Penicillium oxalicum produces an integrated, extracellular cellulase and xylanase system, strictly regulated by several transcription factors. However, the understanding of the regulatory mechanism of cellulase and xylanase biosynthesis in P. oxalicum is limited, particularly under solid-state fermentation (SSF) conditions. In our study, deletion of a novel gene, cxrD (cellulolytic and xylanolytic regulator D), resulted in 49.3 to 2,230% enhanced production of cellulase and xylanase, except for 75.0% less xylanase at 2 days, compared with the P. oxalicum parental strain, when cultured on solid medium containing wheat bran plus rice straw for 2 to 4 days after transfer from glucose. In addition, the deletion of cxrD delayed conidiospore formation, leading to 45.1 to 81.8% reduced asexual spore production and altered mycelial accumulation to various extents. Comparative transcriptomics and real-time quantitative reverse transcription-PCR found that CXRD dynamically regulated the expression of major cellulase and xylanase genes and conidiation-regulatory gene brlA under SSF. In vitro electrophoretic mobility shift assays demonstrated that CXRD bound to the promoter regions of these genes. The core DNA sequence 5'-CYGTSW-3' was identified to be specifically bound by CXRD. These findings will contribute to understanding the molecular mechanism of negative regulation of fungal cellulase and xylanase biosynthesis under SSF. IMPORTANCE Application of plant cell wall-degrading enzymes (CWDEs) as catalysts in biorefining of lignocellulosic biomass into bioproducts and biofuels reduces both chemical waste production and carbon footprint. The filamentous fungus Penicillium oxalicum can secrete integrated CWDEs, with potential for industrial application. Solid-state fermentation (SSF), simulating the natural habitat of soil fungi, such as P. oxalicum, is used for CWDE production, but a limited understanding of CWDE biosynthesis hampers the improvement of CWDE yields through synthetic biology. Here, we identified a novel transcription factor CXRD, which negatively regulates the biosynthesis of cellulase and xylanase in P. oxalicum under SSF, providing a potential target for genetic engineering to improve CWDE production.
Raw-starch-digesting glucoamylases (RSDGs) from filamentous fungi have great commercial values in starch processing; however, the regulatory mechanisms associated with their production in filamentous fungi remain unknown. Penicillium oxalicum HP7-1 isolated by our laboratory secretes RSDG with suitable properties but at low production levels. Here, we screened and identified novel regulators of RSDG gene expression in P. oxalicum through transcriptional profiling and genetic analyses. Penicillium oxalicum HP7-1 transcriptomes in the presence of glucose and starch, respectively, used as the sole carbon source were comparatively analyzed, resulting in screening of 23 candidate genes regulating the expression of RSDG genes. Following deletion of 15 of the candidate genes in the parental P. oxalicum strain ∆PoxKu70, enzymatic assays revealed five mutants exhibiting significant reduction in the production of raw-starch-digesting enzymes (RSDEs). The deleted genes (POX01907, POX03446, POX06509, POX07078, and POX09752), were the first report to regulate RSDE production of P. oxalicum. Further analysis revealed that ∆POX01907 lost the most RSDE production (83.4%), and that POX01907 regulated the expression of major amylase genes, including the RSDG gene POX01356/PoxGA15A, a glucoamylase gene POX02412, and the α-amylase gene POX09352/Amy13A, during the late-stage growth of P. oxalicum. Our results revealed a novel essential regulatory gene POX01907 encoding a transcription factor in controlling the production of RSDE, regulating the expression of an important RSDG gene POX01356/PoxGA15A, in P. oxalicum. These results provide insight into the regulatory mechanism of fungal amylolytic enzyme production.
