Efficient secretory production of large‐size heterologous enzymes in Bacillus subtilis: A secretory partner and directed evolution

Secretory production of recombinant proteins provides a simple approach to the production and purification of target proteins in the enzyme industry. We developed a combined strategy for the secretory production of three large‐size heterologous enzymes with a special focus on 83‐kDa isoamylase (IA) from an archaeon Sulfolobus tokodaii in a bacterium Bacillus subtilis. First, a secretory protein of the B. subtilis family 5 glycoside hydrolase endoglucanase (Cel5) was used as a fusion partner, along with the NprB signal peptide, to facilitate secretory production of IA. This secretory partner strategy was effective for the secretion of two other large enzymes: family 9 glycoside hydrolase from Clostridium phytofermentas and cellodextrin phosphorylase from Clostridium thermocellum. Second, the secretion of Cel5‐IA was improved by directed evolution with two novel double‐layer Petri‐dish‐based high‐throughput screening (HTS) methods. The high‐sensitivity HTS relied on the detection of high‐activity Cel5 on the carboxymethylcellulose/Congo‐red assay. The second modest‐sensitivity HTS focused on the detection of low‐activity IA on the amylodextrin‐I2 assay. After six rounds of HTS, a secretory Cel5‐IA level was increased to 234 mg/L, 155 times the wild‐type IA with the NprB signal peptide only. This combinatory strategy could be useful to enhance the secretory production of large‐size heterologous proteins in B. subtilis. A strategy was developed for efficient protein secretion in Bacillus subtilis via a combination of a SPnprB‐Cel5‐based fusion protein and directed evolution. The 52‐kDa Cel5, as a general secretory protein helper, exhibits the facilitation effect on secretion of three large‐size heterologous enzymes. Through six‐round directed evolution with two novel HTSs, the best mutant 6‐1 produced up to 234 mg/L fusion protein by a factor of 24.0 compared with the wild‐type Cel5‐IA.