title: Improving freeze-tolerance of baker's yeast through seamless gene deletion of NTH1 and PUT1.
abstract: Baker's yeast strains with freeze-tolerance are highly desirable to maintain high leavening ability after freezing. Enhanced intracellular concentration of trehalose and proline in yeast is linked with freeze-tolerance. In this study, we constructed baker's yeast with enhanced freeze-tolerance by simultaneous deletion of the neutral trehalase-encoded gene NTH1 and the proline oxidase-encoded gene PUT1. We first used the two-step integration-based seamless gene deletion method to separately delete NTH1 and PUT1 in haploid yeast. Subsequently, through two rounds of hybridization and sporulation-based allelic exchange and colony PCR-mediated tetrad analysis, we obtained strains with restored URA3 and deletion of NTH1 and/or PUT1. The resulting strain showed higher cell survival and dough-leavening ability after freezing compared to the wild-type strain due to enhanced accumulation of trehalose and/or proline. Moreover, mutant with simultaneous deletion of NTH1 and PUT1 exhibits the highest relative dough-leavening ability after freezing compared to mutants with single-gene deletion perhaps due to elevated levels of both trehalose and proline. These results verified that it is applicable to construct frozen dough baker's yeast using the method proposed in this paper.
authors.affiliation: Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin Industrial Microbiology Key Laboratory, College of Biotechnology, Tianjin University of Science and Technology, No. 29, 13th Avenue, Tianjin Economic and Technological Development Area, Tianjin, 300457, China.|Tianjin Food Safety and Low Carbon Manufacturing Collaborative Innovation Center, Tianjin, 300457, People's Republic of China.
authors.forename: Jian
authors.initials: J
authors.lastname: Dong
authors.affiliation: Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin Industrial Microbiology Key Laboratory, College of Biotechnology, Tianjin University of Science and Technology, No. 29, 13th Avenue, Tianjin Economic and Technological Development Area, Tianjin, 300457, China.|Tianjin Food Safety and Low Carbon Manufacturing Collaborative Innovation Center, Tianjin, 300457, People's Republic of China.
authors.forename: Didi
authors.initials: D
authors.lastname: Chen
authors.affiliation: Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin Industrial Microbiology Key Laboratory, College of Biotechnology, Tianjin University of Science and Technology, No. 29, 13th Avenue, Tianjin Economic and Technological Development Area, Tianjin, 300457, China.
authors.forename: Guanglu
authors.initials: G
authors.lastname: Wang
authors.affiliation: Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin Industrial Microbiology Key Laboratory, College of Biotechnology, Tianjin University of Science and Technology, No. 29, 13th Avenue, Tianjin Economic and Technological Development Area, Tianjin, 300457, China.|Tianjin Food Safety and Low Carbon Manufacturing Collaborative Innovation Center, Tianjin, 300457, People's Republic of China.
authors.forename: Cuiying
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin Industrial Microbiology Key Laboratory, College of Biotechnology, Tianjin University of Science and Technology, No. 29, 13th Avenue, Tianjin Economic and Technological Development Area, Tianjin, 300457, China.|Tianjin Food Safety and Low Carbon Manufacturing Collaborative Innovation Center, Tianjin, 300457, People's Republic of China.
authors.forename: Liping
authors.initials: L
authors.lastname: Du
authors.affiliation: Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin Industrial Microbiology Key Laboratory, College of Biotechnology, Tianjin University of Science and Technology, No. 29, 13th Avenue, Tianjin Economic and Technological Development Area, Tianjin, 300457, China.|Tianjin Food Safety and Low Carbon Manufacturing Collaborative Innovation Center, Tianjin, 300457, People's Republic of China.
authors.forename: Shanshan
authors.initials: S
authors.lastname: Liu
authors.affiliation: Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin Industrial Microbiology Key Laboratory, College of Biotechnology, Tianjin University of Science and Technology, No. 29, 13th Avenue, Tianjin Economic and Technological Development Area, Tianjin, 300457, China.|Tianjin Food Safety and Low Carbon Manufacturing Collaborative Innovation Center, Tianjin, 300457, People's Republic of China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin Industrial Microbiology Key Laboratory, College of Biotechnology, Tianjin University of Science and Technology, No. 29, 13th Avenue, Tianjin Economic and Technological Development Area, Tianjin, 300457, China. xdg@tust.edu.cn.|Tianjin Food Safety and Low Carbon Manufacturing Collaborative Innovation Center, Tianjin, 300457, People's Republic of China. xdg@tust.edu.cn.
authors.forename: Dongguang
authors.initials: D
authors.lastname: Xiao
mesh_terms: D000595:Amino Acid Sequence; D005615:Freezing; D017353:Gene Deletion; D005819:Genetic Markers; D050296:Microbial Viability; D011392:Proline / Q000378:metabolism; D011394:Proline Oxidase / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D014198:Trehalase / Q000235:genetics / Q000378:metabolism*
chemical_list: D005819:Genetic Markers; D029701:Saccharomyces cerevisiae Proteins; C554167:URA3 protein, S cerevisiae; D011392:Proline; D011394:Proline Oxidase; C000614366:NTH1 protein, S cerevisiae; D014198:Trehalase
keywords: Baker’s yeast; Proline; Seamless gene deletion; Trehalose