Engineering CRISPR Systems For Deletion And Repression Of A Class 1 Integron And Development Of Integron-Based Recombination Tool In Escherichia Coli

Class 1 integrons are important DNA elements that have substantially evolved food-borne microbial antibiotic resistance,as they can integrate and express exogenous antibiotic resistance gene(ARG)cassettes through site-specific recombination to bring about novel antibiotic resistance.Since class 1 integrons typically locate in mobile genetic elements(MGEs)such as conjugative plasmids and transposons,they lead to horizontal gene transfer(HGT)of ARGs.Presumably,class 1 integrons facilitate the spread of antibiotic resistance and even the evolution of multidrug resistant bacteria.These events pose a huge threat to human health.To date,no strategies can efficiently cope with the above-mentioned problems caused by class 1 integrons.In this study,CRISPR systems were exploited to delete and repress the class 1 integron on the broad-host-range conjugative plasmid R388 to alleviate microbial multidrug resistance(MDR).Considering class 1 integrons can capture and express gene cassettes,we developed an integron-based gene recombination tool through CRISPR and?Red systems.We also engineered a recombinant platform Escherichia coli strain which can utilize citric acid as carbon source to sustain growth.Moreover,the CRISPR interference-guided modulation of glucose pathways led to significantly improved aconitic acid production in E.coli.The main research contents were outlined as follows:1.A CRISPR/Cas9 system was constructed to specifically knock out the plasmid R388class 1 integron in E.coli.The knockout effect was evaluated by investigating the transformation efficiency of CRISPR plasmids,the minimum inhibitory concentration(MIC)of relevant antibiotics,the copy number of R388 class 1 integron,as well as the DNA sequence of target site.Results showed that the transformation of CRISPR plasmids into E.coli not only reduced antibiotic resistance which is originally due to the R388 class 1 integron but also led to 99%resensitization rate of transformants to trimethoprim(TMP)and sulfamethoxazole(SUL).Surprisingly,the transformation quality of CRISPR plasmids was negatively correlated with the resensitization rate of TMP and SUL.Another interesting finding is that the MIC values of TMP and SUL in transformatns varied substantially.One plausible explanation for this is that the CRISPR/Cas9-mediated DNA double-strand breaks(DSBs)reduced the copy number of R388 class 1 integron.In addition,CRISPR/Cas9 system showed off-target in six transformants,in which 45%of the R388 class 1 integron lost 17 bp in target sequence.It is speculated that the above mutation might prevent the integron from being recognized by CRISPR/Cas9 system.2.Considering the genetic information on plasmid R388 may vanish due to DSBs when CRISPR/Cas9 is used to delete the R388 class 1 integron,we constructed a CRISPR interference(CRISPRi)system to knock down the class 1 integron-mediated expression of ARGs.Using CRISPRi to block plasmid R388 class 1 integron,reverse transcription and quantitative PCR(RT-q PCR)revealed that the CRISPRi strains exhibited 36.7?97.4%and20.8?83.8%decrease in the transcriptional levels of the ARG cassette dfr B2 and the3'-conserved segment ARG sul1,respectively.Moreover,the transcriptional inhibition on ARGs led to an increase in the sensitivity of CRISPRi strains to relevant antibiotics.Microplate alamar Blue assay(MABA)showed that the CRISPRi systems resulted in 87.5%and 50?96.875%decreases in half-maximal inhibitory concentrations(IC₅₀)for TMP and SUL,respectively.The increased sensitivity to above two antibiotics in turn led to halted growth of E.coli recombinants upon exposure to the same antibiotics.The aforementioned evaluation of CRISPRi system revealed that the g RNA R3 and R6 outperformed other g RNAs in repressing dfr B2 and sul1 expression,and the knockdown efficiency is titratable by varying the concentration of inducer anhydrotetracycline(a Tc).Overall,the-35 region of Pc promoter on int I1 template strand and the 31 bp downstream of the Pc on int I1 non-template strand are ideal targets for CRISPRi to knock down the class 1 integron-mediated ARGs expression.3.Apart from