title: Mutation of a regulator Ask10p improves xylose isomerase activity through up-regulation of molecular chaperones in Saccharomyces cerevisiae.
abstract: Economically feasible bioconversion of lignocelluloses into fuels and chemicals is dependent on efficient utilization of all available sugars in lignocellulosic biomass, including hextose and pentose. Previously, we constructed a xylose fermenting strain of Saccharomyces cerevisiae through metabolic engineering and enhanced its xylose utilization capability through evolutionary engineering. However, the key mechanism of improved xylose utilization and xylose isomerase activity was not identified. In this study, we applied the concept of inverse metabolic engineering to identify the factors involved in improving xylose utilization. Genomic sequencing of the evolved strain with fast xylose utilization capability was performed, and mutations possibly affecting xylose utilization were screened. Further genetic analysis of these mutant genes revealed that mutations in ASK10 (both the site-directed mutation ASK10M475Ras well as ASK10 deletion), a stress response regulator-encoding gene, improved growth on xylose and enhanced xylose isomerase activity. We found that mutation of Ask10p did not increase xylose isomerase activity through interacting with the xylose isomerase protein or through directly regulating the xylA gene transcription. Although ASK10 deletion increased the copy number of the plasmid and improved the transcription of xylA, the site-direct mutation ASK10M475Rdid not change the plasmid copy number. Interestingly, we found that both the site-directed mutation ASK10M475Rand ASK10 deletion up-regulated the transcription of molecular chaperone-encoding genes HSP26, SSA1 and HSP104, thereby facilitating the protein folding of xylose isomerase and enhancing xylose isomerase activity. This study revealed the important mechanism of chaperones in xylose isomerase activity regulation, and it provides valuable insights for efficient xylose metabolic strain development.
authors: State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China.
authors: Jin
authors: J
authors: Hou
authors: State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China.
authors: Chunlei
authors: C
authors: Jiao
authors: State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China.
authors: Bo
authors: B
authors: Peng
authors: State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China.
authors: Yu
authors: Y
authors: Shen
authors: State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China. Electronic address: bxm@sdu.edu.cn.
authors: Xiaoming
authors: X
authors: Bao
mesh_terms: D019747:Aldose-Ketose Isomerases / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D024861:Genetic Enhancement / Q000379:methods*; D064688:Metabolic Flux Analysis; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D015854:Up-Regulation / Q000502:physiology; D014994:Xylose / Q000378:metabolism*
chemical_list: C487632:Ask10 protein, S cerevisiae; D018832:Molecular Chaperones; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D014994:Xylose; D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase
keywords: Ask10p; Genomic sequencing; Inverse metabolic engineering; Molecular chaperones; Xylose isomerase