title: 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.
authors.affiliation: Centre for Bacterial Cell Biology, Baddiley-Clark Building, Newcastle University, Newcastle upon Tyne, UK.
authors.forename: Susanne
authors.initials: S
authors.lastname: Pohl
authors.forename: Gaurav
authors.initials: G
authors.lastname: Bhavsar
authors.forename: Joanne
authors.initials: J
authors.lastname: Hulme
authors.forename: Alexandra E
authors.initials: AE
authors.lastname: Bloor
authors.forename: Goksel
authors.initials: G
authors.lastname: Misirli
authors.forename: Matthew W
authors.initials: MW
authors.lastname: Leckenby
authors.forename: David S
authors.initials: DS
authors.lastname: Radford
authors.forename: Wendy
authors.initials: W
authors.lastname: Smith
authors.forename: Anil
authors.initials: A
authors.lastname: Wipat
authors.forename: E Diane
authors.initials: ED
authors.lastname: Williamson
authors.forename: Colin R
authors.initials: CR
authors.lastname: Harwood
authors.forename: Rocky M
authors.initials: RM
authors.lastname: Cranenburgh
mesh_terms: D000942:Antigens, Bacterial / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000032:analysis* / Q000737:chemistry; D001427:Bacterial Toxins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D005110:Extracellular Space / Q000737:chemistry / Q000378:metabolism; D017353:Gene Deletion; D005818:Genetic Engineering / Q000379:methods*; D010447:Peptide Hydrolases / Q000235:genetics / Q000378:metabolism; D020543:Proteome / Q000032:analysis* / Q000737:chemistry; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D020543:Proteome; D011994:Recombinant Proteins; C030325:anthrax toxin; D010447:Peptide Hydrolases
keywords: Anthrax protective antigen; Biomedicine; Gene deletion; Proteases; Recombinant protein; Secretion