| The Gram-positive bacterium Corynebacterium glutamate is not only a food safety strain Generally recognized as safe（GRAS）,but also an important platform strain for industrial biotechnology.C.glutamicum was first isolated as a glutamate production strain,and later to adapt to the production of other amino acids,gradually used to achieve the production of arginine,valine,serine,lysine and other amino acids,and achieved significant results.In this study,a series of mutant plasmid MPs were constructed by screening DNA polymerase Cgl1289 derived from C.glutamicum,combined with the uracil glycosylase inhibitor UGI,cytosine deaminase（pm CDA1）,and adenine deaminase（Tad A-ABE8e）,which achieved efficient mutation rate and extensive mutation spectrum in C.glutamicum.Subsequently,the transcription factors of L-glutamate were excavated based on comparative transcriptomics,then L-glutamate high-throughput detection method was designed,and combined with MPs mutant plasmids to select the high-yield L-glutamate mutant strains and whole genome sequencing analysis the mutants.Then,the high L-glutamate producing strain was built based on whole genome sequencing analysis and metabolic engineering.And finally,by weakening the fermentation by-products and enhancing L-glutamate secretion,then the L-glutamate fermentation level was improved.The main research results are as follows:（1）Screening of endogenous DNA polymerases in C.glutamicum.Exo I contains two highly conserved aspartate and glutamate residues,which are key residues for proofreading exonuclease activity,affecting the high fidelity of DNA replication and repair system.Based on the high conservation of the two amino acid residues in the Exo I region,a multiple sequence alignment identified the Cgl1289 with a highly conserved Exo I region.Subsequently,we mutated the two aspartate residues（D43）and glutamate residues（E45）of Cgl1289 to alanine by site-directed mutation and expressed in C.glutamicum.By calculating the mutation survival rate as well as the mutation rate,it was found that the survival rate of the recombinant strain Cg-MP1 was only 82.4%,and the mutation rateμbpreached 1.2×10-7,a3,000-fold increased relative to the control strain.This indicated that the mutation of the aspartate and glutamate residues to alanine in the Exo I region of Cgl1289 was able to significantly increase the mutation rate.（2）Assembly and optimization of mutant plasmid components.When a mispaired base occurs during DNA replication,the correct base replacement is achieved by the base excision repair system（BER）and DNA polymerase.Thus,when the DNA glycosylase activity is inhibited,the base excision repair system disrupts,thereby increasing the base mutation frequency.Thus,based on the MP1,we expressed the PBS2-derived uracil DNA glycosylase inhibitor（UGI）of the B.subtilis phage in the downstream of Cgl1289M and named MP2.The results showed that the cell survival rate of MP2 was only 35.7%,and theμbpreached3.6×10-7,a 3-fold increased relative to MP1.When the corrected exonuclease activity is lost,the expression of cytosine deaminase（pm CDA1）and adenine deaminase（Tad A-ABE8e）significantly increases DNA replication errors.Therefore,based on MP2,we expressed cytosine deaminase（pm CDA1）and adenine deaminase（Tad A-ABE8e）,and constructed the mutant plasmids MP3,MP4 and MP5,respectively.The results showed that the cell survival rate of MP5 was only 1.7%,and the mutation rateμbpreached 3.84×10-6,a 32-fold increased relative to MP1.Subsequently,through promoter,RBS optimization and optimization of the amount of inducer addition and induction temperature,the optimized mutant plasmid MP5-RBS6 had the highest mutation rate(6.12×10-6),153,000-fold than that of wild-type C.glutamicum.Finally,the temperature-sensitive plasmid replication start site Rep A 101 was used to replace the original plasmid replication start site,and the mutant plasmid MP5T was successfully constructed,which successfully eliminated,terminated the mutations,and built the basis for the construction of strains with stable inheritance.