Engineering strain high in lactoflavin yield and application of engineering strain high in lactoflavin yield

Abstract

The invention discloses a lactoflavin engineering bacterium and a constructing method thereof, and the preservation number of the lactoflavin engineering bacterium is CGMCC NO.16132. Through a geneticengineering means, a lactoflavin auxotroph strain is constructed, then a chromosome integrates and expresses a lactoflavin operon demodulated by bacillus amyloliquefaciens, and the lactoflavin production capacity of the strain is restored; then an expression vector overexpresses the lactoflavin operon demodulated by the bacillus subtilis, so that the lactoflavin production capacity of the straincan be improved; in addition, through point mutation of a ribC gene, the catabolism of lactoflavin is diminished; and finally, a recA gene which is responsible for homologous recombination and DNA repair on the chromosome is inserted for inactivation, and a lactoflavin production chassis cell is constructed. Based on the lactoflavin production chassis cell, through combination of the characteristic that the lactoflavin is a natural flroresence substance, and a strain high in lactoflavin yield is screened out through liquid drop micro-fluidic control. The invention also discloses a method for producing the lactoflavin through fermentation with the bacillus subtilis, and an application of the strain high in lactoflavin yield to lactoflavin production and an application of the strain high inlactoflavin yield to medicines, foods and feeds.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a riboflavin engineering bacterium and a method for producing riboflavin. Background technique [0002] Riboflavin, also known as vitamin B2, is a water-soluble B vitamin that can be synthesized by most microorganisms and plants, while humans and animals can only ingest it from food. Riboflavin mainly exists in the form of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) in organisms, and it is used as a coenzyme or prosthetic group of flavoprotein to participate in the electron transfer and oxidation of the respiratory chain of the body tissue Reduction reaction is an essential nutrient for maintaining normal metabolism and physiological functions of the body. Riboflavin is widely used in the fields of medicine, food and feed. Due to the wide use of riboflavin, the demand for riboflavin at home and abroad is still showing an increasing trend. [0003] Microbial fe...

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Problems solved by technology

[0004] In Bacillus subtilis, more than 20 steps of reactions catalyzed by the pentose phosphate pathway, purine synthesis pathway, and riboflavin synthesis pathway are required to convert glucose into riboflavin. The entire reaction process requires a variety of metabolic pathways. Precursor substances (such as ribulose 5-phosphate, GTP, glutamine, glycine, aspartic acid, etc.) are involved, and there are complex regulatory mechanisms in the purine synthesis pathway and riboflavin synthesis pathway, including a variety of transcription Initiation repression mechanism, attenuation mechanism of leader mRNA transcription and feedback inhibition at the enzyme level, all of which increase the difficulty of strain modification

In recent years, although many genetically engineered strains of Bacillus subtilis with high riboflavin production have been obtained through metabolic engineering, due to the limitations of our understanding of microbial physiology and complex metabolic network regulation mechanisms, genetic engineering technology has been used to continue to rationally transform the strains , it is difficult to obtain strains with a further substantial increase in riboflavin production; and traditional mutation breeding, with a large workload and lack of efficient and rapid screening methods, has greatly affected Bacillus subtilis in improving riboflavin production capacity. degree of limitation

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