expression of integrated genes,integrons can capture gene cassettes.In this study,CRISPRi system was engineered to inhibit the class 1 integron-mediated integration of ARGs in E.coli.Upon bacterial conjugation,the integrons that have captured the ARG cassettes aad A1 and aad B could migrate from the donor strain E.coli C600 to the recipient strain E.coli J53 with the assistance of plasmid R388.According to the horizontal transfer rate of ARGs,the integration efficiency of ARG cassettes was calculated.Using CRISPRi to block plasmid R388 class 1 integron,conjugation assay revealed that the integration efficiency of both aad A1 and aad B cassettes was reduced from 10^-2to 10^-3?10^-5.Especially,the class 1 integrons-mediated integration of ARGs was related to the int I1expression.RT-q PCR results showed that the CRISPRi system downregulated the transcriptional level of int I1 by 70?96%.Combining the int I1 transcription and the ARGs integration efficiency to assess the CRISPRi performance,the g RNA R3 showed the highest inhibition on the integration of aad A1 and aad B cassettes.More importantly,this CRISPRi system is reversible,genetically stable,and titratable by varying the concentration of the inducer a Tc.Overall,the 31 bp downstream of the Pc promoter on int I1 non-template strand is an ideal target for CRISPRi to inhibit the class 1 integron-mediated ARGs integration.4.Given the ability of class 1 integrons to capture and express gene cassettes,an integron recombination tool was developed in E.coli.Using the CRISPR/Cas9 and?Red systems,the R388 class 1 integron was integrated into E.coli BL21(DE3)genome,resulting in E.coli BINT.Meanwhile,the gene cassette engineering vector p Li-int I1-cassette was constructed through molecular cloning.Upon IPTG induction,the p Li-int I1-cassette was able to produce the desired gene cassette which might be captured by the E.coli BINT genome integron.To achieve this,the feasibility of the integron-based gene recombination tool in E.coli was investigated using egfp as the reporter.Results showed that the integron recombination tool was compatible with the native expression system in E.coli.Next,the citrate transporter coding gene cit S was integrated into E.coli BINT genome by this recombination tool,resulting in a platform strain E.coli BINT-cit S which can survive using citrate as carbon source.The E.coli BINT-cit S was then harnessed for bioproduction of aconitic acid.5.Given the high efficiency of CRISPRi system,it was redesigned to moderately down-regulate E.coli isocitrate dehydrogenase(IDH,encoded by icd A)and pyruvate kinase(PK,encoded by pyk A and pyk F)to overproduce aconitic acid.Through independent and simultaneous inhibition of IDH and PK,we investigated bacterial growth,glucose consumption,gene expression,enzyme activity and levels of metabolites.Results showed that the CRISPRi strain targeting both icd A and pyk F could effectively reconcile the rate of tricarboxylic acid(TCA)cycle and glycolysis,and thus produced more aconitic acid than the control strain.In shake-flask and fed-batch conditions,this two-target CRISPRi strain produced 60-fold(362.80±22.05 mg/L)and 15-fold(623.80±20.05 mg/L)of aconitic acid relative to the control strain,respectively.Furthermore,this CRISPRi strain maintained low levels of acetate and lactate.However,in bioreactor cultivation,citrate was substantially formed during the late stage of fermentation.To enhance citrate utilization and aconitic acid biosynthesis,thw two-target CRISPRi system was transformed into E.coli BINT-cit S.In a 5L bioreactor,the recombinant strain produced 730.85±29.75 mg/L aconitic acid.Overall,the engineered CRISPR have deleted and repressed R388 class 1 integron,which provides valuable insights for future development of antimicrobial agents for MDR treatment.Additionally,using CRISPR and?Red systems,the integron-based gene recombination tool has been constructed and used to build a platform strain E.coli BINT-cit S,expanding the application of integrons in prokaryotes.Finally,the developed CRISPRi system has improved aconitic acid production by coordinating glycolysis and TCA cycle in E.coli BINT-cit S.This study provides insights for high-level production of the intermediate metabolites in central pathways.