Effects Of Interference The Expression Of Biosynthetic Genes On L-threonine Synthesis

L-threonine,an essential amino acid,is widely used in the food,pharmaceutical and feed industries.With the increasing demand for L-threonine,increasing the synthesis efficiency of L-threonine and reducing the production cost have become an important topic in metabolic engineering research.In this study,Escherichia coli THRD,L-threonine engineering strain,was used to study the effect of regulating the expression level of genes on the L-threonine synthesis by using CRISPRi(Clustered Regularly Interspaced Short Palindromic Repeats interference)for achieving a balanced metabolic network and improving the synthesis efficiency of L-threonine.In this study,a CRISPRi metabolic interference system based on arabinoglycan induction was established.The dcas9 gene in the metabolic interference system integrated into the genome and regulated by the arabinose promoter.At the same time,the pGRB plasmid expresses the corresponding sgRNA to interfere genes.The above-mentioned interference system regulated the transcription levels of 11 genes in EMP,TCA and branch metabolic pathways,and to study the effect of gene expression level changes on the synthesis efficiency of L-threonine.The results of fermentation showed that the transcriptional levels of the interfering genes gltA,pfkA and zwf significantly increased the synthesis efficiency of L-threonine.The L-threonine titer of the corresponding strains were 64.7 g/L,64.1 g/L and 60.2 g/L,respectively,which increased by 27.1%,25.9%and 18.3%compared with the original strain(50.9 g/L).The yield on glucose were 0.38 g/g,0.38 g/g and 0.40 g/g,respectively,which were 11.8%,11.8%and 17.6%higher than the control strains(0.34 g/g).The transcriptional levels of the interfering genes ackA,pck and pta also increased the accumulation of L-threonine moderately.The L-threonine titer of the corresponding strains were 57.2 g/L,56.9 g/L and 56.0 g/L,respectively,which increased by 12.4%,11.8%and 10.0%compared with the control strain(50.9 g/L).The sgRNAs of gltA,pfkA and zwf permutations and combinations were construct polygenic interfering plasmids to study the effect of simultaneous regulation of polygenes on the synthesis efficiency of L-threonine.The results of fermentation showed that the transcriptional levels of the interfering simultaneously genes zwf,gltA and zwf,pfkA,gltA were helpful to improve the synthesis efficiency of L-threonine.The L-threonine titer of the corresponding strains were 56.8 g/L and 59.5 g/L,respectively.which increased by 12.9%and 18.3%compared with the original strain(50.9 g/L).The yield on glucose were 0.38 g/g and 0.37 g/g,respectively,which were 11.8%and 8.8%higher than the control strains(0.34 g/g).The interference system needs to add arabinose for induction expression.This study further analyzed the add time and amount of the inducer arabinose for L-threonine synthesis efficiency.The results of fermentation showed that the synthesis efficiency of L-threonine significantly increased after fermentation for 2-4 h.The titer of L-threonine(59.4 g/L)was higher than 0 h(52.7 g/L)and 6 h(54.6 g/L)increased by 12.7%and 8.1%,respectively.Changing the amount of inducer added had no significant effect on the L-threonine synthesis efficiency.This interference system expresses the corresponding sgRNA by plasmid.In order to avoid the instability of plasmid expression and reduce the burden of bacteria,this study further integrated the sgRNA expression module into the genome to construct a plasmid-free interference strain.The specific sgRNA of gltA,pfkA and zwf controlled by PT7 promoter and Ptrc promoter,respectively,and the corresponding interference-integrating strains were constructed and subjected to fermentation culture test.The results of fermentation showed that the L-threonine synthesis efficiency of the plasmid-free interference strain was not significantly improved compared with the original strain and the corresponding plasmid-interfering strain.The effects of specific sgRNAs expressed by the two promoters on L-threonine synthesis were studied by comparing the titer of THRD-5 and THRD-8.The results of fermentation showed that the specific sgRNA expressed by the PT7 promoter had a positive effect on the L-threonine synthesis efficiency over the Ptrc promoter,and the L-threonine yield of THRD-5(57.4 g/L)was 5.1%higher than THRD-8(54.6 g/L).