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

By constructing an endogenous PUTR library of *Pseudomonas putida* KT2440, we achieved synergistic regulation of transcription and translation, solved the problem of precise expression regulation of rhamnolipid synthesis in *Pseudomonas putida* KT2440, improved the synthesis efficiency of rhamnolipids, and made it suitable for industrial fermentation and the production of biosurfactants.

CN122012568APending Publication Date: 2026-05-12ZHEJIANG UNIV OF TECH
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Patent Information

Application Number
CN202610239201.X
Authority / Receiving Office
CN · China
Patent Type
Applications(China)
Current Assignee / Owner
Filing Date
2026-02-28
Publication Date
2026-05-12

AI Technical Summary

Technical Problem

How to achieve precise, stable and predictable expression regulation of multiple key genes in *Pseudomonas putida* KT2440 to efficiently synthesize rhamnolipids, and solve the problems of unexpected homologous recombination and insufficient stability in traditional methods.

Method used

We constructed an endogenous PUTR library of Pseudomonas putida KT2440, and screened and characterized promoter-5′UTR combinatorial elements using transcriptomics data to achieve synergistic regulation of transcription and translation, thereby optimizing the expression of key genes in the rhamnolipid synthesis pathway.

Benefits of technology

The PUTR library exhibits high genetic stability, making it suitable for long-term industrial fermentation. It significantly improves the synthesis efficiency of rhamnolipids, provides a tool for fine-tuning complex metabolic pathways, and is applicable to the synthesis of high-value-added biosurfactants.

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Abstract

The invention discloses a pseudomonas putida KT2440 endogenous PUTR library, a construction method and engineering bacteria for efficiently synthesizing rhamnolipid. KT2440 is used as host bacteria, key genes are synthesized by heterologous expression of rhamnolipid, recombinant plasmids are constructed, and the host bacteria are introduced to obtain engineering strains; carrying out fermentation culture on the wild type and the engineering strain under the same condition, and carrying out comparative transcriptomics analysis on samples in different growth stages; on the basis of transcriptome data and the key gene, acquiring a PUTR candidate element at the upstream of a starting site of a key gene sequence, connecting the PUTR candidate element to a carrier genome containing fluorescent protein, and introducing into a host bacterium competent cell to obtain a genetic engineering strain; and screening different PUTR elements to optimize key genes in a rhamnolipid synthesis route to obtain the engineering bacteria. The carbon flow distribution is accurately regulated and controlled, the synthesis efficiency of the rhamnolipid is remarkably improved, and the engineering applicability and practical value of the rhamnolipid in synthesis of the high-added-value biosurfactant are verified.
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