Protein Secretion

Bacillus subtilis and its close relatives secrete proteins directly into the growth medium efficiently and to high concentrations. The productivity of Bacillus species for the production of extracellular proteins has been improved by a variety of techniques including random and directed mu-tagenesis, recombinant techniques, and growth regimes.

Yields of tens of gram per liter are expected from industrial strains secreting native Bacillus proteins (55-58).

The secretion process is highly specific, involving functional sequences on the secretory protein and recognition between the secretory proteins and the host's protein secretion machinery (secretory preprotein translocase). For convenience, the process in B. subtilis can be divided into three stages: intracellular processes, involving the targeting of secretory proteins to the cytoplasmic membrane-secretory translocase; translocation across the cytoplasmic membrane; and posttranslocational stages, involving the processing of the secreted protein, its folding into the native conformation, and passage through the cell wall into the growth medium.

A major limiting step in the production of native secretory proteins is the level of transcription of their genes. The transcription of genes encoding native secretory proteins is tightly controlled and involves a number of complex, often interacting, regulatory networks, including transcrip-tional activators (e.g., DegQ, DegR, DegU, and SenS) and repressors (e.g., AbrB, Hpr, and Sin). For example, the nu-cleotide sequence upstream of apr, encoding the major alkaline protease of B. subtilis, includes binding sites for Hpr, Sin, SacU/Q, and AbrB (48).

Most Bacillus secretory proteins are synthesized as precursors with amino-terminal extensions known as signal peptides, or pre-peptides. The signal peptide is required to target the precursor to the cytoplasmic membrane and for initiating the interaction with the secretory translocase. During or shortly after translocation across the membrane, the signal peptide is removed through the action of signal peptidases. Bacillus signal peptides vary in length from 18 to 35 amino acid residues; their average length of about 30 residues is 5 to 7 residues longer than their counterparts in gram-negative bacteria and eukaryotes. The C-terminal end of the signal peptide contains the signal pep-tidase recognition sequence. Lipoproteins, which remain attached at the trans side of the membrane after translocation, are cleaved by a prolipoprotein-specific signal pep-tidase that has a different recognition sequence (47).

Many exported Bacillus proteins are synthesised as pre-pro-proteins. The pro-peptide, which is located between the signal peptide (pre-) and the mature protein, can vary in length from about 8 (e.g., B. subtilis a-amylase) to more than 200 amino acids (various neutral proteases). Propeptides have been implicated in the folding of the mature protein into its native conformation following translocation (47,59). Cleavage of the pro-peptide occurs after translocation, autocatalytically in the case of proteases or via extracellular proteases in the case of other secretory proteins (60). Long (8-20 residues) hydrophobic domains, flanked by positively charged residues, are usually absent from secretory proteins because such domains act as "stop transfer" or "membrane anchor" sequences. Similarly, few positively charged residues are found in the amino terminus of the mature protein as these reduce export efficiency.

Surprisingly little is known about early events in the B. subtilis secretory pathway. Secretion-specific molecular chaperones, required to maintain secretory precursor proteins in an unfolded conformation, to prevent their aggregation, and to target them to the secretory machinery in the membrane, have not been detected in B. subtilis. This could indicate that secretion is cotranslational, and indeed elements of the signal recognition particle-like (SRP-like) pathway have been found in this bacterium (47). The secretory translocase complex appears to be similar to that of E. coli. The SecAEY complex is primarily responsible for the ATP-dependent transport of proteins across the membrane, and homologs of other components of the E. coli translocase, such as SecD, F, and G, also appear to be present, in the former two cases as a SecDF fusion. A unique feature of B. subtilis strain 168 is the presence of five type I signal peptidases and a single type II lipoprotein signal peptidase; some industrial strains contain plasmids specifying additional type I signal peptidases.

The release of the translocated mature protein from the membrane is usually accompanied by its folding to the native conformation. For many B. subtilis secretory proteins, this requires folding factors such as Fe3+ and Ca2+ and/or the extracytoplasmic lipoprotein PrsA. Efficient folding of the mature protein on the trans side of the membrane appears to be important for avoiding or reducing electrostatic interactions with negatively charged components of the cell wall and degradation by extracellular proteases (61,62).

Although there are limits to the amounts of native proteins secreted by bacilli, such proteins are usually capable of being produced at commercially significant levels. In contrast, the extracellular production of heterologous proteins, especially from eukaryotic sources, is frequently inefficient. The reasons for this are not fully understood but are likely to include that the inherent incompatibilities with the secretory apparatus and their susceptibility to the many extracellular proteases, cell associated and released, produced by these bacteria (61,63).

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