ABSTRACT: De novo assembly of Candida sojae genome, an unexplored xylitol-producing yeast with potential for 2G ethanol and food industries improvements
Project description:We previously reported that a recombinant Candida utilis strain expressing a Candida shehatae xylose reductase K275R/N277D, a C. shehatae xylitol dehydrogenase, and xylulokinase from Pichia stipitis produced ethanol from xylose. However, its productivity was low. In this study, metabolomic (CE-TOF MS) and transcriptomic (microarray) analyses were performed to characterize xylose metabolism by the engineered C. utilis and to identify key genetic changes contributing to efficient xylose utilization. Metabolomic analysis revealed that the xylose-fermenting strain accumulated more pentose phosphate pathway intermediates, more NADH, and more glycolytic intermediates upstream of glyceraldehyde 3-phosphate than wild-type. Transcriptomic analysis of the strain grown on xylose indicated a significant increase in expression of genes encoding tricarboxylic acid cycle enzymes, respiratory enzymes, and enzymes involved in ethanol oxidation. To decrease the NADH/NAD+ ratio and increase ethanol yield from the fermentation of xylose, ADH1 encoding NADH-dependent alcohol dehydrogenase was overexpressed. The resultant strain exhibited a 17% increase in ethanol production and a 22% decrease in xylitol accumulation relative to the control.
Project description:Centromeres are chromosomal regions that serve as platforms for kinetochore assembly and spindle attachments, ensuring accurate chromosome segregation during cell division. Despite functional conservation, centromeric sequences are diverse and usually repetitive across species, making them challenging to assemble and identify. Here, we describe centromeres in the model oomycete Phytophthora sojae by combining long-read sequencing-based genome assembly and chromatin immunoprecipitation for the centromeric histone CENP-A followed by high-throughput sequencing (ChIP-seq). P. sojae centromeres cluster at a single focus in the nucleus at different life stages and during nuclear division. We report a highly contiguous genome assembly of the P. sojae reference strain, which enabled identification of 15 highly enriched CENP-A binding regions as putative centromeres. By focusing on 10 intact regions, we demonstrate that centromeres in P. sojae are regional, spanning 211 to 356 kb. Most of these regions are transposon-rich, poorly transcribed, and lack the euchromatin mark H3K4me2 but are embedded within regions with the heterochromatin marks H3K9me3 and H3K27me3.
Project description:The Mellet-boinot is a promising process to be applied for second-generation ethanol production by wild yeasts. However, the impact of this process on the physiology and fermentative performance of the xylose-fermenting yeast Spathaspora passalidarum during second-generation ethanol production, remains elusive. Therefore, we have conducted a deep transcriptomic analysis of S. passalidarum during five consecutive fed-batches and cell recycles, to determine the differences and global responses of differentially expressed genes (DEGs) during the Melle-Boinot process. A physiological adaptation was observed resulting in an increase on ethanol yield, ethanol volumetric productivity and ethanol titer. Furthermore, a decrease of the subproduct xylitol was also observed. A transcriptional regulation was achieved from the third cell recycle onwards and this regulation was maintained afterwards. Analysing the DEGs during the recycles showed an up-regulation of genes involved in ATP synthesis, N-Glycan biosynthesis, oxidative phosphorylation and purine metabolism, indicating as important mechanisms for adaptation throughout recycles due increased ethanol concentration. Moreover, the TCA cycle anabolic pathway, gluconeogenesis, glycogen and trehalose biosynthesis, fatty acid and sterol biosynthesis were also affected mainly due ethanol concentration and osmotic pressure implying that cell energy was generated towards the production of cell wall components in order to S. passalidarum cells to thrive in consecutive recycles. Taken together these results demonstrate that the Melle-Boinot process is a worthy strategy to be applied on 2G ethanol fermentation by native yeasts. Furthermore, we highlight major microbial molecular strategies for xylose conversion providing relevant insights for further metabolic engineering aiming to improve 2G bioethanol production.
Project description:A recombinant C. utilis strain expressing Candida shehatae xylose reductase K275R/N277D (NADH-preferring), C. shehatae xylitol dehydrogenase and Pichia stipitis xylulokinase produce ethanol from xylose. Here, we report the transcriptional-profiling in the engineered C. utilis strain grown on xylose using DNA microarray. Transcriptome analysis indicated that expression of genes encoding the tricarboxylic acid cycle, respiration enzymes and the ethanol consumption were increased significantly when cells were cultivated on xylose. Gene expression in Candida utilis cells grown on glucose or xylose was measured at 10.5 and 24 hours, respectively. Two or three independent experiments were performed at each time for each experiment.
Project description:This experiment contains Phytophthora sojae samples and RNA-seq data from experiment E-GEOD-29561 (https://www.ebi.ac.uk/arrayexpress/experiments/E-GEOD-29651/) to understand gene expression during the P. sojae life cycle. The transcriptome of the oomycete plant pathogen Phytophthora sojae was profiled at 5 different developmental stages: mycelia (MY), zoosporangia (SP), zoospores (ZO), cysts (CY) and germinating cysts (GC); based on a 3'-tag digital gene expression (DGE) protocol. More than 90 million clean sequence tags were generated and compared to the P. sojae genome and its 19,027 predicted genes. A total of 14,969 genes were detected, of which 10,044 were deemed reliable because they mapped to unambiguous tags. A web-based server named the Phytophthora Transcriptional Database (PTD) has been established.
Project description:Examination of soybean hypocotyls, G. max cv. Harosoy (Rps7), at 3, 6, 12, 24 and 48 hours after inoculation with P. sojae, race 2, isolate P6497 Patterns of Gene Expression Upon Infection of Soybean Plants by Phytophthora sojae. P. Moy, D. Qutob, B. P. Chapman, I. Atkinson, and M. Gijzen. Pages 1051-1062. Publication no. M-2004-0728-01R. Molecular Plant-Microbe Interactions, October 2004, Volume 17, Number 10. Keywords: time-course