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EP-4741505-A1 - NON-CODING SRNA IN CORYNEBACTERIUM GLUTAMICUM AND USE THEREOF

EP4741505A1EP 4741505 A1EP4741505 A1EP 4741505A1EP-4741505-A1

Abstract

Provided is a non-coding small RNA (sRNA) from Corynebacterium glutamicum and use thereof. An RNA sequence of the non-coding sRNA has at least 90% or more sequence identity to a transcription product of a DNA sequence shown in SEQ ID NO.1. Experimental results demonstrate that overexpression of the non-coding sRNA in the recombinant bacterium can significantly increase yields of the branched-chain amino acids such as L-leucine, L-isoleucine, and L-valine and the sugar-to-acid conversion rate, while reducing generation of the byproduct acids. Regarding raw materials, relatively low-cost molasses is selected as a carbon and nitrogen source, corn steep liquor with a high impurity content is discarded, and molasses is converted via enzymatic hydrolysis into a nutrient containing more carbon and nitrogen sources utilizable by strains. In addition, to control a carbon-to-nitrogen ratio in a fermentation broth, a more precise feeding process is selected, which satisfies fermentation requirements of strains and effectively avoids excessively high or low nitrogen content in the fermentation broth, thereby achieving goals of increasing yield and sugar-to-acid conversion rate and reducing the byproduct acid production.

Inventors

  • FAN, Chao
  • LIU, JUN
  • QI, Jiakun
  • CAI, Zhaoning
  • HONG, Hao
  • CHEN, JIANBIN
  • WU, WENZHONG

Assignees

  • Innobio Corporation Limited
  • Innobio (Hei Long Jiang) Limited

Dates

Publication Date
20260513
Application Date
20250321

Claims (20)

