Accessory enzymes of hypercellulolytic Penicillium funiculosum facilitate complete saccharification of sugarcane bagasse

BACKGROUND: Sugarcane bagasse (SCB) is an abundant feedstock for second-generation bioethanol production. This complex biomass requires an array of carbohydrate active enzymes (CAZymes), mostly from filamentous fungi, for its deconstruction to monomeric sugars for the production of value-added fuels and chemicals. In this study, we evaluated the repertoire of proteins in the secretome of a catabolite repressor-deficient strain of Penicillium funiculosum, PfMig188, in response to SCB induction and examined their role in the saccharification of SCB. RESULTS: A systematic approach was developed for the cultivation of the fungus with the aim of producing and understanding arrays of enzymes tailored for saccharification of SCB. To achieve this, the fungus was grown in media supplemented with different concentrations of pretreated SCB (0-45 g/L). The profile of secreted proteins was characterized by enzyme activity assays and liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 280 proteins were identified in the secretome of PfMig188, 46% of them being clearly identified as CAZymes. Modulation of the cultivation media with SCB up to 15 g/L led to sequential enhancement in the secretion of hemicellulases and cell wall-modifying enzymes, including endo-β-1,3(4)-glucanase (GH16), endo-α-1,3-glucanase (GH71), xylanase (GH30), β-xylosidase (GH5), β-1,3-galactosidase (GH43) and cutinase (CE5). There was ~ 122% and 60% increases in β-xylosidase and cutinase activities, respectively. There was also a 36% increase in activities towards mixed-linked glucans. Induction of these enzymes in the secretome improved the saccharification performance to 98% (~ 20% increase over control), suggesting their synergy with core cellulases in accessing the recalcitrant region of SCB. CONCLUSION: Our findings provide an insight into the enzyme system of PfMig188 for degradation of complex biomass such as SCB and highlight the importance of adding SCB to the culture medium to optimize the secretion of enzymes specific for the saccharification of sugarcane bagasse

Metabolite of the pesticide DDT and incident type 2 diabetes in urban India

BACKGROUND: Previous epidemiological studies, largely conducted in high-income countries and cross-sectional, have suggested a relatively strong association between exposure to dichlorodiphenyldichloroethylene (DDE), a metabolite of the pesticide dichlorodiphenyltrichloroethane (DDT), and type 2 diabetes. DDT is widely used in India and the prevalence of type 2 diabetes there is increasing, but the association between these factors has not been explored to date. OBJECTIVE: The objective was to estimate the association of the p,p' isomer of DDE with incident type 2 diabetes in India. METHODS: A nested case-control study was conducted in a representative prospective cohort of adults from two cities in India. Participants were enrolled in 2010-11 (n = 12,271) and followed for annual assessment of chronic diseases including type 2 diabetes. Baseline plasma samples from incident cases of diabetes (n = 193) and sex-city-matched controls (n = 323) were selected for analysis of p,p-DDE. Odds ratios (OR) and 95% confidence intervals (CI) were estimated using conditional logistic regression. RESULTS: At baseline, cases had higher p,p-DDE concentrations: geometric mean (95% CI) 330 (273-399) ng/g lipid compared to 223 (189-262) ng/g lipid among controls. Delhi participants had higher p,p-DDE concentrations: 579 (521-643) ng/g lipid compared to 122 (102-145) ng/g lipid in Chennai. In unadjusted models, being in the highest versus lowest quartile of p,p-DDE was associated with a more than doubling of the odds of diabetes: unadjusted OR (95% CI), 2.30 (1.19, 4.43). However, this effect was no longer significant after adjustment for age: adjusted (95% CI), 0.97 (0.46, 2.06). DISCUSSION: Results suggest that levels of p,p'-DDE in Delhi are exceptionally high, but we did not observe a significant association between p,p-DDE and incident type 2 diabetes. As this is the first study to evaluate this association in India, more studies are needed to inform our understanding of the association in this context, including potential routes of exposure

WITHDRAWN: Identification of an endogenous redox partner for lytic polysaccharide monooxygenase-based oxidative cleavage of polysaccharides.

