Bioinformatic Analyses to Uncover Genes Involved in Trehalose Metabolism in the Polyploid Sugarcane

Trehalose-6-phosphate (T6P) is an intermediate of trehalose biosynthesis that plays an essential role in plant metabolism and development. Here, we comprehensively analyzed sequences from enzymes of trehalose metabolism in sugarcane, one of the main crops used for bioenergy production. We identified protein domains, phylogeny, and in silico expression levels for all classes of enzymes. However, post-translational modifications and residues involved in catalysis and substrate binding were analyzed only in trehalose-6-phosphate synthase (TPS) sequences. We retrieved 71 putative full-length TPS, 93 trehalose-6-phosphate phosphatase (TPP), and 3 trehalase (TRE) of sugarcane, showing all their conserved domains, respectively. Putative TPS (Classes I and II) and TPP sugarcane sequences were categorized into well-known groups reported in the literature. We measured the expression levels of the sequences from one sugarcane leaf transcriptomic dataset. Furthermore, TPS Class I has specific N-glycosylation sites inserted in conserved motifs and carries catalytic and binding residues in its TPS domain. Some of these residues are mutated in TPS Class II members, which implies loss of enzyme activity. Our approach retrieved many homo(eo)logous sequences for genes involved in trehalose metabolism, paving the way to discover the role of T6P signaling in sugarcane.

Parallel evolution of trehalose production machinery in anhydrobiotic animals via recurrent gene loss and horizontal transfer

Trehalose is a versatile non-reducing sugar. In some animal groups possessing its intrinsic production machinery, it is used as a potent protectant against environmental stresses, as well as blood sugar. However, the trehalose biosynthesis genes remain unidentified in the large majority of metazoan phyla, including vertebrates. To uncover the evolutionary history of trehalose production machinery in metazoans, we scrutinized the available genome resources and identified bifunctional trehalose-6-phosphate synthase-trehalose-6-phosphate phosphatase (TPS–TPP) genes in various taxa. The scan included our newly sequenced genome assembly of a desiccation-tolerant tardigrade Paramacrobiotus sp. TYO, revealing that this species retains TPS–TPP genes activated upon desiccation. Phylogenetic analyses identified a monophyletic group of the many of the metazoan TPS–TPP genes, namely ‘pan-metazoan’ genes, that were acquired in the early ancestors of metazoans. Furthermore, coordination of our results with the previous horizontal gene transfer studies illuminated that the two tardigrade lineages, nematodes and bdelloid rotifers, all of which include desiccation-tolerant species, independently acquired the TPS–TPP homologues via horizontal transfer accompanied with loss of the ‘pan-metazoan’ genes. Our results indicate that the parallel evolution of trehalose synthesis via recurrent loss and horizontal transfer of the biosynthesis genes resulted in the acquisition and/or augmentation of anhydrobiotic lives in animals.

HbxB Is a Key Regulator for Stress Response and β-Glucan Biogenesis in Aspergillus nidulans

Homeobox transcription factors are conserved in eukaryotes and act as multi-functional transcription factors in filamentous fungi. Previously, it was demonstrated that HbxB governs fungal development and spore viability in Aspergillus nidulans. Here, the role of HbxB in A. nidulans was further characterized. RNA-sequencing revealed that HbxB affects the transcriptomic levels of genes associated with trehalose biosynthesis and response to thermal, oxidative, and radiation stresses in asexual spores called conidia. A phenotypic analysis found that hbxB deletion mutant conidia were more sensitive to ultraviolet stress. The loss of hbxB increased the mRNA expression of genes associated with β-glucan degradation and decreased the amount of β-glucan in conidia. In addition, hbxB deletion affected the expression of the sterigmatocystin gene cluster and the amount of sterigmatocystin. Overall, these results indicated that HbxB is a key transcription factor regulating trehalose biosynthesis, stress tolerance, β-glucan degradation, and sterigmatocystin production in A.nidulans conidia.

Comparative Genome Analysis of Carbohydrate-Active Enzymes and Virulence Factors in Lichen-Associated Variovorax Sp. PAMC28711

Background: The genus Variovorax sp. PAMC28711 is a cold-adapted microorganism, isolated from Antarctica lichen Himantormia. The complete genomes of six Variovorax species were analyzed and compared along with the strain PAMC28711. The genomic information was collected from NCBI as well as PATRIC databases. Likewise, CAZyme annotation (dbCAN2 Meta server) was performed in order to predict the CAZyme family responsible for trehalose synthesis and trehalose degradation enzymes. The trehalose metabolic pathway was analyzed via the KEGG database. Bioinformatics tools such as OrthoANI software were used to analyze similar genes in different strains under the same genus. Likewise, MEGA X was used for evolutionary and conserved genes.Results: The complete genome of genus V. sp. PAMC28711 was found to comprise CAZyme families GH (10), GT (9), CB (1), AA5 (1), and CE (1). The three trehalose synthetic pathways (OtsA/OtsB, TS, and TreY/TreZ) and trehalose degradation pathway (TreF) were identified only in V. sp. PAMC28711 among the different strains of Variovorax studied, whereas one to two pathways of trehalose biosynthesis, but not trehalose degradation pathways are involved in other Variovorax strains. The strain PAMC28711 comprises of cytoplasmic trehalase (TreF) as a trehalose degrading enzyme that belongs to the CAZyme family GH37, which is not identified in other strains of Variovorax.Conclusions: To date, although the genus V. sp. PAMC28711 has not been reported to exhibit CAZyme activities such as trehalase, and no microorganism expressed different virulence factors, the results based on PATRIC database showed that the strain carried a few virulence genes. Further, this study provides additional information regarding trehalase as one of the factors facilitating bacterial survival under extreme environments and this enzyme has showed potential application in biotechnology fields.

