity leads to the activation of the UPR signaling system for restoring ER homeostasis. Furthermore, activation of the UPR pathways’ modulating lipid metabolism in cells triggers lipogenesis, which leads to higher accumulation of lipids. Based on our results, calcium might have an important function in activating UPR pathways, as a lipid-triggering effect under acidic pH was observed when calcium was removed from the culture medium. In addition to the UPR-based hypothesis, there are two other hypotheses explaining the lipid-triggering effect of calcium deficiency. One is related to the mediation of antilipolytic pathways through a calcium-sensing receptor (CaSR) triggered by the low cellular availability of calcium ions. This results in enhanced lipid accumulation in cells [21
]. The second hypothesis is based on the importance of calcium ions in the basal sensitivity of the sterol-sensing mechanism of the sterol response element binding protein (SREBP) pathway [19
]. Reduction in the calcium concentration in the ER changes the distribution of intracellular sterol/cholesterol, resulting in the enhancement of SREBP activation and triggering the synthesis of neutral lipids. Currently, there is no clear evidence showing which of the hypotheses are valid for fungal cells, and more in-depth investigation is needed to understand the role of calcium in the lipogenesis of oleaginous fungi. Moreover, it is worth exploring whether there is a link between polyphosphate and lipid accumulation, and whether calcium simultaneously affects both accumulation mechanisms.
In addition to the observations related to the calcium involvement in the accumulation of polyphosphate and lipids in Mucoromycota fungi, several other interesting observations arose in this study. When harvesting and washing the fungal biomass, it was observed that the biomass of A. rouxii
and the two M. circinelloides
strains had a yellow coloration, indicating possible high content of carotenoids (Figure 8
). The biomass obtained from Ca0-Pi0.5 media showed the highest pigmentation. It is interesting that the two strains of the same species, M. circinelloides
, showed different metabolic responses. Biomass production and lipid accumulation in the calcium-deficient Pi0.5 condition notably differed for these strains. Furthermore, the strain M. circinelloides
FRR 5020 showed higher carotenoid production than M. circinelloides
VI 04473 did (Figure 8
). The ability of carotenoid production by M. circinelloides
FRR 5020 is most likely the cause of the difference in metabolic behavior of this strain compared to M. circinelloides
Carotenoid production for M. circinelloides
has been reported previously [39
], with the main factors triggering carotenoid production being light, temperature, and aeration [41
]. To the authors’ knowledge, this is the first indication of the triggering effect of calcium deficiency on carotenoid production. Since assessment of carotenoids was outside the scope of this study, no further analysis on estimating carotenoid content was conducted. Due to the fact that Ca availability influenced several co-products in fungal biomass, namely lipids, polyphosphates, carotenoids, and possibly chitin/chitosan, Ca availability can be used to optimize the co-production potential and, therefore, the economic feasibility of Mucoromycota fungal fermentation.