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CN-122012569-A - Efficient synthetic engineering bacterium for rhamnolipid based on glucose metabolism reconstruction and construction method thereof

CN122012569ACN 122012569 ACN122012569 ACN 122012569ACN-122012569-A

Abstract

The invention discloses a rhamnolipid efficient synthesis engineering bacterium based on glucose metabolism reconstruction and a construction method thereof, wherein the construction method takes pseudomonas putida KT2440 or a flag gene cluster knockout strain thereof as an original strain, optimizes the genome thereof through genetic modification, introduces recombinant plasmids to obtain the efficient rhamnolipid synthesis engineering strain, and the modification comprises at least one of (1) heterologous expression rhamnolipid synthesis related genes, (2) substitution of rmlA genes by utilizing genes rmlA, (3) knockout of related genes encoding Gad, (4) heterologous expression of galP genes encoding glucose transport proteins, (5) heterologous expression of glf genes encoding glucose transport proteins and (6) overexpression of glk genes encoding glucose kinase. The invention has the beneficial effects of realizing the efficient directional distribution of glucose to the synthetic route of rhamnolipid and obviously improving the synthetic efficiency of rhamnolipid.

Inventors

  • LIU ZHIQIANG
  • WU ZIDAN
  • PANG AIPING
  • HUANG LIANGGANG
  • ZHANG BO
  • ZHENG YUGUO

Assignees

  • 浙江工业大学

Dates

Publication Date
20260512
Application Date
20260228

Claims (10)

  1. 1. A construction method of a rhamnolipid efficient synthetic engineering bacterium based on glucose metabolism reconstruction is characterized by taking pseudomonas putida KT2440 or a flag gene cluster knockout strain thereof as an original strain, optimizing the genome of the original strain through genetic modification, and then introducing a recombinant plasmid pBBR1MCS5-rhlAB-rmlBDA x C into the genetic modification strain to obtain a target engineering strain, wherein the genetic modification comprises at least one of the following (1) to (6): (1) Heterologous expression of rhamnolipid synthesis-related genes; (2) Replacement of rmlA gene with mutant rmlA; (3) Knocking out related genes encoding a gluconic acid-2-dehydrogenase complex enzyme system; (4) Heterologous expression of a galP gene encoding a glucose transporter; (5) Heterologous expression of a glf gene encoding a glucose transporter; (6) The glk gene encoding glucokinase is overexpressed.
  2. 2. The construction method according to claim 1, wherein the rhamnolipid synthesis related gene is derived from pseudomonas aeruginosa PAO1, including rhlA, rhlB, rmlB, rmlC and/or rmlD genes, the gene rmlA is derived from Salmonella ENTERICA LT2, and the rhamnolipid synthesis related gene and the gene rmlA are both driven by Lac promoter.
  3. 3. The construction method according to claim 2, wherein the Lac promoter has a nucleotide sequence shown in SEQ ID No.1, the rhlA gene has a nucleotide sequence shown in SEQ ID No.2, the rhlB gene has a nucleotide sequence shown in SEQ ID No.3, the mutant rmlA has a nucleotide sequence shown in SEQ ID No.4, the rmlB gene has a nucleotide sequence shown in SEQ ID No.5, the rmlC gene has a nucleotide sequence shown in SEQ ID No.6, the rmlD gene has a nucleotide sequence shown in SEQ ID No.7, the rhlAB gene has a nucleotide sequence shown in SEQ ID No.8, and the rmlBDAC gene has a nucleotide sequence shown in SEQ ID No. 9.
  4. 4. The method according to claim 1, wherein the gene encoding the glucono-2-dehydrogenase complex enzyme system is at least one selected from the group consisting of gene pp_3382, gene pp_3383, gene pp_3384, gene pp_3623 and gene pp_4232.
  5. 5. The construction method according to claim 4, wherein the nucleotide sequence of the gene PP_3382 is shown in SEQ ID NO.10, the nucleotide sequence of the gene PP_3383 is shown in SEQ ID NO.11, the nucleotide sequence of the gene PP_3384 is shown in SEQ ID NO.12, the nucleotide sequence of the gene PP_3623 is shown in SEQ ID NO.13, and the nucleotide sequence of the gene PP_4232 is shown in SEQ ID NO. 14.
  6. 6. The construction method according to claim 1, wherein the galP gene is derived from ESCHERICHIA COLI W to 3110 and has a nucleotide sequence shown in SEQ ID NO.15, the glf gene is derived from Zymomonas mobilis and has a nucleotide sequence shown in SEQ ID NO.16, and the glk gene has a nucleotide sequence shown in SEQ ID NO. 17.
  7. 7. A genetically engineered bacterium obtainable by the construction method according to any one of claims 1 to 6.
  8. 8. The use of the genetically engineered bacterium obtained by the construction method of any one of claims 1 to 6 or the genetically engineered bacterium of claim 7 in the preparation of rhamnolipid by fermentation.
  9. 9. A production method of rhamnolipid is characterized by comprising the steps of fermenting and culturing genetically engineered bacteria obtained by the construction method of any one of claims 1-6 or the genetically engineered bacteria of claim 7 to obtain rhamnolipid.
  10. 10. The production method according to claim 9, characterized by comprising: inoculating the single colony of the selected genetically engineered bacteria into LB liquid medium, culturing at 30-37 ℃ and 150-200 rpm for 12-15 h serving as seed liquid, and Inoculating the seed solution into a fermentation culture medium with an inoculum size of 1%, culturing for 2-4 days at 30-37 ℃ and 150-200 rpm, taking the fermentation solution, and detecting the yield of rhamnolipid.

