title: Enhanced heterologous protein productivity by genome reduction in Lactococcus lactis NZ9000.
abstract: BACKGROUND
  The implementation of novel chassis organisms to be used as microbial cell factories in industrial applications is an intensive research field. Lactococcus lactis, which is one of the most extensively studied model organisms, exhibits superior ability to be used as engineered host for fermentation of desirable products. However, few studies have reported about genome reduction of L. lactis as a clean background for functional genomic studies and a model chassis for desirable product fermentation.
  
  
  RESULTS
  Four large nonessential DNA regions accounting for 2.83% in L. lactis NZ9000 (L. lactis 9 k) genome (2,530,294 bp) were deleted using the Cre-loxP deletion system as the first steps toward a minimized genome in this study. The mutants were compared with the parental strain in several physiological traits and evaluated as microbial cell factories for heterologous protein production (intracellular and secretory expression) with the red fluorescent protein (RFP) and the bacteriocin leucocin C (LecC) as reporters. The four mutants grew faster, yielded enhanced biomass, achieved increased adenosine triphosphate content, and diminished maintenance demands compared with the wild strain in the two media tested. In particular, L. lactis 9 k-4 with the largest deletion was identified as the optimum candidate host for recombinant protein production. With nisin induction, not only the transcriptional efficiency but also the production levels of the expressed reporters were approximately three- to fourfold improved compared with the wild strain. The expression of lecC gene controlled with strong constitutive promoters P5 and P8 in L. lactis 9 k-4 was also improved significantly.
  
  
  CONCLUSIONS
  The genome-streamlined L. lactis 9 k-4 outcompeted the parental strain in several physiological traits assessed. Moreover, L. lactis 9 k-4 exhibited good properties as platform organism for protein production. In future works, the genome of L. lactis will be maximally reduced by using our specific design to provide an even more clean background for functional genomics studies than L. lactis 9 k-4 constructed in this study. Furthermore, an improved background will be potentially available for use in biotechology.
authors.affiliation: Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Nankai University, Tianjin, China.|Department of Food and Environmental Sciences, University of Helsinki, Helsinki, Finland.
authors.forename: Duolong
authors.initials: D
authors.lastname: Zhu
authors.affiliation: Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Nankai University, Tianjin, China.
authors.forename: Yuxin
authors.initials: Y
authors.lastname: Fu
authors.affiliation: Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Nankai University, Tianjin, China.
authors.forename: Fulu
authors.initials: F
authors.lastname: Liu
authors.affiliation: Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Nankai University, Tianjin, China.
authors.forename: Haijin
authors.initials: H
authors.lastname: Xu
authors.affiliation: Department of Food and Environmental Sciences, University of Helsinki, Helsinki, Finland.
authors.forename: Per Erik Joakim
authors.initials: PE
authors.lastname: Saris
authors.affiliation: Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Nankai University, Tianjin, China. mingqiangqiao@aliyun.com.|College of Life Sciences, Nankai University, Room 301, Tianjin, China. mingqiangqiao@aliyun.com.
authors.forename: Mingqiang
authors.initials: M
authors.lastname: Qiao
mesh_terms: D005818:Genetic Engineering / Q000379:methods*; D016680:Genome, Bacterial; D013294:Lactococcus lactis / Q000235:genetics* / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D011994:Recombinant Proteins
keywords: Chassis; Heterologous; Lactococcus lactis; LecC; Microbial cell factories; Red fluorescent protein