| Corynebacterium glutamicum（C.glutamicum）is an industrially important microbial cell factory that is widely used in the production of various high value-added compounds.In recent years,the development of synthetic biology techniques and the emergence of related tools have advanced the construction of efficient cell factories for microorganisms and expanded the substrate and product spectrum of C.glutamicum.Diamines,such as malondiamine,butanediamine,pentanediamine and hexanediamine are widely used as platform compounds in industry and are of interest because they can be condensed with dicarboxylic acids to form polyamide products with different performance advantages.Compared with chemically synthesized diamines,bio-based diamine products can reduce the dependence on petroleum resources,which has the advantages of low carbon,environmental protection and sustainable development.However,the construction of high-yield strains of diamines using traditional microbial metabolic engineering methods requires extensive optimization and reconstruction of the metabolic network,and the whole process is time-consuming and laborious.The development of diamine biosensors can help achieve high-throughput screening and selection of target phenotypes and accelerate the design-build-test（DBT）cycle of cell factories.In addition,gene editing technology plays an indispensable role in the construction of high-yield strains or chassis cells,but the gene editing method in the non-model strain C.glutamicum ATCC 14067 is not yet well established.Based on this,in this study,a CRISPR/Cpf1-RecT-based genome fine modification system was established in C.glutamicum ATCC 14067,and the construction of a butanediamine chassis strain was realized using this method,which enriched the gene editing technology reserve of C.glutamicum.Then,the endogenous and heterologous transcription factor-based diamine sensors were developed and designed in C.glutamicum,and the optimized sensor systems were capable of quantitative detection of butanediamine synthesis and identification of high-yield strains in C.glutamicum,providing a high-throughput screening and evolutionary tool for the construction of efficient cell factories for diamine compounds.The specific research contents and results are as follows:（1）Establishment of CRISPR/Cpf1-RecT system and construction of butanediamine producing strainsFirst,we explored the applicability of the CRISPR/Cpf1-RecT system in C.gulamicum ATCC 14067,and then tested the PAM sequences preference of Cpf1.The results showed that the optimal PAM sequences in ATCC 14067 were TTTC,TTTG,GTTG and CTTC,and the accurate editing efficiency of target genes was more than 91.6%.Combined with CRISPR/Cpf1-RecT and Rec ET-Cre/lox P gene editing technologies,metabolic engineering of ATCC 14067 was performed.The arg CJBD operon promoter Parg C of L-ornithine synthesis pathway was replaced with a strong constituent promoter Peftu,and the ornithine aminotransferase gene（arg F）of L-ornithine synthesis pathway of L-arginine was knocked out.After overexpressing ornithine decarboxylase from Escherichia coli,the recombinant strain PUTP△had a putrescine accumulation of 28.7 m M at the shake flask level.On this basis,the proline codon CCT at position 7 of spermidine acetyltransferase A Sna A was replaced by TAA,which stopped Sna A translation in advance.The recombinant strain PUTP△D accumulated 60.0 m M of putrescine,which was 109%higher than that of PUTP△.（2）Development and design of a diamine biosensor based on the endogenous transcriptional factor CgmRFirstly,it was identified that CgmR,a transcriptional regulator of endogenous butanediamine transport pathway,can regulate the expression of reporter proteins in response to the promoter Pcgm A by responding to the ligand diamines butylenediamine,pentanediamine,hexanediamine and heptanediamine.Then the reporter protein and the expression levels of the CgmR of p Sen Put were optimized,and the utility factor of the obtained the sensor circuit was 330.To further improve the sensitivity of the sensor to butanediamine,random mutation of CgmR and high-throughput screening by FACS were performed,and the obtained sensor p Sen Put I152T,whose lower limit of detection of the experimental assay（LOD）was≤0.2 m M,the response threshold K value was 11.4 m M,and the utility factor was 720.p Sen Put I152Twas transferred into four different butanediamine producing strains to verify the practicability of the sensor.The results showed that the fluorescence intensity of the strains could indicate the butanediamine production capacity of the strains.FACS was used for high-throughput screening of the four mixed strains,and80.77%of the selected cells were identified as the strain with the highest accumulation of butanediamine.This indicates that the constructed diamine biosensor can be used for quantitative monitoring of intracellular metabolites and screening of high yield strains.（3）Development and design of a specific butanediamine biosensor based on the exogenous transcriptional factor Puu REndogenous transcription factor sensors may form mutual interference with endogenous metabolic networks in practical applications,and the CgmR-based diamine sensor p Sen Put responds to a wide range of diamines,which may limit its effective use in metabolic engineering.To address this issue,a specific butanediamine biosensor p Sen Puu R based on the transcriptional factor Puu R derived from Escherichia coli was established in C.gulamicum.p Sen Puu R only responded significantly to butanediamine,and did not respond to intermediate amino acids or diamines above C4.To further improve the signal-to-noise ratio and sensitivity of p Sen Puu R in response to butanediamine,the genetic components of the sensor,including the response promoter,the reporter protein and the promoter controlling the expression of Puu R were optimized.To validate the tuned butanediamine sensor for metabolic engineering applications,ornithine decarboxylases（ODCs）were first mined and initially screened by combining phylogenetic tree and molecular docking strategies.Then,the optimized butanediamine sensor was transferred into butanediamine producing strains containing different ODCs,and the correlation between fluorescence and butanediamine in the intracellular and extracellular of the recombinant strains was measured.The results showed that the intracellular and extracellular of butanediamine all showed strong Spearman correlation with the fluorescence of the recombinant strains.The yield of butanediamine from ornithine decarboxylase ODC5 obtained from initial screening was close to that of probe enzyme,and 70 m M butanediamine could be produced from fermentation supernatant. |
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