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CN-121975707-A - Recombinant chassis cell for high-yield ergothioneine and construction method and application thereof

CN121975707ACN 121975707 ACN121975707 ACN 121975707ACN-121975707-A

Abstract

The invention discloses a recombinant chassis cell for high-yield ergothioneine, and a construction method and application thereof, belonging to the technical field of genetic engineering. And integrating the key synthetic gene (egtA, egtB, egtC, egtD) into the rrn gap of the high expression site by using a CRISPR-Cas9 homologous recombination technology. The native promoters were replaced with strong promoters Ptac or T7, and adjustable expression modules (different strength promoters+RBS strength libraries) were set for each key gene to optimize expression ratios. By inhibiting or deleting metabolic branches competing with EGT synthesis, key metabolic nodes competing with EGT precursors are identified and expression is attenuated metJ, sdaA, tnaA using CRISPRi or promoter substitution. And (3) carrying out directional transformation and high-throughput screening on the catalytic sites of key enzymes (such as EgtB and EgtC), and finally realizing high-flow synthesis and high yield of the EGT in cells.

Inventors

  • WANG XIAOXIA
  • YOU XIAOSHAN
  • Lin Rumeng
  • YU CHUNHUA
  • XU JIE

Assignees

  • 仙池生物医药(杭州千岛湖)有限公司

Dates

Publication Date
20260505
Application Date
20251127

Claims (10)

  1. 1. A recombinant chassis cell for high-yield ergothioneine is characterized in that ESCHERICHIA COLI MG1655 is taken as an engineering chassis strain, a synthetic gene cluster of ergothioneine derived from Mycobacterium smegmatis is integrated in a genome, and expression of metJ gene, sdaA gene and tnaA gene is inhibited by a CRISPRi system; The ergothioneine synthesis gene cluster comprises egtA genes, egtB mutant genes, egtC genes and egtD genes; The egtB mutant is a G146N/V398Y double mutant, and the catalytic efficiency is improved by 3.4 times compared with that of a wild type.
  2. 2. The recombinant chassis cell of claim 1, wherein the expression cassette of the synthetic gene cluster is regulated by a strong promoter and an optimized RBS sequence, and a groEL/groES, dnaK/DnaJ folding partner co-expression module is integrated upstream of the gene cluster, wherein the strong promoter is P_T7 or Ptac.
  3. 3. The recombinant chassis cell of claim 1, wherein the egtA, egtB, egtC, egtD gene is codon optimized to have a GC content of 54.2%.
  4. 4. A method of constructing the recombinant chassis cell of claim 1, comprising the steps of: Cloning the gene of egtA, egtB mutant, egtC and egtD optimized by the codon, and constructing a recombinant expression vector pTrc-EGT; integrating the expression cassette into a chassis cell using CRISPR-Cas9 mediated homologous recombination; Constructing an expression vector pCas-HG containing Cas9-sgRNA, and introducing the expression vector into a chassis cell; positive recombinant strains are obtained through Zeocin resistance screening, qPCR transcription verification and sequencing confirmation.
  5. 5. The method according to claim 4, wherein the egtB mutant is constructed by site-directed mutagenesis PCR, replacing the His146 site of the wild-type egtB gene with Asn and Phe398 site with Tyr, respectively.
  6. 6. A method for producing ergothioneine by fermentation is characterized in that the recombinant chassis cell as defined in claim 1 is used as a production strain, and glucose is used as a substrate for feed fermentation.
  7. 7. The method of claim 6, wherein the fermentation condition is glucose concentration of 30 g/L, pH of 6.0-7.2, temperature of 26-30 ℃, dissolved oxygen of 25-35%, and OD600 approximately equal to 3.0 at induction.
  8. 8. The method of claim 6, wherein the fermentation period is 40-80 hours.
  9. 9. The method of claim 6, wherein the ergothioneine yield is greater than or equal to 8.3 g/L and the purity is greater than or equal to 98.3%.
  10. 10. Use of the recombinant chassis cell of high-yielding ergothioneine according to claim 1 in the production of ergothioneine in the food, cosmetic or pharmaceutical field.

