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CN-122012568-A - Pseudomonas putida KT2440 endogenous PUTR library, construction method and engineering bacteria for efficiently synthesizing rhamnolipid

CN122012568ACN 122012568 ACN122012568 ACN 122012568ACN-122012568-A

Abstract

The invention discloses a pseudomonas putida KT2440 endogenous PUTR library, a construction method and an engineering bacterium for efficiently synthesizing rhamnolipid, which take KT2440 as a host bacterium, heterologously express rhamnolipid synthesis key genes, construct recombinant plasmids, introduce the recombinant plasmids into the host bacterium to obtain an engineering strain, ferment and culture the wild type and the engineering strain under the same condition, sample and carry out comparative transcriptome analysis in different growth stages, acquire PUTR candidate elements at the upstream of a key gene sequence starting site based on transcriptome data and key genes, connect the PUTR candidate elements to a carrier genome containing fluorescent protein, introduce the PUTR candidate elements into host bacterium competent cells to obtain the genetic engineering strain, and screen different PUTR elements to optimize key genes in rhamnolipid synthesis paths to obtain the engineering bacterium. The invention realizes the accurate regulation and control of carbon flow distribution, obviously improves the synthesis efficiency of rhamnolipid, and verifies the engineering applicability and practical value of the rhamnolipid in the synthesis of high-added-value biosurfactants.

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 method for constructing an endogenous PUTR library of pseudomonas putida KT2440, comprising the steps of: (1) Taking pseudomonas putida KT2440 as a host bacterium, and heterologously expressing a rhamnolipid synthesis key gene in the host bacterium to construct a recombinant plasmid pBBR1MCS5-rhlAB; (2) Introducing the recombinant plasmid pBBR1MCS5-rhlAB constructed in the step (1) into host bacteria to obtain recombinant engineering strain P.putida KT2440/pBBR1MCS5-rhlAB; (3) Transcriptome data acquisition, namely fermenting and culturing the wild pseudomonas putida KT2440 and the recombinant engineering strain P.putida KT2440/pBBR1MCS5-rhlAB under the same fermentation and culture conditions, respectively sampling at different growth stages, and comparing transcriptome analysis; (4) Obtaining a PUTR candidate element based on the transcriptome data analysis result and the selected key gene, wherein the PUTR candidate element is positioned at the upstream of the sequence start site of the key gene; (5) Constructing a recombinant expression vector, wherein the candidate PUTR candidate element is operably connected to a vector genome capable of expressing a fluorescent protein in a host bacterium competent cell to obtain the recombinant expression vector; (6) And (3) characterization of the PUTR library, namely introducing the recombinant expression vector obtained in the step (5) into competent cells of host bacteria to obtain a genetic engineering strain containing the recombinant expression vector, and determining fluorescent protein expression intensities at different growth stages and thallus growth conditions of the corresponding genetic engineering strain to determine the Pseudomonas putida KT2440 endogenous PUTR library.
  2. 2. The method for constructing a pseudomonas putida KT2440 endogenous PUTR library according to claim 1, wherein said PUTR candidate element is selected from at least one of the nucleotide sequences set forth in SEQ ID No.1 to SEQ ID No. 80.
  3. 3. The method for constructing an endogenous PUTR library of Pseudomonas putida KT2440 according to claim 1, wherein the key genes comprise rhlA genes and/or rhlB genes, wherein the rhlA genes and the rhlB genes are derived from Pseudomonas aeruginosa PAO1 and driven by Lac promoter.
  4. 4. The method for constructing Pseudomonas putida KT2440 endogenous PUTR library according to claim 3, wherein the nucleotide sequence of the Lac promoter is shown as SEQ ID NO.81, the nucleotide sequence of the rhlA gene is shown as SEQ ID NO.82, and the nucleotide sequence of the rhlB gene is shown as SEQ ID NO. 83.
  5. 5. The method for constructing pseudomonas putida KT2440 endogenous PUTR library according to claim 1, wherein said fluorescent protein is selected from the group consisting of green fluorescent protein, enhanced green fluorescent protein and super-folded fluorescent protein.
  6. 6. A pseudomonas putida KT2440 endogenous PUTR library constructed according to the construction method of any one of claims 1-5.
  7. 7. A recombinant genetically engineered strain constructed based on the pseudomonas putida KT2440 endogenous PUTR library of claim 6.
  8. 8. The method for constructing a recombinant genetic engineering strain according to claim 8, comprising the steps of: (a) Taking pseudomonas putida KT2440 as a host bacterium, and heterologously expressing dTDP-L-rhamnose synthesis key genes in the host bacterium to construct a recombinant expression plasmid pBBR1MCS 5-Plac-rhlAB-Plac-rmlBDAC for rhamnolipid synthesis; (b) Introducing the recombinant expression plasmid constructed in the step (a) into pseudomonas putida KT2440 to obtain a recombinant genetic engineering strain P.putida KT2440/pBBR1MCS5-rhlAB for synthesizing rhamnolipid; (c) Screening PUTR combinations with different expression intensities at plasmid levels based on the Pseudomonas putida KT2440 endogenous PUTR library, and carrying out combination optimization on the expression levels of rhlAB genes and rmlBDAC genes; (d) The Pseudomonas putida KT2440 is used as an initial strain, an adaptive PUTR element is screened, and the native promoters of aceE and gltA are replaced on the genome.
  9. 9. The method for constructing a recombinant genetic engineering strain according to claim 8, wherein the dTDP-L-rhamnose synthesis key gene is derived from pseudomonas aeruginosa PAO1, including rmlA gene, rmlB gene, rmlC gene and rmlD gene, and the genes are driven by Lac promoter or PUTR element.
  10. 10. The method for constructing a recombinant genetic engineering strain according to claim 9, wherein the nucleotide sequence of the rmlA gene is shown as SEQ ID NO.84, the nucleotide sequence of the rmlB gene is shown as SEQ ID NO.85, the sequence of the rmlC gene is shown as SEQ ID NO.86, the nucleotide sequence of the rmlD gene is shown as SEQ ID NO.87, the nucleotide sequence of the aceE gene is shown as SEQ ID NO.88, and the nucleotide sequence of the gltA gene is shown as SEQ ID NO. 89.

