Energy and sugar signaling during hypoxia.

The major consequence of hypoxia is a dramatic reduction in energy production. At the onset of hypoxia, both oxygen and ATP availability decrease. Oxygen and energy sensing therefore converge to induce an adaptive response at both the transcriptional and translational levels. Oxygen sensing results in stabilization of the transcription factors that activate hypoxia-response genes, including enzymes required for efficient sugar metabolism, allowing plants to produce enough energy to ensure survival. The translation of the resulting mRNAs is mediated by SnRK1, acting as an energy sensor. However, as soon as the sugar availability decreases, a homeostatic mechanism, detecting sugar starvation, dampens the hypoxia-dependent transcription to reduce energy consumption and preserves carbon reserves for regrowth when oxygen availability is restored.

The SnRK1-eIFiso4G1 signaling relay regulates the translation of specific mRNAs in Arabidopsis under submergence.

Cellular responses to oxygen deprivation are essential for survival during energy crises in plants and animals. Hypoxia caused by submergence results in reprogramming of translation dynamic in plants, but the molecular mechanisms are not well understood. Here we show that Arabidopsis Snf1-related protein kinase 1 (SnRK1) phosphorylates the translation initiation factor eIFiso4G to regulate translation dynamic under submergence. In Arabidopsis, there are two eIFiso4G genes, eIFiso4G1 and eIFiso4G2, which belong to the eIF4G family. Both eIFiso4Gs were phosphorylated by SnRK1 under submergence. Interestingly, the eIFiso4G1 knockout mutant, but not the eIFiso4G2 mutant, became more sensitive to submergence, implying that eIFiso4G1 is involved in regulating submergence tolerance in Arabidopsis. Comparison of RNA sequences in the polysome fraction and the RNAs immunoprecipitated by eIFiso4G1 from Col-0 and the SnRK1 and eIFiso4G1 mutants revealed that lack of eIFiso4G1 phosphorylation disrupts the translation of specific mRNAs under submergence. Taken together, our findings suggest that the SnRK1-eIFiso4G1 relay controls the translation of an array of genes under hypoxia, including core hypoxia response genes and genes related to stress response and biosynthetic process.

Quantitative phosphoproteomics of protein kinase SnRK1 regulated protein phosphorylation in Arabidopsis under submergence.

SNF1 RELATED PROTEIN KINASE 1 (SnRK1) is proposed to be a central integrator of the plant stress and energy starvation signalling pathways. We observed that the Arabidopsis SnRK1.1 dominant negative mutant (SnRK1.1 (K48M) ) had lower tolerance to submergence than the wild type, suggesting that SnRK1.1-dependent phosphorylation of target proteins is important in signalling pathways triggered by submergence. We conducted quantitative phosphoproteomics and found that the phosphorylation levels of 57 proteins increased and the levels of 27 proteins decreased in Col-0 within 0.5-3h of submergence. Among the 57 proteins with increased phosphorylation in Col-0, 38 did not show increased phosphorylation levels in SnRK1.1 (K48M) under submergence. These proteins are involved mainly in sugar and protein synthesis. In particular, the phosphorylation of MPK6, which is involved in regulating ROS responses under abiotic stresses, was disrupted in the SnRK1.1 (K48M) mutant. In addition, PTP1, a negative regulator of MPK6 activity that directly dephosphorylates MPK6, was also regulated by SnRK1.1. We also showed that energy conservation was disrupted in SnRK1.1 (K48M) , mpk6, and PTP1 (S7AS8A) under submergence. These results reveal insights into the function of SnRK1 and the downstream signalling factors related to submergence.

Systematic screening of glycosylation- and trafficking-associated gene knockouts in Saccharomyces cerevisiae identifies mutants with improved heterologous exocellulase activity and host secretion.

BACKGROUND: As a strong fermentator, Saccharomyces cerevisiae has the potential to be an excellent host for ethanol production by consolidated bioprocessing. For this purpose, it is necessary to transform cellulose genes into the yeast genome because it contains no cellulose genes. However, heterologous protein expression in S. cerevisiae often suffers from hyper-glycosylation and/or poor secretion. Thus, there is a need to genetically engineer the yeast to reduce its glycosylation strength and to increase its secretion ability. RESULTS: Saccharomyces cerevisiae gene-knockout strains were screened for improved extracellular activity of a recombinant exocellulase (PCX) from the cellulose digesting fungus Phanerochaete chrysosporium. Knockout mutants of 47 glycosylation-related genes and 10 protein-trafficking-related genes were transformed with a PCX expression construct and screened for extracellular cellulase activity. Twelve of the screened mutants were found to have a more than 2-fold increase in extracellular PCX activity in comparison with the wild type. The extracellular PCX activities in the glycosylation-related mnn10 and pmt5 null mutants were, respectively, 6 and 4 times higher than that of the wild type; and the extracellular PCX activities in 9 protein-trafficking-related mutants, especially in the chc1, clc1 and vps21 null mutants, were at least 1.5 times higher than the parental strains. Site-directed mutagenesis studies further revealed that the degree of N-glycosylation also plays an important role in heterologous cellulase activity in S. cerevisiae. CONCLUSIONS: Systematic screening of knockout mutants of glycosylation- and protein trafficking-associated genes in S. cerevisiae revealed that: (1) blocking Golgi-to-endosome transport may force S. cerevisiae to export cellulases; and (2) both over- and under-glycosylation may alter the enzyme activity of cellulases. This systematic gene-knockout screening approach may serve as a convenient means for increasing the extracellular activities of recombinant proteins expressed in S. cerevisiae.

