Efficient fermentation production method for L-sorbose

Abstract

The invention discloses an efficient fermentation production method for L-sorbose. The method comprises the following steps of: with an immobilized gene engineering strain as a production seed, inoculating the immobilized gene engineering strain into a seed medium, filling 50mL of liquid into a 500mL shake flask, and carrying out multiplication culture at the temperature of 30 DEG C and the rotary speed of 200rpm for 20-24h; and inoculating the cultured immobilized seed into a fermentation medium according to the inoculum size of 15%, and carrying out semi-continuous fed-batch fermentation in a 1-L fermentation tank at the temperature of 30 DEG C, the rotary speed of 500rpm and the aeration rate of 2.5vvm for 21 days. By using the method, the sorbitol is used as a unique carbon source, and after the fermentation is carried out for 16h, the conversion rate of D-sorbose is up to over 90% which is increased by 37% in comparison with a contrast strain; and the sorbitol is used as a unique carbon source, so that the 21-day semi-continuous fed-batch fermentation of an immobilized cell can be finished, and the fermentation production method can be used for reducing the preparation cost of 20 batches of seeds in comparison with a free strain fermentation way.

Description

technical field [0001] The present invention relates to a method for high-efficiency fermentation and production of L-sorbose, in particular to a method for strengthening engineering by adding poly(A / T) tails to the gene base of sorbitol dehydrogenase by means of molecular biology to enhance the stability of its mRNA Transcription and expression of the sorbitol dehydrogenase gene, and then strengthen the oxidative dehydrogenation pathway of sorbitol, thereby regulating the rapid orientation of sorbitol to target metabolites, and using immobilized genetically engineered bacteria for semi-continuous fed-batch fermentation, Sorbitol sugar method. Background technique [0002] L-sorbose, a ketohexose, is the C-2 and C-3 epimers of D-fructose, with a molecular weight of 180.16. As an important chemical raw material, L-sorbose is mainly used as a precursor for the production of vitamin C (VC). At present, VC is mainly produced in the industry by "Leys method" and "two-step ferme...

AI Technical Summary

Problems solved by technology

Among them, the fermentation process with D-sorbitol as the substrate is the only method for industrial production, but the existing technology has a long fermentation cycle, resulting in high production costs and difficult control of product quality.

[0003] At present, domestic and foreign research on the production of L-sorbose by microbial fermentation mainly focuses on the optimization and control of fermentation conditions, but simple fermentation optimization cannot shorten the fermentation cycle well, metabolic engineering as a rational strain Transformation method can more effectively achieve high yield of target product

Although there are few reports on the use of metabolic engineering methods to enhance the accumulation of L-sorbose, in view of the fact that sorbitol dehydrogenase (sldhAB) is the key enzyme for Gluconobacter oxydans to convert D-sorbitol into sorbose, and G.oxydans belongs to Prokaryotes have poor mRNA stability. If a certain number of bases A(T) are added after the stop codon of the sldhAB gene, the mRNA can be enhanced by transcription with a poly(A / T) tail at the 3' end The stability of mRNA, and thus the enhancement of this sorbitol oxidative dehydrogenation pathway, should enable efficient dehydrogenation of D-sorbitol to L-sorbose, thus being an effective strategy to further enhance the performance of G.oxydans in sorbose production