Method for high-throughput screening of alpha-keto acid high-producing strains

Abstract

The invention discloses a method for high-throughput screening of alpha-keto acid high-producing strains, and belongs to the technical field of bioengineering. The method comprises the steps that protein engineering modification is carried out on the L-amino acid deaminase PmiLAAD derived from proteus mirabilis, a high-throughput screening method is used, a recombinant strain and a strain mutant of the L-amino acid deaminase PmiLAAD containing proteus mirabilis are cultured in a 96-well plate, transfer induction is carried out, cells of the L-amino acid deaminase PmiLAAD containing proteus mirabilis are centrifugally collected to convert L-amino acids (L-valine, L-methionine and L-phenylalanine) to generate corresponding alpha-ketonic acid, developing is carried out by utilizing 2, 4-dinitrophenylhydrazine, a light absorption value is read at 520nm, shake flask verification is carried out on a bacterial strain with a large light absorption value, and screening is carried out to obtain the bacterial strain with significantly improved alpha-keto acid yield.

Description

technical field [0001] The invention relates to a method for high-throughput screening of α-keto acid high-yield bacterial strains, belonging to the technical field of bioengineering. Background technique [0002] Keto acid is a kind of organic acid with biological activity. At the same time, keto acid is also a bifunctional organic compound containing both carboxyl and ketone groups in the molecule. α-keto acid is a keto acid with carboxyl group on the α carbon atom. α-keto acids are important intermediates in drug synthesis and biosynthesis of organic compounds. They have been widely used in the fields of medicine, chemical synthesis, cosmetics, feed and food. They are a class of organic acids that play an important role in living organisms. [0003] At present, the main production methods of α-keto acids are chemical synthesis methods, mainly carbonylation method, Grignard reagent method and Heine method. However, although carbonylation method has the advantages of easy a...

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Problems solved by technology

[0007] Although high-throughput technology has been applied in various fields, no one has applied high-throughput screening technology to screen α-keto acid high-yielding strains. At present, when using high-throughput screening technology to screen high-α-keto acid-producing strains, the main The following problems and challenges are faced: First, the raw materials used in the production of α-keto acids are amino acids. When the amount of substrates is gradually increased, amino acids will not be soluble in the buffer, and it is impossible to prepare a high-concentration substrate mother solution. difficult problem; secondly, when using high-throughput screening technology to screen α-ketoacid high-yielding strains, the whole process from cultivating mutant strains to transforming substrate amino acids needs to be operated in a 96-well plate. Adding an inducer is obviously not practical, and it is necessary to find a fermentation medium that can only change the induction temperature without adding an inducer during induction; finally, use high-throughput screening technology to screen the problems faced by strains with high α-ketoacid production The keto acid produced by converting the substrate amino acid needs to be developed with a specific reagent, and the yield of keto acid must be easily judged by the color. Therefore, it is necessary to find a suitable chromogenic agent and determine the usage and dosage of the chromogenic agent. Another major challenge encountered in high-throughput screening

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