Description

Recombinant chassis cell for high-yield ergothioneine and construction method and application thereof Technical Field The invention belongs to the technical field of genetic engineering, and particularly relates to a recombinant chassis cell for high-yield ergothioneine, and a construction method and application thereof. Background Ergothioneine is a sulfur-containing amino acid derivative, found in ergot at the earliest, and is widely found in fungi, actinomycetes, and some cyanobacteria. The molecular structure of the modified polyurethane contains sulfinyl (thione) and imidazole rings, so that the modified polyurethane has extremely strong antioxidant capacity and free radical scavenging activity. Ergothioneine cannot be synthesized by itself in mammals and needs to be taken up by food. Research shows that EGT can effectively protect mitochondrial function, reduce oxidative damage, delay cell aging, regulate immune system and improve skin photoaging resistance. Therefore, the modified starch is widely applied to the fields of functional foods, nutritional health products, pharmaceutical auxiliary materials, high-end cosmetic raw materials and the like, and the market demand is rapidly growing. The expression of key enzyme for EGT synthesis is enhanced through genome editing, a competitive pathway is restrained to promote substrate flow, and protein engineering is utilized to carry out directional mutation modification on the key enzyme catalytic site, so that the synthesis efficiency and purity of EGT are obviously improved. The technology breaks through the yield bottleneck of traditional fungus fermentation, builds a high-efficiency biosynthesis platform capable of being stably expressed in escherichia coli, and provides a brand-new technical path for industrialized and sustainable production of the EGT. At present, the industrial production of ergothioneine mainly depends on the fermentation mode of natural fungi or actinomycetes, but the method generally has the problems of long growth period, low yield, complex extraction, high cost and the like, and is difficult to meet the requirements of large-scale and stable production. In recent years, researchers have attempted to reconstruct the synthetic pathway of EGT in highly operable hosts (e.g., escherichia coli, pichia pastoris) by metabolic engineering in order to achieve efficient biological production. However, the existing synthesis system still has multiple bottlenecks, which restrict the improvement of yield and conversion efficiency. First, key enzymes (e.g., egtB, egtC) in the EGT synthesis pathway have low catalytic rates, and insufficient substrate affinity and substrate specificity, which are core bottlenecks that limit metabolic flux. Second, histidine and a sulfur source in the host cell are involved in multiple metabolic pathways (e.g., glutathione and methionine synthesis) simultaneously, resulting in a large split of EGT synthesis substrates and limited product accumulation. In addition, the expression level and folding efficiency of different synthetases are not coordinated, which is easy to cause accumulation of intermediates, metabolic blocking or increase of byproducts, and the overall conversion efficiency is reduced. In heterologous expression systems, EGT synthases are prone to misfolding or inactivation problems, affecting enzymatic activity and system stability. On the other hand, the current fermentation process is at laboratory shake flask level, and dynamics control and feeding optimization of a system are not available, so that large-scale production is difficult to realize. In summary, the prior art cannot realize efficient, stable and economical synthesis of ergothioneine in a controllable host, and there is an urgent need to construct a novel bio-manufacturing system that simultaneously strengthens the key enzyme activity, suppresses competition pathways and optimizes metabolic balance, so as to break through the yield bottleneck and meet the industrial application requirements. Disclosure of Invention This section is intended to outline some aspects of embodiments of the application and to briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section as well as in the description of the application and in the title of the application, which may not be used to limit the scope of the application. The present invention has been made in view of the above and/or problems occurring in the prior art. Therefore, the invention aims to overcome the defects in the prior art and provide a recombinant chassis cell for high-yield ergothioneine. In order to solve the technical problems, the invention provides a recombinant chassis cell for high-yield ergothioneine, which is characterized in that ESCHERICHIA COLI MG1655 is taken as an engineering chassis strain, a synthetic gene cluster of ergothioneine derived from Mycobacterium smegmatis is integrated in a genome, and expression of metJ