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: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:Strand-specific RNA-seq libraries were constructed for two samples, including (I) wild-type strain NBRC0988 grown in YEP medium containing 2% w/v glucose;(II) wild-type strain NBRC0988 grown in YEP medium containing 2% w/v xylose. For preparation of RNA samples, NBRC0988 cells grown overnight were inoculated into 100 ml liquid Yeast Extract Peptone Dextrose (YEPD) medium with the initial inoculation amount of OD600= 0.1, and cultured for 15 hours at 30℃ and 250 rpm. The cells were collected by centrifugation at 6,000g for 5 minutes. After washing twice with phosphate buffer saline (PBS), they were inoculated into new 100 mL YEP medium containing 2% w/v glucose or xylose.After flask culturing at 30°C and 250 rpm for an additional 5 hours, the yeast cells were collected by centrifugation for total RNA isolation and Illumina RNA-seq library construction. Total RNA for samples were isolated using TRIzol reagent (Invitrogen, Grand Island, USA), then used for high-throughput RNA sequencing. The 150-nt paired-end strand-specific RNA-seq libraries (SS_lib_type RF) were generated commercially at Novogene Biotechnology Co. Ltd (Tianjin, China) by using Illumina’s novaseq 6000 platform (Illumina, San Diego, USA).