| Corynebacterium glutamicum is a food-safe gram-positive bacterium isolated from soil.It is widely used in the production of amino acids and other products.In recent years,with the development of synthetic biology,artificial cell factories using Corynebacterium glutamicum as the chassis for the synthesis of various bio-energy and fine chemical products have attracted more and more attention.However,it will face many undesirable factors such as high temperature,pH,organic solvents,toxic by-products and mechanical damage in the fermentation process.The stress of these undesirable factors will affect the normal physiological function of microbial cells,which in turn will lead to a reduction in the conversion efficiency of microbial cells and the output of target products will be decreased.Among them,pH is an important factor affecting the growth of microorganisms.During fermentation,improper pH regulation will cause a sharp fluctuation in the pH of the fermentation system,which in turn will cause acid-base stress and greatly affect the growth of microbial cells as well as the accumulation of target products.Therefore,two strategies of irrational ARTP mutagenesis and rational synthetic biological technology were mainly used to achieve acid-base tolerance and self-regulation abilities of Corynebacterium glutamicum with robustness improved.The specific research results are as follows:(1)With ARTP mutagenesis of Corynebacterium glutamicum ATCC13032 and high-throughput screening,18 strains capable of tolerating pH 5.0,25 strains resistant to pH 11.0 were obtained.Through shake flask fermentation,rescreening and verification of the passage stability,a strain icgTAC02 which can grow well and stable under pH 5.0 condition,and an alkali-resistant strain icgTAI02 which grow well and stable under pH 11.0 condition are obtained finally.(2)In Corynebacterium glutamicum,?-galactosidase was used to excavate and characterized the acid-base correspondence and strength of the acid-base-responsive promoter P-asr and P-atp2 which were previously excavated and verified in E.coli(E.coli.k12).The results showed that P-asr has the highest intensity at pH 6.5;P-atp2 has the highest intensity at pH 10.0.(3)The endogenous acid-generating genes PQO,aceE,Idh and the alkali-generating genes glsA and gad from Corynebacterium glutamicum were excavated through KEGG,NCBI and other databases and gene circuits for intelligent pH adjustment were constructed in Corynebacterium acidum.According to the fermentation results under different pH conditions,in engineering strains with alkaline control circuits,P-atp2-PQO,P-atp2-aceE,P-atp2-Idh can quickly respond to the alkaline environment and produce acid in time when the pH was 10.0.Under the alkali environment;in engineering strains with acidic regulation circuits,P-asr-glsA,P-asr-gadA could quickly respond to the acidic environment and produce alkali in time when the pH is 6.5 to neutralize the acid in the environment for achieving pH self-intelligence regulation.(4)In order to verify the compatibility of the two acid-base regulation circuits,the acid-base regulation circuits was integrated into Corynebacterium glutamicum by constructing a dual plasmids system which was characterized by the growth of the cells and the production of glutamic acid.The results showed that when the pH of the culture medium is not artificially controlled,the yield of L-glutamic acid in wild strain was 6.1 g/L and the amount of ammonia was 15.0 mL.But the yield of L-glutamic acid in engineering strains was 7.1 g / L which was 16.4% higher than that of wild strain and the amount of ammonia is 11.0 mL which was 26.6% lower than that of wild strain.These results showed that the pH regulation circuit had a certain regulation ability in the fermentation process. |
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