title: Production of LYZL6, a novel human c-type lysozyme, in recombinant Pichia pastoris employing high cell density fed-batch fermentation.
abstract: Lysozyme acts as an important defensive factor in innate immunity due to its well-recognized bacteriolytic activity. Here we describe the production and performance of human lysozyme-like 6 (LYZL6), a novel human c-type lysozyme homolog. A synthetic codon-optimized cDNA encoding the intact amino acid sequence of LYZL6 was cloned and expressed in Pichia pastoris SMD1168. Bioactive LYZL6 was successfully produced as a single major secreted protein with a molecular weight of 15 kDa, and exhibited bacteriolytic activity against Micrococcus lysodeikticus. The expression conditions were optimized, and the highest expression level of LYZL6 occurred when the recombinant strain was induced with 1.5% methanol under pH 4.5 at 24°C for 96 h. When high cell density fermentation of the recombinant P. pastoris was performed using a fed-batch strategy for totally 125 h in a 30 L fermenter, the dry cell weight and the extracellular lysozyme activity were increased to 116.3 g/L and 2340 U/mL, respectively. The LYZL6 protein concentration was 331 mg/L of fermentation supernatant, and the specific activity of LYZL6 towards M. lysodeikticus was 7069 U/mg. Therefore, we proved that LYZL6 is an antibacterial protein, suggesting a potential application of LYZL6 as an antimicrobial agent, and Pichia expression system for LYZL6 was successful and industrially promising.
authors.affiliation: State Key Laboratory of Genetic Engineering, Fudan University, 220 Hadan Road, Shanghai 200433, China.
authors.forename: Xiaoyu
authors.initials: X
authors.lastname: Zhou
authors.affiliation: State Key Laboratory of Genetic Engineering, Fudan University, 220 Hadan Road, Shanghai 200433, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Yu
authors.affiliation: State Key Laboratory of Genetic Engineering, Fudan University, 220 Hadan Road, Shanghai 200433, China.
authors.forename: Jianjun
authors.initials: J
authors.lastname: Tao
authors.affiliation: State Key Laboratory of Genetic Engineering, Fudan University, 220 Hadan Road, Shanghai 200433, China. Electronic address: longyu@fudan.edu.cn.
authors.forename: Long
authors.initials: L
authors.lastname: Yu
mesh_terms: D000595:Amino Acid Sequence; D061249:Batch Cell Culture Techniques; D019149:Bioreactors; D003001:Cloning, Molecular; D003062:Codon; D004790:Enzyme Induction; D005285:Fermentation; D015870:Gene Expression; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D000432:Methanol / Q000378:metabolism / Q000494:pharmacology; D008837:Micrococcus / Q000187:drug effects; D008969:Molecular Sequence Data; D009113:Muramidase / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D010843:Pichia / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D016415:Sequence Alignment
chemical_list: D003062:Codon; D011994:Recombinant Proteins; C507694:LYZL6 protein, human; D009113:Muramidase; D000432:Methanol
keywords: Bacteriolytic activity; Expression optimization; Fed-batch fermentation; Lysozyme-like; Pichia pastoris