Complementary function of two transketolase isoforms from Moniliella megachiliensis in relation to stress response

Two TKL-encoding isogenes (MmTKL1 and MmTKL2) were cloned and sequenced from M. megachiliensis, a hyper-osmotolerant basidiomycetous yeast-like fungus. The amino acid sequences of the MmTKL1 and MmTKL2 proteins exhibited lower levels of identity to that of S. cerevisiae TKL1 (28 and 26%, respectively) than to those of other basidiomycetous fungi. Strikingly, several amino acid residues known to be involved in TPP binding were substituted in the MmTKL proteins compared to the corresponding residues of ScTKL1. However, all these substitutions were conservative, representing amino acids of the same family with hydrophobic side chains. Thus, despite sequence divergence, MmTKL1 and MmTKL2 were expected to possess functions similar to those of the TKLs of S. cerevisiae.

The MmTKL1 and MmTKL2 proteins were found to exhibit strongest homologies (61–66% identity) to TKL proteins of Cryptococcus or Puccinia species. Pathogenic Cryptococcus (Wong et al. 1990) and Aspergillus (Wong et al. 1989) have been reported to accumulate high levels of mannitol in response to hyper-osmotic environments, a strategy that enhances survival when infecting host cells. S. cerevisiae or Candida glycerinogenes also are known to accumulate glycerol in response to conditions of hyper-osmosis, with glycerol serving as an osmo-regulatory-compatible solute (O’ Rourke et al. 2002; Chen et al. 2008). Under hyper-osmotic conditions, the S. cerevisiae Hog1 protein (the downstream-most protein kinase of the HOG (high osmolarity glycerol) pathway is activated via phosphorylation and rapidly translocates to the nucleus (Edmunds and Mahadevan 2004). Upon translocation to the nucleus, phosphorylated Hog1 (in cooperation with other transcription factors) stimulates transcription of the genes encoding GPD1 (glycerol-3-phosphate dehydrogenase 1) and GPP2 (glycerol-3 phosphate phosphatase 2) via STREs located upstream of the corresponding genes, resulting in glycerol biosynthesis (Alepuz et al. 2001; Ansell et al. 1997). Hence, STREs are believed to play an essential role in the osmotic stress response in yeast cells. We previously reported that M. megachiliensis possesses three erythrose reductase isogenes (ER1, ER2, ER3) and two transaldolase isogenes (TAL1, TAL2); the products of these genes are known to be involved in erythritol biosynthesis (Kobayashi et al. 2013, Iwata et al. 2015). Among these genes, ER3 and TAL2 harbor putative STREs within 1000 bp upstream of the respective ORFs. In the present work, we noted that the MmTKL1 ORF is preceded (at ?556 and ?220 bp) by two upstream putative STREs; in contrast, the MmTKL2 ORF appears to lack STREs within 1000 bp upstream of the initiation codon. Hence, we postulate that MmTKL1 is involved in regulation of the osmotic-stress response via the PPP. On the other hand, two putative AP-1 elements were found upstream of the MmTKL2 ORF, but not proximal to MmTKL1. Since AP-1 has been shown in other organisms to mediate responses to oxidative stress (Toone and Jones 1999), we hypothesize that MmTKL2 is involved in the oxidative stress response. Similar results have been obtained for MmTAL1 (Iwata et al. 2015).

In addition to stress response, the function of MmTKL1 and MmTKL2 was evaluated based on another criterion: nutritional requirement. The erythrose-4-phosphate generated by TKLs can be converted (via the PPP; in microorganisms, fungi, and plants) to AAAs by way of the shikimic acid pathway (Hermann and Weaver 1999). Notably, a S. cerevisiae tkl1 deletion mutant is not able to grow in synthetic complete medium lacking AAAs. Our transformation tests demonstrated that MmTKL2 (but not MmTKL1) can partially complement the AAA auxotrophy of a S. cerevisiae tkl1 mutant. We have confirmed this result, including demonstration that the MmTKL1 plasmid is indeed present in the S. cerevisiae transformant (data not shown). The reason for this failure to complement is unknown, but may reflect the absence of heterologous expression of MmTKL1 in the yeast background under the plate assay conditions. Meanwhile, we are not convinced that MmTKL1 and MmTKL2 are orthlogs of TKL1 and TKL2 of S. cerevisiae, respectively, because number of MmTKL is not strictly defined. In fact, we have found three TKL homologues in M. megachiliensis draft genome sequence decoded, and obtained two of them, MmTKL1 and MmTKL2, as shown in this study. It is unclear that putative third TKL gene may complement S. cerevisiae TKL function. Analyses to determine MmTKL1 expression in the transformant and further, putative third gene will be needed.

