Enhanced secretion of recombinant proteins via signal recognition particle (SRP)‐dependent secretion pathway by deletion of rrsE in Escherichia coli

Abstract

Although signal recognition particle (SRP)-dependent secretion pathway, which is characterized by co-translational translocation, helps prevent cytoplasmic aggregation of proteins before secretion, its limited capacity for the protein secretion is a major hurdle for utilizing the pathway as an attractive route for secretory production of recombinant proteins. Therefore, we developed an Escherichia coli mutant, whose efficiency of secretion via the SRP pathway was dramatically increased. First, we developed a novel FACS-based screening system by combining a periplasmic display system (PECS) and direct fluorescent labeling with the organoarsenic compound, FlAsH-EDT2 . With this screening system, transposon-insertion library of E. coli was screened, and then we isolated mutants which exhibited higher protein production through the SRP pathway than the parental strain. From the genetic analysis, we found that all isolated mutants had the same mutation-disruption of the 16S rRNA gene (rrsE). The positive effect of rrsE deficiency on protein secretion via the SRP pathway was successfully demonstrated using various model proteins including endogenous SRP-dependent proteins, antibodies, and G protein-coupled receptor. For the large-scale production of IgG and GPCR, we performed fed-batch cultivation with the rrsE-deficient mutant, and very high yields of IgG (0.4 g/L) and GPCR (1.4 g/L) were obtained. Biotechnol. Bioeng. 2016;113: 2453-2461. © 2016 Wiley Periodicals, Inc.

High‐yield production of recombinant CRM197, a non‐toxic mutant of diphtheria toxin, in the periplasm of Escherichia coli

“…Limited capacity of the SRP pathway has been reported previously, and several studies have attempted to overcome it by genetically engineering SRP pathway components or selecting mutants with increased secretion capacity (see and references therein). Recently, Blattner et al reported that E. coli mutants with reduced genomes are better suited than wild‐type E. coli strains such as BL21 for production of periplasmic CRM197 : they reported a yield of 1.96 g/L with the OmpA signal sequence (Sec‐dependent) in an E. coli K‐12 derivative containing no less than 74 mutations (mostly gene deletions).…”

“…The periplasm provides an oxidizing environment where disulfide bond formation is catalyzed by a dedicated enzymatic system (Dsb) . Periplasmic production provides several other benefits such as N‐terminal authenticity after cleavage of the signal peptide, as well as less proteolysis and a higher recombinant protein purity . The most widely used secretion systems for periplasmic expression of recombinant proteins in E. coli are the Sec and signal recognition particle (SRP) pathways, which share the same translocon (SecYEG) but target proteins to the secretion machinery in different ways.…”

“…Compared to the Sec pathway – which drives the post‐translational translocation of unfolded proteins –, translocation via the SRP pathway occurs in a co‐translational fashion. From a biotechnological perspective, this provides the benefit that premature cytoplasmic folding and aggregation can be efficiently prevented .…”

Biocatalytic synthesis of lactosucrose using a recombinant thermostable β-fructofuranosidase from Arthrobacter sp. 10138

“…Due to the specificity of the signal peptide, the secretory pathways of different kinds of proteins in E. coli are different. Signal recognition particle (SRP)-dependent secretion pathway, which is characterized by co-translational translocation, helps prevent cytoplasmic aggregation of proteins before secretion [24]. It was found that the torT signal peptide dependent on the SRP pathway had the best secretory capacity.…”

Developing Gram-negative bacteria for the secretion of heterologous proteins

“…Lee et al developed a method to measure the amount of protein accumulated in the periplasm using the fluorescent arsenical hairpin binder (FlAsH) tag and periplasmic expression with cytometric screening, (PECS)-FlAsH [96]. This screen specifically measures export to the periplasm, though it is also a useful technique for improving protein secretion via two-step systems because export can be a bottleneck in protein production [97, 98].…”