title: A novel methanol-free Pichia pastoris system for recombinant protein expression.
abstract: BACKGROUND
  As one of the most popular expression systems, recombinant protein expression in Pichia pastoris relies on the AOX1 promoter (PAOX1) which is strongly induced by methanol. However, the toxic and inflammatory nature of methanol restricts its application, especially in edible and medical products. Therefore, constructing a novel methanol-free system becomes necessary. The kinases involved in PAOX1activation or repression by different carbon sources may be promising targets.
  
  
  RESULTS
  We identified two kinase mutants: Δgut1 and Δdak, both of which showed strong alcohol oxidase activity under non-methanol carbon sources. Based on these two kinases, we constructed two methanol-free expression systems: Δgut1-HpGCY1-glycerol (PAOX1induced by glycerol) and Δdak-DHA (PAOX1induced by DHA). By comparing their GFP expression efficiencies, the latter one showed better potential. To further test the Δdak-DHA system, three more recombinant proteins were expressed as examples. We found that the expression ability of our novel methanol-free Δdak-DHA system was generally better than the constitutive GAP promoter, and reached 50-60 % of the traditional methanol induced system.
  
  
  CONCLUSIONS
  We successfully constructed a novel methanol-free expression system Δdak-DHA. This modified expression platform preserved the favorable regulatable nature of PAOX1, providing a potential alternative to the traditional system.
authors: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China.
authors: Wei
authors: W
authors: Shen
authors: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China.
authors: Ying
authors: Y
authors: Xue
authors: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China.
authors: Yiqi
authors: Y
authors: Liu
authors: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China.
authors: Chuixing
authors: C
authors: Kong
authors: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China.
authors: Xiaolong
authors: X
authors: Wang
authors: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China.
authors: Mengmeng
authors: M
authors: Huang
authors: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China.
authors: Menghao
authors: M
authors: Cai
authors: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China.
authors: Xiangshan
authors: X
authors: Zhou
authors: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China.|Shanghai Collaborative Innovation Center for Biomanufacturing (SCICB), Shanghai, 200237, China.
authors: Yuanxing
authors: Y
authors: Zhang
authors: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China. mianzhou@ecust.edu.cn.
authors: Mian
authors: http://orcid.org/0000-0003-4226-6299
authors: M
authors: Zhou
mesh_terms: D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism; D004098:Dihydroxyacetone / Q000378:metabolism / Q000494:pharmacology; D055786:Gene Knockout Techniques; D005990:Glycerol / Q000378:metabolism / Q000494:pharmacology; D005991:Glycerol Kinase / Q000235:genetics / Q000378:metabolism; D000432:Methanol / Q000494:pharmacology; D010843:Pichia / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D000429:Alcohol Oxidoreductases; C019643:alcohol oxidase; D005991:Glycerol Kinase; D004098:Dihydroxyacetone; D005990:Glycerol; D000432:Methanol
keywords: AOX1 promoter; DAK; Dihydroxyacetone; GUT1; Pichia pastoris; Recombination protein expression