Simplified Construction of Engineered Bacillus subtilis Host for Improved Expression of Proteins Harboring Noncanonical Amino Acids

The
UAG-based genetic code expansion (GCE) enables site-specific
incorporation of noncanonical amino acids (ncAAs) harboring novel
chemical functionalities in specific target proteins. However, most
GCE studies were done in several whole-genome engineered chassis cells
whose hundreds of UAG stop codons were systematically edited to UAA
to avoid readthrough in protein synthesis in the presence of GCE.
The huge workload of removing all UAG limited the application of GCE
in other microbial cell factories (MCF) such as Bacillus
subtilis, which has 607 genes ended with UAG among
its 4245 coding genes. Although the 257 essential genes count only
6.1% of the genes in B. subtilis, they
transcribe 12.2% of the mRNAs and express 52.1% of the proteins under
the exponential phase. Here, we engineered a strain named Bs-22 in
which all 22 engineerable UAG stop codons in essential genes were
edited to UAA via CRISPR/Cas9-mediated multiple-site engineering to
minimize the negative effect of GCE on the expression of essential
genes. Besides the process of constructing GCE-compatible B. subtilis was systematically optimized. Compared
with wild-type B. subtilis (Bs-WT),
the fluorescence signal of the eGFP expression could enhance 2.25-fold
in Bs-22, and the production of protein tsPurple containing l-(7-hydroxycoumarin-4-yl) ethylglycine (Cou) was increased 2.31-fold
in Bs-22. We verified that all purified tsPurple proteins from Bs-22
contained Cou, indicating the excellent fidelity of the strategy.
This proof-of-concept study reported efficient overexpression of ncAA-rich
proteins in MCF with minimized engineering, shedding new light on
solving the trade-off between efficiency and workload.