CRISPR-Cpf1-assisted Gene Editing And Metabolic Engineering Of 4-hydroxyisoleucine Biosynthesis In Corynebacterium Glutamicum

Corynebacterium glutamicum is the industrial pillar of white biotechnology.To improve the efficiency of genetic modification of C.glutamicum genome,a CRISPR-Cpf1-assisted gene editing system was established in this thesis.4-hydroxyisoleucine（4-HIL）is a kind of amino acid derivative with great medicinal value.In previous study,the exogenous isoleucine dioxygenase（IDO）was expressed in an L-isoleucine（Ile）-producing strain and the de novo synthesis of 4-HIL was realized.In this thesis,the influence of ddh and/or lysE knockout on the overall transcription level of genes for 4-HIL production was analyzed,and the anabolic pathway of 4-HIL was engineered by using the newly established gene editing system according to the analysis of transcriptome data.The main research contents and results are as follows.（1）In order to improve the editing efficiency,a pCS plasmid which can kill false positive strains in SacB system was constructed based on CRISPR-Cpf1 system,and its lethal efficiency was tested.The lethal rate of pCS plasmid to C.glutamicum carrying kan box on the genome was as high as 98.6%.Then a gene editing system assisted by CRISPR-Cpf1 was established,and compared with the traditional SacB editing method by replacing odh A promoter.Finally,the screening efficiency of the second-round exchange strain was improved to over 95.0%.（2）In order to reduce the accumulation of L-lysine,the synthesis of Lys was modified by metabolic engineering.We analyzed the transcriptome of ddh and/or lysE knockout strains,and found that ddh knockout enhanced Ile synthesis and succinylacylase branching pathway of Lys,resulting in unchanged Lys accumulation and increased 4-HIL titer of ddh single knockout stain SL09.Although the knockout of lysE also enhanced the synthesis of Lys,the export of Lys was blocked,and the excessive consumption of succinyl-Co A led to the insufficient supply ofα-ketoglutaric acid（α-ketoglutarate,α-KG）,and the insufficient supply ofα-KG led to the decrease of 4-HIL and Lys production.Different Terminators were inserted into the upstream of dapD gene of strain SL09 to weaken Lys synthesis.The results showed that the weakening of dapD by terminator could not effectively reduce the accumulation of Lys.The expression of lysE and gdh was weakened by promoter engineering,and ido was integrated while cg2564 was knocked out,and then pIL-_TD5I^M plasmid carrying P_{brn FE}D5-ido was transferred into mutant strains.The Lys titer of lysE and gdh weakening strains decreased by 30.3%and 23.8%,respectively,and the titer of 4-HIL decreased by 23.6%and 7.9%,respectively.Knockout of cg2564 did not reduce the accumulation of Lys,but reduced the titer of 4-HIL by 26%.（3）In order to obtain 4-HIL producing strain with stable expression of ido^M,ido^M was integrated into ldh A-pyk2 locus and IScgl locus.Firstly,Thr176Cys mutation was introduced into ido^U to obtain ido^M,and ido^M expression was regulated by Ile inducible promoter P_{brn FE}N.The SC-_TD5I^M strain carrying P_{brn FE}D5-ido^M was obtained,and its 4-HIL titer was 140.9mmol·L^-1.Then,the ido^M controlled by P_{brn FE}D5 or P_{ldh A} was integrated into the ldh A-pyk2locus of strain SC00.The 4-HIL titer of strain SC01 integrated with P_{brn FE}D5-ido^M was the highest,which was 14.8 mmol·L^-1,and 170.1 mmol·L^-1 Ile was still left.Through homologous recombination mediated by suicide plasmid p BS and thereafter screening with increasing kanamycin concentration,P_{brn FE}D5-ido^M was integrated into the IScgl locus with multiple copies.The final multi-copy integrated strain SCM12 accumulated 43.7 mmol·L^-1 4-HIL,which was 3 times higher than that of SC01.