In the presence of 20% glucose in M. megachiliensis, endogenous MmTKL1 expression peaked at 40 min after osmotic stress loading before subsequently gradually decreasing through 120 min. In contrast, MmTKL2 showed an approximately constant expression level during this osmotic stress interval. Similar results were obtained for gene expression profiles under NaCl-induced osmotic stress. In contrast, distinct results were obtained under conditions of short-term (120-min) oxidative stress, with the level of MmTKL1 expression remaining relatively low while MmTKL2 transcript exhibited marked accumulation. These results implicate MmTKL1 as a major mediator of the response to hyper-osmotic stress; in contrast, MmTKL2 is inferred to be a major mediator in the response to oxidative stress. The oxidative stressor used here (menadione) is metabolized to semiquinone by the oxido-reductase system of the cell, and then subsequently converted to a quinone that generates reactive oxygen species (ROS) (Yamashoji et al. 1991). ROS often induce oxidative damage and impair cell survival (Yashiki and Yamashoji 1996). S. cerevisiae TKL1 reportedly is induced by oxidative agents like hydrogen peroxide or acetoaldehyde (Jamieson 1998). Furthermore, TKL mediated by Yap1p and Skn7p in S. cerevisiae has been reported to contribute to the regulation of glutathione and NADPH for cell redox homeostasis (Carter et al. 2005; Slekar et al. 1996). We postulate that the ROS generated by menadione degradation similarly induces MmTKL2 expression in M. megachiliensis, such that this isoform of TKL contribute to the regulation of glutathione and NADPH for eliminating ROS in this yeast-like fungus.

We used 72-h growth in medium containing 20% glucose to model long-term exposure to hyper-osmotic conditions; these conditions parallel those used in industrial fermentation for production of erythritol. Under these conditions, MmTKL1 expression rapidly increased starting at 12 h and reached a maximum at 48 h, while MmTKL2 expression increased throughout the 72-h experiment. A clear correlation was observed between MmTKL1 expression and erythritol accumulation under conditions of hyper-osmotic glucose stress; no such correlation was observed between MmTKL2 expression and erythritol production. As postulated for short-term stress, MmTKL1 expression appears to be associated with the long-term response to osmotic stress in this organism. The expression of MmTKL2 increased with time during long-term osmotic stress, and this isogene may contribute to elimination of ROS that accumulate during long-term stress in high-glucose culture, which is more or less similar to oxidative stress caused by menadione. Regarding stress responses, compensatory behavior of isogenes is known to apply to S. cerevisiae GPD1 and GPD2, which encode isoforms of a key enzyme of glycerol biosynthesis (Ansell et al. 1997).

Based on the results obtained here, we consider that MmTKL1 is involved in the M. megachiliensis response to osmotic stress. In contrast, MmTKL2 appears to be involved in the response to oxidative stress, while also contributing to the AAA supply that is essential for growth in synthetic and minimal media. Intriguingly, S. cerevisiae also has been reported to possess two TKL isogenes, TKL1 and TKL2. Based on mutant phenotype, TKL1 is presumed to contribute to the supply of AAAs; the function of the ScTKL2 isogene presumed complementary of TKL1 remains unclear.

In summary, our results suggest that MmTKL1 and MmTKL2 may play distinct and complementary roles in M. megachiliensis defense against environmental stress, mediated by induction of erythritol production. To our knowledge, the results obtained in our study are the first instance of complementary function of TKL isogenes in association with stress response. We are now going to analyze the detailed mechanism of erythritol biosynthesis involved in ROS elimination in stress response of M. megachiliensis.