Construction Of Metabolic Pathway For Synthesis Of 3-HP In Escherichia Coli

As a platform compound with high economic value,3-hydroxypropionic acid(3-HP)can be used to produce a series of bulk chemicals.It can be used not only as a precursor for the synthesis of acrylic acid,malonic acid and other chemicals,but also as raw materials for the production of many green materials.It is widely used in medicine,chemical industry,materialsand other fields.With the development of genetic engineering technology,the use of related technologies to transform bacteria has become one of the important means of industrial production of 3-HP.In this paper,glucose was used as carbon source,Escherichia coli with clear genetic background was selected as host strain to construct malonyl-Co A pathway,and the metabolic pathway was modified to achieve the purpose of efficient synthesis of 3-HP.The main contents are as follows:(1)Using the plasmid containing the target fragment preserved in the laboratory as the template,several pairs of primers containing homologous fragments were designed,and the recombinant plasmid p A2s-mcr containing malonyl-Co A reductase mcr gene was constructed by recombinant ligation kit.After gene sequencing and protein expression were verified,the recombinant plasmid p A2s-mcr and laboratory preserved plasmid p E7a-acc were cotransformed into E.coli BL21(DE3)competent cells to construct recombinant strain WY1.After fermentation and culture,its 3-HP concentration was verified by HPLC.The content of 3-HP in the fermentation broth of the recombinant strain was about 0.94 g/L.(2)In order to increase the yield of 3-HP,CRISPR/Cas9 gene editing technique was used to knock out the genes encoding phosphate transacetylase(pta),pyruvate dehydrogenase(pox B),lactate dehydrogenase(ldh A)and acetate kinase(ack A)in metabolic bypass,and the corresponding single gene deletion Escherichia coli was constructed to inhibit the metabolic pathway of acetyl-Co A intermediate into other substances.The plasmid p E7a-acc and p A2s-mcr were transferred into the gene editing strains together to construct the recombinant strain WY2～WY4.The preliminary fermentation showed that the 3-HP yield of the recombinant strain WY2 with ldh A deletion was higher than that of WY1,up to 1.10 g/L.(3)In order to reduce the metabolic burden caused by plasmid maintenance and replication,gene editing strain BL21Δldh AΔpta: :119-acc DAΔpox B: :CPA1-acc BC(Q1Z2)was constructed by using CRISPR/Cas9 gene editing technique,which achieved the purpose of over-expression of acetyl-Co A carboxylase at the genomic level while knocking out the pta/pox B gene encoding by-product synthesis pathway.(4)To improve the activity of key enzyme MCR and further increase the yield of 3-HP,the mcr gene was divided into two segments(N/C),and the mutation sites(N940V/K1106W/S1114R)which was known to increase enzyme activity were introduced into mcr-C.The recombinant plasmid p LB1s-mcr-N-C* was transferred into Q1Z2 to obtain the recombinant strain WY6.The results of fermentation culture showed that the recombinant strain could synthesize the 3-HP of1.30 g/L.Compared with the WY1 strain,the titer was 38% higher than it,and compared with the initial strain DB1,the titer was increased by about 4 times.