title: Effect of Co-overexpression of Nisin Key Genes on Nisin Production Improvement in Lactococcus lactis LS01.
abstract: Nisin is a small antimicrobial peptide produced by several subset strains of Lactococcus lactis. To improve nisin yield in the producer L. lactis LS01, we proposed a successive fusion of nisA with nisRK and nisFEG into a single shuttle expression vector pMG36e under the control of the native strong constitutive promoter p32. Subsequently, the recombinant vectors were transplanted into the producer cell through electroporation. Nisin productivity was determined through sodium dodecyl sulfate-polyacrylamide gel electrophoresis and bioactivity assays. Expression of nisin peptide was detected by agar diffusion bioassay, and the transcriptional levels of the target genes involved in nisin biosynthesis were investigated via semi-quantitative reverse transcription PCR expression analysis using 16S ribosomal RNA (rRNA) as an internal control. Results suggested that the introduction of empty plasmid did not affect nisin production of L. lactis LS01, whereas by our rational construction and screening, the engineered strain co-overexpressing nisA, nisRK, and nisFEG achieved a maximum increment in bioactive nisin production with a yield of 2470 IU/ml in shake flasks and 4857 IU/ml in 1.0-l fermenters, which increased by approximately 66.3 and 52.6% (P < 0.05), respectively, compared with that of the original strain under the given fermentation conditions. Meanwhile, the transcriptional analysis revealed that the expression of most of these multicopy genes except nisE at transcriptional level were upregulated in the two recombinant strains (LS01/pAR and LS01/pARF), possibly contributing to the improved nisin production. Therefore, this study would provide a potential strategy to improve the economic benefits of nisin manufacture for large-scale industrial production.
authors.affiliation: Institute of Biochemical and Biotechnological Drugs, College of Pharmacy, Shandong University, Jinan, 250012, People's Republic of China.
authors.forename: Zhi-Jian
authors.initials: ZJ
authors.lastname: Ni
authors.affiliation: Shandong Academy of Pharmaceutical Science, Jinan, 250101, People's Republic of China.
authors.forename: Xiao-Yuan
authors.initials: XY
authors.lastname: Zhang
authors.affiliation: Institute of Biochemical and Biotechnological Drugs, College of Pharmacy, Shandong University, Jinan, 250012, People's Republic of China.|Shandong Academy of Pharmaceutical Science, Jinan, 250101, People's Republic of China.
authors.forename: Fei
authors.initials: F
authors.lastname: Liu
authors.affiliation: Shandong Academy of Pharmaceutical Science, Jinan, 250101, People's Republic of China.
authors.forename: Miao
authors.initials: M
authors.lastname: Wang
authors.affiliation: Shandong Academy of Pharmaceutical Science, Jinan, 250101, People's Republic of China.
authors.forename: Rong-Hua
authors.initials: RH
authors.lastname: Hao
authors.affiliation: Institute of Biochemical and Biotechnological Drugs, College of Pharmacy, Shandong University, Jinan, 250012, People's Republic of China.|Shandong Academy of Pharmaceutical Science, Jinan, 250101, People's Republic of China.
authors.forename: Pei-Xue
authors.initials: PX
authors.lastname: Ling
authors.affiliation: Institute of Biochemical and Biotechnological Drugs, College of Pharmacy, Shandong University, Jinan, 250012, People's Republic of China. mars_pharma@163.com.|Shandong Academy of Pharmaceutical Science, Jinan, 250101, People's Republic of China. mars_pharma@163.com.
authors.forename: Xi-Qiang
authors.initials: XQ
authors.lastname: Zhu
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D013294:Lactococcus lactis / Q000235:genetics* / Q000378:metabolism*; D009561:Nisin / Q000096:biosynthesis* / Q000235:genetics; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic
chemical_list: D001426:Bacterial Proteins; D009561:Nisin
keywords: Co-overexpression; Key genes; Lactococcus lactis; Nisin; Yield