Optimization of linear plasmid expression system for protein production and secretion in Bacillus thuringiensis

Bacillus thuringiensis, a safe bacterium widely used in agriculture for the biocontrol of pests, has great potential for protein production. The linear plasmid expression system, bacterial orthogonal DNA replication system constructed based on B. thuringiensis prophage GIL16, can achieve stable and high levels of gene expression in the absence of external selection pressure, facilitating development of B. thuringiensis chassis cells. However, the regulatory elements of gene expression and protein secretion suitable for the B. thuringiensis expression system are still lacking. Therefore, the development and optimization of different genetic tools are required. We constructed a promoter library containing 107 different-strength promoters (covering persistently high/intermediate/low level) by transcriptomic analysis of the cell at different growth stages and a signal peptide library (59 signal peptides from Bacillus subtilis and four endogenous signal peptides from B. thuringiensis) to enrich the genetic toolbox using alpha-lactalbumin (α-LA) as the characterization product. Then, a high-throughput microfluidic screening platform based on BacORep and self-assembled split fluorescent protein was developed to further optimize expression elements, resulting in an improved α-LA-producing B. thuringiensis. Finally, the maximum copy number of linear plasmids was 9.3 times higher than that of the original. The titer of α-LA reached 107.7 mg/L in a 3 L bioreactor, which was comparable to the highest yield reported in Komagataella phaffii. We substantially expanded the synthetic biology toolbox for linear plasmid expression systems and provided a strategy for creating efficient prokaryotic expression system.