title: Enhanced production of Thermomyces lanuginosus lipase in Pichia pastoris via genetic and fermentation strategies.
abstract: This study attempted to enhance the expression level of Thermomyces lanuginosus lipase (TLL) in Pichia pastoris using a series of strategies. The tll gene was first inserted into the expression vector pPIC9 K and transformed into P. pastoris strain GS115. The maximum hydrolytic activity of TLL reached 4,350 U/mL under the optimal culture conditions of a 500 mL shaking flask containing 20 mL culture medium with the addition of 1.2 % (w/v) methanol, cultivation for 144 h at pH 7.0 and 27 °C. To further increase the TLL expression and copy number, strains containing two plasmids were obtained by sequential electroporation into GS115/9k-TLL #3 with a second vector, either pGAPZαA-TLL, pFZα-TLL, or pPICZαA-TLL. The maximum activity of the resultant strains GS115/9KTLL-ZαATLL #40, GS115/9KTLL-FZαATLL #46 and GS115/9KTLL-GAPTLL #45 was 6,600 U/mL, 6,000 U/mL and 4,800 U/mL, respectively. The tll copy number in these strains, as assessed by real-time quantitative PCR, was demonstrated to be seven, five, and three, respectively, versus two copies in GS115/9k-TLL #3. When a co-feeding strategy of sorbitol/methanol was adopted in a 3-L fermenter, the maximum TLL activity of GS115/9k-TLL #3 increased to 27,000 U/mL after 130 h of fed-batch fermentation, whereas, the maximum TLL activity was 19,500 U/mL after 145 h incubation when methanol was used as the sole carbon source.
authors: Key Laboratory of Molecular Biophysics of Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, 430074, People's Republic of China.
authors: Zhonggang
authors: Z
authors: Fang
authors: Li
authors: L
authors: Xu
authors: Dujie
authors: D
authors: Pan
authors: Liangcheng
authors: L
authors: Jiao
authors: Ziming
authors: Z
authors: Liu
authors: Yunjun
authors: Y
authors: Yan
mesh_terms: D001203:Ascomycota / Q000201:enzymology*; D003001:Cloning, Molecular; D005285:Fermentation; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D018628:Gene Dosage; D015870:Gene Expression; D005822:Genetic Vectors; D006868:Hydrolysis; D008049:Lipase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D000432:Methanol / Q000378:metabolism; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D013012:Sorbitol / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D013012:Sorbitol; D008049:Lipase; D000432:Methanol