Abstract
BackgroundThe 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.ResultsFour 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.ConclusionsThe 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.
Highlights
The implementation of novel chassis organisms to be used as microbial cell factories in industrial applications is an intensive research field
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
Design and construction of platform L. lactis strain for heterologous protein production To construct the streamlined-genome mutants, we initially searched the prophage, prophage-like, and transposon genes in L. lactis 9 k genome sequences because prophages and transposons may not always be necessary for the strain cultured in the laboratory or industrial condition
Summary
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. 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. Bacteria are commonly used as microbial cell factories for metabolic engineering and desirable product fermentation at the laboratory scale and in industrial applications [1, 2]. Shen and co-workers achieved high-titer anaerobic 1-butanol synthesis in E. coli [5]. Moon and co-workers achieved production of glucaric acid from a synthetic pathway in recombinant E. coli [6]. The minimized E. coli displayed some convenience as a host to express target products, but several disadvantages were observed, such as the formation of endotoxins and inclusion in intracellular protein
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