title: β-Glucan synthase gene overexpression and β-glucans overproduction in Pleurotus ostreatus using promoter swapping.
abstract: Mushroom β-glucans are potent immunological stimulators in medicine, but their productivities are very low. In this study, we successfully improved its production by promoter engineering in Pleurotus ostreatus. The promoter for β-1,3-glucan synthase gene (GLS) was replaced by the promoter of glyceraldehyde-3-phosphate dehydrogenase gene of Aspergillus nidulans. The homologous recombination fragment for swapping GLS promoter comprised five segments, which were fused by two rounds of combined touchdown PCR and overlap extension PCR (TD-OE PCR), and was introduced into P. ostreatus through PEG/CaCl2-mediated protoplast transformation. The transformants exhibited one to three fold higher transcription of GLS gene and produced 32% to 131% higher yield of β-glucans than the wild type. The polysaccharide yields had a significant positive correlation to the GLS gene expression. The infrared spectra of the polysaccharides all displayed the typical absorption peaks of β-glucans. This is the first report of successful swapping of promoters in filamentous fungi.
authors: College of Life Sciences, Henan Agricultural University, Zhengzhou, People's Republic of China.
authors: Ran
authors: R
authors: Chai
authors: Cuiwei
authors: C
authors: Qiu
authors: Dongren
authors: D
authors: Liu
authors: Yuancheng
authors: Y
authors: Qi
authors: Yuqian
authors: Y
authors: Gao
authors: Jinwen
authors: J
authors: Shen
authors: Liyou
authors: L
authors: Qiu
mesh_terms: D000595:Amino Acid Sequence; D015870:Gene Expression; D015966:Gene Expression Regulation, Fungal; D023061:Gene Order; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D059765:Homologous Recombination; D008969:Molecular Sequence Data; D020076:Pleurotus / Q000235:genetics* / Q000378:metabolism*; D011134:Polysaccharides / Q000096:biosynthesis; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D047071:beta-Glucans / Q000378:metabolism*
chemical_list: D011134:Polysaccharides; D012333:RNA, Messenger; D047071:beta-Glucans; D005964:Glucosyltransferases; C040232:glucan synthase
references: Gene. 1987;56(1):117-24
references: 2824287
references: Fungal Biol. 2010 May-Jun;114(5-6):507-13
references: 20943162
references: Nat Protoc. 2007;2(4):924-32
references: 17446874
references: Proc Natl Acad Sci U S A. 2005 Sep 6;102(36):12678-83
references: 16123130
references: Nat Protoc. 2008;3(9):1452-6
references: 18772872
references: Chem Pharm Bull (Tokyo). 1991 Mar;39(3):798-800
references: 2070468
references: Int J Med Mushrooms. 2012;14(1):21-6
references: 22339704
references: Curr Microbiol. 2012 Jul;65(1):85-90
references: 22538468
references: Microb Cell Fact. 2012 Mar 02;11:30
references: 22380540
references: FEMS Yeast Res. 2012 Mar;12(2):197-214
references: 22129153
references: Nucleic Acids Res. 1991 Jul 25;19(14):4008
references: 1861999
references: Metab Eng. 2012 May;14(3):233-41
references: 22629571
references: J Biotechnol. 1991 Jan;17(1):19-33
references: 1367494
references: Biotechnol J. 2013 Jan;8(1):46-58
references: 22890821
references: Nutr Rev. 2009 Nov;67(11):624-31
references: 19906249
references: Genetics. 2011 Nov;189(3):705-36
references: 22084422
references: Appl Microbiol Biotechnol. 2009 Sep;84(4):641-8
references: 19633836
references: Methods Enzymol. 1999;300:437-56
references: 9919545
references: Exp Biol Med (Maywood). 2004 May;229(5):393-406
references: 15096651
references: Bioessays. 2006 Aug;28(8):799-808
references: 16927300
references: Microbiology (Reading). 2001 Apr;147(Pt 4):781-794
references: 11283274
references: Gene. 1989 Apr 15;77(1):61-8
references: 2744488
references: Mycol Res. 2007 Jun;111(Pt 6):635-52
references: 17590323
references: Appl Microbiol Biotechnol. 2006 Sep;72(2):211-22
references: 16791589
references: Nucleic Acids Res. 2004 Jan 22;32(2):e19
references: 14739232
references: Gene. 1997 Feb 20;186(1):37-44
references: 9047342
references: J Sci Food Agric. 2012 Jul;92(9):1826-32
references: 22234986
references: Appl Microbiol Biotechnol. 2010 Jul;87(4):1255-70
references: 20532762
references: Biotechniques. 1990 Sep;9(3):298, 300
references: 2223068
references: Appl Microbiol Biotechnol. 2004 Dec;66(2):217-25
references: 15558276