Description

Pseudomonas putida KT2440 endogenous PUTR library, construction method and engineering bacteria for efficiently synthesizing rhamnolipid Technical Field The invention belongs to the technical field of genetic engineering and protein engineering transformation, and particularly relates to a pseudomonas putida KT2440 endogenous PUTR library, a construction method and engineering bacteria for efficiently synthesizing rhamnolipid. Background Pseudomonas putida KT2440 (Pseudomonas putida KT 2440) is a gram-negative soil bacterium that is widely recognized as an engineered host of great potential in the fields of synthetic biology and industrial biotechnology due to its good biosafety (no known virulence factors), strong environmental adaptation capability, and highly flexible metabolic network. The strain can tolerate various toxic compounds, organic solvents and wide-range pH and temperature conditions, and contains abundant metabolic enzyme systems and substance transport systems in genome, so that the strain supports the efficient utilization of various carbon sources. Based on the advantages, pseudomonas putida KT2440 has been used for synthesizing various high-added-value chemicals and biological materials, including polyhydroxyalkanoates, biosurfactants, short-chain alcohols, aromatic compound intermediates, bioplastic precursors and the like, and has wide application prospects in the fields of industrial fermentation, green chemistry, environmental remediation and the like. Rhamnolipid (Rhamnolipids) is a glycolipid biosurfactant consisting of rhamnose and fatty acid, has excellent surface activity, biodegradability and biocompatibility, and has important application value in the fields of petroleum exploitation, environmental remediation, foods, cosmetics, medicines and the like. Traditional rhamnolipid is mainly synthesized naturally by conditional pathogenic bacteria such as pseudomonas aeruginosa and the like, has biosafety risks, and limits the large-scale application of the rhamnolipid in industry. In contrast, pseudomonas putida KT2440 has definite safety attribute and good industrial applicability, but the strain does not have natural synthesis capability of rhamnolipid, and efficient and stable rhamnolipid biosynthesis can be realized by introducing a rhamnolipid synthesis pathway in a heterologous way and performing systematic metabolism and expression regulation. Therefore, how to realize accurate, stable and predictable expression regulation and control of a plurality of key genes in the host becomes one of the core technical problems restricting efficient synthesis of rhamnolipid. Along with the development of synthetic biology and metabolic engineering, precise regulation and control of gene expression become key factors for metabolic pathway optimization and efficient product synthesis. Researchers have developed a variety of gene expression regulation means, such as CRISPR/dCas9 regulation, RNA interference, and RBS engineering, etc., for regulating metabolic flux and balancing cell growth with target product synthesis. Wherein, the promoter is used as a core element of transcription regulation, and the expression intensity of downstream proteins is directly influenced by controlling the transcription level of mRNA. At present, although the expression intensity range of a synthetic promoter library constructed based on mutation can be expanded to a certain extent, the sequence homology is higher, and the problems of unexpected homologous recombination, insufficient stability and the like exist. In contrast, the natural promoter derived from the host genome has the characteristics of high sequence diversity and large transcription strength span, and is more beneficial to realizing long-term stable and predictable expression regulation and control. Further research shows that the requirement of complex metabolic pathways on expression fineness is often difficult to meet by purely relying on promoters to regulate transcription levels. The 5' untranslated region (5 ' Untranslated Region,5' UTR) plays a key role in regulating mRNA stability, ribosome binding efficiency and translation initiation rate, and has an important influence on the final protein expression level. Therefore, the Promoter and the 5' UTR are designed and screened as a whole, and a combined regulatory element of the Promoter-5' UTR (Promoter-5 ' UTR, PUTR) is constructed, so that the coordinated regulation and control are realized at the two layers of transcription and translation, and the metabolic flux is regulated more accurately. Although synthetic promoter libraries and UTR regulatory strategies have been more systematically studied and applied in model microorganisms (e.g., E.coli, B.subtilis), their suitability in P.putida is still limited, especially with regard to the systematic mining, characterization and engineering of the host endogenous PUTR assembly elements. Therefore, development of a screening and construct