Ethylene modulates translation dynamics in Arabidopsis under submergence via GCN2 and EIN2.

General translational repression is a key process that reduces energy consumption under hypoxia. Here, we show that plant stress-activated general control nonderepressible 2 (GCN2) was activated to regulate the reduction in polysome loading during submergence in Arabidopsis. GCN2 signaling was activated by ethylene under submergence. GCN2 activity was reduced in etr1-1, but not in ein2-5 or eil1ein3, under submergence, suggesting that GCN2 activity is regulated by a noncanonical ethylene signaling pathway. Polysome loading was not reduced in ein2-5 under submergence, implying that ethylene modulates translation via both EIN2 and GCN2. Transcriptomic analysis demonstrated that EIN2 and GCN2 regulate not only general translational repression but also translational enhancement of specific mRNAs under submergence. Together, these results demonstrate that during submergence, entrapped ethylene triggers GCN2 and EIN2 to regulate translation dynamics and ensure the translation of stress response proteins.

A highly efficient ß-glucosidase from the buffalo rumen fungus Neocallimastix patriciarum W5.

BACKGROUND: Cellulose, which is the most abundant renewable biomass on earth, is a potential bio-resource of alternative energy. The hydrolysis of plant polysaccharides is catalyzed by microbial cellulases, including endo-ß-1,4-glucanases, cellobiohydrolases, cellodextrinases, and ß-glucosidases. Converting cellobiose by ß-glucosidases is the key factor for reducing cellobiose inhibition and enhancing the efficiency of cellulolytic enzymes for cellulosic ethanol production. RESULTS: In this study, a cDNA encoding ß-glucosidase was isolated from the buffalo rumen fungus Neocallimastix patriciarum W5 and is named NpaBGS. It has a length of 2,331 bp with an open reading frame coding for a protein of 776 amino acid residues, corresponding to a theoretical molecular mass of 85.1 kDa and isoelectric point of 4.4. Two GH3 catalytic domains were found at the N and C terminals of NpaBGS by sequence analysis. The cDNA was expressed in Pichia pastoris and after protein purification, the enzyme displayed a specific activity of 34.5 U/mg against cellobiose as the substrate. Enzymatic assays showed that NpaBGS was active on short cello-oligosaccharides from various substrates. A weak activity in carboxymethyl cellulose (CMC) digestion indicated that the enzyme might also have the function of an endoglucanase. The optimal activity was detected at 40°C and pH 5 ~ 6, showing that the enzyme prefers a weak acid condition. Moreover, its activity could be enhanced at 50°C by adding Mg2+ or Mn2+ ions. Interestingly, in simultaneous saccharification and fermentation (SSF) experiments using Saccharomyces cerevisiae BY4741 or Kluyveromyces marxianus KY3 as the fermentation yeast, NpaBGS showed advantages in cell growth, glucose production, and ethanol production over the commercial enzyme Novo 188. Moreover, we showed that the KY3 strain engineered with the NpaNGS gene can utilize 2 % dry napiergrass as the sole carbon source to produce 3.32 mg/ml ethanol when Celluclast 1.5 L was added to the SSF system. CONCLUSION: Our characterizations of the novel ß-glucosidase NpaBGS revealed that it has a preference of weak acidity for optimal yeast fermentation and an optimal temperature of ~40°C. Since NpaBGS performs better than Novo 188 under the living conditions of fermentation yeasts, it has the potential to be a suitable enzyme for SSF.

Functional characterization of cellulases identified from the cow rumen fungus Neocallimastix patriciarum W5 by transcriptomic and secretomic analyses.

BACKGROUND: Neocallimastix patriciarum is one of the common anaerobic fungi in the digestive tracts of ruminants that can actively digest cellulosic materials, and its cellulases have great potential for hydrolyzing cellulosic feedstocks. Due to the difficulty in culture and lack of a genome database, it is not easy to gain a global understanding of the glycosyl hydrolases (GHs) produced by this anaerobic fungus. RESULTS: We have developed an efficient platform that uses a combination of transcriptomic and proteomic approaches to N. patriciarum to accelerate gene identification, enzyme classification and application in rice straw degradation. By conducting complementary studies of transcriptome (Roche 454 GS and Illumina GA IIx) and secretome (ESI-Trap LC-MS/MS), we identified 219 putative GH contigs and classified them into 25 GH families. The secretome analysis identified four major enzymes involved in rice straw degradation: ß-glucosidase, endo-1,4-ß-xylanase, xylanase B and Cel48A exoglucanase. From the sequences of assembled contigs, we cloned 19 putative cellulase genes, including the GH1, GH3, GH5, GH6, GH9, GH18, GH43 and GH48 gene families, which were highly expressed in N. patriciarum cultures grown on different feedstocks. CONCLUSIONS: These GH genes were expressed in Pichia pastoris and/or Saccharomyces cerevisiae for functional characterization. At least five novel cellulases displayed cellulytic activity for glucose production. One ß-glucosidase (W5-16143) and one exocellulase (W5-CAT26) showed strong activities and could potentially be developed into commercial enzymes.