Establishment And Application Of Promoter Library And CRISPR-Cas9n Mediated Genome Editing For Bacillus Subtilis

Synthetic biology approaches to the design and construction of de novo biological systems.The implementation of synthetic biology concept of engineered biological systems depends on the availability of standardized biological elements and gene editing tools.Construction of promoter library for Bacillus subtilis.We designed a synthetic promoter library based on consensus sequences by analyzing microarray transcriptome data of B.subtilis 168.A total of 5000 library promoters were screened by report gene gfp,and the resulting library of synthetic promoters had strengths ranging from 2% to 94% of P43.The tandem promoter cluster were designed via tandem dual-promoter,which increased by 2.77-fold in term of strength compared with promoter P43.Furthermore,214 potential promoters spanning a gradient of strengths were isolated and analyzed.Promoter strength closely reflected the transcriptional and translational levels of gfp gene.Linear regression curve and probability distribution of sequence structure were obtained by model-based Multivariate Statistics and Partial Least Squares analysis of promoter sequences.We developed an analysis and reconstruction strategy based on position/nucleotide combinations,and the strength of reconstructed promoter Pm increased by 1.2-fold than that of P43.Meanwhile,the reconstruction strategy based probability distribution was applied to construct promoter library in corynebacterium glutamicum,and the mean strength of reconstructed library was increased by 39% compared to initial library.Finally,the synthetic promoter library was employed in the production of inosine and acetoin by repressing and overexpressing the relevant metabolic pathways,yielding a 7-fold and 44%increase relative to the respective control strains.Development and application of CRISPR/Cas9n mediated genome editing for B.subtilis.We established a CRISPR/Cas9n based multiplex genome editing system for rapid,efficient and iterative genome editing of B.subtilis.The time required for each editing cycle is 3 days by optimization of plasmid elimination and assembly process of g RNA and donor DNA.This system achieved nearly 100% efficiency for single site mutation,at least 90% efficiency for short fragment deletions and insertions(1-6 kb gene deletions or 1-2 kb gene insertions),82% efficiency for 8 kb gene deletions and 23.6% efficiency for large DNA fragment deletion.The system showed obvious advantages in multiplex editing based on activity of Cas9n,which achieved at least 90% efficiency for double-point mutations and 49% efficiency for three simultaneous point mutations.The efficiency for multiplex gene editing was further improved by regulating the nick repair mechanism mediated by ligD gene,which finally led to roughly 65% efficiency for introducing three point mutations on the chromosome.Finally,we applied CRISPR/Cas9n mediated multiplex gene editing to optimize the riboflavin operon by combinatorial modification of RBS sequence.Compared to the control strain,the riboflavin peoduction of improved strain increased by 59% in a single editing cycle.The increase of ribA gene expression and the decrease of ribB gene expression were concluded by simulating the secondary structure of RBS region in highest production strains,which provides theoretical support for explaining the mechanism of riboflavin metabolism.