Toward Antibody Production in Genome-Minimized Bacillus subtilis Strains

Area of Science:

• Biotechnology
• Microbial Engineering
• Protein Production

Background:

• Bacillus subtilis is a well-established bacterial cell factory for industrial enzyme production.
• Producing complex proteins like antibody formats in B. subtilis has been challenging due to folding and disulfide bond requirements.

Purpose of the Study:

• To investigate the feasibility of producing functional antibody fragments (scFvs, scFabs) using genome-reduced Midi- and MiniBacillus strains.
• To optimize the workflow for high-quality antibody fragment secretion.

Main Methods:

• Selection of optimal chassis strains, expression vectors, signal peptides, and growth media.
• Upscaling scFv production to 1 L bioreactor level.
• Utilizing mass spectrometry, biolayer interferometry, and other biophysical techniques to verify antibody functionality.

Main Results:

• MidiBacillus strains successfully secreted functional antibody fragments.
• Secreted antibody fragments exhibited native folding and correct disulfide bonding.
• Antibody fragments demonstrated thermostability and functionality, as exemplified by a human C-reactive protein-binding scFv.

Conclusions:

• Genome-reduced Bacillus subtilis strains are suitable for high-quality antibody fragment secretion.
• This advancement enables the production of complex therapeutic proteins using microbial systems.