Intramembrane protease RasP boosts protein production in Bacillus

Abstract

BackgroundThe microbial cell factory Bacillus subtilis is a popular industrial platform for high-level production of secreted technical enzymes. Nonetheless, the effective secretion of particular heterologous enzymes remains challenging. Over the past decades various studies have tackled this problem, and major improvements were achieved by optimizing signal peptides or removing proteases involved in product degradation. On the other hand, serious bottlenecks in the protein export process per se remained enigmatic, especially for protein secretion at commercially significant levels by cells grown to high density. The aim of our present study was to assess the relevance of the intramembrane protease RasP for high-level protein production in B. subtilis.ResultsDeletion of the rasP gene resulted in reduced precursor processing and extracellular levels of the overproduced α-amylases AmyE from B. subtilis and AmyL from Bacillus licheniformis. Further, secretion of the overproduced serine protease BPN’ from Bacillus amyloliquefaciens was severely impaired in the absence of RasP. Importantly, overexpression of rasP resulted in threefold increased production of a serine protease from Bacillus clausii, and 2.5- to 10-fold increased production of an AmyAc α-amylase from Paenibacillus curdlanolyticus, depending on the culture conditions. Of note, growth defects due to overproduction of the two latter enzymes were suppressed by rasP-overexpression.ConclusionHere we show that an intramembrane protease, RasP, sets a limit to high-level production of two secreted heterologous enzymes that are difficult to produce in the B. subtilis cell factory. This finding was unexpected and suggests that proteolytic membrane sanitation is key to effective enzyme production in Bacillus.

Relative contributions of non-essential Sec pathway components and cell envelope-associated proteases to high-level enzyme secretion by Bacillus subtilis

“…The present study was aimed at a systematic examination of the impact of non-essential secretion machinery components of B. subtilis on the secretion of two α-amylases, namely AmyE and AmyL and the serine-protease BPN' . To exclude differential effects on the secretion of these three reporter proteins due to the usage of different expression or secretion signals, the amyE, amyL and bpn' genes were inserted in the chromosomal aprE locus, transcribed from the aprE promoter, and provided with the aprE signal sequence that directs Sec-dependent secretion [26]. The use of the strong DegU-controlled aprE promoter has the additional advantage that it is highly activated in a so-called degU32(Hy) mutant background, where DegU is constitutively phosphorylated [41].…”

“…The use of the strong DegU-controlled aprE promoter has the additional advantage that it is highly activated in a so-called degU32(Hy) mutant background, where DegU is constitutively phosphorylated [41]. Accordingly, strains containing these expression modules and the degU32(Hy) mutation can secrete high levels of AmyE, AmyL or BPN' into the growth medium [26]. This is exemplified in Fig.…”

“…Several factors are not directly involved in the protein export process but are, nonetheless, needed for its optimal performance. These include potential signal peptide peptidases, like TepA, SppA and RasP, [24][25][26], and quality control proteases like HtrA, HtrB and WprA [27][28][29][30][31][32]. TepA, SppA and RasP have been implicated in degradation of cleaved signal peptides, and in keeping the membrane clear from mistranslocated or misassembled proteins [24][25][26].…”

“…These include potential signal peptide peptidases, like TepA, SppA and RasP, [24][25][26], and quality control proteases like HtrA, HtrB and WprA [27][28][29][30][31][32]. TepA, SppA and RasP have been implicated in degradation of cleaved signal peptides, and in keeping the membrane clear from mistranslocated or misassembled proteins [24][25][26]. HtrA, HtrB and WprA remove aggregated or malfolded proteins from the membrane-cell wall interface or the cell wall and they may contribute to folding of translocated proteins as well [27][28][29][30][31][32].…”

EngineeringBacillusfor efficient production of heterologous protein: current progress, challenge and prospect

“…Neef et al . () implied that the overexpression of RasP led to a threefold increase of a serine protease and a 10‐fold increase of α‐amylase production in B. subtilis . Moreover, cell membrane phospholipid bilayer and isoelectric point (PI) of heterologous proteins were two important factors that influence protein secretion.…”