Design,Construction And Application Of Crispr Based Gene Editing And Expression Regulation Systems In Bacillus Subtilis

Bacillus subtilis,which is not only a model microorganism of gram-positive bacteria but also a food safety strain,has been widely used both in basic science research and industrial fermentation.In recent years,with the development of synthetic biology and metabolic engineering,numerous tools for gene editing and expression regulation have been built in B.subtilis,including various CRISPR-Cas（Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein）based systems.Due to the extremely complex and extensive interactions of metabolic regulatory networks in the cell,it is difficult to meet the requirement of new generation microbial cell factories by just modifying or regulating a single gene locus.However,those gene editing tools built in B.subtilis still possess great deficiencies in the number of operable sites and editing efficiency.Although there have been several preliminary attempts in expression regulation of multiple-gene,systematic combinatorial optimization regulation has not been carried out,and process is usually achieved by artificial induction,which greatly increases the complexity of operations in production.In this study,B.subtilis was selected as the research object.Based on CRISPR-Cas system,a series of convenient,efficient,and easy-to-use tools for multi-gene editing and expression regulation were designed and constructed,and that were successfully applied in the metabolic engineering of B.subtilis.The main findings are as follows:（1）A xylose induced CRISPRi system has been built in B.subtilis,and that was used in combinational dynamic regulation of the competitive pathways related to N-acetylglucosamine（GlcNAc）synthesis.Firstly,a xylose induced CRISPRi system,which can achieve a 70.1-fold repression by using GFP as the reporter,was built from the CRISPR-Cas9 of Streptococcus pyogenes.Then this tool was applied in the combinational optimization and regulation of the three major competitive pathways（HMP,EMP,and PSP）of GlcNAc,and 27 sg RNA arrays have been designed and assembled.Employing this regulation method promoted the co-utilization of xylose and glucose,and GlcNAc titer of strain BNX122 possessed the best combination improved by 21.4%to 18.7 g/L.After further optimizing the addition time and amount of xylose,the GlcNAc titer improved to 20.5 g/L.In addition,RT-q PCR was performed in the engineered strains which approved that carbon catabolite repression（CCR）of B.subtilis have been relieved by applying the above regulatory strategy.Finally,the GlcNAc titer and productivity in a 3-L fed-batch bioreactor reached 103.1 g/L and 1.17 g/L/h,which were 5.0-fold and 2.7-fold of that in shake flask culture,respectively.（2）A biosensor-CRISPRi based autonomous dual-control（ADC）system has been designed,and the feedback genetic circuits was built using the ADC system to achieve the programable multi-module coordinated regulation of GlcNAc synthesis related metabolic networks.Firstly,14 hybrid promoters were designed in B.subtilis based on the regulation mechanism of transcription factor GamR.Promoter P_vg6,P_{gam A},and P_sg2,which can respond to intracellular glucosamine-6-phosphate（GlcN6P）concentration and has different dynamic range,were chosen to be the GlcNAc biosensor.Next,by coupling the“NOT”gate derived from CRISPRi,the“positive-negative”conversion of biosensor signals was realized,and the ADC system which can simultaneously activate and repress different genes was further developed.Then feedback genetic circuits was designed and constructed using the ADC system to perform the autonomous dynamic up-regulation and down-regulation of the synthetic module and competitive modules of GlcNAc,respecitively.After further optimization and matching of these modules,both the titer and yield of GlcNAc in shake flask improved by 53%to 28.0 g/L and 0.37 g/g,respectively.Lastly,the GlcNAc titer and yield in a 15-L fed-batch bioreactor reached 131.6 g/L and 0.38 g/g,which approved that the above genetic circuits still have good stability and robustness in large-scale bioreactor.（3）A CRISPR-Cpf1 based multi-gene editing system has been built in B.subtilis,and the synthetic pathways of GlcNAc were quickly reconstructed from the beginning in B.subtilis 168.Firstly,using CRISPR-Cpf1 system derived from Francisella novicida U112,a gene editing tool that could modify multi-loci simultaneously was designed and constructed in B.subtilis.The Natronobacterium gregoryi Argonaute（Ng Ago）protein has been included in this system,which significantly improved the number of operational targets and editing efficiency.Besides,the SOMACA（synthetic oligos mediated assembly of crRNA array）method was developed to rapid assemble any crRNA arrays from non-phosphorylated primers.Next,by using six non-essential protease genes of B.subtilis as editing targets,double genes in-frame knocking out,multiple point mutations（up to six）,or single gene insertion was achieved at a time with 100%efficiency.Finally,through the integrated expression of exogenous gene GNA1,the feedback inhibition relief and overexpression of key regulatory locus glm S,and the inactivation of the degradation pathway and by-product generation pathway of GlcNAc,the synthetic pathways of GlcNAc were quickly reconstructed from the beginning in B.subtilis 168.（4）A CRISPR-Cpf1 based multi-gene regulation system with dual regulation function（up-regulation and down-regulation）was constructed in B.subtilis,and the synthesis of endogenous product acetoin was significantly improved by using that.Firstly,a CRISPR-Cpf1 based xylose induced CRISPRi system was built by modifying the CRISPR-Cas9 based CRISPRi system.Then an isopropyl-beta-D-thiogalactopyranoside（IPTG）induced CRISPRi system was constructed,and the promoters of Cpf1 and crRNA array were optimized to reduce the leakage expression of the system,subsequently.The relation between IPTG concentration and repression strength was explored,and multi-gene repression was also performed using a designed crRNA array.Next,10 transcriptional activators from different sources were chosen and fused into the C-terminus of d Cpf1.Between them the fusion protein d Cpf1-Rem A can make a 58%improvement on the expression of target gene,and this fusion can simultaneously activate and repress different genes.Finally,a crRNA array was designed and built to down-regulate the key genes in consumption pathways of acetoin（bdh A and aco A）and synthetic pathways of by-products acetate and lactate（ldh and pta）,and to up-regulate the key gene in the synthetic pathways of acetoin and the gene encoding related transcriptional activator（als SD and als R）.Installing this crRNA array improved acetate titer by 44.8%to 25.8 g/L.