A new and efficient phosphate starvation inducible expression system for Lactococcus lactis

Abstract

A new expression system for Lactococcus lactis was developed. The system is based on a phosphate starvation inducible pstF promoter of L. lactis MG1363. Intracellular beta-galactosidase and secreted alpha-amylase were produced using this tightly regulated system. No evidence of regulatory sites in regions of the 5'-end of the pstF coding sequence was found. High expression levels of the beta-galactosidase gene were obtained using the original pstF RBS in a phosphate-depleted medium. The results suggested that with the phosphate starvation inducible system, it is possible to achieve expression levels comparable to the ones obtained with the widely used nisin-controlled gene expression system (NICE). A specific beta-galactosidase activity of 670 microkat g(-1) using a phosphate-depleted medium and an alpha-amylase activity of 3.6 microkat l(-1) in a bioreactor cultivation were produced. The advantages of the current expression system include that no prior removal of phosphate from the medium in bioreactor scale is required, and no additions of inducing agents are needed. Furthermore, the system can be operated in L. lactis without introduction of regulatory genes into the host.

The pstSCAB operon for phosphate uptake is regulated by the global regulator GlxR in Corynebacterium glutamicum

“…Immediately after the shift to low-phosphate medium, the operon encoding the high affinity phosphate uptake system PstSCAB is induced, followed by induction of genes for uptake and utilization of organophosphates and, thirdly, genes for extracellular enzymes to gain access to phosphate from non-transportable organophosphates (Ishige et al, 2003). The immediate induction of genes for an ATP-dependent high affinity ABC-type uptake system for inorganic phosphate is a common response to phosphate starvation of several bacteria: Escherichia coli (Baek and Lee, 2007), Bacillus subtilis (Allenby et al, 2005), Mycobacterium tuberculosis (Rifat et al, 2009), Lactococcus lactis (Siren et al, 2008), and Streptomyces coelicolor (Sola-Landa et al, 2005).…”

A novel manganese starvation-inducible expression system forLactobacillus plantarum

“…Our results demonstrated that in addition to the reported phosphate starvation promoter system for Lactococcus and Bacillus species (Kerovuo et al ., ; Sirén et al ., ), another starvation promoter system based on manganese ions can be applied to suitable hosts such as Lactobacilli . A similar system was developed for L. lactis using promoter and repressor protein of a zinc uptake system (Llull & Poquet, ).…”

“…Other types of auto‐inducing expression systems were developed for L. lactis and Bacillus subtilis in recent years. These are based on phosphate or metal starvation transporter systems (Kerovuo et al ., ; Sirén et al ., ). Because phosphate is an essential component for bacterial growth, the promoters of these transport proteins are turned on under starvation conditions, and this can be used for recombinant protein expression.…”

Tuning Gene Expression by Phosphate in the Methanogenic Archaeon Methanococcus maripaludis

“…This approach has been applied in bioproduction under the limitation of different nutrients, such as phosphate, nitrogen, and glucose . For instance, the production of α-amylase in bioreactor-cultivated Lactococcus lactis and phytase in fed-batch-grown Bacillus licheniformis during phosphate-limited stationary phase shows the adaptability of this expression system to different cultivation methods. Similarly, nitrogen-limited inducible expression systems have been developed for the production of C 12 –C 16 fatty alcohols from glucose in continuously cultivated Escherichia coli and lipid accumulation in microalgae …”