title: Secretory expression of a phospholipase A2 from Lactobacillus casei DSM20011 in Kluyveromyces lactis.
abstract: The pla2 gene encoding a phospholipase A2 (EC 3.1.1.4) of Lactobacillus casei DSM20011 was cloned and expressed in the yeast Kluyveromyces lactis GG799 successfully for the first time. The structural pla2 gene fused in frame with the K. lactis secretion signal α-mating factor was integrated into the LAC4 locus and expressed under the control of the LAC4 promoter. sPLA2 activity was detected in the culture supernatant during shake flask culture of K. lactis/pKLAC1-pla2. In comparison with the control strain K. lactis/pKLAC1, SDS-PAGE analysis revealed a 17-kDa recombinant protein band in K. lactis/pKLAC1-pla2, which was consistent with the predicted molecular weight of the mature protein. Real-time quantitative PCR analysis indicated that the copy number of the integrated pla2 gene ranged from 2 to 6 and positively correlated with sPLA2 activity. When the inducer galactose was used as the carbon source, the sPLA2 activity in the culture supernatant of the recombinant that harbored six pla2 gene copies reached 1.96 ± 0.15 U/mL. The influence of the culture composition and conditions on the recombinant sPLA2 activity in shake flask culture were also studied. When the recombinant was cultured at 30°C in a YPD medium culture volume of 70 mL in a 250-mL shake flask with an initial pH of 7.0, the sPLA2 activity reached 2.16 ± 0.18 U/mL.
authors.affiliation: National Engineering Laboratory for Cereal Fermentation Technology of Jiangnan University, 1800 Lihu Road, Wuxi 214122, China; Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 LiHu Road, Wuxi 214122, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China; National Engineering Laboratory for Cereal Fermentation Technology of Jiangnan University, 1800 Lihu Road, Wuxi 214122, China. Electronic address: zhangl@jiangnan.edu.cn.
authors.forename: Liang
authors.initials: L
authors.lastname: Zhang
authors.affiliation: National Engineering Laboratory for Cereal Fermentation Technology of Jiangnan University, 1800 Lihu Road, Wuxi 214122, China.
authors.forename: Guiyang
authors.initials: G
authors.lastname: Shi
mesh_terms: D003470:Culture Media / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005690:Galactose / Q000378:metabolism; D018628:Gene Dosage; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007716:Kluyveromyces / Q000235:genetics / Q000378:metabolism*; D007780:Lacticaseibacillus casei / Q000201:enzymology* / Q000235:genetics; D000072235:Mating Factor; D008970:Molecular Weight; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D054467:Phospholipases A2 / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D060888:Real-Time Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013696:Temperature
chemical_list: D003470:Culture Media; D010455:Peptides; D011994:Recombinant Proteins; D000072235:Mating Factor; D054467:Phospholipases A2; D005690:Galactose
keywords: Copy number; Heterologous expression; Kluyveromyces lactis; Lactobacillus casei; Real-time quantitative PCR; Secretory phospholipase A(2)