title: Construction of efficient xylose utilizing Pichia pastoris for industrial enzyme production.
abstract: BACKGROUND
  Cellulosic biomass especially agricultural/wood residues can be utilized as feedstock to cost-effectively produce fuels, chemicals and bulk industrial enzymes, which demands xylose utilization from microbial cell factories. While previous works have made significant progress in improving microbial conversion of xylose into fuels and chemicals, no study has reported the engineering of efficient xylose utilizing protein expression systems for the purpose of producing industrial enzymes.
  
  
  RESULTS
  In this work, using Pichia pastoris as an example, we demonstrated the successful engineering of xylose metabolizing ability into of protein expression systems. A heterologous XI (xylose isomerase) pathway was introduced into P. pastoris GS115 by overexpressing the Orpinomyces spp. XI or/and the endogenous XK (xylulokinase) gene, and evolutionary engineering strategies were also applied. Results showed that the XI pathway could be functionally expressed in P. pastoris. After 50 generation of sequential batch cultivation, a set of domesticated recombinant P. pastoris strains with different performance metrics on xylose were obtained. One evolved strain showed the highest xylose assimilation ability, whose cell yield on xylose can even be comparable to that on glucose or glycerol. This strain also showed significantly increased β-mannanase production when cultured on xylose medium. Furthermore, transcription analysis of xylose pathway genes suggested that overexpression of XI and XK might be the key factors affecting effective xylose assimilation.
  
  
  CONCLUSIONS
  To our best knowledge, this study is the first work demonstrating the construction of efficient xylose utilizing P. pastoris strains, thus providing a basis for using cellulosic biomass for bulk industrial enzyme production.
authors.affiliation: CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China. real_lpf@163.com.
authors.forename: Pengfei
authors.initials: P
authors.lastname: Li
authors.affiliation: National Engineering Laboratory for Industrial Enzymes, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China. bbmm0412@sina.com.
authors.forename: Hongbing
authors.initials: H
authors.lastname: Sun
authors.affiliation: CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China. chenwenzhu0207@163.com.|Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, China. chenwenzhu0207@163.com.
authors.forename: Zao
authors.initials: Z
authors.lastname: Chen
authors.affiliation: CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China. yli@im.ac.cn.
authors.forename: Yin
authors.initials: Y
authors.lastname: Li
authors.affiliation: CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China. zhutc@im.ac.cn.
authors.forename: Taicheng
authors.initials: T
authors.lastname: Zhu
mesh_terms: D019747:Aldose-Ketose Isomerases / Q000235:genetics / Q000378:metabolism; D018533:Biomass; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D020085:Neocallimastigales / Q000201:enzymology; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D014994:Xylose / Q000378:metabolism; D044902:beta-Mannosidase / Q000235:genetics / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins; D014994:Xylose; D017853:Phosphotransferases (Alcohol Group Acceptor); C032700:xylulokinase; D044902:beta-Mannosidase; D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase