title: Cloning and molecular characterization of a gene coding D-xylulokinase (CmXYL3) from Candida maltosa.
abstract: OBJECTIVE
  To clone and identify a gene (CmXYL3) coding D-xylulokinase from Candida maltosa Xu316 and understand its physiological function.
  
  
  RESULTS
  Based on the conserved regions of the known D-xylulokinase-encoding genes, a pair of degenerate primers was designed to clone the CmXYL3 gene from C. maltosa Xu316. The coding region and sequences flanking the CmXYL3 gene were obtained by PCR-based DNA walking method. Southern blotting analysis suggested that there is a single copy of the CmXYL3 gene in the genome. The open reading frame starting from ATG and ending with TAG stop codon encoded 616 amino acids with a calculated molecular mass of 68889.743 Da. The CmXYL3 gene under the control of the GPD1 promoter was heterologously expressed in Saccharomyces cerevisiae deficient in D-xylulokinase (deltaScXKS1::LEU2) activity, and restored growth on D-xylulose. The specific activity of D-xylulokinase varied during xylose fermentation and was correlated with aeration level. After growth on different pentoses and pentitols as sole carbon sources, the highest specific activity of D-xylulokinase was observed on D-xylose.
  
  
  CONCLUSIONS
  The CmXYL3 gene isolated from C. maltosa Xu316 encodes a novel D-xylulokinase that plays a pivotal role in xylulose metabolism.
  
  
  CONCLUSIONS
  This is the first report that describes the isolation and cloning of D-xylulokinase gene (CmXYL3) from C. maltosa Xu316. D-xylulokinase is pivotal for growth and product formation during xylose metabolism. Better understanding of the biochemical properties and the physiological function of D-xylulokinase will contribute to optimizing fermentation conditions and determining the strategies for metabolic engineering of C. maltosa Xu316 for further improvement of xylitol yield and productivity.
authors.affiliation: Centre of Microbial Biotechnology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, PR China.
authors.forename: C
authors.initials: C
authors.lastname: Guo
authors.forename: P
authors.initials: P
authors.lastname: He
authors.forename: D
authors.initials: D
authors.lastname: Lu
authors.forename: A
authors.initials: A
authors.lastname: Shen
authors.forename: N
authors.initials: N
authors.lastname: Jiang
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern / Q000379:methods; D002175:Candida / Q000201:enzymology / Q000235:genetics*; D003001:Cloning, Molecular; D017931:DNA Primers; D015870:Gene Expression; D005800:Genes, Fungal; D016681:Genome, Fungal; D008969:Molecular Sequence Data; D009172:Mycology / Q000379:methods; D016366:Open Reading Frames; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics* / Q000378:metabolism; D017422:Sequence Analysis, DNA
chemical_list: D017931:DNA Primers; D017853:Phosphotransferases (Alcohol Group Acceptor); C032700:xylulokinase