Mining Of Expression Elements,Development Of CRISPR Genetic Systems And Its Applications In Bacillus Amyloliquefaciens

Bacillus amyloliquefaciens is generally recognized as food safe（GRAS）microbial host,which is widely used in agriculture,industry,food industry and medicine.As a closely related species of Bacillus subtilis,it has not been fully developed and applied as a high-efficiency expression host for target protein.The restriction-modification system makes it difficult for exogenous plasmid DNA to be transformed,and the low transformation efficiency and the lack of fast and efficient genetic editing tools further hinder its research progress.In this paper,B.amyloliquefaciens LB1ba02,an industrialα-amylase Amy X producing strain,was taken as the research object.Subsequently,the endogenous expression elements were mined based on transcriptome RNA-seq.Furthermore,a series of applicable CRISPR genetic operating systems were established,including CRISPR/Cas9n gene knockout and integration,CRISPR/Cas9n-AID base editing,and CRISPR/d Cpf1-ωtranscription regulatory system.Based on these systems,the construction of food grade efficient expression host,the modification of restriction-modification system,the global optimization of protein secretion pathway and the growth characteristics research were carried out.The specific contents and results are as follows:（1）The whole genome and RNA-seq of B.amyloliquefaciens LB1ba02 was sequenced and five endogenous strong promoters(P_{deoc C},P_{ssr A},P_{gud B},P_{gnt K} and P₃₄₁₃₅)were screened by RNA-seq.In addition,six rearranged promoters with GFP expression 1.62-2.08 times higher than P₄₃were screened by rearrangement of non-conserved regions of strong promoters.The screened and rearranged single promoters were used for the expression of Amy X.Furthermore,the combination research of 12 double promoters showed that P₄₃-P_{gnt K} had the best effect,and the expressed GFP and Amy X activity were 3.56 and 1.23 times higher than P₄₃,respectively.This indicates that the expression intensity of dual promoters is significantly higher than that of single promoter.（2）A single temperature-sensitive plasmid CRISPR/Cas9n genome editing system was established in B.amyloliquefaciens LB1ba02.Compared with the traditional homologous recombination knockout,this system required shorter time and greatly improved editing efficiency,and the editing efficiency of single gene wpr A reached 93%.Further,the type II restriction-modification system in B.amyloliquefaciens LB1ba02 was removed by this method,which simplified the transformation process.In addition,our editing system successfully achieved rapid gene integration in the genome.（3）The CRISPR/Cas9n-AID single base editing system was further implemented based on CRISPR/Cas9n system in B.amyloliquefaciens LB1ba02.Our base editing system has a 6nt editing window consisting of an all-in-one temperature-sensitive plasmid that facilitates multiple rounds of genome engineering.The total editing efficiency reached 100%and it achieved simultaneous editing of three loci with an efficiency of 53.3%.Besides,we generated4 genes（apr E,npr E,wpr A,and bam HIR）mutant strain,LB1ba02△4.The mutant strain secreted 1.25-fold moreα-amylase into the medium than the wild-type strain.（4）To increase the diversity of the CRISPR system in B.amyloliquefaciens,we adapted a nuclease-deficient mutant d Cpf1（D917A）of Cpf1 and developed a CRISPR/d Cpf1 assisted multiplex gene regulation system for the first time.A 73.9-fold inhibition efficiency and an optimal 1.8-fold activation effect at the-327 bp site upstream of the TSS were observed in this system.In addition,this system achieved the simultaneous activation of the expression of three genes（sec E,sec DF,and prs A）by designing a cr RNA array.On this basis,we constructed a cr RNA activation library for the proteins involved in the Sec pathway,and screened 7 proteins that could promote the secretion of extracellular proteins.Among them,the most significant effect was observed when the expression of molecular motor transporter Sec A was activated.Not only that,we constructed cr RNA arrays to activate the expression of two or three proteins in combination.The results showed that the secretion efficiency of Amy X and GFP was further increased to 1.46 and 9.8-fold when Sec A and Csa A were simultaneously activated in shake flask fermentation.（5）Based on CRISPRi,a genome wide cr RNA knockout library was further designed and a high-throughput screening system was developed in combination with flow cytometry to study the growth characteristics of B.amyloliquefaciens LB1ba02.The whole-genome cr RNA library was first verified by 5-fluorouracil resistance screening,and further used in the verification of essential genes.Subsequently,7 gene sites（opp D,flil,tua A,prm A,sig O,hsl U and GE03231）affecting the growth characteristics of B.amyloliquefaciens LB1ba02 were screened through the high-throughput protocol.Among them,the Opp system had the greatest impact on the growth.When the expression of polycistron opp A-opp B-opp C-opp D-opp F was inhibited,the cell growth was the fastest and the cell concentration was the highest.Inhibition of other sites could also promote the rapid growth of bacteria in different degrees;however,the autolysis rate of the late growth stage increased after inhibition of the putative protein gene site GE03231.Therefore,the whole genome cr RNA inhibitory library can be well applied to B.amyloliquefaciens LB1ba02 and can be further applied to other studies.