Metabolic Engineering Breeding Of Vitamin B₁₂ High-yield Strain

Vitamin B₁₂(Vitamin B₁₂,VB₁₂)is also called cobalamin,and its appearance is dark red because it contains metallic cobalt.Viscera of animal origin contains a large amount of vitamin B₁₂,which is also widely present in foods such as milk,fish and egg yolks.The plant does not contain vitamin B₁₂.The human body cannot synthesize vitamin B₁₂,but many microorganisms such as rhizobia,actinomycetes and intestinal bacteria can synthesize it.Vitamin B₁₂is one of the most expensive varieties of vitamin products.It can participate in the activities of blood cell development,and it also has its presence in the metabolic activities of organisms.From the human infant period to the adult period,vitamin B₁₂plays a vital role.At the same time,it is also effective in preventing and treating pernicious anemia,migraine headaches,endooral inflammation and other diseases.The international demand for it has always been at the forefront of the vitamin family,but the output is often at the bottom of the vitamin series.In industry,vitamin B₁₂is mainly produced by common strains such as Propionibacterium freudenreichii and Pseudomonas denitrificans.In this study,the experimental strain Ensifer adhaerens D4-2 is a strain preserved by the"Key Laboratory of Microbial Diversity Research and Application of Hebei Province",which can produce vitamin B₁₂by aerobic fermentation process.Based on the comprehensive analysis of its vitamin B₁₂metabolic pathways and amino acid metabolic pathways,the strategy for the selection of this vitamin B₁₂-producing bacteria and the target genes that need genetic manipulation were determined.That is,overexpression of the cob A gene can promote the downward metabolism of uroporphyrinogen III and avoid the accumulation of uroporphyrinogen III;knock out the cys G gene to prevent the flow of precorrin-2 to other metabolisms;knock out the ilv A gene,To prevent the flow of threonine to other metabolic pathways,thereby increasing the production of vitamin B₁₂.Accordingly,in this study,E.adhaerens D4-2 was used as the starting strain,and the gene was overexpressed through the expression vector p BBR1mcs-5;based on two homologous recombinations,the sucrose lethal gene sac B was used as a selection marker to perform a seamless gene knockout,So as to obtain the vitamin B₁₂metabolic engineering strain.First,a modified strain overexpressing the cob A gene was constructed to obtain the strain Strain 1(D4-2 p BBR1mcs-cob A);on the basis of the strain Strain 1,the cys G gene was superimposed to knock out the strain 2(D4-2 p BBR1mcs-cob A?cys G);On the basis of Strain 2,knock out the ilv A gene to obtain the strain Strain 3(D4-2 p BBR1mcs-cob A?cys G?ilv A);shake flask fermentation for the starting strain of E.adhaerens D4-2 and the 3 mutant strains constructed in the experiment Cultivation,HPLC detection of vitamin B₁₂content.Among them,the vitamin B₁₂output of strain D4-2 was 0.230±0.001 mg/m L;the vitamin B₁₂content of Strain 1 was 0.244±0.001 mg/m L,which was 6%higher than that of strain D4-2;the vitamin B₁₂content of Strain 2 was 0.276±0.001 mg/m L,which is 20%higher than the D4-2 strain;the vitamin B₁₂content of Strain 3 is 0.308±0.001 mg/m L,which is34%higher than the D4-2 strain.In this study,the content of vitamin B₁₂in the starting strain E.adhaerens D4-2 was increased by genetic engineering,and a set of metabolic engineering operating systems suitable for gene overexpression and gene knockout of E.adhaerens was developed to transform E.adhaerens.Increasing vitamin B₁₂content provides another way.