The ability to adapt to changing environmental conditions is crucial for living organisms, as it enables them to successfully compete in natural niches, a process which generally depends upon protein phosphorylation-mediated signaling transduction. In the present study, protein kinase PoxMKK1, an ortholog of mitogen-activated protein kinase kinase Ste7 in Saccharomyces cerevisiae, was identified and characterized in the filamentous fungus Penicillium oxalicum. Deletion of PoxMKK1 in P. oxalicum ΔPoxKu70 led the fungus to lose 64.4–88.6% and 38.0–86.1% of its plant-polysaccharide-degrading enzyme (PPDE) production on day 4 after a shift under submerged- and solid-state fermentation, respectively, compared with the control strain ΔPoxKu70. In addition, PoxMKK1 affected hypha growth and sporulation, though this was dependent on culture formats and carbon sources. Comparative transcriptomics and real-time quantitative reverse transcription PCR assay revealed that PoxMKK1 activated the expression of genes encoding major PPDEs, known regulatory genes (i.e., PoxClrB and PoxCxrB) and cellodextrin transporter genes (i.e., PoxCdtD and PoxCdtC), while it inhibited the essential conidiation-regulating genes, including PoxBrlA, PoxAbaA and PoxFlbD. Notably, regulons modulated by PoxMKK1 and its downstream mitogen-activated protein kinase PoxMK1 co-shared 611 differential expression genes, including 29 PPDE genes, 23 regulatory genes, and 16 sugar-transporter genes. Collectively, these data broaden our insights into the diverse functions of Ste7-like protein kinase, especially regulation of PPDE biosynthesis, in filamentous fungi.
Objective The objective of this study was the visualization of hot spots and evolving trends in research on the association between vitamin D and infections through the use of bibliometric analysis. Methods Based on 3046 relevant articles collected in the Web of Science Core Collection for the period of 2001–2021, the data were processed using CiteSpace software. GraphPad software was used for some of the graphics. Results A total of 3,046 literature were retrieved, with an average citation frequency of 27.89 times. The number of published papers in the direction of “Immunology” (453 articles, 14.9%) and “Infectious diseases” (312 articles, 10.2%) is much higher. The United States presents the highest publication count (890, 29.2%) and shows a strong leadership in this field. Country burst shows that since 2015, many developing countries and low-income countries have carried out enthusiastic research in this regard, including China, Pakistan, and Iran. As for institutions, the League of European Research Universities produces a larger proportion of articles (220, 7.2%). In terms of authors, Martineau AR and Camargo CA have the highest number of published articles, contributing 30 (0.99%) and 28 articles (0.92%), respectively. Major studies are supported by the United States Department of Health Human Services funding (394, 12.9%). According to the keyword co-occurrence diagram, the 10 most frequent keywords from 2001 to 2021 are “vitamin D”, “infection”, “d deficiency”, “risk”, “association”, “expression”, “disease”, “d supplementation”, “vitamin d deficiency”, and “children”. The top 10 cited articles in 2021 are all related to COVID-19, suggesting it is a hotspot in recent times. Conclusion Research on the association between vitamin D and infection has grown rapidly since 2012 and is generally developing well. While developed Western countries continue to be leading roles in this field, research trends in developing countries are also very promising. It is demonstrated that the relationship between vitamin D and respiratory infections, especially respiratory viruses and the more recently COVID-19, has received a lot of attention in the last two decades, suggesting that this is the hotspot and frontier of research issue.
Rapeseed meal is severely restricted in its utilization as unconventional animal feed due to anti-nutritive compounds, such as glucosinolate, that are degraded to toxic nitriles such as 3-butenenitrile and 4-pentenenitrile in animals. Few studies on nitrilases that can degrade glucosinolate-derived nitriles have been reported thus far. In the present study, a nitrilase gene
Short tandem repeat (STR) analysis, such as the AmpFlSTR® Identifiler® Plus kit, is a standard, PCR-based human genotyping method used in the field of forensics. Misidentification of cell line and tissue DNA can be costly if not detected early; therefore it is necessary to have quality control measures such as STR profiling in place. A major issue in large-scale research studies involving archival formalin-fixed paraffin embedded (FFPE) tissues is that varying levels of DNA degradation can result in failure to correctly identify samples using STR genotyping. PCR amplification of STRs of several hundred base pairs is not always possible when DNA is degraded. The Sample ID Plus® panel from Sequenom allows for human DNA identification and authentication using SNP genotyping. In comparison to lengthy STR amplicons, this multiplexing PCR assay requires amplification of only 76–139 base pairs, and utilizes 47 SNPs to discriminate between individual samples. In this study, we evaluated both STR and SNP genotyping methods of sample identification, with a focus on paired FFPE tumor/normal DNA samples intended for next-generation sequencing (NGS). The ability to successfully validate the identity of FFPE samples can enable cost savings by reducing rework.
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