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.

Efficient Large-Scale and Scarless Genome Engineering Enables the Construction and Screening of Bacillus subtilis Biofuel Overproducers.

Bacillus subtilis is a versatile microbial cell factory that can produce valuable proteins and value-added chemicals. Long fragment editing techniques are of great importance for accelerating bacterial genome engineering to obtain desirable and genetically stable host strains. Herein, we develop an efficient CRISPR-Cas9 method for large-scale and scarless genome engineering in the Bacillus subtilis genome, which can delete up to 134.3 kb DNA fragments, 3.5 times as long as the previous report, with a positivity rate of 100%. The effects of using a heterologous NHEJ system, linear donor DNA, and various donor DNA length on the engineering efficiencies were also investigated. The CRISPR-Cas9 method was then utilized for Bacillus subtilis genome simplification and construction of a series of individual and cumulative deletion mutants, which are further screened for overproducer of isobutanol, a new generation biofuel. These results suggest that the method is a powerful genome engineering tool for constructing and screening engineered host strains with enhanced capabilities, highlighting the potential for synthetic biology and metabolic engineering.