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

Xurui Jin,Yifei Lu,Jiezhong Deng,Jing Wang,Fuquan Hu,Zhigang Huang,Yanlan Yu,Qiwen Hu,Hongxiang Yan

doi:10.1186/s12934-017-0770-1

Abstract

BackgroundLactococcus 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.ResultsOur 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.ConclusionsBy 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.

Highlights

Lactococcus lactis is a food grade probiotics and widely used to express heterologous proteins
L. lactis has been broadly used as an “efficient cell factory” for recombinant protein production [5] because of the following properties: (i) As a generally regarded as safe (GRAS) microorganism [1], L. lactis elicits weak immune responses against itself and does
PNZ8148 [3, 23], pMG36e [15, 24], pAMJ399 [19,20,21], and pLEB590 [25, 26]. This approach is limited by several disadvantages. (i) In these vectors, antibioticresistant genes, which are banned for use in humans, are commonly employed as selective markers. (ii) Food-grade selective markers, such as nisin resistance gene, have been applied in L. lactis

Summary

Introduction

Lactococcus lactis is a food grade probiotics and widely used to express heterologous proteins. Target genes are knocked into the L. lactis genome through double-crossover recombination to express heterologous proteins stably. An efficient heterologous gene knock-in reporter system was developed in L. lactis NZ9000. Heterologous proteins can be expressed in L. lactis by encoding their genes harbored in vectors, such as. (ii) Food-grade selective markers, such as nisin resistance gene (nsr), have been applied in L. lactis. Most of the foodgrade selective markers cannot be used in Escherichia coli Plasmids containing these food-grade selective markers can only be constructed in L. lactis, but the efficiency of constructing plasmids in L. lactis is much lower than that in E. coli. As an efficient alternative approach, the knock-in of target genes into L. lactis chromosome through double-crossover recombination is performed to stably express heterologous proteins without antibiotic-selective markers

Methods

Results

Conclusion