Font Size: a A A

Enhancement Of L-histidine Synthesis In E.coli By Increasing ATP Supply

Posted on:2021-09-13Degree:MasterType:Thesis
Country:ChinaCandidate:Y ZhangFull Text:PDF
GTID:2481306317967299Subject:Fermentation engineering
Abstract/Summary:PDF Full Text Request
L-Histidine is a semi-essential amino acid in the human body.It is widely used in medicine,food and feed industries,and also used in the production of tobacco products and cosmetics.At present,the production of L-histidine is mainly based on the proteolytic method,but this method has problems such as complicated operation steps,large environmental pollution,and high production costs.Microbial fermentation has been successfully applied to the industrial production of a variety of amino acids.It has the advantages of short fermentation cycle,low cost,mild action conditions,easy process control and high yield.The L-histidine metabolism pathway is lengthy and coupled with the purine synthesis pathway in microorganisms,and ATP is an important precursor of the L-histidine synthesis pathway,so it is possible to increase the supply of ATP and increase L-histidine production.In this study,CRISPR/Cas9 mediated gene editing technology was used to explore the effect of L-histidine on the existing uridine producing strain of E.coli in the laboratory by enhancing the supply of ATP in E.coli.The content and results are as follows:First,the shake flask fermentation medium was screened and optimized for the starting strain HIS0.Three mediums A,B and C were selected and tested with strain HIS0 for shake flask fermentation.The medium B was selected from the aspects of L-histidine production and bacterial biomass.Comparing the three medium formulations,it was found that the medium B lacked Mg2+.The medium B was optimized by changing the amount of Mg2+added.Finally,the amount of Mg2+was determined to be 1 g/L.After optimization,the L-histidine accumulation in the shake flask medium reached 3.45 g/L,the OD600 of the bacteria reached 31.0.Second,strengthen the AMP synthesis pathway.E.coli's own inosine cyclic hydrolase-encoding gene(purH)was integrated into the genome of the starting bacteria HISO,and double and triple copies were made to construct strains HIS1 and HIS2.The shake flask fermentation results showed that the L-histidine production of the HIS 1 strain increased to 3.72 g/L,while the L-histidine production and the OD600 of the HIS2 strain significantly decreased.Obviously,two copies of the purH gene could meet the metabolic pathway requirements.The strain HIS1 was constructed by simultaneously integrating the adenylate succinate synthetase(purA)and adenylate succinate lyase(purB)genes from Bacillus subtilis to construct the strain HIS4.The shake flask fermentation results showed that the increase of L-histidine production was 4.71 g/L,which was 26.6%higher than that of HIS1 strain,which proved that the strategy of increasing the accumulation of L-histidine by enhancing the AMP synthesis pathway was feasible.Third,in order to enhance the ATP synthesis pathway,some branch metabolism is blocked.(1)The HIS5 strain was constructed by knocking out the ATP hydrolase-encoding gene(mazG)on the genome of the HIS4 strain.The shake flask fermentation results showed that HIS5 increased the production of L-histidine by 15.3%compared with HIS4 strain.(2)Simultaneously knock out ushA,surE,yrfG and yjjG genes on the genome of HIS5 strain to construct HIS9 strain.The shake flask fermentation results showed that HIS9 increased the production of L-histidine by 17.6%compared with HIS5 strain.(3)The HIS9 strain was constructed by simultaneously knocking out the AMP ribozyme encoding gene(amn)and the pyrimidine/purine-5'-nucleotide ribozyme encoding gene(ygdH)on the genome of the HIS9 strain.The shake flask fermentation results showed that HIS11 increased L-histidine production by 37.3%compared with HISO strain.(4)The apt and hpt genes were knocked out simultaneously on the genome of HIS11 strain,and the results showed that it was not conducive to L-histidine accumulation.Finally,enhance the dynamic regulation between AMP,ADP and ATP.Adenylate kinase-encoding genes(adk)with different expression intensities were integrated on the strain HIS11 genome to construct HIS 15 and HIS 16 strains.The shake flask fermentation results showed that compared with HIS11,the L-histidine production of the HIS 15 strain did not increase significantly,while the L-histidine production of the HIS 16 strain decreased.A nucleoside diphosphate kinase-encoding gene(NDK)was integrated into the genome of strain HIS 15 to construct HIS 17.Then the gene encoding pyruvate kinase ?/?(pykF and pykA)was integrated into its genome to construct the HIS 19 strain.The shake flask fermentation results showed that the production of L-histidine increased by 12.6%compared with HIS 15 strain,while the growth of HIS 19 strain was negatively affected and the production of L-histidine was significantly reduced.Through the study of this subject,with the help of metabolic engineering transformation strategies,ATP supply was enhanced,and the metabolic flux of the L-histidine synthesis pathway was increased,laying a foundation for the industrialization of L-histidine production strains.
Keywords/Search Tags:Escherichia coli, metabolic engineering, L-histidine, ATP synthesis, CRISPR/Cas9
PDF Full Text Request
Related items