Tricarboxylic acid cycle-free Escherichia coli chassis bacteria as well as construction method and application thereof

Abstract

The invention discloses tricarboxylic acid cycle-free Escherichia coli chassis bacteria as well as a construction method and application thereof. The preparation method of the Escherichia coli chassis bacteria comprises the following steps: increasing the expression quantity and / or activity of tetrahydropyridine dicarboxylic acid: N-acetyltransferase, N-acetyl diaminopimelic acid deacetylase, N-acetyl diaminopimelic acid aminotransferase, O-acetyl homoserine thiolase and homoserine: O-acetyltransferasein host bacteria (Escherichia coli or mutant Escherichia coli), reducing the expression quantity and / or activity of tetrahydropicolinic acid: N-succinyltransferase and homoserine: O-succinyltransferase in the host bacteria, and obtaining the tricarboxylic acid cycle-free Escherichia coli chassis bacteria. Experiments prove that the Escherichia coli chassis bacteria can reduce the carbon loss rate and improve the capability of synthesizing a target product by taking central metabolic intermediates (such as acetyl coenzyme A, pyruvic acid and alpha-ketoglutaric acid) as precursors. The tricarboxylic acid cycle-free Escherichia coli chassis bacteria and the preparation method have an important application value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an Escherichia coli chassis bacterium without tricarboxylic acid cycle and its construction method and application. Background technique [0002] The tricarboxylic acid cycle (TCA cycle) is an important part of the central carbon metabolism of Escherichia coli. The biosynthesis of numerous bio-based chemicals is dependent on the tricarboxylic acid cycle intermediate metabolite α-ketoglutarate. At the same time knockout the α-ketoglutarate dehydrogenase gene (sucA) and the isocitrate lyase gene (aceA) in the tricarboxylic acid cycle of Escherichia coli, forcing more α-ketoglutarate to flow to the target product synthesis, is The best way to facilitate the synthesis of target products. [0003] When Escherichia coli grows aerobically with glucose as the only carbon source in the inorganic salt basal medium, the tricarboxylic acid cycle undertakes the function of completel...

AI Technical Summary

Problems solved by technology

If the α-ketoglutarate dehydrogenase gene (sucA) and the isocitrate lyase gene (aceA) are knocked out at the same time, or the sucA is knocked out, the metabolism of the tricarboxylic acid cycle is disrupted, which often leads to slow growth of the strain, especially in inorganic Aerobic growth on glucose as sole carbon source is limited in salt-based media

At present, there are two main strategies to solve: one is to dynamically regulate the expression of α-ketoglutarate dehydrogenase gene (sucA), and to perform switch regulation in cell growth and synthesis of target products. The regulatory elements are too complex, which often affects the synthesis pathway of the target product, and the process is complicated and difficult for industrial application; the second is to use α-ketoglutarate-dependent dioxygenase to reconstruct the tricarboxylic acid cycle, and combine the production and engineering of the target product It is associated with the growth of bacteria, which has high requirements for the activity of α-ketoglutarate-dependent dioxygenase, and is not suitable for the production of products with certain cytotoxicity

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