title: Scarless gene deletion in methylotrophic Hansenula polymorpha by using mazF as counter-selectable marker.
abstract: With increasing application of Hansenula polymorpha in fundamental research and biotechnology, many more genetic manipulations are required. However, these have been restricted for the finiteness of selectable markers. Here, MazF, a toxin protein from Escherichia coli, was investigated as a counter-selectable marker in H. polymorpha. The lethal effect of MazF on yeast cells suggested that it is a candidate for counter-selection in H. polymorpha. Markerless or scarless gene deletion in H. polymorpha was conducted based on selectable markers cassette mazF-zeoR, in which the zeocin resistance cassette and mazF expression cassette were used as positive and counter-selectable markers, respectively. For markerless deletion, the target region can be replaced by CYC1TT via two-step homologous recombination. For scarless deletion, the innate upstream region (5'UP) of target genes rather than CYC1TT mediates homologous recombination to excise both selectable markers and 5' sequence of target genes. Moreover, scarless deletion can be accomplished by using short homologous arms for the effectiveness of mazF as a counter-selectable marker. The applicability of the strategies in markerless or scarless deletion of PEP4, LEU2, and TRP1 indicates that this study provides easy, time-efficient, and host-independent protocols for single or multiple genetic manipulations in H. polymorpha.
authors.affiliation: CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Panpan
authors.initials: P
authors.lastname: Song
authors.affiliation: CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Sha
authors.initials: S
authors.lastname: Liu
authors.affiliation: CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Xuena
authors.initials: X
authors.lastname: Guo
authors.affiliation: CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Xuejing
authors.initials: X
authors.lastname: Bai
authors.affiliation: CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China. Electronic address: hexp@im.ac.cn.
authors.forename: Xiuping
authors.initials: X
authors.lastname: He
authors.affiliation: CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Borun
authors.initials: B
authors.lastname: Zhang
mesh_terms: D001427:Bacterial Toxins / Q000235:genetics; D001709:Biotechnology; D004268:DNA-Binding Proteins / Q000235:genetics; D004722:Endoribonucleases / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics; D017353:Gene Deletion; D018390:Gene Targeting / Q000379:methods; D005798:Genes, Bacterial; D005800:Genes, Fungal; D005804:Genes, Lethal; D005819:Genetic Markers; D008957:Models, Genetic; D010843:Pichia / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism
chemical_list: D001427:Bacterial Toxins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D005819:Genetic Markers; C422955:MazF protein, E coli; D004722:Endoribonucleases
keywords: Counter-selectable marker; Escherichia coli; Hansenula polymorpha; Markerless gene deletion; MazF; Scarless gene deletion