title: Molecular cloning and overexpression of an endo-β-1,4-xylanase gene from Aspergillus niger in industrial Saccharomyces cerevisiae YS2 strain.
abstract: The aim of this study was to endow an industrial strain of Saccharomyces cerevisiae with the ability to overexpress the xylanase by constructing a homology-driven integration vector. The total mRNA from a xylanase-producing strain of Aspergillus niger IME-216 was extracted and used as the template for the production of endo-β-1,4-xylanase cDNA by reverse transcription. The fusion fragment containing the phosphoglycerate kinase promoter, α-factor signal peptide, xylanase gene encoding the mature peptide, and CYC1 terminator was first generated by overlap extension polymerase chain reaction. Then, the vector pUPX was constructed by inserting the fusion fragment into the S. cerevisiae plasmid pUG6. Then, A 2.2-kb rDNA sequence was further cloned and attached to the SalI-digested pUPX to obtain the integration plasmid pUPXR. The pUPXR was linearized by KpnI, transformed into the industrial strain S. cerevisiae YS2 using the lithium acetate method and integrated into the S. cerevisiae chromosome. The maximum yield of the recombinant xylanase produced by the engineered S. cerevisiae strain YS2_2 was 74.8 U per microliter, which was about 1.5-fold higher than the original 50 U per microliter by Aspergillus niger IME-216 strain under the flask culture at 28 °C for 72 h. The findings of our study can be used for further development of industrial S. cerevisiae strain for producing interested enzymes, or improving the achievement of metabolism, for example, simultaneous fermentation of glucose and xylose to producing bioethanol.
authors.affiliation: Engineering Research Center of Industrial Microbiology of Ministry of Education, and College of Life Sciences, Fujian Normal University, Fuzhou, 350108, People's Republic of China. baoyutian@yahoo.com.cn
authors.forename: Baoyu
authors.initials: B
authors.lastname: Tian
authors.forename: Yan
authors.initials: Y
authors.lastname: Xu
authors.forename: Wanling
authors.initials: W
authors.lastname: Cai
authors.forename: Qingeng
authors.initials: Q
authors.lastname: Huang
authors.forename: Yuanyuan
authors.initials: Y
authors.lastname: Gao
authors.forename: Xin
authors.initials: X
authors.lastname: Li
authors.forename: Jianzhong
authors.initials: J
authors.lastname: Huang
mesh_terms: D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D043364:Endo-1,4-beta Xylanases / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D005285:Fermentation; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D005822:Genetic Vectors; D030781:Organisms, Genetically Modified / Q000235:genetics / Q000378:metabolism; D012331:RNA, Fungal / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D014170:Transformation, Genetic
chemical_list: D005656:Fungal Proteins; D012331:RNA, Fungal; D012333:RNA, Messenger; D011994:Recombinant Proteins; D043364:Endo-1,4-beta Xylanases