Adenosine engineering bacterium as well as construction method and application thereof

Abstract

The invention provides an adenosine engineering bacterium as well as a construction method and application thereof. The construction method comprises the following steps: integrating purine nucleosideoperon purEKBCSQLFMNHD in a genome of E.coli w3110, and performing starting by a strong promoter Ptrc; in addition, integrating an adenosine succinate synthase gene purA on an escherichia coli genome, and performing starting by a Ptrc promoter; integrating a nucleotide phosphatase gene yfkN at an mbhA site, and performing starting by the promoter Ptrc; besides, deleting the activity of IMP dehydrogenase, AMP nuclease, adenosine deaminase and purine nucleoside phosphorylase, so that an adenosine synthesis path is reconstructed, and a foundation is laid for producing purine substances by utilizing escherichia coli; and in addition, producing adenosine through shake-flask fermentation by using a genetically engineered bacterium, performing fermentation for 24h to produce 4.3 g / L adenosine, and optimizing culture medium components for producing the adenosine through large intestine fermentation, so that the optimal adenosine fermentation effect is achieved. A foundation is laid for subsequent fermentation production of adenosine.

Description

technical field [0001] The invention mainly relates to the technical field of genetic engineering, in particular to an adenosine engineering bacterium and its construction method and application. Background technique [0002] Adenosine, chemically named 9-β-D-ribofuranosyl adenine, is a compound formed by linking N-9 of adenine and C-1 of D-ribose through β-glucosidic bonds, and its phosphate is adenosine acid. Adenosine is an important nucleotide derivative, which is the product of dephosphorylation of adenine nucleotide. Adenosine can directly enter the myocardium and undergo phosphorylation to generate adenosine acid, participate in myocardial energy metabolism, and at the same time participate in the expansion of coronary vessels and increase coronary blood flow. It is widely used in the treatment of heart insufficiency, cerebral arteriosclerosis and muscle atrophy. In addition, adenosine is an inhibitory neurotransmitter and plays an important role in neurotransmissi...

AI Technical Summary

Problems solved by technology

However, most of the bacteria used for adenosine fermentation on the market are Bacillus subtilis, which have relatively high requirements on the medium during the fermentation process, need to provide a large amount of nitrogen source, sometimes need to add a large amount of corn steep liquor, and the fermentation cycle is as long as 50 -More than 60 hours, which greatly increases the cost of fermentation and is easy to cause bacterial contamination

However, the genetic engineering of Bacillus subtilis is more difficult, and it is not easy to carry out system-directed molecular transformation.

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