This article has been withdrawn by the authors. Figs 4B and 6C were inappropriately presented

Metabolomic Profiling Revealed Diversion of Cytidinediphosphate-Diacylglycerol and Glycerol Pathway towards Denovo Triacylglycerol Synthesis in Rhodosporidium toruloides

Oleaginous yeast Rhodosporidium toruloides has great biotechnological potential and scientific interest, yet the molecular rationale of its cellular behavior to carbon and nitrogen ratios with concurrent lipid agglomeration remains elusive. Here, metabolomics adaptations of the R. toruloides in response to varying glucose and nitrogen concentrations have been investigated. In preliminary screening we found that 5% glucose (w/v) was optimal for further analysis in Rhodosporidium toruloides 3641. Hereafter, the effect of complementation to increase lipid agglomeration was evaluated with different nitrogen sources and their concentration. The results obtained illustrated that the biomass (13 g/L) and lipid (9.1 g/L) production were maximum on 5% (w/v) glucose and 0.12% (NH4)2SO4. Furthermore, to shed lights on lipid accumulation induced by nitrogen-limitation, we performed metabolomic analysis of the oleaginous yeast R. toruloides 3641. Significant changes were observed in metabolite concentrations by qualitative metabolomics through gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS), which were mapped onto the governing metabolic pathways. Notable finding in this strain concerns glycerol and CDP-DAG metabolism wherein reduced production of glycerol and phospholipids induced a bypass leading to enhanced de-novo triacylglyceride synthesis. Collectively, our findings help in understanding the central carbon metabolism of R. toruloides which may assist in developing rationale metabolic models and engineering efforts in this organism

Biophysical and structural studies reveal marginal stability of a crucial hydrocarbon biosynthetic enzyme acyl ACP reductase

Abstract Acyl-ACP reductase (AAR) is one of the two key cyanobacterial enzymes along with aldehyde deformylating oxygenase (ADO) involved in the synthesis of long-chain alkanes, a drop-in biofuel. The enzyme is prone to aggregation when expressed in Escherichia coli, leading to varying alkane levels. The present work attempts to investigate the crucial structural aspects of AAR protein associated with its stability and folding. Characterization by dynamic light scattering experiment and intact mass spectrometry revealed that recombinantly expressed AAR in E. coli existed in multiple-sized protein particles due to diverse lipidation. Interestingly, while thermal- and urea-based denaturation of AAR showed 2-state unfolding transition in circular dichroism and intrinsic fluorescent spectroscopy, the unfolding process of AAR was a 3-state pathway in GdnHCl solution suggesting that the protein milieu plays a significant role in dictating its folding. Apparent standard free energy $$\left( {\Delta {\text{G}}_{{{\text{NU}}}}^{{{\text{H}}_{2} {\text{O}}}} } \right)$$ Δ G NU H 2 O of ~ 4.5 kcal/mol for the steady-state unfolding of AAR indicated borderline stability of the protein. Based on these evidences, we propose that the marginal stability of AAR are plausible contributing reasons for aggregation propensity and hence the low catalytic activity of the enzyme when expressed in E. coli for biofuel production. Our results show a path for building superior biocatalyst for higher biofuel production

C1QA and COMP: plasma-based biomarkers for early diagnosis of pancreatic neuroendocrine tumors

Pancreatic Neuroendocrine tumors (PanNET) are challenging to diagnose and often detected at advanced stages due to a lack of specific and sensitive biomarkers. This study utilized proteomics as a valuable approach for cancer biomarker discovery; therefore, mass spectrometry-based proteomic profiling was conducted on plasma samples from 12 subjects (3 controls; 5 Grade I, 4 Grade II PanNET patients) to identify potential proteins capable of effectively distinguishing PanNET from healthy controls. Data are available via ProteomeXchange with the identifier PXD045045. 13.2% of proteins were uniquely identified in PanNET, while 60% were commonly expressed in PanNET and controls. 17 proteins exhibiting significant differential expression between PanNET and controls were identified with downstream analysis. Further, 5 proteins (C1QA, COMP, HSP90B1, ITGA2B, and FN1) were selected by pathway analysis and were validated using Western blot analysis. Significant downregulation of C1QA (p = 0.001: within groups, 0.03: control vs. grade I, 0.0013: grade I vs. grade II) and COMP (p = 0.011: within groups, 0.019: control vs grade I) were observed in PanNET Grade I &amp; II than in controls. Subsequently, ELISA on 38 samples revealed significant downregulation of C1QA and COMP with increasing disease severity. This study shows the potential of C1QA and COMP in the early detection of PanNET, highlighting their role in the search for early-stage (Grade-I and Grade-II) diagnostic markers and therapeutic targets for PanNET.Validerad;2023;Nivå 2;2023-12-05 (joosat);License full text: CC BY Funder: Department of Science and Technology, Govt of India (Grant Number: EEQ/2018/000697)</p