  1. A non-coding small RNA (sRNA) from Corynebacterium glutamicum, wherein an RNA sequence of the non-coding sRNA has at least 90% sequence identity to a transcription product of a DNA sequence set forth in SEQ ID NO:1.
  2. The non-coding sRNA according to claim 1, wherein a DNA sequence encoding the non-coding sRNA is set forth in SEQ ID NO:1.
  3. A vector comprising the DNA sequence encoding the non-coding sRNA of claim 2.
  4. A recombinant bacterium expressing the DNA sequence encoding the non-coding sRNA of claim 2, wherein a bacterial strain of the recombinant bacterium is Corynebacterium glutamicum.
  5. The recombinant bacterium according to claim 4, wherein the Corynebacterium glutamicum is selected from Corynebacterium glutamicum IBBH-15, Corynebacterium glutamicum IBCL-1, Corynebacterium glutamicum IBCVQ, Corynebacterium glutamicum CICC21756, Corynebacterium glutamicum ATCC13002, or any combination thereof.
  6. A method for constructing a recombinant bacterium, the method comprising: obtaining a recombinant strain by transferring a recombinant vector comprising a target DNA fragment into a host strain; wherein the target DNA fragment has at least 90% sequence identity to a DNA sequence set forth in SEQ ID NO:1.
  7. The method according to claim 6, wherein a DNA sequence of the target DNA fragment is set forth in SEQ ID NO:1.
  8. A fermentation method for a recombinant bacterium, the method comprising: obtaining a seed culture by inoculating at least one recombinant strain into a seed medium for cultivation; inoculating the seed culture into a fermentation medium for fermentation cultivation; and in the fermentation cultivation, increasing production of branched-chain amino acids by adjusting at least one of: an added amount of inducer, a dissolved oxygen level, pH, and nutrient supply.
  9. The method according to claim 8, wherein the at least one recombinant strain comprises a DNA sequence encoding a non-coding sRNA, and the DNA sequence has at least 90% sequence identity to a DNA sequence set forth in SEQ ID NO:1.
  10. The method according to claim 8 or 9, wherein the recombinant bacterium is an L-leucine-producing strain, the L-leucine-producing strain including at least one of Corynebacterium glutamicum mutant strains IBBH-15, IBCLQ-257, and IBCLQ-257e.
  11. The method according to claim 8 or 9, wherein the seed medium comprises glucose at an initial concentration of 2.5-3.5 wt% and a molasses hydrolysate at an initial concentration of 1-2 wt%, the fermentation medium comprises glucose at an initial concentration of 3.5-4.5 wt% and a molasses hydrolysate at a concentration of 0.5-1.1 wt%, and the molasses hydrolysate is selected from at least one of a beet molasses hydrolysate and a cane molasses hydrolysate.
  12. The method according to any one of claims 8-11, wherein a dissolved oxygen level in the fermentation cultivation is 5-10%; the method further comprising: in the fermentation cultivation, after a cell OD562 increases to 19-21, increasing pH to 6.85-6.95, and when a residual sugar concentration decreases to 1.5-2.5 wt%, starting to feed a glucose solution having a concentration of 40-50 wt% and a molasses hydrolysate having a concentration of 40-50 wt%, controlling a flow rate ratio of the glucose solution to the molasses hydrolysate to be 5:1-10:1, and controlling a ratio of the molasses hydrolysate to the fermentation medium to be 2.5-3.5 wt%; and after the cell OD562 increases to 29-31, increasing the pH to 7.05-7.15, after the cell OD562 increases to 39-41, increasing the pH to 7.15-7.25, and controlling the residual sugar concentration to be 1.5-2.5 wt% during the fermentation process.
  13. The method according to claim 8, wherein the recombinant bacterium is an L-isoleucine-producing strain, the L-isoleucine-producing strain comprising at least one of Corynebacterium glutamicum mutant strains IBCIL-253, IBCL-1, and IBCIL-253k.
  14. The method according to claim 8 or 13, wherein the seed medium comprises glucose at an initial concentration of 4.5-5.5 wt% and a molasses hydrolysate at an initial concentration of 2.5-3.5 wt%, the fermentation medium comprises glucose at an initial concentration of 9.5-10.5 wt% and a molasses hydrolysate at a concentration of 1.5-2.5 wt%, and the molasses hydrolysate is selected from at least one of a beet molasses hydrolysate and a cane molasses hydrolysate.
  15. The method according to any one of claims 8, 13, and 14, wherein a dissolved oxygen level in the fermentation cultivation is 8-12%; the method further comprising: in the fermentation cultivation, after a cell OD562 increases to 39-41, increasing pH to 6.85-6.95; when a residual sugar concentration decreases to 5.5-6.5 wt%, starting to feed an inorganic salt supplementary solution and controlling a fermentation broth osmolality to be 690-710 mosm/L; and when a residual sugar concentration decreases to 1.5-2.5 wt%, starting to feed a supplementary solution A and a supplementary solution B, the supplementary solution A comprising a glucose solution at a concentration of 40-50 wt%, a potassium dihydrogen phosphate solution at a concentration of 0.1 wt%, and a magnesium sulfate solution at a concentration of 0.05 wt%, the supplementary solution B comprising a molasses hydrolysate at a concentration of 40-50 wt%, controlling a flow rate ratio of the supplementary solution A to the supplementary solution B to be 4:1-7:1, controlling a ratio of the molasses hydrolysate to the fermentation medium to be 3.5-4.5 wt%, and continuing to feed the inorganic salt supplementary solution to control the fermentation broth osmolality to be 890-910 mosm/L.
  16. A separation and purification method for a fermentation product, the method comprising: redissolving the fermentation product using p-toluenesulfonic acid, wherein a molar ratio of the fermentation product to the p-toluenesulfonic acid is 1:1-1:2; performing a hydrogen bond network reconstruction on the redissolved fermentation product using 0.5-1.5 M inorganic acid; decolorizing the fermentation product after cooling crystallization for 30-50 min using 1-2% malic acid, 0.1-0.2% tartaric acid, and 0.5-1.5‰ activated carbon; and purifying the decolorized fermentation product by ion exchange resin and evaporation crystallization to obtain a purified fermentation product.
  17. The method according to claim 16, wherein after ion exchange resin and before evaporation crystallization, the method further comprises: eluting and recovering p-toluenesulfonic acid using 0.3-0.8 M hydrochloric acid, and regenerating the resin using 2.5-3.5 M hydrochloric acid.
  18. The method according to claim 16 or 17, wherein after the hydrogen bond network reconstruction and before decolorization, the method further comprises: cooling the fermentation product after the hydrogen bond network reconstruction to 9-11°C at a cooling rate of 8-12°C/h, and performing seed crystallization for 0.5-1.5 h.
  19. The method according to any one of claims 16-18, wherein before redissolution, the method further comprises: performing complex precipitation on a fermentation broth containing the fermentation product using a composition of polypeptides and metal ions to precipitate the fermentation product.
  20. The method according to any one of claims 16-19, wherein the fermentation product is a Corynebacterium glutamicum fermentation product.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority of the Chinese application filed on March 22, 2024, with the application number 202410335441.0, the entire contents of which are incorporated herein by reference. TECHNICAL FIELD The present disclosure relates to the field of genetic engineering technologies, and in particular to a non-coding small RNA (sRNA) from Corynebacterium glutamicum and its use. BACKGROUND Branched-chain amino acids (c) possess a variety of physiological functions and have very broad applications in industries such as food, animal feed, cosmetics, and medicine. In the early stage, BCAAs were mainly produced by chemical synthesis; however, the chemical synthesis had a low yield and caused severe environmental pollution, thus being gradually eliminated. Subsequently, microbial fermentation attracted widespread attention. At present, strains used for producing the BCAAs mainly include Brevibacterium flavum, Escherichia coli, and Corynebacterium glutamicum. The biosynthetic pathway of the BCAAs is lengthy and has a complex and precise self-adjustment mechanism. In natural strains, the biosynthetic pathway of amino acids is strictly controlled by metabolism to ensure that amino acids are produced only for intrinsic needs of cells. Therefore, production of the BCAAs through strain modification has attracted increasing attention. With technological advancements, metabolic modification is gradually becoming a main manner for obtaining high-yield amino acid strains. However, the industrial stability of the strains subjected to extensive metabolic modification is not ideal. Although metabolic engineering modification can improve the yield of the amino acids to some extent, as primary metabolites, on one hand, weakening of a synthesis pathway of by-products and metabolic burden caused by adding synthesis modules weakens growth of the strains; on the other hand, key enzymes on the synthesis pathway are mostly subjected to feedback inhibition from intermediate metabolites and final products. A balance between sufficient precursors and alleviation of the feedback inhibition cannot be stably achieved in industrial production. In summary, due to a lack of in-depth understanding and application of fine adjustable mechanisms of microorganisms, limitations exist in optimizing performance of the strains, enhancing stability, and further increasing yield of target products. Bacterial non-coding small RNAs (sRNAs) are a class of RNA regulators discovered in prokaryotes such as bacteria in recent years. The non-coding sRNAs do not encode proteins, have a length of 50-500 nucleotides, and are extensively involved in regulation of a plurality of life activities in vivo. Bacterial sRNAs are important regulatory factors of bacterial metabolism, virulence, and adaptation to environmental stress, and play an important role in gene expression adjustment in response to environmental changes. However, an action mechanism of non-coding sRNAs in regulating the amino acid biosynthetic pathway is still unclear, and technologies for targeted design and utilization of sRNAs for high-efficiency and precise regulation are lacking. Therefore, it is necessary to provide a non-coding sRNA from Corynebacterium glutamicum and its use, so as to efficiently, low-costly, and continuously separate and purify fermented BCAAs, increasing product purity, reducing impurity content, and achieving recycling of key raw materials. SUMMARY One or more embodiments of the present disclosure provide a non-coding small RNA (sRNA) from Corynebacterium glutamicum. An RNA sequence of the non-coding sRNA has at least 90% sequence identity to a transcription product of a DNA sequence set forth in SEQ ID NO: 1. In some embodiments, a DNA sequence encoding the non-coding sRNA is set forth in SEQ ID NO:1. One or more embodiments of the present disclosure provide a vector comprising the DNA sequence encoding the non-coding sRNA. One or more embodiments of the present disclosure provide a recombinant bacterium expressing the DNA sequence encoding the non-coding sRNA, wherein a bacterial strain of the recombinant bacterium is Corynebacterium glutamicum. In some embodiments, the Corynebacterium glutamicum is selected from Corynebacterium glutamicum IBBH-15, Corynebacterium glutamicum IBCL-1, Corynebacterium glutamicum IBCVQ, Corynebacterium glutamicum CICC21756, Corynebacterium glutamicum ATCC13002, or any combination thereof. One or more embodiments of the present disclosure provide a method for constructing a recombinant bacterium. The method comprises the following step. A recombinant strain is obtained by transferring a recombinant vector comprising a target DNA fragment into a host strain, wherein the target DNA fragment has at least 90% sequence identity to a DNA sequence set forth in SEQ ID NO: 1. In some embodiments, a DNA sequence of the target DNA fragment is set forth in SEQ ID NO: 1. One or more embodiments of the present