BackgroundLignocellulolytic enzymes are the main enzymes to saccharify lignocellulose from renewable plant biomass in the bio-based economy. The production of these enzymes is transcriptionally regulated by multiple transcription factors. We previously engineered Penicillium oxalicum for improved cellulase production via manipulation of three genes in the cellulase expression regulatory network. However, the potential of combinational engineering of multiple regulators and their targets at protein abundance and activity levels has not been fully explored.ResultsHere, we verified that a point mutation XlnRA871V in transcription factor XlnR enhanced the expression of lignocellulolytic enzymes, particularly hemicellulases, in P. oxalicum. Then, overexpression of XlnRA871V with a constitutive PDE_02864 promoter was combined with the overexpression of cellulase transcriptional activator ClrB and deletion of carbon catabolite repressor CreA. The resulted strain RE-7 showed 8.9- and 51.5-fold increased production of cellulase and xylanase relative to the starting strain M12, respectively. Further overexpression of two major cellulase genes cbh1-2 and eg1 enabled an additional 13.0% improvement of cellulase production. In addition, XlnRA871V led to decreased production of β-glucosidase and amylase, which could be attributed to the reduced transcription of corresponding enzyme-encoding genes.ConclusionsThe results illustrated that combinational manipulation of the involved transcription factors and their target genes was a viable strategy for efficient production of lignocellulolytic enzymes in filamentous fungi. The striking negative effect of XlnRA871V mutation on amylase production was also highlighted.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-017-0783-3) contains supplementary material, which is available to authorized users.

Adenosine 5′-triphosphate (ATP) is a central extracellular signaling agent involved in various physiological and pathological processes. However, precise measurements of the temporal and spatial components of ATP dynamics are lacking due primarily to the limitations of available methods for ATP detection. Here, we report on the first effort to design a self-phosphorylating DNAzyme (SPDz) sensor for fluorescence imaging of ATP. In response to ATP, SPDz sensors exhibit subsecond response kinetics, extremely high specificity, and micromolar affinities. In particular, we demonstrate cell-surface-anchored SPDz sensors for fluorescence imaging of both stress-induced endogenous ATP release in astrocytes and mechanical stimulation-evoked ATP release at the single-cell level. We also validated their utility for visualizing the rapid dynamic properties of ATP signaling upon electrical stimulation in astrocytes. Thus, SPDz sensors are robust tools for monitoring ATP signaling underlying diverse cellular processes.

To understand the early signaling steps that regulate cold responses in rice, two-dimensional difference gel electrophoresis (2-D DIGE) 1 was used to study early cold-regulated proteins in rice seedlings. Using mass spectrometry, 32 spots, which represent 26 unique proteins that showed an altered expression level within 5 min of cold treatment were identified. Among these proteins, Western blot analyses confirmed that the cellular phospholipase D ␣1 (OsPLD␣1) protein level was increased as early as 1 min after cold treatment. Genetic studies showed that reducing the expression of OsPLD␣1 makes rice plants more sensitive to chilling stress as well as cold acclimation increased freezing tolerance. Correspondingly, cold-regulated proteomic changes and the expression of the coldresponsive C repeat/dehydration-responsive element binding 1 (OsDREB1) family of transcription factors were inhibited in the pld␣1 mutant. We also found that the expression of OsPLD␣1 is directly regulated by OsDREB1A. This transcriptional regulation of OsPLD␣1 could provide positive feedback regulation of the cold signal transduction pathway in rice. OsPLD␣1 hydrolyzes phosphatidylcholine to produce the signal molecule phosphatidic acid (PA). By lipid-overlay assay, we demonstrated that the rice cold signaling proteins, MAP kinase 6 (OsMPK6) and OsSIZ1, bind directly to PA. Taken together, our results suggest that OsPLD␣1 plays a key role in transducing cold signaling in rice by producing PA and regulating OsDREB1s' expression by OsMPK6, OsSIZ1, and possibly other PA-binding proteins. Molecular &