A host-vector toolbox for improved secretory protein overproduction in Bacillus subtilis

Area of Science:

• * Biotechnology
• * Molecular Biology
• * Microbial Engineering

Background:

• * Diverse target proteins in biotechnology necessitate flexible expression systems for functional overproduction.
• * Identifying optimal heterologous gene expression strategies requires suitable host-vector systems with diverse genetic circuits.

Purpose of the Study:

• * To design and construct a novel Bacillus subtilis expression toolbox for the overproduction and secretion of potentially toxic enzymes.
• * To validate the utility of this toolbox for expressing challenging eukaryotic proteins.

Main Methods:

• * Development of a 60-vector expression toolbox combining promoter variants, secretion signals, and plasmid backbones.
• * Utilization of a custom Bacillus subtilis strain deficient in proteases, sporulation, autolysis, and secondary metabolism.
• * Testing the system with Saccharomyces cerevisiae sulfhydryl oxidase (Sox) and human interleukin-1β.

Main Results:

• * Successful construction of a versatile Bacillus subtilis gene expression toolbox.
• * Demonstrated secretory overproduction of two challenging eukaryotic proteins (Sox and interleukin-1β) using the toolbox.
• * Developed and tested fermentation strategies for acetoin-controlled overproduction of heterologous proteins.

Conclusions:

• * The Bacillus subtilis host-vector system is suitable for extracellular production of relevant eukaryotic proteins.
• * The developed toolbox offers a robust platform for biotechnological applications.
• * Optimized fermentation strategies enhance the overproduction of heterologous proteins.