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CN-121992046-A - Process for preparing tryptophane and its new use

CN121992046ACN 121992046 ACN121992046 ACN 121992046ACN-121992046-A

Abstract

The invention discloses a production method and new application of tryptophane. Co-culturing Streptomyces lungii and Candida albicans on a solid culture medium to obtain tryptophane. The compound has remarkable anti-tumor activity, has the IC 50 of 154.5 mug/mL, 144.3 mug/mL, 122.6 mug/mL and 110.7 mug/mL for human lung cancer, mouse colon cancer, human liver cancer and human breast cancer cells, and has important value as a key intermediate of a plurality of medicaments. The invention utilizes a microbiological synthesis method to produce the chromanol and improve the yield, and introduces the synthesis gene of the chromanol into the escherichia coli C41 to successfully express the chromanol, and after further optimizing the synthesis path of the chromanol, the yield of the chromanol is improved to 149.76 mg/L. The invention combines the microorganism co-culture and the genetic engineering technology for synthesizing the color alcohol, and has profound significance for producing the color alcohol by microorganism fermentation and promoting the synthesis to be converted into the green energy-saving direction.

Inventors

  • LI YUE
  • TAN HUARONG
  • XIA XIULEI

Assignees

  • 首都医科大学附属北京口腔医院

Dates

Publication Date
20260508
Application Date
20251219

Claims (10)

  1. 1. A method for producing color alcohol by co-culturing microorganisms, which is characterized in that Streptomyces lungiensis and Candida albicans are co-cultured, and the color alcohol is produced by inducing the expression of silenced color alcohol synthesis genes in Candida albicans (Candida albicans) by Streptomyces lungiensis (Streptomyces longshengensis).
  2. 2. The method according to claim 1, wherein the tryptophane is obtained by co-culturing streptomyces lungigensis with candida albicans on a solid medium; preferably, the solid medium is MS medium.
  3. 3. The method according to claim 1 or 2, wherein the Streptomyces lungiensis has a preservation number of CGMCC 4.1101 and the Candida albicans has a preservation number of CGMCC 2.4159.
  4. 4. The engineering bacterium of the escherichia coli for producing the color alcohol is characterized in that after the color alcohol synthesis gene derived from candida albicans Candida albicansSC and 5314 are subjected to codon optimization, the color alcohol synthesis gene is introduced into the escherichia coli through plasmids or is integrated on a chromosome of the escherichia coli (ESCHERICHIA COLI) through genetic engineering means; among them, the color alcohol synthesis genes derived from Candida albicans SC5314 are aro9CA, aro10CA and adh1CA.
  5. 5. The engineering bacterium according to claim 4, wherein the nucleotide sequences of the optimized genes aro9CA, aro10CA and adh1CA are shown in SEQ ID NO. 1-3 respectively.
  6. 6. The engineering bacterium according to claim 5, wherein the optimized genes aro9CA, aro10CA and adh1CA are sequentially connected in series and then constructed on a pET expression vector, and introduced into escherichia coli; Preferably, the expression vector is pET28a.
  7. 7. The engineered bacterium of any one of claims 4-6, wherein the starting strain is escherichia coli C41.
  8. 8. Use of the engineering bacteria of any one of claims 4-7 in the fermentation production of tryptophane.
  9. 9. A process for producing tryptophane, which comprises fermenting the engineering bacterium of any one of claims 4 to 7 in a medium containing tryptophan, and separating the tryptophane from the fermentation product.
  10. 10. New use of tryptophane in preparing antitumor drug; The tumor comprises lung cancer, liver cancer, colon cancer and breast cancer.

Description

Process for preparing tryptophane and its new use Technical Field The invention relates to the field of biological medicine, in particular to a production method and new application of tryptophane. Background The bacteria (mainly actinomycetes) and fungi have huge natural product libraries, and the microbial natural products are used as key source springs of novel active micromolecular medicaments, have huge development and application values, and are widely applied to the fields of clinic, agriculture, animal husbandry and the like. With the rapid development of the third generation whole genome sequencing technology, researchers have been provided with great potential for synthesizing various secondary metabolites, genome mining technology further reveals that a large number of uncharacterized or unexpressed' silent metabolic pathways exist in bacteria and fungi, and products are called silent secondary metabolites, so that the novel bacterial strain is gradually a precious resource [1] for novel drug mining along with the reduction of newly discovered antibiotic types and the enhancement of bacterial strain drug resistance. Thus, activating secondary metabolic pathways in microorganisms is a central task in the discovery of novel secondary metabolites. The microbial co-culture is one of effective ways for excavating active natural products, and has important scientific significance for evaluating the activity of the products and improving the yield of the valuable products. In natural environment, ecological relationship of microorganisms is complicated, and they are related to each other through various modes of co-location, intergrowth, symbiosis, antagonism and the like. Standard laboratory culture often ignores this complexity, resulting in reduced secondary metabolite production. The co-culture technology can simulate natural ecology, excite microorganisms to secrete active metabolites, and becomes a leading-edge strategy [2] for excavating new molecules. The activation mechanism is also more complex, such as ① nutrient acquisition, in which the diffused small molecule is used as a precursor or substrate to induce another strain to produce new metabolites, ② competition and signaling, exogenous molecules are used as chemical defense means to produce antibiotics or signaling molecules to participate in competition, ③ physical contact, direct cell-to-cell contact can activate silent gene cluster expression, but the specific mechanism is not clear [3]. Studies have shown that bacterial-fungal interactions can be broadly divided into 3 classes: ① bacterial-bacterial co-culture to activate natural products: huang Fenzhi bacillus and streptomyces coelicolor co-culture under conditions of limited iron element, which trigger the synthesis [4];② of actinorhodin (Actinorhodin) in streptomyces coelicolor in competition for iron, which is an important source of novel active metabolites. For example, pham et al co-cultures basidiomycetes Phellinus orientoasiaticus and Xylodon flaviporus, targeting isolation of 3 new sesquiterpenes (Cyclohumulanoid Sesquiterpenes) and five known analogues [5];③, a mixed fungal and bacterial culture, were more complex, and co-culture of Fusarium Majorana with Streptomyces lividans on solid rice media was able to induce the production of four novel naphthoquinone dimers (fusatricinones A-D) and one novel sequoyipyrone derivative dihydrolateropyrone, [6]. Disclosure of Invention The invention aims to provide a method for producing tryptophane by co-culture of microorganisms. The invention also aims to provide the escherichia coli engineering bacteria for producing the color alcohol, a construction method thereof and application thereof in fermentation production of the color alcohol. It is a further object of the present invention to provide a novel use of tryptophane. In order to achieve the object of the invention, in a first aspect, the invention provides a method for producing chromanol by co-culturing microorganisms, which comprises the steps of co-culturing Streptomyces lunjunction with Candida albicans, and inducing the expression of a silenced chromanol synthesis gene in Candida albicans (Candida albicans) by using Streptomyces lunjunction (Streptomyces longshengensis) to produce chromanol. Further, the tryptophane is obtained by co-culturing Streptomyces lungii and Candida albicans on a solid medium. Preferably, the solid medium is MS medium (mannitol 20 g/L, soybean powder 20 g/L, agar powder 1.5% -2% (w/w)). Preferably, the preservation number of the Streptomyces lungientus is CGMCC 4.1101, and the preservation number of the Candida albicans is CGMCC 2.4159. Both strains are purchased from China general microbiological culture collection center (CGMCC). In a second aspect, the present invention provides an engineering bacterium of escherichia coli producing color alcohol, wherein a color alcohol synthesis gene derived from candida albicans Candida albicans SC and 5314 is