Tie-Jun Chen1, Zhe Chi2, Hong Jiang1, Guang-Lei Liu2, Zhong Hu3, Zhen-Ming Chi4. 1. College of Marine Life Sciences, Ocean University of China, Yushan Road, No. 5, Qingdao, China. 2. College of Marine Life Sciences, Ocean University of China, Yushan Road, No. 5, Qingdao, China; Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, 266003 Qingdao, China. 3. Department of Biology, Shantou University, Shantou 515063, China. 4. College of Marine Life Sciences, Ocean University of China, Yushan Road, No. 5, Qingdao, China; Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, 266003 Qingdao, China. Electronic address: chi@ouc.edu.cn.
Abstract
BACKGROUND: Pullulan and glycogen have many applications and physiological functions. However, to date, it has been unknown where and how the pullulan is synthesized in the yeast cells and if cell wall structure of the producer can affect pullulan and glycogen biosynthesis. METHODS: The genes related to cell wall integrity were cloned, characterized, deleted and complemented. The cell wall integrity, pullulan biosynthesis, glycogen accumulation and gene expression were examined. RESULTS: In this study, the GT6 and GT7 genes encoding different α1,2 mannosyltransferases in Aureobasidium melanogenum P16 were cloned and characterized. The proteins deduced from both the GT6 and GT7 genes contained the conserved sequences YNMCHFWSNFEI and YSTCHFWSNFEI of a Ktr mannosyltransferase family. The removal of each gene and both the two genes caused the changes in colony and cell morphology and enhanced glycogen accumulation, leading to a reduced pullulan biosynthesis and the declined expression of many genes related to pullulan biosynthesis. The swollen cells of the disruptants were due to increased accumulation of glycogen, suggesting that uridine diphosphate glucose (UDP-glucose) was channeled to glycogen biosynthesis in the disruptants, rather than pullulan biosynthesis. Complementation of the GT6 and GT7 genes in the corresponding disruptants and growth of the disruptants in the presence of 0.6 M KCl made pullulan biosynthesis, glycogen accumulation, colony and cell morphology be restored. GENERAL SIGNIFICANCE: This is the first report that the two α1,2 mannosyltransferases were required for colony and cell morphology, glycogen accumulation and pullulan biosynthesis in the pullulan producing yeast.
BACKGROUND:Pullulan and glycogen have many applications and physiological functions. However, to date, it has been unknown where and how the pullulan is synthesized in the yeast cells and if cell wall structure of the producer can affect pullulan and glycogen biosynthesis. METHODS: The genes related to cell wall integrity were cloned, characterized, deleted and complemented. The cell wall integrity, pullulan biosynthesis, glycogen accumulation and gene expression were examined. RESULTS: In this study, the GT6 and GT7 genes encoding different α1,2 mannosyltransferases in Aureobasidium melanogenum P16 were cloned and characterized. The proteins deduced from both the GT6 and GT7 genes contained the conserved sequences YNMCHFWSNFEI and YSTCHFWSNFEI of a Ktr mannosyltransferase family. The removal of each gene and both the two genes caused the changes in colony and cell morphology and enhanced glycogen accumulation, leading to a reduced pullulan biosynthesis and the declined expression of many genes related to pullulan biosynthesis. The swollen cells of the disruptants were due to increased accumulation of glycogen, suggesting that uridine diphosphate glucose (UDP-glucose) was channeled to glycogen biosynthesis in the disruptants, rather than pullulan biosynthesis. Complementation of the GT6 and GT7 genes in the corresponding disruptants and growth of the disruptants in the presence of 0.6 M KCl made pullulan biosynthesis, glycogen accumulation, colony and cell morphology be restored. GENERAL SIGNIFICANCE: This is the first report that the two α1,2 mannosyltransferases were required for colony and cell morphology, glycogen accumulation and pullulan biosynthesis in the pullulan producing yeast.
Authors: Marek Kieliszek; Katarzyna Bierla; Javier Jiménez-Lamana; Anna Maria Kot; Jaime Alcántara-Durán; Kamil Piwowarek; Stanisław Błażejak; Joanna Szpunar Journal: Int J Mol Sci Date: 2020-07-25 Impact factor: 5.923