Proteomic analysis of Bacillus subtilis strains engineered for improved production of heterologous proteins

Abstract

The use of bacterial systems for recombinant protein production has advantages of simplicity, time and cost over competing systems. However, widely used bacterial expression systems (e.g. Escherichia coli, Pseudomonas fluorescens) are not able to secrete soluble proteins directly into the culture medium. This limits yields and increases downstream processing time and costs. In contrast, Bacillus spp. secrete native enzymes directly into the culture medium at grams-per-litre quantities, although the yields of some recombinant proteins are severely limited. We have engineered the Bacillus subtilis genome to generate novel strains with precise deletions in the genes encoding ten extracytoplasmic proteases that affect recombinant protein secretion, which lack chromosomal antibiotic resistance genes. The deletion sites and presence of single nucleotide polymorphisms were confirmed by sequencing. The strains are stable and were used in industrial-scale fermenters for the production of the Bacillus anthracis vaccine protein, protective antigen, the productivity of which is extremely low in the unmodified strain. We also show that the deletion of so-called quality control proteases appears to influence cell-wall synthesis, resulting in the induction of the cell-wall stress regulon that encodes another quality control protease.

Degradation of Extracytoplasmic Catalysts for Protein Folding in Bacillus subtilis

“…The lower-M w form of FeuA in the medium of strain BRB14 is the result of FeuA cleavage by an asyet-unidentified protease. For example, this could be the HtrC protease, which was shown to be upregulated in the BRB14 strain (40), but it might also be another as-yet-unidentified protease that is still present in the strain (4,18). Similar to FeuA, two forms of YclQ were released into the medium, but in this case, the lower-M w form was completely absent from the medium of strain BRB14 (Fig.…”

“…In particular, the strains lacking seven or more protease genes (i.e., BRB07, -08, -11, -12, and -14) were recently shown to be suitable for industrial-scale fermentations to produce the protective antigen of Bacillus anthracis (40). This secreted protein and potential vaccine candidate is highly susceptible to proteolytic degradation and cannot be produced in the parental strain 168.…”

“…Remarkably, only one significant pleiotropic effect was detected for the 10-fold protease mutant BRB14, which involved the induction of a cell wall stress regulon that encodes the quality control protease HtrC (YyxA/YycK). HtrC is a homologue of HtrA and HtrB, and therefore, the htrC induction most likely reflects an attempt of the cells to compensate for the absence of HtrA and HtrB (40).…”

Bacillus anthracis toxins

“…These systems secrete more than 10 mg/L, but in B. subtilis and other bacilli, the proteins are often degraded by extracellular proteases [100,101]. Expression in B. subtilis strains deleted of 6-12 extracellular proteases greatly improved yields of intact PA [101,102].…”

The Blueprint of a Minimal Cell: MiniBacillus

“…Of note, the nonessential DnaK/ DnaJ chaperone is also present in M. mycoides JCVI-syn3.0, whereas the universally conserved essential GroES/GroEL chaperonin is not (10). In addition, viability of B. subtilis requires the presence of one intramembrane quality control protease, and we selected HtrB for this activity (120,121).…”