（3）Breeding of glutamate-producing chassis cells.First,the mutant plasmid MP5T was used in C.glutamicum 13032 to obtain an acid-tolerant mutant strain NS-A1,which could accumulate L-glutamate 35.3 g/L in 5 L bioreator.Subsequently,through comparative transcriptomic analysis and EMSA experiments,it was also found that RosR can bind to the promoter PGlnAand regulate the expression of its downstream genes.Based on the interaction between RosR and the promoter PGlnA,we successfully constructed the L-glutamate biosensor PGlnA-biosensor.However,the biosensor PGlnA-biosensor cannot specifically respond to L-glutamate.In order to further specifically detect L-glutamate in the subsequent mutation screening process,we used glutamate oxidase（LGOX）specificity oxidation of L-glutamate generates hydrogen peroxide,and the LGOX chromogenic method is constructed.Finally,combined with the MP5T mutant plasmid and the L-glutamate high-throughput screening method,and using the acid-tolerant mutant strain NS-A1 as the chassis cell,the L-glutamate high-producing mutant strain was further bred.Among them,the mutant NSH187 could accumulate L-glutamate 55.8 g/L in 5 L bioreator,and the yield was 0.372 g/g,and could be inherited stably.Finally,through the whole genome sequencing analysis of the TOP 10L-glutamate high-producing strain and three acid-tolerant mutant strains obtained during the mutation process.It was found that some single-base mutations involved the L-glutamate synthesis pathway,which in turn affected the biosynthesis of L-glutamate.（4）Systematic metabolic engineering to create L-glutamate high-yielding strains.First,by adding double copies of gdh to the genome of NSH187 to construct CG1 to enhance the activity of glutamate dehydrogenase.Using the promoter Ptufto enhance the expression of ICD and the promoter Pdap Ato enhance the expression of GAPDH and GAPDHm,and then construct CG4 to provide sufficient NADPH supply for GDH.Then,on the basis of CG4,the RBS sequence with weak translation initiation rate was used to attenuate the expression of odh A,and the promoter Psodwas used to enhance the expression of glt A.The engineered bacteria CG5 and CG6 were constructed respectively.Among them,CG6 could accumulate L-glutamate 78.7 g/L,yield 0.436 g/g,in 5 L bioreator.By integrating PYC,PC and PEPC mutation sites on the basis of CG6,and using the promoter Psodto enhance the expression of ppc,CG7,CG8 and CG9 were constructed,respectively.Among them,CG9 was able to accumulate L-glutamate 96.2 g/L,yield 0.443 g/g,in 5 L bioreator.Finally,on the basis of CG9,and using the promoter Psodto enhance the expression of ppgk and ilo T1,the engineered strain G01 could accumulate L-glutamate 104.2 g/L in a 5 L bioreator with a yield of 0.452g/g,and the accumulation of by-products such as acetic acid and lactic acid was reduced compared with CG9.（5）The transcription factor RosR regulates the glutamate metabolic synthesis network.Recombinant strain G01 also still had the accumulation of by-products,such as L-glutamine,L-valine,L-alanine,acetate and lactate.Based on the expression of RosR,it could inhibit the synthesis of L-glutamine,and could significantly promote L-glutamate accumulation.To further attenuate the accumulation of G01 by-products and promote L-glutamate biosynthesis,we therefore overexpressed RosR in G01.Comparative transcriptomic analysis combined with EMSA experiments showed that the transcription factor RosR could also bind to many promoters related to L-glutamate synthesis-related pathways.By analyzing the interaction between the transcription factor RosR and DNA-binding Motif,it was found that the Ser72and His76 residues in RosR can recognize the DNA-binding Motif sequence,which in turn regulates the transcription level of downstream genes in the promoter region and participates in the L-glutamate metabolic network,and significantly weakened the accumulationthe of by-products L-glutamine,L-arginine,L-proline,L-leucine,L-lysine,L-alanine,acetate,lactate and succinate.Finally,we optimized the extracellular secretion of L-glutamate by excising the C-terminal 110 amino acids in NCgl1221 and the integration of the mutant Msc CG2I269V/F149Lon the genome of G01,and constructed engineering strain G05.At the same time,RosR was overexpressed in G05,recombinant strain G05/RosR could accumulate L-glutamate 152.1 g/L with a yield of 0.565 g/g in 5 L bioreator for 48 h,which was 16.5%and 4.4%higher than that of G01/RosR. |
