Development and evaluation of an efficient heterologous gene knock-in reporter system in Lactococcus lactis

• Main Authors: Yifei Lu, Hongxiang Yan, Jiezhong Deng, Zhigang Huang, Xurui Jin, Yanlan Yu, Qiwen Hu, Fuquan Hu, Jing Wang
• Format: Article
• Language: English
• Published: BMC 2017-09-01
• Series: Microbial Cell Factories
• Subjects: Lactococcus lactis; NZ9000; Knocked-in heterologous gene; Knock-in reporter system; lacZ
• Online Access: http://link.springer.com/article/10.1186/s12934-017-0770-1

Abstract Background Lactococcus lactis is a food grade probiotics and widely used to express heterologous proteins. Generally, target genes are knocked into the L. lactis genome through double-crossover recombination to express heterologous proteins stably. However, creating marker-less heterologous genes knocked-in clones is laborious. In this study, an efficient heterologous gene knock-in reporter system was developed in L. lactis NZ9000. Results Our knock-in reporter system consists of a temperature-sensitive plasmid pJW and a recombinant L. lactis strain named NZB. The pJW contains homologous arms, and was constructed to knock-in heterologous genes at a fixed locus of NZ9000 genome. lacZ (β-galactosidase) gene was knocked into the chromosome of NZ9000 as a counter-selective marker through the plasmid pJW to generate NZB. The engineered NZB strain formed blue colonies on X-Gal plate. The desired double-crossover mutants formed white colonies distinctive from the predominantly blue colonies (parental and plasmid-integrated clones) when the embedded lacZ was replaced with the target heterologous genes carried by pJW in NZB. Conclusions By using the system, the heterologous gene knocked-in clones are screened by colony phenotype change rather than by checking colonies individually. Our new knock-in reporter system provides an efficient method to create heterologous genes knocked-in clones.