Description

Efficient synthetic engineering bacterium for rhamnolipid based on glucose metabolism reconstruction and construction method thereof Technical Field The invention belongs to the technical field of genetic engineering reconstruction, and relates to a rhamnolipid efficient synthesis engineering bacterium based on glucose metabolism reconstruction and a construction method thereof. Background Rhamnolipid (Rhamnolipids) is a glycolipid biosurfactant formed by connecting a rhamnose group and beta-hydroxy fatty acid through glycosidic bond and ester bond, has excellent surface tension reducing and interfacial tension reducing capabilities, and simultaneously has good biodegradability, biocompatibility and antibacterial activity. Because the rhamnolipid has renewable sources and is environment-friendly, the rhamnolipid has wide application prospect in the fields of food, medicine, cosmetics, agriculture, petroleum exploitation, environmental remediation and the like, and is one of the biosurfactants with the most intensive research and application at present. Currently, rhamnolipids are mainly obtained by fermentation from pseudomonas aeruginosa (Pseudomonas aeruginosa). However, the strain has potential pathogenicity and low biological safety level, and is strictly limited in the fields of industrial production, food, medicine and the like. In addition, the synthesis of rhamnolipid in pseudomonas aeruginosa is influenced by a complex regulation network, so that the yield fluctuation is large, and the stable and controllable large-scale production is not facilitated. Therefore, the construction of engineering strains with high safety and clear genetic background and suitable for industrial application becomes an important development direction for green production of rhamnolipid. Pseudomonas putida (Pseudomonas putida) KT2440 is an industrial chassis strain which is widely researched and applied, has the advantages of strong metabolic capability, good environmental adaptability, mature genetic operation system, lack of virulence factors and the like, and has good potential in the synthesis of various bio-based chemicals and high added value products. However, pseudomonas putida KT2440 is not a natural rhamnolipid producing bacterium, lacks key functional genes and complete metabolic modules necessary for rhamnolipid synthesis in the genome, and cannot synthesize rhamnolipid in a natural state. Therefore, if KT2440 is used as a host to achieve biosynthesis of rhamnolipids, the relevant synthetic pathway must be introduced and reconstructed by genetic engineering means. The biosynthesis process of rhamnolipids relies on three key metabolic modules coupled to each other, the sugar module, the fatty acid module and the rhamnolipid synthesis module. Wherein glucose is used as a carbon source, not only provides a carbon skeleton for the synthesis of rhamnose groups, but also generates acetyl-CoA through central carbon metabolism, and further generates a lipid chain precursor through a fatty acid de novo synthesis pathway. Therefore, the way glucose is taken up and its metabolic partition within the cell determines the material and energy supply efficiency between the sugar and fatty acid modules, with central carbon metabolism acting as a pivotal regulator in the overall rhamnolipid synthesis network. In pseudomonas putida KT2440, glucose is metabolized primarily by the periplasmic space oxidation pathway and the ATP-dependent active transport pathway. A large amount of glucose is oxidized to gluconic acid and its derivatives before entering the cytoplasm, and is secreted out of the cell with a certain proportion of intermediate products, resulting in loss of carbon source. Meanwhile, the ATP dependent glucose transport system can obviously increase the energy burden of cells under the high-flux metabolism condition, and reduce the distribution ratio of carbon sources to the synthesis path of target products. This mode of glucose metabolism, which is directed towards the formation of environmental adaptations, presents a significant disadvantage in engineering systems aimed at the efficient synthesis of rhamnolipids. Therefore, an engineering method is needed to take the pseudomonas putida KT2440 as a host, reconstruct a glucose metabolism network through a system, and simultaneously, endow the rhamnolipid synthesis capability, reduce the loss of carbon sources and energy consumption and improve the directional distribution efficiency of central carbon metabolism to the rhamnolipid synthesis path so as to overcome the defects in the prior art. Therefore, the construction of the efficient synthetic engineering bacteria of the rhamnolipid based on glucose metabolism reconstruction breaks through the bottleneck of the prior art, and the industrial production process of the rhamnolipid is forced to be urgent. Disclosure of Invention In order to solve the problem of a rhamnolipid production engineering strain in the prior ar