Characteristics and Expression Analyses of Trehalose-6-Phosphate Synthase Family in Prunus mume Reveal Genes Involved in Trehalose Biosynthesis and Drought Response

Trehalose and its key synthase (trehalose-6-phosphate synthase, TPS) can improve the drought tolerance of plants. However, little is known about the roles of trehalose and the TPS family in Prunus mume response to drought. In our study, we discovered that the trehalose content in leaf, root, and stem tissues significantly increased in P. mume in response to drought. Therefore, the characteristics and functions of the TPS family are worth investigating in P. mume. We identified nine TPS family members in P. mume, which were divided into two sub-families and characterized by gene structure, promoter elements, protein conserved domains, and protein motifs. We found that the Hydrolase_3 domain and several motifs were highly conserved in Group II instead of Group I. The distinctions between the two groups may result from selective constraints, which we estimated by the dN/dS (ω) ratio. The ω values of all the PmTPS family gene pairs were evaluated as less than 1, indicating that purity selection facilitated their divergence. A phylogenetic tree was constructed using 92 TPSs from 10 Rosaceae species, which were further divided into five clusters. Based on evolutionary analyses, the five clusters of TPS family proteins mainly underwent varied purity selection. The expression patterns of PmTPSs under drought suggested that the TPS family played an important role in the drought tolerance of P. mume. Combining the expression patterns of PmTPSs and the trehalose content changes in leaf, stem, and root tissues under normal conditions and drought stress, we found that the PmTPS2 and PmTPS6 mainly function in the trehalose biosynthesis in P. mume. Our findings not only provide valuable information about the functions of trehalose and TPSs in the drought response of P. mume, but they also contribute to the future drought breeding of P. mume.

Genome Wide Analysis Reveals the Role of VadA in Stress Response, Germination, and Sterigmatocystin Production in Aspergillus nidulans Conidia

In the Aspergillus species, conidia are asexual spores that are infectious particles responsible for propagation. Conidia contain various mycotoxins that can have detrimental effects in humans. Previous study demonstrated that VadA is required for fungal development and spore viability in the model fungus Aspergillus nidulans. In the present study, vadA transcriptomic analysis revealed that VadA affects the mRNA expression of a variety of genes in A. nidulans conidia. The genes that were primarily affected in conidia were associated with trehalose biosynthesis, cell-wall integrity, stress response, and secondary metabolism. Genetic changes caused by deletion of vadA were related to phenotypes of the vadA deletion mutant conidia. The deletion of vadA resulted in increased conidial sensitivity against ultraviolet stress and induced germ tube formation in the presence and absence of glucose. In addition, most genes in the secondary metabolism gene clusters of sterigmatocystin, asperfuranone, monodictyphenone, and asperthecin were upregulated in the mutant conidia with vadA deletion. The deletion of vadA led to an increase in the amount of sterigmatocystin in the conidia, suggesting that VadA is essential for the repression of sterigmatocystin production in conidia. These results suggest that VadA coordinates conidia maturation, stress response, and secondary metabolism in A. nidulans conidia.

A hybrid Embden–Meyerhof–Parnas pathway provides a synthetic link between sugar and phosphate metabolism

The fundamental Embden–Meyerhoff–Paranas (EMP) pathway for sugar catabolism, anabolism, and energy metabolism has been reconstituted with non-oxidative glycolysis (NOG). Although carbon conservation was achieved via NOG, the energy metabolism was significantly limited. Herein, we showed the construction of a hybrid EMP that replaced the first phase of the EMP in Corynebacterium glutamicum with NOG and revealed a metabolic link of carbon and phosphorus metabolism. In accordance with synthetic glucose kinase activity and phosphoketolase on the hybrid EMP, cell growth was completely recovered in the C. glutamicum pfkA mutant strain where the first phase of EMP was eliminated. Notably, we have revealed a phosphate-replenishing pathway that involved trehalose biosynthesis for the generation of inorganic phosphate (Pi) sources in the hybrid EMP when external Pi supply was limited. Thus, the re-designed hybrid EMP pathway with balanced carbon and phosphorus states provides an efficient microbial platform for biochemical production.

Kramers’ Theory and the Dependence of Enzyme Dynamics on Trehalose-Mediated Viscosity

The disaccharide trehalose is accumulated in the cytoplasm of some organisms in response to harsh environmental conditions. Trehalose biosynthesis and accumulation are important for the survival of such organisms by protecting the structure and function of proteins and membranes. Trehalose affects the dynamics of proteins and water molecules in the bulk and the protein hydration shell. Enzyme catalysis and other processes dependent on protein dynamics are affected by the viscosity generated by trehalose, as described by the Kramers’ theory of rate reactions. Enzyme/protein stabilization by trehalose against thermal inactivation/unfolding is also explained by the viscosity mediated hindering of the thermally generated structural dynamics, as described by Kramers’ theory. The analysis of the relationship of viscosity–protein dynamics, and its effects on enzyme/protein function and other processes (thermal inactivation and unfolding/folding), is the focus of the present work regarding the disaccharide trehalose as the viscosity generating solute. Finally, trehalose is widely used (alone or in combination with other compounds) in the stabilization of enzymes in the laboratory and in biotechnological applications; hence, considering the effect of viscosity on catalysis and stability of enzymes may help to improve the results of trehalose in its diverse uses/applications.